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Environmental Health Criteria 229

SELECTED NITRO- AND NITRO-OXY-POLYCYCLIC AROMATIC HYDROCARBONS

First draft prepared by Drs J. Kielhorn, U. Wahnschaffe and I. Mangelsdorf, Fraunhofer Institute of Toxicology and Aerosol Research, Hanover, Germany

Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals.

World Health Organization

Geneva, 2003

The International Programme on Chemical Safety (IPCS), established in 1980, is a joint venture of the United Nations Environment Programme (UNEP), the International Labour Organization (ILO) and the World Health Organization (WHO). The overall objectives of the IPCS are to establish the scientific basis for assessment of the risk to human health and the environment from exposure to chemicals, through international peer review processes, as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemicals.

The Inter-Organization Programme for the Sound Management of Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and Agriculture Organization of the United Nations, WHO, the United Nations Industrial Development Organization, the United Nations Institute for Training and Research and the Organisation for Economic Co-operation and Development (Participating Organizations), following recommendations made by the 1992 UN Conference on Environment and Development to strengthen cooperation and increase coordination in the field of chemical safety. The purpose of the IOMC is to promote coordination of the policies and activities pursued by the Participating Organizations, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment.

WHO Library Cataloguing-in-Publication Data

Selected nitro- and nitro-oxy-polycyclic aromatic hydrocarbons.

(Environmental health criteria ; 229)

1.Polycyclic hydrocarbons, Aromatic - toxicity 2.Polycyclic hydrocarbons,

Aromatic - adverse effects 3.Environmental exposure 4.Risk assessment

I.International Programme for Chemical Safety II.Series

ISBN 92 4 157229 9        (NLM classification: QD 341.H9)

ISSN 0250-863X

The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available.

©World Health Organization 2003

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The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters.

CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FOR
SELECTED NITRO- AND NITRO-OXY-POLYCYCLIC AROMATIC HYDROCARBONS

PREAMBLE

ACRONYMS AND ABBREVIATIONS

1. SUMMARY

1.1 Identity, physical and chemical properties, and analytical methods

1.2 Sources of human and environmental exposure

1.3 Environmental transport, distribution and transformation

1.3.1 Environmental transport and distribution

1.3.2 Biotransformation

1.3.3 Abiotic degradation

1.4 Environmental levels and human exposure

1.4.1 Indoor air

1.4.2 Food and beverages

1.4.3 Other products

1.4.4 Occupational exposure

1.5 Kinetics and metabolism in laboratory animals and humans

1.6 Effects on laboratory mammals and in vitro test systems

1.7 Effects on humans

1.8 Effects on other organisms in the laboratory and field

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS

2.1 Identity

2.2 Physical and chemical properties

2.3 Conversion factors

2.4 Analytical methods

2.4.1 Sampling

2.4.2 Extraction

2.4.3 Cleanup

2.4.4 Analytical separation and detection

2.4.4.1 Difficulties in analysis

2.4.4.2 Complex mixtures

2.4.4.3 Analysis of nitro-oxyPAHs

2.4.5 Use of bioassay (mutagenicity) fractionation and chemical analysis

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

3.1 Industrially produced nitroPAHs

3.1.1 Production levels and processes

3.1.2 Uses of commercially produced nitroPAHs

3.2 Other sources of nitroPAHs

3.2.1 Direct sources of nitroPAHs from combustion processes

3.2.1.1 Diesel exhaust

3.2.1.2 Diesel compared with gasoline exhaust

3.2.1.3 Aeroplane emissions

3.2.1.4 Emissions from combustion of heating oils

3.2.1.5 Fumes from cooking oils

3.2.1.6 Other combustion sources

3.2.2 Atmospheric formation of nitroPAHs

3.2.2.1 Reactions of gas-phase PAHs (and nitroPAHs) with the hydroxyl radical (daytime reactions)

3.2.2.2 Reactions of gas-phase PAHs (and nitroPAHs) with the nitrate radical (nighttime reactions)

3.3 Oxygen-containing nitroPAHs

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

4.1 Transport and distribution between media

4.1.1 Distribution and transport in the atmosphere

4.1.1.1 Distribution of nitroPAHs between fine and coarse fractions of inhalable atmospheric particulates

4.1.2 Distribution and transport in the hydrosphere

4.1.3 Adsorption onto soils and sediments

4.1.4 Bioaccumulation

4.1.5 Biomagnification

4.2 Transformation

4.2.1 Biotransformation

4.2.1.1 Bacteria

4.2.1.2 Fungi

4.2.1.3 Plants

4.2.1.4 Aquatic animals

4.2.2 Abiotic degradation

4.2.2.1 Direct photolysis

4.2.2.2 Other atmospheric transformations

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

5.1 Environmental levels

5.1.1 Air

5.1.1.1 Ambient air

5.1.1.2 Indoor air

5.1.2 Water

5.1.3 Soil, sewage sludge, sediment and incinerator ash

5.1.4 Food and beverages

5.1.4.1 Food

5.1.4.2 Beverages

5.1.5 Other sources

5.1.5.1 Carbon black and toners

5.1.5.2 Cigarette smoke

5.2 General population exposure

5.3 Occupational exposure

6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS

6.1 Overview of the metabolism of nitroPAHs

6.2 1-Nitropyrene metabolism in mammals

6.2.1 Absorption

6.2.2 Distribution

6.2.3 Metabolism

6.2.3.1 Introduction

6.2.3.2 Identification of metabolites

6.2.3.3 Cytochrome P450-mediated ring C-oxidative pathway

6.2.3.4 Nitroreduction pathway

6.2.3.5 Human and rodent intestinal microflora

6.2.3.6 Suggested metabolic pathway

6.2.4 Elimination and excretion

6.2.4.1 Elimination

6.2.4.2 Excretion

6.2.4.3 Biliary excretion and enterohepatic circulation

6.2.5 Reaction with body components

6.2.5.1 Protein binding

6.2.5.2 DNA adducts

6.2.6 Biomonitoring studies

6.3 Mononitropyrenes (1-, 2- and 4-nitropyrene) — a comparison

6.3.1 Faecal and urinary excretion

6.3.2 Metabolism

6.3.3 DNA adducts

6.4 2-Nitrofluorene

6.4.1 Absorption, distribution and elimination

6.4.2 Metabolism/mechanism of action

6.4.2.1 Metabolites

6.4.2.2 DNA adducts

6.4.2.3 Haemoglobin adducts

6.5 Dinitropyrenes (1,3-, 1,6- and 1,8-dinitropyrene)

6.6 Mononitrobenzo[a]pyrenes (1-, 3- and 6-nitrobenzo[a]pyrene)

6.7 The nitrofluoranthene family

6.8 2- and 9-nitroanthracene

6.9 6-Nitrochrysene

6.10 K- and H-ras mutations in tumours produced by nitroPAHs

6.11 Human enzymes expected to be involved in nitroPAH metabolism

7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

7.1 Acute toxicity

7.1.1 1-Nitronaphthalene

7.1.2 2-Nitronaphthalene

7.1.3 5-Nitroacenaphthene

7.1.4 2-Nitrofluorene

7.1.5 3,9-Dinitrofluoranthene

7.1.6 1-Nitropyrene

7.2 Short-term and long-term exposure (non-neoplastic effects)

7.2.1 1-Nitronaphthalene

7.2.2 5-Nitroacenaphthene

7.2.3 2-Nitrofluorene

7.2.4 1-Nitropyrene

7.2.5 1,3-Dinitropyrene

7.2.6 1,6-Dinitropyrene

7.2.7 1,8-Dinitropyrene

7.2.8 6-Nitrochrysene

7.2.9 1- and 3-nitrobenzo[a]pyrene

7.2.10 1,6-Dinitrobenzo[a]pyrene

7.3 Skin and eye irritation and sensitization

7.4 Reproductive toxicity, embryotoxicity and teratogenicity

7.5 Mutagenicity and related end-points

7.5.1 In vitro genotoxicity studies

7.5.1.1 Salmonella typhimurium microsome assay

7.5.1.2 Comparison of the mutagenic potency of nitroPAHs in the Salmonella microsome assay

7.5.1.3 Studies into the pathways of microbial metabolism

7.5.1.4 Relationship between mutagenic potency in S. typhimurium and the chemical structure of nitroPAHs

7.5.1.5 Bacterial test systems other than the Salmonella microsome assay

7.5.1.6 Eukaryotic test systems

7.5.1.7 High potency in the Salmonella microsome assay in relation to gene mutation results from other in vitro assays

7.5.1.8 Assessment of data on genotoxicity in vitro

7.5.2 In vivo genotoxicity studies

7.5.2.1 Comparison with in vitro results

7.5.3 Genotoxicity of oxygen-containing nitroPAHs

7.5.3.1 3-Nitrobenzanthrone

7.5.3.2 Nitrodibenzopyranones

7.5.3.3 Nitropyrene lactones

7.5.3.4 Comparison of mutation frequency at hprt versus tk locus in human B-lymphoblastoid cell lines

7.5.4 Summary of the genotoxicity of nitroPAHs

7.5.5 Mutagenicity of complex mixtures

7.5.5.1 Difficulties encountered when interpreting the mutagenicity of complex mixtures compared with individual compounds

7.5.5.2 Mutagenicity of diesel engine exhaust

7.5.5.3 Mutagenic effect of urban air samples

7.5.5.4 Bioassay-directed chemical analysis of airborne particulate matter using a human cell mutagenicity assay

7.5.5.5 DNA adducts

7.5.5.6 Mutagenic contribution of selected nitroPAHs from their occurrence in air samples multiplied by the mutagenicity in the Salmonella mutagenicity test

7.5.5.7 Municipal waste incinerator emissions

7.5.5.8 Mutagenicity of soils exposed to automobile exhaust

7.5.6 Effects of antimutagenic compounds on nitroPAH mutagenicity

7.6 Carcinogenicity

7.6.1 Route of administration

7.6.2 Adequacy of data

7.6.3 Type of induced tumours

7.6.4 Ranking of carcinogenic potency in comparative studies on nitroPAHs

7.6.4.1 Comparison of the carcinogenicity of nitroPAHs with parent PAHs

7.6.4.2 Comparison of the dose

7.6.5 Carcinogenicity of oxygen-containing nitroPAHs

7.6.6 Carcinogenicity of the metabolites

7.6.7 Carcinogenic potency of nitroPAHs in diesel exhaust

7.6.8 Genotoxicity in vivo and in vitro versus carcinogenicity

7.6.9 Potency equivalency factors for nitroPAHs

7.6.10 Mechanisms of carcinogenesis

7.7 Special studies: Target organ effects

7.7.1 1-Nitronaphthalene

7.7.2 2-Nitronaphthalene

7.7.3 1-Nitropyrene

8. EFFECTS ON HUMANS

8.1 General population exposure

8.2 Occupational exposure

8.3 Indicators of exposure to nitroPAHs in diesel exhaust

8.3.1 Biomonitoring of exposure/effect

8.3.1.1 DNA adducts

8.3.1.2 Protein adducts

8.3.1.3 1-Nitropyrene metabolites

8.3.1.4 Immunochemical determination

8.3.2 Biomarkers of susceptibility

8.3.2.1 Cytochrome P450

8.3.2.2 Influence of polymorphisms on biomarkers

9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

9.1 Laboratory experiments

9.1.1 Aquatic species

9.1.2 Biotransformation studies in aquatic species

9.1.3 DNA damage in aquatic species

9.2 Field observations

10. EVALUATION OF HUMAN HEALTH RISKS AND EFFECTS ON THE ENVIRONMENT

10.1 Evaluation of human health risks

10.1.1 Exposure levels

10.1.1.1 NitroPAHs

10.1.1.2 Nitroketones

10.1.1.3 Nitrolactones

10.1.2 Fate in the body

10.1.2.1 NitroPAHs

10.1.2.2 Nitroketones

10.1.2.3 Nitrolactones

10.1.3 Toxic effects

10.1.3.1 Non-neoplastic effects

10.1.3.2 Genotoxicity

10.1.3.3 Neoplastic effects

10.1.4 Evaluation of nitroPAHs, nitroketones and nitrolactones that seem to be of importance in the environment

10.2 Evaluation of effects on the environment

10.3 General considerations

10.4 Overall evaluation

11. RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH AND THE ENVIRONMENT

12. RECOMMENDATIONS FOR FURTHER RESEARCH

13. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

REFERENCES

RESUME

RESUMEN

NOTE TO READERS OF THE CRITERIA MONOGRAPHS

Every effort has been made to present information in the criteria monographs as accurately as possible without unduly delaying their publication. In the interest of all users of the Environmental Health Criteria monographs, readers are requested to communicate any errors that may have occurred to the Director of the International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland, in order that they may be included in corrigenda.

* * *

The WHO Environmental Health Criteria Programme is financially supported by the US Environmental Protection Agency, European Commission, German Federal Ministry of the Environment, Nature Conservation, and Nuclear Safety, and Japanese Ministry of Health, Labour and Welfare.

Environmental Health Criteria

PREAMBLE

Objectives

In 1973, the WHO Environmental Health Criteria Programme was initiated with the following objectives:

  1. to assess information on the relationship between exposure to environmental pollutants and human health, and to provide guidelines for setting exposure limits;
  2. to identify new or potential pollutants;
  3. to identify gaps in knowledge concerning the health effects of pollutants;
  4. to promote the harmonization of toxicological and epidemiological methods in order to have internationally comparable results.

The first Environmental Health Criteria (EHC) monograph, on mercury, was published in 1976, and since that time an ever-increasing number of assessments of chemicals and of physical effects have been produced. In addition, many EHC monographs have been devoted to evaluating toxicological methodology, e.g. for genetic, neurotoxic, teratogenic and nephrotoxic effects. Other publications have been concerned with epidemiological guidelines, evaluation of short-term tests for carcinogens, biomarkers, effects on the elderly and so forth.

Since its inauguration, the EHC Programme has widened its scope, and the importance of environmental effects, in addition to health effects, has been increasingly emphasized in the total evaluation of chemicals.

The original impetus for the Programme came from World Health Assembly resolutions and the recommendations of the 1972 UN Conference on the Human Environment. Subsequently the work became an integral part of the International Programme on Chemical Safety (IPCS), a cooperative programme of UNEP, ILO and WHO. In this manner, with the strong support of the new partners, the importance of occupational health and environmental effects was fully recognized. The EHC monographs have become widely established, used and recognized throughout the world.

The recommendations of the 1992 UN Conference on Environment and Development and the subsequent establishment of the Intergovernmental Forum on Chemical Safety with the priorities for action in the six programme areas of Chapter 19, Agenda 21, all lend further weight to the need for EHC assessments of the risks of chemicals.

Scope

The criteria monographs are intended to provide critical reviews on the effects on human health and the environment of chemicals and of combinations of chemicals and physical and biological agents. As such, they include and review studies that are of direct relevance for the evaluation. However, they do not describe every study carried out. Worldwide data are used and are quoted from original studies, not from abstracts or reviews. Both published and unpublished reports are considered, and it is incumbent on the authors to assess all the articles cited in the references. Preference is always given to published data. Unpublished data are used only when relevant published data are absent or when they are pivotal to the risk assessment. A detailed policy statement is available that describes the procedures used for unpublished proprietary data so that this information can be used in the evaluation without compromising its confidential nature (WHO (1990) Revised Guidelines for the Preparation of Environmental Health Criteria Monographs. PCS/90.69, Geneva, World Health Organization).

In the evaluation of human health risks, sound human data, whenever available, are preferred to animal data. Animal and in vitro studies provide support and are used mainly to supply evidence missing from human studies. It is mandatory that research on human subjects is conducted in full accord with ethical principles, including the provisions of the Helsinki Declaration.

The EHC monographs are intended to assist national and international authorities in making risk assessments and subsequent risk management decisions. They represent a thorough evaluation of risks and are not, in any sense, recommendations for regulation or standard setting. These latter are the exclusive purview of national and regional governments.

Content

The layout of EHC monographs for chemicals is outlined below.

Selection of chemicals

Since the inception of the EHC Programme, the IPCS has organized meetings of scientists to establish lists of priority chemicals for subsequent evaluation. Such meetings have been held in Ispra, Italy, 1980; Oxford, United Kingdom, 1984; Berlin, Germany, 1987; and North Carolina, USA, 1995. The selection of chemicals has been based on the following criteria: the existence of scientific evidence that the substance presents a hazard to human health and/or the environment; the possible use, persistence, accumulation or degradation of the substance shows that there may be significant human or environmental exposure; the size and nature of populations at risk (both human and other species) and risks for environment; international concern, i.e., the substance is of major interest to several countries; adequate data on the hazards are available.

If an EHC monograph is proposed for a chemical not on the priority list, the IPCS Secretariat consults with the Cooperating Organizations and all the Participating Institutions before embarking on the preparation of the monograph.

Procedures

The order of procedures that result in the publication of an EHC monograph is shown in the flow chart. A designated staff member of IPCS, responsible for the scientific quality of the document, serves as Responsible Officer (RO). The IPCS Editor is responsible for layout and language. The first draft, prepared by consultants or, more usually, staff from an IPCS Participating Institution, is based on extensive literature searches from reference databases such as Medline and Toxline.

Flow Chart

EHC PREPARATION FLOW CHART

The draft document, when received by the RO, may require an initial review by a small panel of experts to determine its scientific quality and objectivity. Once the RO finds the document acceptable as a first draft, it is distributed, in its unedited form, to well over 150 EHC contact points throughout the world who are asked to comment on its completeness and accuracy and, where necessary, provide additional material. The contact points, usually designated by governments, may be Participating Institutions, IPCS Focal Points or individual scientists known for their particular expertise. Generally some four months are allowed before the comments are considered by the RO and author(s). A second draft incorporating comments received and approved by the Director, IPCS, is then distributed to Task Group members, who carry out the peer review, at least six weeks before their meeting.

The Task Group members serve as individual scientists, not as representatives of any organization, government or industry. Their function is to evaluate the accuracy, significance and relevance of the information in the document and to assess the health and environmental risks from exposure to the chemical. A summary and recommendations for further research and improved safety aspects are also required. The composition of the Task Group is dictated by the range of expertise required for the subject of the meeting and by the need for a balanced geographical distribution.The three cooperating organizations of the IPCS recognize the important role played by nongovernmental organizations. Representatives from relevant national and international associations may be invited to join the Task Group as observers. Although observers may provide a valuable contribution to the process, they can speak only at the invitation of the Chairperson. Observers do not participate in the final evaluation of the chemical; this is the sole responsibility of the Task Group members. When the Task Group considers it to be appropriate, it may meet in camera.

All individuals who as authors, consultants or advisers participate in the preparation of the EHC monograph must, in addition to serving in their personal capacity as scientists, inform the RO if at any time a conflict of interest, whether actual or potential, could be perceived in their work. They are required to sign a conflict of interest statement. Such a procedure ensures the transparency and probity of the process.

When the Task Group has completed its review and the RO is satisfied as to the scientific correctness and completeness of the document, it then goes for language editing, reference checking and preparation of camera-ready copy. After approval by the Director, IPCS, the monograph is submitted to the WHO Office of Publications for printing. At this time, a copy of the final draft is sent to the Chairperson and Rapporteur of the Task Group to check for any errors.

It is accepted that the following criteria should initiate the updating of an EHC monograph: new data are available that would substantially change the evaluation; there is public concern for health or environmental effects of the agent because of greater exposure; an appreciable time period has elapsed since the last evaluation.

All Participating Institutions are informed, through the EHC progress report, of the authors and institutions proposed for the drafting of the documents. A comprehensive file of all comments received on drafts of each EHC monograph is maintained and is available on request. The Chairpersons of Task Groups are briefed before each meeting on their role and responsibility in ensuring that these rules are followed.

WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR SELECTED NITRO- AND NITRO-OXY-POLYCYCLIC AROMATIC HYDROCARBONS

Members

Professor D. Anderson, Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, United Kingdom (Chairperson)

Professor J. Arey, Air Pollution Research Center, University of California, Riverside, California, USA

Dr R.P. Bos, Department of Pharmacology & Toxicology, UMC St. Radboud, University of Nijmegen, Nijmegen, The Netherlands

Dr A. Cecinato, Istituto sull’Inquinamento Atmosferico-CNR, CP10 Monterotondo Stazione, Rome, Italy

Dr K. El-Bayoumy, Division of Cancer Etiology & Prevention, American Health Foundation, Valhalla, New York, USA (Vice-Chairperson)

Dr P.C. Howard, Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas, USA (Co-Rapporteur)

Dr J. Kielhorn, Chemical Risk Assessment, Fraunhofer Institute of Toxicology and Aerosol Research, Hanover, Germany (Co-Rapporteur)

Professor M. Kirsch-Volders, Laboratory of Cell Genetics, Free University of Brussels, Brussels, Belgium

Dr I. Mangelsdorf, Chemical Risk Assessment, Fraunhofer Institute of Toxicology and Aerosol Research, Hanover, Germany

Dr S. Pavittranon, Toxicology and Environmental Laboratory, National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nontaburi, Thailand

Dr H. Tokiwa, Department of Environmental Health Science, Kyushu Women’s University, Kitakyushu, Japan

Dr U. Wahnschaffe, Consultant, Uetze, Germany

Professor Z. Yuxin, Institute of Occupational Medicine, Chinese Academy of Preventive Medicine, Beijing, People’s Republic of China

Secretariat

Mr T. Ehara, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland

Mrs P. Harlley, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland

WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR SELECTED NITRO- AND NITRO-OXY-POLYCYCLIC AROMATIC HYDROCARBONS

The first and second drafts of this monograph were prepared by the authors, Drs J. Kielhorn, U. Wahnschaffe and I. Mangelsdorf.

A WHO Task Group on Environmental Health Criteria for Selected Nitro- and Nitro-oxy-Polycyclic Aromatic Hydrocarbons met at the Fraunhofer Institute of Toxicology and Aerosol Research, in Hanover, Germany, on 26–30 November 2001. The group reviewed the draft and the peer review comments, revised the draft and made an evaluation of the risks for human health and environment from exposure to selected nitro- and nitro-oxy-polycyclic aromatic hydrocarbons.

Dr P. Jenkins and Mr T. Ehara of the IPCS central unit were responsible for the scientific aspects of the monograph, and Ms. Marla Sheffer was responsible for the technical editing.

The efforts of all, especially the Fraunhofer Institute of Toxicology and Aerosol Research, which helped in the preparation and finalization of the monograph, are gratefully acknowledged.

ACRONYMS AND ABBREVIATIONS

BaP

benzo[a]pyrene

bw

body weight

CAS

Chemical Abstracts Service

cDNA

complementary (or copy) DNA

CHO

Chinese hamster ovary

CYP

cytochrome P450

D2

no. 2 diesel fuel

dA

deoxyadenosine

dA-C8-2-AP

N-(deoxyadenosin-8-yl)-2-aminopyrene

DCM

dichloromethane

dG

deoxyguanosine

dG-C8-AAF

N-(deoxyguanosin-8-yl)-2-acetylaminofluorene

dG-C8-AF

N-(deoxyguanosin-8-yl)-2-aminofluorene

dG-C8-1-amino-6-NP

N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene

dG-C8-1-amino-8-NP

N-(deoxyguanosin-8-yl)-1-amino-8-nitropyrene

dG-C8-AP

N-(deoxyguanosin-8-yl)-1-aminopyrene

dG-C8-2-AP

N-(deoxyguanosin-8-yl)-2-aminopyrene

dG-C8-4-AP

N-(deoxyguanosin-8-yl)-4-aminopyrene

dG-N2-AAF

C3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene

dG-1-nitroBaP-DE

10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene

dG-3-nitroBaP-DE

10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]pyrene

6-dG-N2-1-aminoBaP

6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene

6-dG-N2-3-aminoBaP

6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene

dI

deoxyinosine

DMSO

dimethyl sulfoxide

DNA

deoxyribonucleic acid

DNP

dinitropyrene

EC

electron capture

EC50

median effective concentration

ECD

electron capture detector

ED50

median effective dose

EHC

Environmental Health Criteria monograph

EI

electron impact

ELISA

enzyme-linked immunosorbent assay

EPA

Environmental Protection Agency (USA)

FAO

Food and Agriculture Organization of the United Nations

FTP

Federal Test Procedure (USA)

GC

gas chromatography

GPC

semipreparative gel permeation chromatography

GST

glutathione S-transferase

Hb

haemoglobin

HCFC-22

chlorodifluoromethane

HDD

heavy-duty diesel

HPLC

high-performance liquid chromatography

IARC

International Agency for Research on Cancer

ILO

International Labour Organization

i.m.

intramuscular

i.p.

intraperitoneal

IPCS

International Programme on Chemical Safety

i.v.

intravenous

JECFA

Joint FAO/WHO Expert Committee on Food Additives

JMPR

Joint FAO/WHO Meeting on Pesticide Residues

Koc

organic carbon/water partition coefficient

Kow

n-octanol/water partition coefficient

LC50

median lethal concentration

LC100

lethal concentration for 100% of test organisms

LD50

median lethal dose

LOAEL

lowest-observed-adverse-effect level

LOEL

lowest-observed-effect level

LPG

liquefied petroleum gas

MA

metabolic activation

MN

micronucleus induction

MS

mass spectrometry

MS/MS

tandem mass spectrometry

MTD

maximum tolerated dose

MW

molecular weight (relative molecular mass)

NADH

nicotinamide adenine dinucleotide

NADPH

reduced nicotinamide adenine dinucleotide

NAT

N-acetyltransferase

NB

nitrobenzanthrone

nd

not detected

NICI

negative ion chemical ionization

NIST

National Institute of Standards and Technology (USA)

nitroPAH

nitro-polycyclic aromatic hydrocarbon

NMR

nuclear magnetic resonance

NOAEL

no-observed-adverse-effect level

NPD

nitrogen–phosphorus detector

NP-LC

normal-phase high-performance liquid chromatography

NPR

NADPH-cytochrome P450 reductase

OCC

oxidation catalytic converter

OECD

Organisation for Economic Co-operation and Development

OH-AAF

N-acetyl-2-aminofluoren-x-ol

OH-2-NF

2-nitrofluorenol

PAH

polycyclic aromatic hydrocarbon

PCB

polychlorinated biphenyl

PEF

potency equivalency factor

PICI

positive ion chemical ionization

PM2.5

particulate matter <2.5 µm in diameter

PM10

particulate matter <10 µm in diameter

p.o.

per os

ppb

part per billion

ppm

part per million

ppt

part per trillion

PUF

polyurethane foam

RO

Responsible Officer

RP-LC

reversed-phase high-performance liquid chromatography

s.c.

subcutaneous

SCE

sister chromatid exchange

SEOM

solvent extractable organic matter

SOF

soluble organic fraction

SPE

solid-phase extraction

SRM

Standard Reference Material

TA98AT+

N-hydroxyarylamine O-acetyltransferase-overproducing S. typhimurium strain

TA98NR+

nitroreductase-overproducing S. typhimurium strain

TA98AT–

N-hydroxyarylamine O-acetyltransferase-deficient S. typhimurium strain

TA98NR–

nitroreductase-deficient S. typhimurium strain

TEA

thermal energy analyser

TID

thermionic detector

TLC

thin-layer chromatography

UDS

unscheduled DNA synthesis

UN

United Nations

UNEP

United Nations Environment Programme

UV

ultraviolet

WHO

World Health Organization

XOC

extractable organic component

1. SUMMARY

1.1 Identity, physical and chemical properties, and analytical methods

Nitro-polycyclic aromatic hydrocarbons (nitroPAHs) are derivatives of polycyclic aromatic hydrocarbons (PAHs), which contain two or more fused aromatic rings made of carbon and hydrogen atoms. NitroPAHs occur in the environment as a mixture together with parent PAHs and hundreds of other organic compounds. NitroPAHs are usually present in much smaller quantities than PAHs.

NitroPAHs in the environment occur either in the vapour phase or adsorbed to particulate matter. NitroPAHs are insoluble or sparingly soluble in water but mostly soluble in organic solvents.

The sampling of nitroPAHs is similar to that of PAHs. Ambient air is sampled by collecting particulate matter on special filters by means of high-volume samplers. Vapour-phase nitroPAHs are commonly collected on solid sorbents such as polyurethane foam.

Solvent extraction is followed by cleanup using liquid chromatography with silica gel or alumina, high-performance liquid chromatography (HPLC) or solid-phase extraction. The nitroPAH fraction must be separated from the PAH fraction and oxygenated PAH fraction by HPLC on silica. Methods used for the separation and detection of nitroPAHs include gas chromatography with a variety of detectors, HPLC with fluorescence, chemiluminescence or electrochemical detector, and mass spectrometric techniques. Analysis is dependent on the standards available.

Another approach to analysis of complex mixtures is bioassay-directed chemical analysis, where mutagenically active fractions are bioassayed and characterized until the major class or specific compounds potentially responsible for the mutagenicity are identified. The use of bacterial tester strains selectively sensitive to nitroarenes has led to the identification of nitroPAHs as potent mutagens in complex mixtures from diverse sources. Synthetic standards are required for this type of analysis.

The nitroketone 3-nitrobenzanthrone and nitrolactones, such as 2- and 4-nitrodibenzopyranone, are nitro-oxy compounds, which have been detected together with nitroPAHs and are analysed by similar methods.

1.2 Sources of human and environmental exposure

NitroPAHs originate primarily as direct or indirect products of incomplete combustion. Only a few nitroPAHs are produced industrially; commercially produced nitronaphthalenes and 5-nitroacenaphthene, for example, are used primarily as chemical intermediates.

NitroPAHs originate from PAHs (generally adsorbed on particu-late matter and themselves products of incomplete combustion) by at least two distinct processes: (1) through nitration during combustion processes (e.g., in vehicle exhaust, particularly diesel, but also gasoline and aircraft emissions; industrial emissions; domestic residential heating/cooking; wood burning) and (2) through atmospheric formation from PAHs by either gas-phase reactions — daytime hydroxyl radical addition to the PAH followed by reaction with nitrogen dioxide and loss of a water molecule and nighttime nitrate radical addition to the PAH followed by reaction with nitrogen dioxide and loss of nitric acid — or heterogeneous gas–particle interaction of parent PAHs adsorbed onto particles with nitrating agents.

The distribution of nitroPAH isomers in samples of ambient air has been found to be significantly different from that in direct emissions from combustion. 2-Nitrofluoranthene and 2-nitropyrene are ubiquitous components of particulate matter, although they are not directly emitted from most combustion sources. The nitroPAH profile, or the relative quantities of certain "marker" PAHs, is a pointer to the source of formation of the nitroPAH. The most abundant nitro isomers of pyrene, fluorene and fluoranthene observed in diesel exhaust are 1-nitropyrene, 2-nitrofluorene and 3-nitrofluoranthene, whereas the isomers formed from the hydroxyl radical reactions of these PAHs are 2-nitropyrene, 3-nitrofluorene and 2-nitrofluoranthene.

The majority of ambient nitroPAHs are now thought to be formed in the atmosphere from the gas-phase reactions of PAHs with four rings or less.

Many mono- and some di- and trinitroPAH isomers have been identified and quantified in various samples of diesel exhaust, 1-nitropyrene usually being the most abundant. 1-Nitropyrene is the "marker" nitroPAH for diesel exhaust, and its presence in ambient air samples is a sign of pollution by diesel vehicle traffic. Diesel fuel, engine types and catalytic traps are continually being modified, so the various studies of nitroPAHs in diesel exhaust cannot be directly compared. In general, the mass emission of particles, emissions of particle-bound PAHs and nitroPAHs, and mutagenic activity levels generally decreased with the use of either particulate traps or catalytic converters.

The concentration of 1-nitropyrene was much less in gasoline exhaust particles than in diesel exhaust particles, but the concentrations of 1,3-, 1,6- and 1,8-dinitropyrenes were found to be almost the same in gasoline and diesel exhaust particles.

There is evidence of the presence of nitroPAHs in jet aeroplane exhaust.

NitroPAHs have been detected in the emissions of kerosene heaters, fuel gas and liquefied petroleum gas (LPG) burners, which are used in many countries for heating and cooking at home.

3-Nitrobenzanthrone has been detected in diesel exhaust particulate and in urban air samples. 2-Nitrodibenzopyranone and 4-nitrodibenzopyranone as well as nitropyrene lactones have been observed in ambient particulate matter.

1.3 Environmental transport, distribution and transformation

1.3.1 Environmental transport and distribution

NitroPAHs can be transported in the vapour phase or adsorbed onto particulate matter. Those with liquid-phase vapour pressures greater than 10–4 Pa at ambient air temperature (i.e., two- to four-ring PAHs and two-ring nitroPAHs) will exist at least partially in the gas phase.

Owing to their low aqueous solubility or insolubility, nitroPAHs are not expected to be transported in water. Data available give high values for sorption coefficients (log Koc), suggesting that nitroPAHs, similar to PAHs, adsorb onto soil and sediments. Leaching into groundwater is thought to be negligible. Some nitroPAHs may be slowly biodegradable under certain conditions.

The values for the n-octanol/water partition coefficient (log Kow) range from 2.5 for 1-nitronaphthalene to 6.3 for 3-nitroperylene, suggesting a potential for bioaccumulation. There were no data available on biomagnification.

1.3.2 Biotransformation

Many anaerobic and aerobic bacteria reduce nitroPAHs to mutagenic aminoPAHs. Nitroreduction by intestinal microflora plays a major role in the metabolism of nitroPAHs in mammals. Although a wide variety of bacteria, fungi and algae have been shown to degrade the parent PAHs containing two to five rings, nitro-substituted PAHs are only slowly degraded by indigenous microorganisms and may persist in soils and sediments. The recalcitrance of high molecular weight nitroPAHs is due in part to the strong adsorption to soil organic matter, low solubility, large molecular size and the polar character of the nitro group.

Time course studies in microcosms showed that 1-nitropyrene was degraded slowly under aerobic and anaerobic conditions in estuarine sediments.

Sphingomonas paucimobilis strain EPA 505 (a soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy) biodegraded 1-nitropyrene to 48.6% after 6 h.

The filamentous fungus Cunninghamella elegans has been shown to oxidatively metabolize, via a cytochrome P450 monooxygenase, a number of nitroPAHs (1-nitropyrene, 2-nitrofluorene, 2- and 3-nitrofluoranthene, 6-nitrochrysene, 1-nitrobenzo[e]pyrene and 6-nitrobenzo[a]pyrene) to products that are less mutagenic than the nitroPAHs themselves.

A plant cell culture derived from alligator weed (Alternanthera philoxeroides) detoxified 1-nitropyrene and 1,3-, 1,6- and 1,8-dinitropyrene, all direct-acting mutagens, when incubated with them, as shown by mutagenicity response in the Salmonella typhimurium TA98 assay.

1.3.3 Abiotic degradation

The photolysis of nitroPAHs has been studied under varied conditions of irradiation. The rate of photolysis depends not only on the conditions of irradiation but also on whether the nitroPAH is in the gaseous phase (e.g., 1-and 2-nitronaphthalene), in solution (type of solvent) or bound to solids/particles. In the latter case, the type and age of the particle seem to influence the photochemistry of the respective nitroPAH. The rate of photodecomposition, identification of photolytic products and resulting loss or gain of metabolic activity as determined by the S. typhimurium assay have been the main end-points studied.

Calculated atmospheric lifetimes of nitroPAHs due to photolysis and gas-phase reactions with hydroxyl and nitrate radicals and with ozone under atmospheric conditions show that the dominant loss process for nitroPAHs (e.g., 1- and 2-nitronaphthalene) is photolysis. Particle oxidation of nitroPAHs by ozone may be the main loss process at night.

1.4 Environmental levels and human exposure

NitroPAHs that have been detected in ambient air include 1- and 2-nitronaphthalene and methylnitronaphthalenes (predominantly in the vapour phase), 2-nitrofluorene, 9-nitroanthracene, 9-nitrophenanthrene, 2-, 3- and 8-nitrofluoranthene, 1- and 2-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene and 6-nitrochrysene.

At remote and forest sites, nitroPAHs were either not detected or detected in the low picogram per cubic metre range (e.g., 17 pg/m3 for 2-nitrofluoranthene; 4 pg/m3 for 1-nitropyrene). The concentration of nitroPAHs in the atmosphere of urban regions depends on the season, the type of heating used and the number and regulation of traffic vehicles. Reported levels in air do not usually exceed 1 ng/m3, although maxima of up to 13 ng/m3 have been reported.

Various studies have been performed monitoring certain isomeric nitroPAHs. Investigators have concentrated on the nitroPAHs that seem to be of quantitative/environmental (e.g., nitroPAHs of relative molecular mass 247: 1-nitropyrene, 2-nitropyrene, 2-nitrofluoranthene) or carcinogenic (e.g., 1-nitropyrene, 2-nitrofluorene, dinitropyrenes) importance.

Studies of daytime/nighttime concentrations of specific isomeric nitroPAHs in certain regions (in particular California, USA) and parallel environmental chamber studies have led to an understanding of the atmospheric formation of certain nitroPAHs (2-nitrofluoranthene and 2-nitropyrene). Concurrent studies of certain nitroPAHs (1-nitropyrene, dinitropyrenes) and traffic volume have confirmed that traffic emission is a source of nitroPAHs.

Most seasonal studies show higher winter/spring concentrations of marker nitroPAHs, which parallels the use of domestic heating, although this is not always the case.

1.4.1 Indoor air

As nitroPAHs have been detected in the emissions of kerosene heaters, fuel gas and LPG burners used for heating and cooking at home, as well as in the fumes of cooking oils, there is therefore a potential indoor exposure to nitroPAHs in poorly ventilated conditions.

Concentrations of polyaromatic compounds, including nitroPAHs, were measured in a study of indoor and outdoor air levels associated with 33 homes located in two US cities: Columbus, Ohio, and Azusa, California. The overall levels were much higher in homes occupied by smokers, but the use of natural gas heating and cooking appliances also appeared to increase the nitroPAH levels slightly.

1-Nitropyrene (4.2–25 600 ng/litre) was detected in 36 of 55 samples of wastewater from oil–water separating tanks of gasoline stations and in used crankcase oil.

1- and 2-nitronaphthalene and 1,3- and 1,5-dinitronaphthalene were detected in river water in Japan at concentrations of 1.3, 11.7, 1.7 and 3.2 ng/litre, respectively. In another water sample, 1-nitropyrene was identified.

There are only limited data on the presence of nitroPAHs in samples of soil, sewage sludge, sediment and incinerator ash (e.g., for 1-nitropyrene, 0.03–0.8 µg/kg dry weight in soil, 0.68 µg/kg in sewage sludge, 25.2 µg/kg in sediment and <0.01–0.89 mg/kg in incinerator ash).

1.4.2 Food and beverages

With the exception of spices, smoked and grilled foods and peanuts, the concentrations of nitroPAHs in foods are below 5 µg/kg.

In a study in the United Kingdom, foodstuffs were monitored for the presence of 9-nitroanthracene and 1-nitropyrene. Twenty-five out of 28 foods contained no detectable levels of these nitroPAHs. 9-Nitroanthracene was tentatively identified in peated malt, at 0.9 µg/kg, and 1-nitropyrene in two samples of tea leaves, at 1.7 and 0.17 µg/kg.

Another survey of nitroPAH levels in various foods in Austria showed mostly detectable levels of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene. The highest concentrations were found in spices, smoked foods and teas, in particular Mate tea, which is roasted. NitroPAHs were also detected in vegetables and fruits, probably due to atmospheric pollution.

1-Nitropyrene was detected in grilled corn, mackerel and (in considerable amounts) pork and yakitori (grilled chicken) grilled with sauce (up to 43 ng/g).

1.4.3 Other products

In 1980, studies showed that extracts of selected xerographic toners and paper photocopies were mutagenic. The fraction of the carbon black B responsible for 80% of the mutagenicity contained 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene. As a result of this finding, the manufacturers modified the production of carbon black B, substantially reducing the levels of nitropyrenes.

1.4.4 Occupational exposure

Occupational exposure to nitroPAHs has been demonstrated in workplaces associated with the use of diesel engines. For example, concentrations of 1-nitropyrene in air were measured in various workplaces associated with the use of diesel engines. The highest levels (42 ng/m3) reported were determined in the breathing zones of the underground workers (drivers of diesel-powered excavators) at an oil shale mine in Estonia.

1.5 Kinetics and metabolism in laboratory animals and humans

1-Nitropyrene and 2-nitrofluorene administered by various routes are rapidly absorbed, and the resulting metabolites are conjugated and excreted. Radiolabelled 1-nitropyrene was found to be widely distributed in the body of rats and mice following administration by all routes. Other nitroPAHs have not been as well studied.

The metabolism of nitroPAHs is complex. It seems that there are at least five metabolic activation pathways through which mutations can be induced by nitroPAHs in bacterial and mammalian systems and/or through which DNA binding occurs. These are 1) nitroreduction; 2) nitroreduction followed by esterification (in particular acetylation); 3) ring oxidation; 4) ring oxidation and nitroreduction; and 5) ring oxidation and nitroreduction followed by esterification. In bacteria, nitroreduction seems to be the major metabolic pathway, whereas the fungus Cunninghamella elegans is an example of a species in which nitroPAHs are metabolized by ring oxidation.

Nitroreduction of nitroPAHs in vivo probably occurs mainly by bacteria in the intestinal tract. In oxidative metabolism, the first step is transformation to phase I primary metabolites such as epoxides, phenols and dihydrodiols, and then to secondary metabolites, such as diol epoxides, tetrahydrotetrols and phenol epoxides. In mammalian systems, the phase I metabolites are then conjugated with glutathione, sulfate or glucuronic acid to form phase II metabolites, which are more polar and water-soluble than the parent hydrocarbons. On reaching the intestine, the conjugated metabolites can be deconjugated by the intestinal microflora and absorbed, entering enterohepatic circulation. Nitroreduction and N-acetylation can occur, resulting in the excretion in urine and faeces of metabolites such as acetylaminopyrenols after 1-nitropyrene administration.

Different cytochrome P450 enzymes may be involved in the metabolism of a specific nitroPAH, and these may differ in the related isomers, resulting in possibly different kinetics and pathways. Cytochrome P450 enzymes responsible for the metabolism of nitroPAHs may vary between species and in different target organs and in different cell types within target organs.

All nitroPAHs do not follow the same activation pathways. Some are mutagenic when reduced to an arylhydroxylamine (e.g., 1-nitropyrene is metabolized mainly by hydroxylation of the aromatic moiety, followed by nitroreduction and N-acetylation); others (e.g., 1,8- and 1,6-dinitropyrene) are reduced to the arylhydroxylamine and then require further O-esterification (in particular O-acetylation) to an acyloxy ester for mutagenicity. Some may be mutagenic only after activation by oxidation to reactive epoxides or dihydrodiol epoxides (as possibly in 6-nitrobenzo[a]pyrene, similar to benzo[a]pyrene, or BaP). The main DNA adducts detected with nitroPAHs in vivo and in vitro are C8-substituted deoxyguanosine adducts; however, N2-substituted deoxyguanosine and C8-substituted deoxyadenosine derivatives have also been detected and may predominate in nitroPAHs with greater hydrocarbon character (e.g., 3-nitrobenzo[a]pyrene and 6-nitrochrysene). DNA adducts of dinitropyrenes are formed only via nitroreduction, presumably owing to the high electron deficiency in the aromatic rings caused by the presence of two nitro groups. The DNA adducts resulting from the nitroreduction of nitroPAHs are better characterized than those arising from oxidative metabolism, although the latter may be of more importance in mammalian metabolism.

1.6 Effects on laboratory mammals and in vitro test systems

Only six nitroPAHs have been tested for acute toxicity. In rats, an LD50 of 86 mg/kg of body weight (kg bw) after intraperitoneal (i.p.) application was reported for 1-nitronaphthalene; in mice, an oral LD50 of 1300 mg/kg bw was reported for 2-nitronaphthalene. In further studies on both substances, systemic effects on the target organs lung and liver were observed after single high doses; however, 2-nitronaphthalene seemed to be less toxic than 1-nitronaphthalene. 5-Nitroacenaphthene at an i.p. dose of 1700 mg/kg bw was lethal to all treated rats. For 2-nitrofluorene, an oral LD50 of 1600 mg/kg bw in mice was reported, whereas gavaging with up to 5000 mg 1-nitropyrene/kg bw resulted in no observable toxic effects. Local inflammation and ulceration were seen in rats after subcutaneous (s.c.) injection of 8 mg 3-nitrofluoranthene/kg bw.

Data on systemic or local non-neoplastic effects caused by short-term or long-term treatment with nitroPAHs are limited, as the end-point of most studies has been carcinogenicity. In most cases, non-neoplastic toxic effects were observed at doses at which carcinogenic responses are also manifested. Systemic non-neoplastic toxic effects, such as reduced body weight or increased mortality, appeared presumably independently of carcinogenic effects in feeding studies with 5-nitroacenaphthene at a dose level of 500 mg/kg bw per day (rat) or 40 mg/kg bw per day (mice) and with 2-nitrofluorene at a dose of 25 mg/kg bw per day (rat). Medium-term exposure via inhalation to 1-nitropyrene resulted in metaplasia of the upper respiratory tract at concentrations of >0.5 mg/m3.

No data are available on skin and eye irritation, sensitization or reproductive toxicity.

Data on genotoxicity in vitro are available on 95 nitroPAHs; for 74 nitroPAHs, however, only one or two end-points, mainly in bacterial test systems, were investigated. A sufficient database, including eukaryotic test systems, has been found only with 21 nitroPAHs. Most of these substances (67 out of 95) showed positive results, but the results were derived from a small database. Clearly positive results were obtained for 19 nitroPAHs, and questionable results for 8 nitroPAHs. With none of the nitroPAHs were clearly negative results obtained.

For 86 nitroPAHs, data on the S. typhimurium microsome test are available. In contrast to the parent PAHs, most nitroPAHs were clearly more effective in the Salmonella microsome test without metabolic activation. There are five nitroPAHs that showed exceptionally high mutagenic potency (>100 000 revertants/nmol) in this test system: 3,7- and 3,9-dinitrofluoranthene, 1,6- and 1,8-dinitropyrene, and 3,6-dinitrobenzo[a]pyrene.

Bacterial nitroreductase and acetyltransferase are involved in the metabolic activation of nitroPAHs, but not all nitroPAHs follow the same metabolic activation pathways. Furthermore, there is no uniform mutagenic effect of the different nitroPAHs, as they produce both frameshift and base pair substitutions in the S. typhimurium microsome test. There is evidence that nitroPAHs with nitro groups perpendicular to the aromatic ring are not as mutagenic as isomers having parallel nitro orientation.

Data on the in vivo genotoxicity of nitroPAHs are available for 15 nitroPAHs. All nitroPAHs that gave positive results in vivo were also positive in vitro. Four nitroPAHs that were positive in in vitro genotoxicity tests revealed inconsistent or inconclusive genotoxicity (2-nitronaphthalene, 5-nitroacenaphthene and 3-nitrofluoranthene) or negative genotoxicity (2,7-dinitrofluorene; limited validity) results in vivo.

3-Nitrobenzanthrone, like 1,6- and 1,8-dinitropyrene, is highly mutagenic in bacteria through nitroreduction and O-esterification. 3-Nitrobenzanthrone is also an effective gene mutagen and causes micronuclei formation in human cells in vitro and in mice in vivo.

2-Nitrodibenzopyranone was reported to be highly mutagenic in the S. typhimurium microsome test in strain TA98 (–S9), being more mutagenic than 2-nitrofluorene and 1-nitropyrene. 1- and 3-nitropyrene lactones have been found to be highly mutagenic in the S. typhimurium microsome test.

Studies on the in vitro genotoxicity of 2-nitrodibenzopyranone in forward mutation assays using two human B-lymphoblastoid cell lines are conflicting. Nitropyrene lactones were found to induce mutations at the tk and hprt loci in both cell lines. Further, they induced kinetochore-positive and -negative micronuclei in the CREST modified micronucleus assay, which detects chromosomal loss and breakage events.

Data on carcinogenic effects are available for 28 nitroPAHs. Although inhalation is the main exposure route in humans, no long-term inhalation study on any nitroPAH is available. Most studies examined the carcinogenic effects of nitroPAHs by oral administration, topical application, pulmonary implantation or intratracheal administration.

Owing to the limitations in experimental design, none of the negative studies confirmed the absence of carcinogenic effects in animals. However, results showed carcinogenic effects in experimental animals for 5-nitroacenaphthene, 2-nitrofluorene, 3-nitrofluoranthene, 3,7- and 3,9-dinitrofluoranthene, 1- and 4-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene and 6-nitrochrysene. Some carcinogenic effects in experimental animals were observed for 2-nitropyrene, 7-nitrobenz[a]anthracene, 2- and 6-nitrobenzo[a]pyrene, 3,6-dinitrobenzo[a]pyrene, 7-nitrodibenz[a,h]anthracene and 3-nitroperylene. For the remaining 10 nitroPAHs tested, not enough data were available with which to evaluate their carcinogenicity in experimental animals.

Besides local effects at the site of injection, nitroPAHs induced mainly systemic tumours in mammary tissue, lung, liver and the haematopoietic system. 6-Nitrochrysene appears to be the most carcinogenic of the nitroPAHs considered here. With systemic effects after s.c. or i.p. injection, 1-nitropyrene was more carcinogenic than the dinitropyrenes. The carcinogenicity of 1-nitropyrene and dinitropyrenes varies, depending on the route of administration.

Nitrated benzo[a]pyrenes are generally less potent carcinogens than the parent compound BaP. However, the mono- or dinitrated pyrenes are more carcinogenic than pyrene. Similar results were presented for 3-nitroperylene compared with perylene and for 6-nitrochrysene compared with chrysene; with local effects after dermal exposure, however, 6-nitrochrysene was less active than chrysene.

Data were available on carcinogenic effects of some metabolites of 2-nitrofluorene, 1-nitropyrene and 6-nitrochrysene. Comparing 2-nitrofluorene with its metabolites in rats, the highest carcinogenic potency was shown by 2-acetylaminofluorene. 1-Nitropyrene was significantly more carcinogenic after oral application in rats than either 1-nitrosopyrene or 1-aminopyrene. In contrast, 1-nitrosopyrene induced a higher incidence of liver tumours in mice after i.p. application than 1-nitropyrene; no effects were observed with ring hydroxylated metabolites. 6-Nitrosochrysene and 6-aminochrysene were inactive, in contrast to the ring hydroxylated metabolites, which showed carcinogenic activity in the liver similar to that of the parent compound 6-nitrochrysene; this indicates that the metabolic activation of 6-nitrochrysene occurs by ring oxidation and/or a combination of ring oxidation and nitroreduction.

1.7 Effects on humans

There are no reports on the effects of individual nitroPAHs on humans. As would be expected, since nitroPAHs occur in complex mixtures in the atmosphere and exhaust, the exact contribution of nitroPAHs to the adverse health consequences of exposure to polluted atmospheres and to exhaust cannot be elucidated.

At present, investigations on the effects of nitroPAHs on human health are being carried out using biomarkers of exposure. Several reports have described the development of methods for and provided data on the evaluation of 1-nitropyrene as a biomarker for occupational exposure to diesel exhaust. Urinary metabolites of PAHs and nitroPAHs were determined in the urine of diesel mechanics using the enzyme-linked immunosorbent assay (ELISA). In another study, metabolites of 1-nitropyrene (namely, N-acetyl-1-aminopyren-6-ol and N-acetyl-1-aminopyren-8-ol) were measured in the urine of workers in a shipping department. Several studies have focused on measuring the haemoglobin and plasma adducts of metabolites of 1-nitropyrene and other nitroPAHs and may provide appropriate biomarkers in future molecular epidemiological investigations.

1.8 Effects on other organisms in the laboratory and field

Data on the acute toxicity of nitroPAHs to aquatic organisms are available only for 1-nitronaphthalene. An LC50 (96 h) of 9.0 mg/litre was reported for the fathead minnow (Pimephales promelas). Furthermore, this nitroPAH inhibited the growth of the ciliate Tetrahymena pyriformis, with an EC50 (60 h) of 17.3 mg/litre.

Some studies have been concerned with the effect of nitroPAHs on the metabolism of some aquatic species — for example, the subcellular and tissue distribution of two- and one-electron NAD(P)H-dependent nitroreductase activity in marine invertebrates from three phyla: mussel (Mytilus edulis), crab (Carcinus maenas) and starfish (Asteria rubens). NADPH-dependent two-electron nitroreductase activity, occurring only under anaerobic conditions, was detected in the microsomal and cytosolic fractions of the major digestive tissues of mussel (digestive gland) and crab, but not in the gills of either species. 1-Aminopyrene was the only metabolite identified. No activity was detectable in the pyloric caeca or stomach region of the starfish. NAD(P)H-dependent one-electron nitroreduction was present in all subcellular fractions of the major digestive tissues of the three species.

In the presence of calf thymus DNA, adducts derived from 1-nitropyrene were detected in vitro using hepatic S9 fractions prepared from fish. The ability of 1-nitropyrene to form DNA adducts was also established in vivo using brown trout (Salmo trutta) and turbot (Scophthalmus maximus). These DNA adducts were comparable to those obtained in Wistar rats treated with 1-nitropyrene.

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS

2.1 Identity

Nitro-polycyclic aromatic hydrocarbons (nitroPAHs) are derivatives of polycyclic aromatic hydrocarbons (PAHs), which contain two or more fused aromatic rings made of carbon and hydrogen atoms, formed as a result of incomplete combustion (see IPCS, 1998). NitroPAHs occur in the environment as a mixture together with parent PAHs and hundreds of other organic compounds (see chapter 3). NitroPAHs are usually present in smaller quantities (by 2 orders of magnitude) than PAHs.

Interest was focused on nitroPAHs in the early 1980s as correlations were found between the presence of nitroPAHs in diesel exhaust and environmental extracts and mutagenic activity. A large number of groups of nitro, oxy and mixed nitro-oxy compounds eluted together in the mutagenic fractions. Analytical methods were developed to separate and identify these compounds and to specify their isometric composition, as the biological action of these compounds also depends on their stereospecificity (see chapters 6 and 7). As it would be impossible to evaluate all these compounds in one document, a decision was made to include mono- and dinitroPAHs (2–5 rings) but, in general, not methylated or hydroxylated nitroPAHs. Some nitro-oxyPAHs are also included: nitroketones (3-nitrobenzanthrone) and selected nitrolactones (e.g., the nitrophenanthrene lactones: 2- and 4-nitrodibenzopyranone [2- and 4-nitro-6H-dibenzo[b,d]pyran-6-one] and nitropyrene lactones), which have recently been shown to be present in the extracts of the polar fractions of diesel exhaust and airborne particulates.

The nomenclature, molecular formula, relative molecular mass and Chemical Abstracts Service (CAS) number of selected nitroPAHs and nitro-oxyPAHs are given in Table 1. The structural formulas of some selected nitroPAHs and nitro-oxyPAHs are shown in Figure 1.

Figure 1

Fig. 1. Structural formulas of some nitroPAHs and some nitro-oxyPAHs.

Table 1. Nomenclature, molecular formulas, relative molecular mass and CAS numbers of selected nitroPAHs and their oxygen-containing derivatives

Parent PAHs

Nitro derivative

Molecular formula

Relative molecular mass

CAS number

Two-ring PAHs

       

Naphthalene

1-Nitronaphthalene

C10H7NO2

173.17

86-57-7

 

2-Nitronaphthalene

"

"

581-89-5

 

1,3-Dinitronaphthalene

C10H6N2O4

218.17

606-37-1

 

1,5-Dinitronaphthalene

"

"

605-71-0

 

1,8-Dinitronaphthalene

"

"

602-38-0

 

2,7-Dinitronaphthalene

"

"

24824-27-9

 

2,3,5-Trinitronaphthalene

C10H5N3O6

263.17

87185-24-8

 

1,3,6,8-Tetranitronaphthalene

C10H4N4O8

308.16

28995-89-3

Three-ring PAHs

       

Acenaphthene

3-Nitroacenaphthene

C12H9NO2

199.21

3807-77-0

 

5-Nitroacenaphthene

"

"

602-87-9

Fluorene

1-Nitrofluorene

C13H9NO2

211.22

22250-99-3

 

2-Nitrofluorene

"

"

607-57-8

 

3-Nitrofluorene

"

"

5397-37-5

 

4-Nitrofluorene

"

"

24237-68-1

 

2,7-Dinitrofluorene

C13H8N2O4

256.22

5405-53-8

Anthracene

2-Nitroanthracene

C14H9NO2

223.23

3586-69-4

 

9-Nitroanthracene

"

"

602-60-8

 

9,10-Dinitroanthracene

C14H8N2O4

268.23

33685-60-8

Phenanthrene

2-Nitrophenanthrene

C14H9NO2

223.23

17024-18-9

 

9-Nitrophenanthrene

"

"

954-46-1

 

2,6-Dinitrophenanthrene

C14H8N2O4

268.23

 

Four-ring PAHs

       

Fluoranthene

1-Nitrofluoranthene

C16H9NO2

247.25

13177-28-1

 

2-Nitrofluoranthene

"

"

13177-29-2

 

3-Nitrofluoranthene

"

"

892-21-7

 

7-Nitrofluoranthene

"

"

13177-31-6

 

8-Nitrofluoranthene

"

"

13177-32-7

 

1,2-Dinitrofluoranthene

C16H8N2O4

292.25

33611-88-0

 

2,3-Dinitrofluoranthene

"

"

105735-66-8

 

2,4-Dinitrofluoranthene

"

"

102493-19-6

 

2,5-Dinitrofluoranthene

"

"

102493-21-0

 

3,4-Dinitrofluoranthene

"

"

 
 

3,7-Dinitrofluoranthene

"

"

105735-71-5

 

3,9-Dinitrofluoranthene

"

"

22506-53-2

 

1,2,4-Trinitrofluoranthene

C16H7N3O6

337.25

102493-20-9

 

1,2,5-Trinitrofluoranthene

"

"

102493-22-1

 

2,3,5-Trinitrofluoranthene

"

"

116331-54-5

Pyrene

1-Nitropyrene

C16H9NO2

247.25

5522-43-0

 

2-Nitropyrene

"

"

789-07-1

 

4-Nitropyrene

"

"

57835-92-4

 

1,3-Dinitropyrene

C16H8N2O4

292.25

75321-20-9

 

1,6-Dinitropyrene

"

"

42397-64-8

 

1,8-Dinitropyrene

"

"

42397-65-9

 

1,3,6-Trinitropyrene

C16H7N3O6

337.25

75321-19-6

 

1,3,6,8-Tetranitropyrene

C16H6N4O8

382.24

28767-61-5

Benz[a]anthracene

7-Nitrobenz[a]anthracene

C18H11NO2

273.29

20268-51-3

Chrysene

2-Nitrochrysene

C18H11NO2

273.29

3989-90-0

 

5-Nitrochrysene

"

"

89455-17-4

 

6-Nitrochrysene

"

"

7496-02-8

Five-ring PAHs

       

Benzo[e]fluoranthene

3-Nitrobenzo[e]fluoranthene

C20H11NO2

297.31

 

Benzo[a]pyrene

1-Nitrobenzo[a]pyrene

C20H11NO2

297.31

70021-997

 

3-Nitrobenzo[a]pyrene

"

"

70021-98-6

 

6-Nitrobenzo[a]pyrene

"

"

63041-90-7

 

3,6-Dinitrobenzo[a]pyrene

C20H10N2O4

342.31

128714-76-1

Benzo[e]pyrene

1-Nitrobenzo[e]pyrene

C20H11NO2

297.31

91259-16-4

 

3-Nitrobenzo[e]pyrene

"

"

81340-58-1

Perylene

3-Nitroperylene

C20H11NO2

297.31

20589-63-3

Dibenz[a,h]anthracene

7-Nitrodibenz[a,h]anthracene

C22 H13 NO2

323.4

 

Six-ring PAHs

       

Benzo[ghi]perylene

4-Nitrobenzo[ghi]perylene

C22H11NO2

321.34

 
 

7-Nitrobenzo[ghi]perylene

"

"

 

Coronene

1-Nitrocoronene

C24H11NO2

345.36

 

Nitro-oxyPAHs

       
 

3-Nitrobenzanthrone

C17H9NO3

275.26

1711-34-9

 

2-Nitrobenzopyranone

C13H7NO4

241.20

6623-66-1

 

3-Nitrobenzopyranone

C13H7NO4

241.20

6638-64-8

 

4-Nitrobenzopyranone

C13H7NO4

241.20

51640-90-5

 

Nitropyrene lactones

C15H7NO4

265.22

 

2.2 Physical and chemical properties

At ambient temperatures, nitroPAHs are yellowish to orange solids that tend to sublime (White, 1985). NitroPAHs in the environment occur in the vapour phase or are absorbed and/or adsorbed to particulate matter, depending upon their vapour pressure and the ambient conditions. Two- to four-ring nitroPAHs are present partially in the vapour phase under certain conditions; for example, 1-nitronaphthalene (in certain climates) occurs mainly in the vapour phase (Arey et al., 1987), whereas 2-nitrofluorene occurs equally in both vapour and particulate phases, and 1-nitropyrene occurs in the particulate phase (Schuetzle & Frazier, 1986).

NitroPAHs are insoluble or sparingly soluble in water but are mostly soluble in organic solvents such as acetone, benzene, dimethyl sulfoxide (DMSO) and methylene chloride.

Table 2 gives details of some environmentally relevant physical and chemical properties of nitroPAHs together with those of the parent PAHs (see IPCS, 1998). Nitrodibenzopyranones have a lower vapour pressure than nitroPAHs and therefore are to be found predominantly in the particulate phase.

Table 2. Physical and chemical properties of nitroPAHs and their parent PAHsa

Parent PAHs;
nitro derivatives

Melting point (°C)

Boiling point (°C) at 101.3 kPa

Vapour pressure
(Pa at 25 °C)

Solubility in water at 25 °C (mg/litre)

Henry’s law constant at 25 °C (kPa·m3/mol)

Log Kowb

Log Kocb

Two-ring PAHs

             

Naphthalene

81

218

10.4

31.7

4.9 × 10–2

3.4

 

1-Nitro-

58–61.5 (56.5c)

330; 314 sublimesd
312e

0.0154 (20 °C)d 3.2 × 10–2 e

34f
9.18e

6.1 × 10–1 e

2.50c
3.19g,h

3.02
2.98e

2-Nitro-

74–79 (76c)

304e

3.2 × 10–2 e

26f
9.24e

6.1 × 10–1 e

2.78c
3.24g,h

3.09
3.03e

1,3-Dinitro-

144–149

       

2.83g,h

 

1,5-Dinitro-

215–219

       

2.58g,h

 

1,8-Dinitro-

171–172

       

2.52g,h

 

2,7-Dinitro-

234

           

1,3,6,8-Tetranitro-

195

       

2.29g

 

Three-ring PAHs

             

Acenaphthene

95

279

2.9 × 10–1

3.93

1.5 × 10–2

3.92

 

3-Nitro-

151

           

5-Nitro-

102
(101–102c)

       

3.36c
3.85g,h

 

Fluorene

115

295

8 × 10–2

1.98

1.0 × 10–2

4.18

 

1-Nitro-

 

326

9.7 × 10–5

0.28

7.2 × 10–2

 

3.76e

2-Nitro-

154–158 (158c)

326

9.7 × 10–5

0.216

9.5 × 10–2

4.08c
3.37g,h

3.16e

3-Nitro-

105–106

326

9.7 × 10–5

0.28

7.2 × 10–2

 

3.76e

4-Nitro-

75–76

326

9.7 × 10–5

0.28

7.2 × 10–2

 

3.76e

2,7-Dinitro-

334

       

3.35g,h

 

Anthracene

216

342

8 × 10–4

0.073

7.3 × 10–2

4.5

 

2-Nitro-

172

       

4.23g

 

9-Nitro-

141–146 (146c)

       

4.16c
4.50g
4.78i

4.69j

9,10-Dinitro-

263, 310

       

4.10c

 

Phenanthrene

100.5

340

1.6 × 10–2

1.29

     

2-Nitro-

119–120

       

4.23g

 

9-Nitro-

116–117

           

2,7-Dinitro-

             

Four-ring PAHs

             

Fluoranthene

108.8

375

1.2 × 10–3

0.26

6.5 × 10–4 (20 °C)

5.22

 

1-Nitro-

         

4.69g

 

2-Nitro-

 

420e

9.9 × 10–7 e

0.019e

1.3 × 10–2 e

 

4.48e

3-Nitro-

156–162 (166c)

       

5.15
4.69g

 

7-Nitro-

144–145

420e

9.9 × 10–7 e

0.017e

1.4 × 10–2 e

4.69g

4.48e

8-Nitro-

158–164

420e

9.9 × 10–7 e

0.017e

1.4 × 10–2 e

4.69g

4.48e

3,4-Dinitro-

279–280k

           

3,7-Dinitro-

203–204k

           

3,9-Dinitro-

275–276
222–224k

           

Pyrene

150.4

393

6.0 × 10–4

0.135

1.1 × 10–3

5.18

 

1-Nitro-

151–152 (153c)

472e

4.4 × 10–6 e

0.017e

6.4 × 10–2 e

5.29c
4.69

4.48e

2-Nitro-

197–199

472e

4.4 × 10–6 e

0.021e

6.4 × 10–2 e

 

3.53e

4-Nitro-

190–192

472e

4.4 × 10–6 e

0.017e

6.4 × 10–2 e

 

4.48e

1,3-Dinitro-

295–297

       

4.44g

 

1,6-Dinitro-

309–310

       

4.44g

 

1,8-Dinitro-

299–300

       

4.44g

 

1,3,6-Trinitro-

         

4.18g

 

1,3,6,8-Tetranitro-

335

       

3.92g

 

Benz[a]-anthracene

160.7

400

2.8 × 10–5

0.014

 

5.61

 

7-Nitro-

161–162c

       

5.34c

 

Chrysene

253.8

448

8.4 × 10–5

0.002

 

5.91

 

2-Nitro-

             

5-Nitro-

             

6-Nitro-

208c

       

5.41c
5.41g

 

Five-ring PAHs

             

Benzo[a]pyrene

178.1

496

7.3 × 10–7

0.004

3.4 × 10–5 (20 °C)

6.50

 

1-Nitro-

             

3-Nitro-

             

6-Nitro-

260c

567e

   

0.012e

6.13c
5.87e

5.66e

3,6-Dinitro-

             

Benzo[e]pyrene

178.7

493

7.4 × 10–7

0.005

 

6.44

 

1-Nitro-

         

5.87g

 

Perylene

277.5

503

 

0.0004

 

5.3

 

3-Nitro-

209c

       

6.34c

 

Six-ring PAHs

             

Coronene

439

525

2 × 10–10

0.000 14

 

5.4

 

1-Nitro-

             

Benzo[ghi]-perylene

278.3

545

1.4 × 10–8

0.000 26

2.7 × 10–5 (20 °C)

7.10

 

4-Nitro-

             

7-Nitro-

             

Nitro-oxyPAHs

             

3-Nitrobenzanthrone

256–257l

           

2-Nitrodibenzo[b,d]-pyranone

 

368e

2.5 × 10–6 e

128e,f

2.9 × 10–6 e

 

2.04e

4-Nitrodibenzo[b,d]-pyranone

 

368e

2.5 × 10–6 e

128e,f

2.9 × 10–6 e

 

2.04e

a

The data for parent PAHs are from IPCS (1998). The data for nitroPAHs are from White (1985), unless stated otherwise.

b

Log Kow = octanol/water partition coefficient; log Koc = sorption coefficient.

c

From Karcher et al. (1991).

d

From IUCLID dataset on 1-nitronaphthalene, 2000.

e

From Yaffe et al. (2001).

f

From Al-Bashir et al. (1994).

g

From Compadre et al. (1990).

h

Experimental values.

i

From Sinks et al. (1997).

j

From Nielsen et al. (1997).

k

From Nakagawa et al. (1987).

l

From Suzuki et al. (1997).

2.3 Conversion factors

Atmospheric concentrations of nitroPAHs are usually expressed as micrograms, nanograms or picograms per cubic metre. At 25 °C and 101.3 kPa, the conversion factors for a compound of given molecular mass are obtained as follows:

ppb = µg/m3 × 24.45/relative molecular mass

µg/m3 = ppb × relative molecular mass/24.45

where ppb is parts per billion, and one billion is 109.

For example, for 1-nitropyrene, 1 ppb = 10.1 µg/m3, and 1 µg/m3 = 0.099 ppb.

2.4 Analytical methods

A direct analysis of nitroPAHs from environmental sources is not possible, as all environmental matrices are very complex. The samples often contain thousands of combustion products, including parent PAHs and other closely related derivatives (in particular oxygenated PAHs such as aldehydes, ketones and carboxylic acids), which tend to co-elute with nitroPAHs under a variety of liquid and gas chromatographic conditions and are present at concentrations 1 or 2 orders of magnitude higher than those of the nitro-substituted compounds (Schuetzle, 1983; Vincenti et al., 1996; see Table 3). An extensive sample cleanup and prefractionation of the sample are tedious but necessary prerequisites for trace analysis of nitroPAHs.

Isomer-specific identification is necessary, as the biological activity depends on the position of the nitro substituent. The source of the nitroPAH (e.g., from combustion or from atmospheric reactions; see chapter 3) may determine the isomeric specificity of the nitroPAH.

Although 1- and 2-nitronaphthalene are expected to be found predominantly in the gas phase, other semivolatile nitroPAHs will be distributed between the particulate and gas phases, depending upon the ambient temperature. Thus, many ambient measurements will underestimate the total nitroPAHs present unless both gas- and particulate-associated species have been measured.

Analysis is hindered by a lack of adequate instrumental sensitivity or selectivity and limited availability of native and isotope-labelled standards (Chiu & Miles, 1996).

Bioassay-directed fractionation (usually using the Ames test or modifications of this with specific Salmonella typhimurium strains) and subsequent chemical characterization have been used for the identification of nitroPAHs in a number of complex mixtures (see section 2.4.5).

As most nitroPAHs are mutagenic, special precautions should be taken, even at ultratrace concentrations.

Table 3. Relative concentrations of various PAH compounds and PAH derivatives in the non-polar and moderately polar fractions of a diesel particle extract, showing only the nitroPAH fraction in detaila

Compound

Fraction concentration

Non-polar fractions

1000

 

Total PAHs, hydrocarbons, alkylbenzenes

1000

 

Moderately polar fractions

1000

 

PAH ketones

147

 

PAH carboxyaldehydes

122.2

 

PAH acid anhydrides

54.1

 

HydroxyPAHs

113.1

 

PAH quinones

71.3

 

NitroPAHs

 

2.9

 
 

Nitrofluorenes

 

0.34

 

Nitro(anthracenes and phenanthrenes)

 

0.71

 

Nitrofluoranthenes

 

0.05

 

Nitropyrenes

 

1.5

 

Methyl nitro(pyrenes and fluoranthenes)

 

0.25

Other oxygenated PAHs

83.4

 

PAH carry-over phthalates, hydrocarbon contaminants

340

 

a Adapted from Schuetzle (1983).

It should be noted that many nitroPAHs, in particular 9-nitro-anthracene, are unstable in the presence of light; therefore, reduced light conditions should be used (see chapter 4).

Details of methods used for the analysis of nitroPAHs in different matrices are given in Table 4. Reviews on the analysis of nitroPAHs are given by White (1985), Vincenti et al. (1996), CONCAWE (1998) and Hayakawa (2000).

Table 4. Analysis of nitroPAHsa

Sample type

Extraction

Cleanup

Analysis

Detector

Detection limitb

References

Air

           

Ambient air

Soxhlet (DCM)

NP-LC

GC

MS in SIM

 

Arey et al. (1987)

Reaction chamber study

Soxhlet (DCM)

NP-LC

GC
RP-LC

MS in SIM
UV

 

Atkinson et al. (1987a)

Ambient air

Ultrasonication (DCM)

Extensive, including
NP-LC

GC

FPD; NPD; MS EI

 

Fernández & Bayona (1992)

Air particulate

Ultrasonication (DCM)

Silica gel

HPLC

Electrochemical

 

Galceran & Moyano (1993)

Air particulate

Ultrasonication (benzene/ethanol)

NP-LC

HPLC (with on-line reduction)

CL

0.3–5 fmol

Murahashi & Hayakawa (1997)

Air particulate

Ultrasonication (DCM)

Silica/cyclohexane

HPLC switching technique
GC
ELISA

FL

HRMS

20 fmol
(1-nitropyrene)

Zühlke et al. (1998)

Diesel exhaust

           

Diesel

Soxhlet

NP-LC

GC

NPD (FL)

 

Schuetzle & Perez (1983)

Diesel particulate

Soxhlet (DCM)

NP-LC

GC
GC

NPD
MS

0.5 ppm
5 ppm

Paputa-Peck et al. (1983)

Diesel or air particulates

Soxhlet (DCM)

Extensive; also
NP-LC

GC
GC

FID
TEA

 

Niles & Tan (1989)

Diesel or air particulates

Ultrasonication (acetone)

SPE and on-line reduction

GC

MS EI

1 ng/g

Scheepers et al. (1994a)

Air and diesel particulates

Soxhlet (DCM)

NP-LC

LC
On-line reduction

Electrochemical FL

60 pg
10 pg

MacCrehan et al. (1988)

Diesel or air particulates and gaseous

DCM

SPE

HRGC

HRMS

low ng/g (diesel particulate); pg/m3 range

Chiu & Miles (1996)

Vehicle or air particulates

Ultrasonication (benzene/ethanol)

Precolumn reduction

HPLC (column switching)

CL with on-line reduction

 

Hayakawa et al. (1999a)

Water

           

River water

 

DCM; florisil column

GC

MS

 

Takahashi et al. (1995)

River water

Concentration by blue chitin column; methanol

 

GC

MS

 

Nagai et al. (1999)

Wastewater

Fractionation into diethyl ether-soluble neutral, acidic and basic fractions

 

HPLC

UV and FL

 

Manabe et al. (1984)

Crankcase oil

Extraction with methanol, concentration and dissolution in water; then as above

 

HPLC

UV and FL

 

Manabe et al. (1984)

Soil, sewage sludge, sediment

         

Urban dust, residual of incinerator, soil

Soxhlet (acetonitrile)

SPE

GC

NPD

 

Librando et al. (1993)

Sewage sludge

Dimethyl formamide

LSE or SPE

GC

FID; GC-MS

 

Bodzek & Janoszka (1995)

River sediment

Ether; sodium hydroxide or
benzene methanol; chloroform

Silica gel column; reduced/acylated

GC

ECD

 

Sato et al. (1985)

Soil

Methanol

Silica gel column; hexane/benzene/
chloroform; HPLC ODS columns

Analytical ODS column for HPLC

Reduction to amino derivatives; fluorescence

Dinitropyrenes 0.7–4 pg

Watanabe et al. (1999)

Soil

Methanol/acetone

SPE; HPLC

GC

MS EI or NICI

30 ng/kg dry weight

Niederer (1998)

Biological samples

         

Rat tissue

Homogenate treated with acetonitrile; evaporated under nitrogen; residue dissolved in methanol; extraction with Blue Rayon

 

HPLC; on-line reduction

Fluorescence

 

van Bekkum et al. (1999)

Human lung specimens

Ultrasonication (DCM)

 

Bioassay-directed chemical analysis, HPLC, GC

MS

 

Tokiwa et al. (1993a, 1998a)

Food and beverages

       

Various food samples

Homogenization; acetonitrile; hexane

Silica gel column; DCM

GC

TEA

12 pg

Dennis et al. (1984)

Various food samples

   

GC
GC

MS
NPD

5 pg

Schlemitz & Pfannhauser (1996a)

Grilled and smoked food samples

   

HPLC; on-line reduction

Fluorescence

 

Schlemitz & Pfannhauser (1996b)

Fish, meat products and cheese

   

HPLC; on-line reduction

Fluorescence

50 ng/kg

Dafflon et al. (2000)

a

Abbreviations:

 

CL

chemiluminescence

MS

mass spectrometry

 

DCM

dichloromethane

MS EI

mass spectrometer electron impact mode

 

ECD

electron capture detector

NICI

negative-ion chemical ionization

 

ELISA

enzyme-linked immunosorbent assay

NPD

nitrogen–phosphorus detection

 

FID

flame ionization detector

NP-LC

normal-phase high-performance liquid chromatography

 

FL

fluorescence detection

ODS

octadecylsilyl

 

FPD

flame photometric detection

ppm

parts per million

 

GC

gas chromatography

RP-LC

reversed-phase high-performance liquid chromatography

 

HPLC

high-performance liquid chromatography

SIM

selective ion monitoring

 

HRGC

high-resolution gas chromatography

SPE

solid-phase microextraction

 

HRMS

high-resolution mass spectrometry

TEA

thermal energy analyser

 

LC

liquid chromatography

UV

ultraviolet

 

LSE

adsorption column chromatography on silica gel

   

b

The percent recovery was not given in most cases.

2.4.1 Sampling

Methods of collecting air particulates include a) Mega sampler with 50% cut-off point of 20 µm and typically 6-h sampling periods (used in Claremont, California, USA); b) an electrostatic precipitator with an impact stage designed for a 15-µm cut-off (used in Aurskog, Norway); and c) specially designed filter baghouses collected over a period of a year (National Institute of Standards and Technology [NIST] Standard Reference Materials [SRMs]) (Ramdahl et al., 1986). A 10-µm size-selective inlet (for particulate matter less than 10 µm in diameter, or PM10) sampler is also reported (Nishioka & Lewtas, 1992) .

For investigations into the formation and photochemistry of nitroPAHs, three different collection media for ambient air sampling have been used: Tenax-GC solid adsorbent, polyurethane foam (PUF) and filters for high-volume sampling (Arey et al., 1991).

Three methods for the sampling of the semivolatile phase of diesel exhaust were compared: cryogenic sampling, adsorbent sampler with XAD-2 and PUF. The PUF technique gave the highest recovery of PAHs and mutagenic activity. The three sampling techniques for the semivolatile phase resulted in extracts with different chemical composition, different mutagenic potency and different mutagenicity profiles (Westerholm et al., 1991).

In general, vapour-phase constituents are collected on solid sorbents such as XAD and PUF, and particles are collected on Teflon-impregnated glass fibre filters (see Table 4).

For sampling of diesel exhaust particulates, a dilution tunnel method is mainly used (Hayakawa, 2000). The exhaust is diluted by the filtered air in the tunnel to simulate the real road conditions, and an aliquot is sampled on the filter. Gaseous substances are trapped in PUF. The sampling and analytical methods are reviewed by Levsen (1988).

Blue cotton bearing covalently linked copper phthalocyanine trisulfonates as a ligand adsorbs polyaromatic compounds and preconcentrates several nitroPAHs in water (Hayatsu, 1992).

2.4.2 Extraction

Extraction of filter and PUF samples can be carried out by dichloromethane (DCM) in a Soxhlet apparatus for 16 h, the Soxhlet body being loosely covered in aluminium foil to exclude light (Chiu & Miles, 1996). DCM was found to be the most efficient solvent for extraction of mutagenic compounds from diesel particles (Montreuil et al., 1992).

Toluene as solvent has also been reported (Spitzer, 1993; Vincenti et al., 1996). Supercritical fluid extraction of nitroPAHs from diesel exhaust particulate matter using carbon dioxide–chlorodifluoromethane (HCFC-22) or carbon dioxide–toluene has been demonstrated (Paschke et al., 1992).

Soil sample extraction has been described using the Soxhlet device using 1:1 (v/v) toluene:methanol (Vincenti et al., 1996) or 5% ethanol in toluene (Spitzer, 1993).

2.4.3 Cleanup

Various procedures for fractionation of particulate extracts have been described:

From open-column liquid chromatography, four main fractions are obtained by eluting with different solvents (in parentheses): aliphatics (hexane), aromatics (hexane/benzene), moderately polar (DCM) and highly polar (methanol) (Lewtas, 1988). The nitroPAHs are to be found in the moderately polar fraction, but together with a number of oxy derivatives (e.g., aldehydes, ketones, quinones), which may interfere with them. Therefore, a more effective fractionation is achieved by NP-LC, but here the nitroPAHs are not collected in a single fraction but are distributed in several fractions (Vincenti et al., 1996). Another approach to separate nitroPAHs from hydroxyPAHs uses a sequential cleanup with silica and alumina (Moyano & Galceran, 1997). Nitrated and hydroxylated PAHs extracted from air particulates could be fractionated from other micropollutants by semipreparative packed-column supercritical fluid chromatography on silica gel (Medvedovici et al., 1998).

Figure 2 shows a schematic diagram of an analytical method used for the cleanup and separation of particulate organic matter (Ciccioli et al., 1996).

Figure 2

 

Fig. 2. Schematic diagram of the analytical method for the cleanup and separation of particulate organic matter (Ciccioli et al., 1996). DCM = dichloromethane; DMSO = dimethyl sulfoxide; GC-FID = gas chromatography with flame ionization detection; GC-MS = gas chromatography/mass spectrometry; HPLC = high-performance liquid chromatography.

 

2.4.4 Analytical separation and detection

The most frequently used techniques for the detection of nitroPAHs are (Vincenti et al., 1996):

A thin-layer chromatography method using plates coated with a silica gel layer has been developed for analysing nitroPAHs. DCM and DCM with n-hexane and methanol were used for the mobile phases. The chromatograms developed using a mixture of n-hexane–DCM (1:1 v/v) were observed under ultraviolet (UV) light before and after being sprayed with a reducing agent (sodium borohydride dissolved in methanol and copper chloride solution). Light at lambda = 254 nm induced green fluorescence for 1-nitropyrene and 1,3-dinitropyrene only and a violet colour for the remaining compounds. Carbon disulfide quenched fluorescence (observed at lambdaexc = 365 nm) for 1-nitronaphthalene, 9-nitroanthracene, 1-nitropyrene and 1,3-dinitropyrene only (Tyrpien, 1993; Tyrpien et al., 1997 ). Janoszka et al. (1997) used acetonitrile/water as the mobile phase. The method is suggested as a simple and quick method for identifying the above nitroPAHs in airborne particulate matter after separation in moderately polar fractions by column chromatography on silica gel. Isomeric nitroPAHs cannot be separated.

More recent developments in this field include a) selective detection of several nitroPAHs by using time-of-flight MS (Bentz et al., 1995; Dotter et al., 1996; Bezabeh et al., 1997); b) a method using supercritical fluid extraction and on-line multidimensional chromatographic methods (NP-LC coupled to a high-resolution GC) and ion trap detector MS (Lewis et al., 1995a,b; Feilberg et al., 2001); and c) particle beam liquid chromatography–MS with NICI mode (Bonfanti et al., 1996). High-resolution NICI is reported to be at least 20 times more sensitive than the low-resolution NICI or EI for determination of nitroPAHs in air samples (Chiu & Miles, 1996). A method has been developed involving the derivatization of nitroPAHs to their corresponding fluorinated derivatives, followed by GC-ECD analysis. The sensitivity of the method is an order of magnitude higher than that of direct GC-ECD analysis of nitroPAHs themselves. This method is suitable for routine monitoring of nitroPAHs in air samples (Jinhui & Lee, 2001).

2.4.4.1 Difficulties in analysis

Owing to small differences in GC retention times for 2- and 3-nitrofluoranthene, the 2-nitrofluoranthene present in some samples was incorrectly reported as 3-nitrofluoranthene in earlier reports. After reanalysis, the original reports were corrected (Ramdahl et al., 1986; Nishioka et al., 1988). Use of more selective stationary phases enabled better separation of isomeric pairs (Ciccioli et al., 1988). For the best separation of all of the nitrofluoranthene and nitropyrene isomers, the use of a DB13 (or SP30-type) column is recommended (A. Cecinato, personal communication, 2002).

Sampling gas-phase PAHs from environmental chambers onto Tenax adsorbent under conditions not typical of ambient atmospheres (e.g., 19 mg nitrogen dioxide/m3, 44 mg nitrogen pentoxide/m3) can lead to artificial formation of nitro derivatives via reactions with the Tenax adsorbent (Zielinska et al., 1986a).

2.4.4.2 Complex mixtures

The chemical analysis of trace levels of organic mutagens in ambient air is more complex than comparable analyses of emissions from specific sources. Source-emitted pollutants account for only part of the whole ambient air sample. Aging of air samples introduces unknown, highly variable chemical and meteorological factors. Further, the concentrations of pollutants are much lower in ambient air than at the sources (Greenberg et al., 1993).

2.4.4.3 Analysis of nitro-oxyPAHs

The procedures followed for investigating nitro-oxyPAHs in emissions and ambient air are, in general, the same as those adopted for nitroPAHs.

2.4.5 Use of bioassay (mutagenicity) fractionation and chemical analysis

Bioassay-directed fractionation closely coupled to chemical characterization has been developed as a method of determining nitroPAHs in complex mixtures (see Figure 3) (Schuetzle & Lewtas, 1986; Lewtas, 1988; Lewtas & Nishioka, 1990; Legzdins et al., 1995; Enya et al., 1997). In this approach, the complex mixture is fractionated, and each fraction is bioassayed; the mutagenic activity for each (HPLC) fraction is plotted in a manner analogous to a conventional chromatogram, and the plot is referred to as a mutagram (mutagenicity profile = mutagram).

Figure 3

Fig. 3. Bioassay-directed chemical analysis scheme for the determination of air particulate matter. The numbers under each fraction represent the percent distribution of mass and mutagenicity in Salmonella tester strains (in parentheses); adapted from Schuetzle & Lewtas (1986) and Lewtas et al. (1990a).

Mutagenically active fractions are further fractionated, bioassayed and characterized until the major class of compounds or specific com-pounds potentially responsible for the mutagenicity are identified. Use of bacterial tester strains selectively sensitive to nitroarenes has led to the identification of nitroPAHs as potent mutagens in complex mixtures from diverse sources.

Non-polar fractions of an extract of diesel particulates (SRM 1650) accounted for less than 2–3% of the mutagenicity in the total extract. The distribution of mutagenicity in the moderately polar and polar fractions was dependent on the source sample (Schuetzle & Lewtas, 1986).

Studies using this bioassay-directed fractionation and chemical characterization (see also section 7.5.5) include, for example, studies of diesel exhaust extracts (Schuetzle et al., 1981; Nishioka et al., 1982; Claxton et al., 1992; Legzdins et al., 1994; Enya et al., 1997; Hayakawa et al., 1997), xerographic toners (Rosenkranz et al., 1980), cigarette smoke (Kier et al., 1974), ambient atmospheric particles (Nishioka et al., 1988; Lewtas et al., 1990a; Arey et al., 1992; Casellas et al., 1995; Hayakawa et al., 1995b) and the metabolites of 1-nitropyrene (Lewtas et al., 1990b).

For example, Casellas et al. (1995) made a detailed chemical analysis of mutagenic fractions using bioassay-directed chemical analysis in urban airborne particulate matter in Barcelona, Spain. The first fractionation of the solvent (DCM) extractable organic matter was achieved by semipreparative gel permeation chromatography (GPC). The second fractionation was achieved with NP-LC. The collected fractions were tested for mutagenicity using the S. typhimurium microsome assay with strains TA98, TA98NR– and TA98AT– (for more details, see chapter 7). The chemical characterization of mutagenic fractions was carried out by an extensive application of capillary GC-MS in the EI and NICI modes. Those fractions exhibiting the highest levels of mutagenicity were subjected to a third level of fractionation by reversed-phase HPLC (RP-LC) and analysed by GC-MS. Two sampling sites in Barcelona were monitored during 1990. Samples (24 h) of air particulate matter over periods of 1 week per season were processed.

The direct mutagenicity in the fractions NP-LC 3 (and 4) isolated from the GPC-2 fraction of airborne particulate matter collected in Barcelona (autumn 1990) seemed to be accounted for by nitrated arenes, 9-nitroanthracene, 2-nitrofluoranthene and 2-nitropyrene. 6-Nitrobenzo[a]pyrene in polar fraction 5 needed application of metabolic activation (+S9) for mutagenicity. In order to evaluate the contribution of nitro derivatives to the total mutagenicity, the NP-LC fractions were tested against TA98NR– and TA98AT–. Generally, a remarkable decrease in mutagenic activity was observed in all fractions; these decreases were more apparent in fractions NP-LC3 to NP-LC7, thus suggesting a significant contribution of nitroarenes to the mutagenicity of these fractions of medium and high polarity. Subfraction NP-LC2 was fractionated further. The expected composition was PAHs, but two nitro derivatives, 2-nitrofluoranthene and 1-nitropyrene, were identified in subfraction RP-LC3, which may be responsible for the high direct-acting mutagenic activity observed in this fraction and in the NP-LC2 fraction (Casellas et al., 1995).

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

NitroPAHs originate primarily as direct or indirect products of incomplete combustion. Only a few nitroPAHs are produced industrially (e.g., nitronaphthalenes and 5-nitroacenaphthene).

3.1 Industrially produced nitroPAHS

3.1.1 Production levels and processes

Treatment of naphthalene with mixed sulfuric/nitric acids at 60 °C yields 95% 1-nitronaphthalene and 5% 2-nitronaphthalene, together with traces of dinitronaphthalene and dinaphthol. Higher temperatures (80–100 °C) result in a mixture of 1,5- and 1,8-dinitronaphthalene in about a 2:3 ratio (Booth, 1991). The dinitro isomers can then be further separated for specific uses.

Nitronaphthalenes are produced in Germany and Japan. The production capacity of dinitronaphthalenes in Japan was 1200 tonnes per year (no year given; Booth, 1991).

3.1.2 Uses of commercially produced nitroPAHs

1-Nitronaphthalene is used almost exclusively for catalytic reduction to 1-naphthylamine. Further uses, such as use as a deblooming agent for petroleum and oils and as a component in the formulation of explosives, are of historical interest only (Booth, 1991).

1,5-Dinitronaphthalene is an intermediate in the production of naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) and 1,5-naphthalenediamine, which is mainly converted to naphthalene 1,5-diisocyanate. It is further used as a sensitizing agent for ammonium nitrate explosives (Booth, 1991).

1,8-Dinitronaphthalene is catalytically hydrogenated to 1,8-naphthalenediamine for use mainly as a colorant intermediate for naphthperinones (Booth, 1991).

5-Nitroacenaphthene is reported to be an intermediate in the synthesis of naphthalimide dyes that are used as fluorescent whitening agents and photochemical agents (Yahagi et al., 1975; IARC, 1978).

3.2 Other sources of nitroPAHs

NitroPAHs in the environment originate from direct emissions from combustion sources and nitration of PAHs, primarily in the atmosphere.

The nitroPAHs emitted from combustion sources are nitroPAHs that would be formed through electrophilic nitration (e.g., 1-nitropyrene and 2-nitrofluorene; see section 3.2.1). NitroPAHs have been observed in vehicle exhaust (particularly diesel), industrial emissions and emissions from domestic residential heating/cooking and wood burning.

NitroPAHs are formed in the atmosphere from PAHs by the following reactions:

These processes are described in more detail in section 3.2.2. Other less important pathways, which are briefly mentioned here, include:

The distribution of nitroPAH isomers in samples of ambient air has been found to be significantly different from that in direct emissions from combustion (compare Table 5 and chapter 5). For example, 2-nitrofluoranthene and 2-nitropyrene are ubiquitous components of particulate matter that have been detected in urban, suburban, forest and remote areas located in Europe, America, Asia and Antarctica (Ciccioli et al., 1996), although they are not directly emitted from most combustion sources (see chapter 5). The nitroPAH profile, or the relative quantities of certain "marker" PAHs, is a pointer to the source of formation of nitroPAHs — for example, markers of direct emissions from combustion, in particular diesel exhaust, are 1-nitropyrene and 2-nitrofluorene, whereas the presence of 2-nitrofluoranthene and 2-nitropyrene points to atmospheric transformation.

Table 5. NitroPAHs detected in diesel emissionsa,b

Parent PAH;
nitro derivative

Concentration (ppm = µg/g, unless otherwise stated)

a)
E

b)
E

c)
P

d)
E

e)
P

f)
E

g)
P

h)
P

i)
E

j)
E

k)
P

l)
E

m)
P µg/m3

n)
µg/km

o)
P pmol/mg

Naphthalene

                             

1-Nitro-

X (nd)

X

X

0.7

0.88

       

0.013

         

2-Nitro-

nd

X

X

 

0.02

       

0.039

         

1,3-Dinitro-

 

X

X

                       

1,5-Dinitro-

X (nd)

X

X

                       

1,8-Dinitro-

X (nd)

                           

Acenaphthene

                             

3-Nitro-

 

X*

                         

Fluorene

                             

1-Nitro-

           

5.91*

 

0.08*

           

2-Nitro-

X

X

X

nd

0.11

4.1

<0.01

0.27

0.001

 

X

27

8.77

0.99

nd

2,5-Dinitro-

X (nd)

                           

2,7-Dinitro-

X (nd)

                       

0.13

 

Anthracene

                             

1-Nitro-

 

X*

 

300*

 

4.6

X*

               

2-Nitro-

X

X

     

10.1

                 

9-Nitro-

X

X

X

   

2.8

6.02

10

1.19

1.36

7

63

 

1.00

 

Phenanthrene

                             

1-Nitro-

         

0.5

                 

2-Nitro-

X

X

     

X

16.5*

 

1.8*

           

3-Nitro-

         

9.3

                 

4-Nitro-

         

0.7

                 

9-Nitro-

         

2.3

0.37

 

0.18

0.27

         

Fluoranthene

                             

1-Nitro-

X (nd)

X

     

4.1

X*

               

2-Nitro-

nd

X

             

0.06

         

3-Nitro-

X (nd)

X

 

1

 

10

0.33

 

1.34

1.47

<1

10

 

0.06

 

7-Nitro-

X (nd)

X

     

1.6

                 

8-Nitro-

X (nd)

X

     

2.0

     

0.25

         

Pyrene

                             

1-Nitro-

75

X

X

590

2.84

43

18; 19.6*

20

16.6; 18.0*

16.4

19

450

0.2 (+1,3-DNP)

3.43

6.49

2-Nitro-

nd

                           

4-Nitro-

nd

               

0.07

       

0.04

1,3-Dinitro-

0.30

X

 

0.6

   

<0.1

 

0.58

0.6

   

with 7-NP

0.12 all DNP

0.17

1,6-Dinitro-

0.40

   

0.6

   

<0.1

 

0.64

1.39

   

0.37

 

0.04

1,8-Dinitro-

0.53

   

0.4

   

<0.1

 

0.49

1.55

       

0.07

Benz[a]anthracene

                             

7-Nitro-

nd

X

 

8*

   

0.45

2.8

1.99

1.62

<1

10

0.61

   

Chrysene

                             

1-Nitro-

X (nd)

         

0.8*

 

2.9*

           

5-Nitro-

         

4.6

                 

6-Nitro-

 

X

       

0.04

 

0.87

0.782

X

4

0.08

 

0.10

Benzo[a]pyrene

                             

1-Nitro-

1- + 3-

   

10*

1.2*

 

1.4*

               

6-Nitro-

X

 

X

   

5.8

1.4

1.6

 

0.641

X

   

0.12

0.30

Benzo[e]pyrene

                             

1-Nitro-

                 

0.83

         

3-Nitro-

                 

2.2

         

Perylene

                             

3-Nitro-

X

 

X

                     

0.64

a

E = extract; P = particulate; DNP = dinitropyrene; 7-NP = 7-nitropyrene; X = detected; * = mixture of isomers; X (nd) = detected by GC/nitrogen–phosphorus detector (NPD) (0.5 µg/g) but not by GC-MS (detection limit 5 µg/g); nd = not detected by either.

b

Samples were as follows:

 

a)

Light-duty diesel particulate extract (Paputa-Peck et al., 1983).

 

b)

Levsen (1988); Schilhabel & Levsen (1989). Thirty-eight nitroPAHs were detected (including methyl-nitroPAHs and mixed isomers, not noted in the table). Identification by comparison with reference compounds or relative retention times reported by Paputa-Peck et al. (1983).

 

c)

Hartung et al. (1984). Exhaust from light-duty diesel test engine.

 

d)

Concentration in extract from VW Rabbit diesel (Nishioka et al., 1982, 1983).

 

e)

Yu et al. (1984).

 

f)

Campbell & Lee (1984); g/g light-duty diesel particulate extract (US Environmental Protection Agency [EPA] recalculated concentrations from mg/g extract to g/g particle using a value of 44% for extractable material).

 

g)

Diesel particulate SRM 1650 (Chiu & Miles, 1996).

 

h)

Diesel particulate SRM 1650 (MacCrehan et al., 1988).

 

i)

Diesel extract SRM 1975 (Chiu & Miles, 1996).

 

j)

SRM 1975 diesel particle extract (DCM extract of diesel particulate matter collected from an industrial diesel-powered forklift) (NIST, 2000) (SRMs are analytical reference samples for quality control, not implied as representative of environmental diesel samples).

 

k)

Heavy-duty diesel particulate matter (SRM 1650) (Niles & Tan, 1989).

 

l)

NitroPAHs extracted from bus soot (Paschke et al., 1992). Bus soot contained much higher concentrations of 1-nitropyrene than standard diesel sample SRM 1650 (450 compared with 20 µg/g); extraction by carbon dioxide–HCFC-22.

 

m)

Particulate nitroPAH concentration; 1988 Cummins LTA engine operated under baseline conditions (no trap) at US EPA steady-state engine mode 9; mean of n = 3; vapour-phase exhaust was >0.025 µg/m3. Details also given with mode 11; with and without ceramic particle trap and with copper fuel additive (Harvey et al., 1994).

 

n)

µg/km US Federal Test Procedure 72 (FTP-72) cycle with hot start, four-cylinder engine, light diesel vehicle (n = 3–6) (Scheepers & Bos, 1992a).

 

o)

pmol/mg; particulate emission from 1995 diesel engine vehicle, idling engine (Hayakawa et al., 1997).

The majority of ambient nitroPAHs are now thought to be formed in the atmosphere from the gas-phase reactions of PAHs with four rings or less (Atkinson & Arey, 1994).

3.2.1 Direct sources of nitroPAHs from combustion processes

3.2.1.1 Diesel exhaust

1) Qualitative and quantitative studies of nitroPAHs in diesel exhaust

NitroPAHs have been detected in particulate exhaust emissions of motor vehicles, in particular diesel exhaust emissions, together with hundreds of other organic compounds (see Table 3; Schuetzle, 1983). Interest was focused on nitroPAHs in the early 1980s because correlations were found between the presence of nitroPAHs in diesel exhaust (and environmental extracts) and mutagenic activity in Salmonella typhimurium (see chapter 7). A large number of groups of nitro, oxy and mixed nitro-oxy compounds eluted together in the mutagenic fractions. Analytical methods were developed to separate and identify as many mononitro- and dinitroPAHs in diesel exhaust as technically possible, first as isomer groups and then using isomer-specific identification (e.g., Schuetzle et al., 1981, 1982; Newton et al., 1982; Xu et al., 1982; Henderson et al., 1983; Paputa-Peck et al., 1983; Campbell & Lee, 1984; Levsen, 1988; Niles & Tan, 1989; Schilhabel & Levsen, 1989; Chiu & Miles, 1996; see also Table 5). The number of nitroPAHs quantified is generally limited due to lack of standards. Investigators have used different samples of diesel exhaust as well as different analytical methods. Further, the concentration of nitroPAHs adsorbed on diesel particulate varies substantially from sample to sample (Levsen, 1988). It is therefore difficult to compare the various nitroPAH profiles. Usually, 1-nitropyrene is the predominant component, and concentrations of 7–165 µg/g particulate have been reported (Levsen, 1988). However, 1-nitropyrene is not always the dominating substance. Especially in heavy-duty diesel, 2-nitrofluorene may exceed 1-nitropyrene by a factor of 1.8–15, with an average of 9.8. When 1-nitropyrene is the dominant substance, a 2-nitrofluorene concentration equal to 15% of that of 1-nitropyrene is common (Beije & Möller, 1988a).

Some studies have focused on measuring concentrations of specific nitroPAHs — for example, 1-nitropyrene as a marker of nitroPAH formation (see below), 2-nitrofluorene (Beije & Möller, 1988a; Möller et al., 1993a) or dinitroPAHs, in particular 1,3-, 1,6- and 1,8-dinitropyrenes, which have been found to be more mutagenic (although not necessarily more carcinogenic; see chapter 7) than mononitroPAHs (Pederson et al., 1984; Hayakawa et al., 1992, 1994) (see Table 6). Dinitropyrenes are formed from the further nitration of 1-nitropyrene and are present at only about 1% of its concentration (Schuetzle & Frazier, 1986). Mononitrofluoranthene and mononitropyrenes were separated and identified in diesel extracts (Ciccioli et al., 1988).

Table 6. Concentrations of 1-nitropyrene and 1,3-, 1,6- and 1,8-dinitropyrene in diesel particulate and gasoline exhausta

NitroPAH

Concentration (µg/g)

a)

b)

c)

d)

e)

f)

g)

1-Nitropyrene

3.9

3.9–116

13

37

0.32

0.44

2.1 (<5)

1,3-Dinitropyrene

<0.005

 

0.07

0.08

0.05

0.06

 

1,6-Dinitropyrene

0.033

0.04–4.47

0.07

0.15

0.06

0.12

 

1,8-Dinitropyrene

0.013

0.04–6.24

0.06

0.23

0.08

0.10

 

a

Samples were as follows:

 

a)

From 1978 Opel diesel (Gibson, 1982, 1983).

 

b)

Range of nitropyrenes detected in diesel particulate from five different motors (1979–1983) under different driving speeds and cycles and collected by different methods (Pederson et al., 1984).

 

c)

Mean from seven diesel vehicles (1983–1991) (Hayakawa et al., 1994).

 

d)

Diesel exhaust from 1980 Isuzu truck (Sera et al., 1994).

 

e)

Gasoline exhaust from 1985 Toyota automobile (Sera et al., 1994).

 

f)

Exhaust from eight gasoline vehicles (year not given) (Hayakawa et al., 1994).

 

g)

On-road emission factors for 1-nitropyrene of 0.49 versus <0.03 µg/km (2.1 versus <5 µg/g, by mass of the extracted particulate matter) were given for heavy-duty diesel trucks and spark ignition gasoline light-duty passenger cars, respectively (Gorse et al., 1983).

2) Formation of nitroPAHs and comparison of emissions

NitroPAHs are formed in vehicle engines from the reactions of PAHs with nitrating species that are provided by the conversion of nitrogen and oxygen at high temperatures in the combustion chamber (Scheepers & Bos, 1992a).

Studies compare the emissions of nitroPAHs from various engines (Henderson et al., 1983), under differing driving conditions (different speeds, loads, etc.) (Schuetzle & Perez, 1983; Draper, 1986; Lorber & Mollenhauer, 1989; Veigl et al., 1994), with and without an oxidation catalytic converter (Johnson et al., 1994; Mitchell et al., 1994; Pataky et al., 1994), with and without catalysed or non-catalysed particulate traps (Westerholm et al., 1986; Bagley et al., 1993; Harvey et al., 1994; Johnson et al., 1994) and using a variety of fuels and/or additives (Harvey et al., 1994; Johnson et al., 1994).

1-Nitropyrene concentrations in exhaust (given as µg/g dust) from heavy-duty trucks during simulated driving cycles are as follows: suburban, 1.94; urban, 3.39; and motorway, 1.94 and 2.73 (Scheepers et al., 1994a). 1-Nitropyrene emission rates in exhaust emissions from a heavy-duty diesel vehicle during transient driving conditions were 1.6 µg/km in the particulate and <0.05 µg/km in the semivolatile phase (Westerholm et al., 1991). Light-duty trucks with oxidation catalysts emitted 1-nitropyrene at 0.36 µg/km with the US Federal Test Procedure 75 (FTP-75) driving cycle and 0.32 and 0.22 µg/km with the European driving cycle (cold and hot start, respectively; Scheepers et al., 1994a).

In a multivariate analysis of exhaust emissions, 10 different fuels were combusted using two different types of heavy-duty diesel engines. The levels of 1-nitropyrene in the exhaust from the different fuels ranged from 0.07 to 2.17 µg/km in one engine and from 0.32 to 7.19 µg/km in the other. It was not found that one fuel gave a high 1-nitropyrene emission in both engine types. There was a negative correlation between 1-nitropyrene and nitrogen oxides and pyrene content, indicating the formation of 1-nitropyrene from the reaction between nitrogen oxides and pyrene (Sjögren et al., 1996).

Studies using low-sulfur fuel showed that, independent of the type of engine and exhaust after-treatment device, the primary effect of reducing sulfur content in diesel fuel was to substantially decrease the sulfate emissions and the number of respirable particles (Bagley et al., 1996). The total particulate matter emissions were not significantly diminished. However, because sulfate particles are so small, decreases in the emissions of these particles do not significantly diminish the emissions of total particulate matter. Using low-sulfur fuel had no significant effect on emissions of semivolatile organic compounds in the vapour phase or of soluble organic compounds in the particulate phase. However, the emission of hydrocarbon gases, the emission of some PAHs in the particulate and vapour phases and the mutagenicity of particulate-phase fractions were significantly elevated under some operating conditions, which may have been due to differences in the hydrocarbon composition of the low-sulfur fuels compared with that of conventional, high-sulfur fuels. This study did not measure nitroPAHs but should be relevant for these compounds (Bagley et al., 1996).

Particulate-associated and vapour-phase emissions of 2-nitrofluorene, 1,3-, 1,6- and 1,8-dinitropyrene, 1-nitropyrene, 3-nitrofluoranthene, 7-nitrobenz[a]anthracene and 6-nitrochrysene were measured with a low-sulfur (0.01% by mass sulfur) fuel and a particulate trap at steady-state mode 9 (Johnson et al., 1994; Table 7). Relatively large amounts of nitroPAHs were found in the vapour phase. In contrast to other studies involving diesel exhausts, it is not clear why the dinitropyrene concentrations that were detected are so high relative to the 1-nitropyrene concentration (see also Harvey et al., 1994, in Table 5). The use of a low-sulfur fuel with the particulate trap is known to alter particle size distributions and partitioning of PAHs (see Table 8).

Table 7. Particulate-associated (SOF) and vapour-phase (XOC) nitroPAH emissions with a low-sulfur fuel and a
particulate trap at steady-state mode 9a

 

Phaseb

Mean nitroPAH level (ng/m3)

   

2-Nitro-
fluorene

1,6/1,8-Dinitropyrene

1,3-Dinitropyrene

1-Nitropyrene

3-Nitro-
fluoranthene

7-Nitro-
benz[a]anthracene

6-Nitrochrysene

Baseline

SOF

420

550

420

120

1020

<24

<21

 

XOC

<25

830

82

77

<38

10

140

Trap

SOF

920

1200

100

≤16

25

≤20

<12

 

XOC

<16

1300

240

130

<24

170

38

a From Johnson et al. (1994).

b SOF = soluble organic fraction; XOC = extractable organic component.

Table 8. Changes in PAH emissions with a non-catalysed particulate trapa

Fuel type

Particulate-associated PAH (%)

Vapour-phase PAH (%)

Overall PAH (%)

Commercial No. 2 (0.32% sulfur)

–25

+15

+2

"Low sulfur" (0.01% sulfur)

–65

+140

+18

a From CONCAWE (1998).

Low-sulfur no. 2 diesel fuel and 100% soy methyl ester biodiesel fuel were tested with and without an oxidation catalytic converter over a light-duty transient test cycle (Bagley et al., 1998). Of the nitroPAH compounds analysed (1-nitropyrene, 2-nitrofluorene, 6-nitrochrysene and 1,3- and 1,6-dinitropyrenes), only 1-nitropyrene was found in quantifiable levels in all particle-associated samples (although 1,3-dinitropyrene may also be present, as it co-elutes with 1-nitropyrene in the method used in this study). The use of the oxidation catalytic converter with low-sulfur no. 2 diesel fuel reduced 1-nitropyrene by 66%, but this was not significant.

From CONCAWE’s review of studies on PAHs in automotive exhaust emissions, it can be said that, in general, after-treatment systems can substantially decrease PAH emissions. Diesel oxidation catalysts may be more effective in reducing the vapour-phase PAHs, whereas particulate traps seem to deal more effectively with PAHs condensed onto particulate matter. The few data on the effects of after-treatment (e.g., catalysts) on nitrated PAHs and the associated mutagenicity of the exhaust are variable, and the results of different studies can be contradictory (CONCAWE, 1998).

However, there does seem to be increasing evidence in recent studies that nitroPAHs, in particular in volatile and semivolatile fractions, are still emitted in diesel exhaust emissions. For example, in a study by Sharp (2000), all of the PAH and nitroPAH compounds investigated were present in the exhaust of all three engines tested (Cummins N14, DDC Series 50 and Cummins B5.9) when operated on a special batch of diesel fuel, blended to meet the stringent specifications required by the US Clean Air Act, at levels well above the US Environmental Protection Agency (EPA) required threshold of 0.5 ng/hp-h (Table 9). Further, Sharp (2000) showed that emissions of PAH and nitroPAH compounds were substantially lower with biodiesel than with conventional diesel fuel. This is not unexpected, when considering that the biodiesel contains no aromatics and no PAH compounds. The catalyst-out nitroPAH data presented an unexpected trend, in that catalyst-out nitroPAH levels were significantly higher than engine-out levels. This trend was most evident with the lighter nitroPAH compounds.

Table 9. PAHs or nitroPAHs measured in exhaust from different types of engine with diesel fuel with and without an oxidation catalysta

PAH or nitroPAH

Mass in exhaust (ng/hp-h)

Cummins N14

DDC 50

Cummins B5.9

No catalyst

No catalyst

Catalyst

No catalyst

Catalyst

Benzo[a]pyrene

1435

1614

226

723

241

2-Nitrofluorene

123

88

90

257

478

1-Nitropyrene

82

83

76

210

2171

7-Nitrobenz[a]anthracene

1.9

1.4

1.4

34

60

6-Nitrochrysene

0.8

0.8

5.8

11

56

6-Nitrobenz[a]pyrene

2.5

11.1

4.2

6.1

6.8

a From Sharp (2000).

Diesel fuel, engine types and catalytic traps/converters are continually being modified, so the various studies of nitroPAHs in diesel exhaust cannot be directly compared. A detailed survey of nitroPAHs in exhaust emissions under various conditions is beyond the scope of this document.

Although diesel use is likely to increase during the coming years (Lloyd & Cackette, 2001), a relatively small number of tests have been conducted to determine the gaseous, semivolatile and particulate organic matter in diesel fuel and exhaust. Very little is known about how this composition changes with different operating conditions and the introduction of new technologies. Further understanding will require significant improvements in the analytical methods and procedures used in emissions testing of diesel engines (Chow, 2001).

3) Diesel engine oil

New diesel engine oil did not contain 1-nitropyrene at a detection limit of 0.1 mg/litre; after a vehicle travel distance of 9000 km, however, 0.5 mg/litre was detected in the used oil. The presence of 1-nitropyrene in diesel engine oil points to another source of environmental contamination (e.g., used engine oil; Jensen et al., 1986).

3.2.1.2 Diesel compared with gasoline exhaust

Concentrations of some nitroPAHs detected in the exhaust particulate from mufflers of gasoline engines were much lower than those from diesel engines — for example, for 1-nitropyrene (0.16 versus 27.7 µg/g tar, respectively) and 2-nitrofluorene (0.16 versus 5.52 µg/g tar, respectively) (Handa et al., 1983). These differences were not as pronounced in a comparative study of 1-nitropyrene in the soluble organic fraction (SOF) of particles from exhaust emissions of in-use gasoline- and diesel-powered passenger cars under simulated driving conditions (mean 27.4 versus 53 µg/g, respectively) (Tejada et al., 1986).

Particulate-associated 1-nitropyrene was emitted at a rate of 0.03–0.05 µg/km driving distance from two three-way catalyst-equipped light-duty gasoline-fuelled vehicles using the US FTP-75 driving cycle under three different driving conditions — cold transient, stabilized and hot transient (Westerholm et al., 1996). (Three-way catalysts for gasoline exhaust seem to be effective in reducing both vapour- and particulate-phase PAHs [CONCAWE, 1998].)

Although the concentration of 1-nitropyrene, for example, was less in gasoline particles than in diesel particles, the concentrations of 1,3-, 1,6- and 1,8-dinitropyrene were found to be almost the same (Hayakawa et al., 1994; Sera et al., 1994; see Table 6).

3.2.1.3 Aeroplane emissions

Studies on the mutagenicity of aircraft (jet aeroplane) particulate extracts suggested the presence of nitroarenes, but no analytical determination was made (McCartney et al., 1986). The mutagenicity derived from idling aircraft was greater than that collected across an active runway. This is in agreement with the finding that particulates from idling aircraft are much richer in PAHs than those collected during simulated landings and takeoffs (Robertson et al., 1980). PAHs emitted during simulated idling are greatly enriched with respect to three- and four-ring structures. The majority of organic pollutants in airports come from idling aircraft. Takeoffs contribute only 1–2% to the total burden (Gelinas & Fan, 1979). In simulation experiments, the particulate-adsorbed PAHs constitute less than 1% of the total PAHs emitted by aircraft, the remainder residing in the gaseous phase (Robertson et al., 1980).

3.2.1.4 Emissions from combustion of heating oils

NitroPAHs have been detected in the emissions of kerosene heaters, fuel gas and liquefied petroleum gas (LPG) burners, which are used in many countries (e.g., Japan, China, Taiwan; also mobile homes in USA) for heating and cooking at home (Tokiwa et al., 1985, 1990a; Kinouchi et al., 1988).

In a study on indoor air concentrations in mobile homes with kerosene heaters, 1-nitronaphthalene, 2- or 3-nitrofluoranthene and 1-nitropyrene were found in the particulate phase (dinitropyrenes were below the detection limit). In the semivolatile organic fraction, from these nitroPAHs, only 1-nitronaphthalene was detected (naphthalene itself was present at a concentration more than 1000-fold higher than the 1-nitronapthalene concentration) (Mumford et al., 1991).

3.2.1.5 Fumes from cooking oils

Fume samples from three different commercial cooking oils frequently used in Taiwan were collected and analysed. As well as several PAHs (benzo[a]pyrene [BaP], benz[a]anthracene and dibenz[a,h]anthracene), two nitroPAHs were also identified. Concentrations of 1-nitropyrene and 1,3-dinitropyrene were, respectively, 1.1 and 0.9 µg/m3 in fumes from lard oil, 2.9 and 3.4 µg/m3 from soybean oil, and 1.5 and 0.4 µg/m3 from peanut oil (Wu et al., 1998). (The incidence of lung cancer in Chinese women is relatively high and is thought to be associated with cooking practices [Ko et al., 1997].)

3.2.1.6 Other combustion sources

Williams et al. (1986) reported significant levels of 18 different species of nitroPAHs in diesel extract but did not find any in coke oven mains, roofing tar vapour or cigarette smoke condensate at a detection level of <50 pg. The lack of 1-nitropyrene, 1-nitronaphthalene and 6-nitrochrysene in mainstream cigarette smoke was also shown by El-Bayoumy et al. (1985).

In another study, coke oven emission extractable organic matter was found to contain 3-nitrophenanthrene and 1-nitropyrene (66 and 27 ng/mg, respectively) in the slightly polar fraction and 9-nitroanthracene and 3-nitrophenanthrene (78 and 23 ng/mg, respectively) in the acidic fraction. However, 9-nitrophenanthrene, 3-nitrofluoranthene, 6-nitrochrysene and 6-nitrobenzopyrene were not detectable. In vitro mammalian cell cultures were used to determine whether DNA adducts derived from individual nitroPAHs or from organic extracts of coke oven emissions can be detected. Using the 32P-postlabelling method, 4-nitropyrene, 6-nitrochrysene and 3-nitrofluoranthene were reported to cause 10–100 DNA adducts per 108 nucleotides. When the extractable organic matter was used, the results suggest that nitroPAH adducts (detected by 32P-postlabelling) were present and may contribute to the genotoxicity of coke oven emissions (Topinka et al., 1998).

Nitroarenes were found to be an important contributor to the mutagenic activity of the emissions from municipal waste incinerators (DeMarini et al., 1996; see also section 7.5.5.7). 1-Nitropyrene was identified in coal fly ash (Harris et al., 1984). NitroPAHs, in particular 2-nitrofluoranthene and 2-nitropyrene, have been detected in the stack emissions of a plant manufacturing carbon electrodes (Ciccioli et al., 1988, 1989).

3.2.2 Atmospheric formation of nitroPAHs

The gas-phase formation of nitroPAHs in the atmosphere was first proposed by Pitts et al. (1985a) to explain the unexpected presence of 2-nitrofluoranthene and 2-nitropyrene in particulate organic matter sampled in the Los Angeles basin in California, USA (see chapter 5). These two nitroPAHs have not been identified in diesel exhaust or other combustion products. The occurrence of these nitroPAHs in different locations of southern California (Arey et al., 1987; Atkinson et al., 1987a; Zielinska et al., 1989a) and Europe (Nielsen & Ramdahl, 1986; Ramdahl et al., 1986; Ciccioli et al., 1989; Feilberg et al., 2001) as well as in forest and remote areas of America and Asia (Ciccioli et al., 1996; see chapter 5 and Table 19) showed their ubiquitous presence. Further studies showing differences in daytime/nighttime concentrations of certain nitroPAHs under various climatic conditions (see Figure 6 in chapter 5) provided further support for the gas-phase formation of nitroPAHs.

A further confirmation of the atmospheric origin of 2-nitrofluoranthene is the finding of its absence in tunnel air (Queensway, Birmingham, United Kingdom) and its abundant presence in the ambient atmosphere of this city (Dimashki et al., 2000; see also Table 10).

Table 10. Measurements of nitroPAHs in the atmosphere showing distribution in particulate and vapour phasesa

Compound

Particulate (ng/m3)

Vapour (ng/m3)

Mean
(n = 25)

Range

Mean
(n = 5)

Range

1-Nitronaphthalene

<0.61x10–4

 

0.089

0.033–0.207

2-Nitronaphthalene

<1.4x10–4

 

0.067

0.027–0.176

9-Nitroanthracene

0.130

0.034–0.520

0.057

0.014–0.177

1-Nitropyrene

0.090

0.019–0.204

<0.22x10–4

 

2-Nitrofluoranthene

0.221

0.046–0.586

<0.21x10–4

 

7-Nitrobenz[a]anthracene

0.033

0.011–0.059

<0.74x10–4

 

a From Dimashki et al. (2000); Birmingham, United Kingdom (November 1995 – February 1996).

Two-ring nitroPAHs are present mainly in the vapour phase; under some climatic conditions, other nitroPAHs are also present in the vapour phase. For example, according to Arey et al. (1987) in a study in California, the four-ring PAHs fluoranthene and pyrene were mainly (>90%) present in the gas phase; even at 0 °C, 30% and 70%, respectively, were measured in the gas phase and were therefore available for reaction. However, the nitrofluoranthenes and nitropyrenes exist almost exclusively in the particulate phase. 2-Nitrofluoranthene and 2-nitropyrene formed in the gas phase by hydroxyl-initiated reactions were observed to condense on airborne particles immediately, so that these particles were not detected in the gas phase (Fan et al., 1995). Ambient data from California suggest that the nitrofluorenes are distributed between the gas and particulate phases (Helmig et al., 1992c). Table 10 shows the concentration of selected nitroPAHs measured in vapour and particulate phases in ambient air.

Laboratory studies were carried out to understand the formation of nitroPAHs in ambient air (e.g., Arey et al., 1986, 1989a, 1990; Zielinska et al., 1989b; Atkinson et al., 1990a,b). From these studies, it was concluded that gas-phase daytime hydroxyl radical- and nighttime nitrate radical-initiated reactions of simple volatile and semivolatile PAHs do form nitroPAH derivatives (Atkinson, 1990).

Both daytime hydroxyl radical-initiated reactions and nighttime nitrate radical-initiated reactions have been shown to produce ambient nitroPAHs. For references summarizing the atmospheric formation of nitroPAHs, see Atkinson & Arey (1994) and Arey (1998).

3.2.2.1 Reactions of gas-phase PAHs (and nitroPAHs) with the hydroxyl radical (daytime reactions)

The photolysis of ozone in the troposphere results in the formation of the hydroxyl radical. The hydroxyl radical-initiated reactions of PAHs lead to the formation of nitroPAHs in low yields (5% or less; see Table 11) — for example, 2-nitrofluoranthene (Arey et al., 1986; Zielinska et al., 1986b; Atkinson et al., 1990a), 2-nitropyrene (Arey et al., 1986; Atkinson et al., 1990a) and 3-nitrofluorene (Helmig et al., 1992c). The proposed mechanism involves hydroxyl radical reaction with the gaseous PAH, followed by nitrogen dioxide addition at the free radical site. Although this reaction occurs in competition with the reaction with oxygen, nitroPAH formation is preferred in the presence of sufficient nitrogen dioxide. The resulting nitroPAH products having a relatively low vapour pressure may then condense out on the surface of ambient particles (Atkinson & Arey, 1994).

This hydroxyl radical-initiated mechanism could also explain the formation of volatile nitroarenes such as 1- and 2-nitronaphthalene from gaseous naphthalene (Atkinson et al., 1987b, 1990a). Ambient measurements indicate that the atmospheric reaction products of the two-ring PAHs, such as nitronaphthalenes, remain predominantly in the gas phase. Phenanthrene is more abundant in ambient air than anthracene, fluoranthene and pyrene. Helmig et al. (1992a,b) observed nitrophenanthrenes at only very low yields (# 1%), although other authors (Wilson et al., 1995) found that 9-nitrophenanthrene was the second most abundant nitroPAH after 1-nitronaphthalene (see Figure 6 in chapter 5).

3.2.2.2 Reactions of gas-phase PAHs (and nitroPAHs) with the nitrate radical (nighttime reactions)

In ambient air, in contrast to environmental chambers, the nitrate radical is formed from the reaction of nitrogen dioxide with ozone. Concentrations of nitrate radical are low during daylight hours because of the rapid photolysis of the nitrate radical (with a photolysis lifetime at solar noon of approximately 5 s) and the rapid reactions of nitric oxide with ozone and nitrate with nitric oxide (Atkinson et al., 1992). At night, however, in the absence of nitric oxide, the concentrations of the nitrate radical and nitrogen pentoxide increase (Atkinson et al., 1986). Average nitrate radical concentrations in the lower troposphere over continental areas during nighttime hours have been estimated as 5 × 108 molecules/cm3 (~20 ppt), but are lower over marine areas (Atkinson & Arey, 1994). In the dark, nitrate radicals react in the gas phase with PAHs to form nitro derivates in significant yield (see Table 11) (Pitts et al., 1985b,c,d; Sweetman et al., 1986; Zielinska et al., 1986b; Arey et al., 1989b; Atkinson et al., 1990a,b; Inazu et al., 1996, 1997).

Table 11. NitroPAHs formed from the gas-phase reactions of PAHs known to be present in ambient air with hydroxyl radicals and nitrate radicals (both in the presence of nitrogen dioxide) and their yieldsa

PAH

Daytime reactions:

Nighttime reactions:

Initiation by hydroxyl radical followed by reaction with nitrogen dioxide

Reaction with nitrate radical

Naphthalene

1-Nitronaphthalene (0.3%)
2-Nitronaphthalene (0.3%)

1-Nitronaphthalene (17%)
2-Nitronaphthalene (7%)

1-Methyl-naphthalene

All 1-methylnitronaphthalene isomers except 1-methyl-2-nitronaphthalene (~0.4%)

All 1-methylnitronaphthalene isomers (~30%)

2-Methyl-naphthalene

All 2-methylnitronaphthalene isomers except 2-methyl-1- and 2-methyl-3-nitronaphthalene (~0.2%)

All 2-methylnitronaphthalene isomers (~30%)

Acenaphthene

5-Nitroacenaphthene
3-Nitroacenaphthene
4-Nitroacenaphthene
(Σ ~0.2%)

4-Nitroacenaphthene (40%)b
3-Nitroacenaphthene (~2%)b
5-Nitroacenaphthene (~1.5%)b

Acenaphthylene

4-Nitroacenaphthylene (2%)

No nitro isomers formed

Fluorene

3-Nitrofluorene (~1.4%)
1-Nitrofluorene (~0.6%)
4-Nitrofluorene (~0.3%)
2-Nitrofluorene (~0.1%)

 

Phenanthrene

Two nitro isomers (not 9-nitrophenanthrene) in trace yields

Four nitro isomers (including 9-nitrophenanthrene) in trace yields

Anthracenec

1-Nitroanthracene, low yield
2-Nitroanthracene, low yield

1-Nitroanthracene, low yield
2-Nitroanthracene, low yield

Pyrene

2-Nitropyrene (~0.5%)
4-Nitropyrene (~0.06%)

4-Nitropyrene (~0.06%)

Fluoranthene

2-Nitrofluoranthene (~3%)
7-Nitrofluoranthene (~1%)
8-Nitrofluoranthene (~0.3%)

2-Nitrofluoranthene (~24%)

a

From Arey (1998); Atkinson & Arey (1994).

b

Yields for the nitrate radical addition pathway to the fused aromatic rings (Arey et al., 1989a).

c

9-Nitroanthracene was observed in both the hydroxyl and nitrate radical reactions, but may not be a product of these reactions, because it is also formed from exposure to nitrogen dioxide/nitric acid.

The proposed mechanism of reaction of naphthalene in nitrogen pentoxide–nitrate–nitrogen dioxide–air mixtures occurs by the initial addition of the nitrate radical to the aromatic rings to form a nitratocyclohexadienyl-type radical, which then either decomposes to reactants or reacts exclusively with nitrogen dioxide (Atkinson & Arey, 1994).

3.3 Oxygen-containing nitroPAHs

3-Nitrobenzanthrone was found by bioassay-directed fractionation of diesel particulates (0.6–6.6 µg/g, depending on load; Enya et al., 1997). 3-Nitrobenzanthrone was also detected in airborne particle extracts from urban samples taken in autumn/winter during the day (nd–5.2 pg/m3) and night (7.7–11.5 pg/m3) (Enya et al., 1997). The day/night differences could be the result of different emissions or different meteorological conditions.

Mutagenic nitropyrene lactones (El-Bayoumy & Hecht, 1986) were identified in environmental chamber simulations of atmospheric reactions of pyrene (Sasaki et al., 1995).

2- and 4-nitrodibenzopyranone were identified in the products of the gas-phase hydroxyl radical-initiated reaction of phenanthrene (Helmig et al., 1992a,b).

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION AND TRANSFORMATION

4.1 Transport and distribution between media

The transport and distribution of nitroPAHs depend on their physicochemical characteristics (see chapter 2), but data for nitroPAHs are scarce. Their behaviour in the environment is expected to be similar to that of the parent PAHs (see IPCS, 1998), for which there is more information.

4.1.1 Distribution and transport in the atmosphere

NitroPAHs are either formed in the atmosphere from PAHs or emitted directly into the atmosphere during combustion processes (see chapter 3). They can be transported in the vapour phase or adsorbed onto particulate matter. Those with liquid-phase vapour pressures greater than approximately 10–4 Pa at ambient air temperature (i.e., two- to four-ring PAHs and two-ring nitroPAHs) will exist at least partially in the gas phase (Atkinson & Arey, 1994). NitroPAHs having a relatively low vapour pressure will condense out on the surface of ambient particles. Based on ambient measurements, 1- and 2-nitronaphthalene are expected to be found predominantly in the gas phase (Arey et al., 1987). This has also been shown for 1-nitronaphthalene in diesel exhaust samples (Feilberg et al., 1999). Concentrations of a number of PAHs and nitroPAHs were measured (Arey et al., 1987; Atkinson & Arey, 1994), and it was shown that at daytime temperatures in, for example, California, USA, the four-ring PAHs fluoranthene and pyrene are mainly (>90%) present in the gas phase; even at 0 °C, 30% and 70%, respectively, were measured in the gas phase and were therefore available for radical-initiated reactions. There is currently no clear understanding of the partitioning of PAHs between the gas and particulate phases or of the size distribution and mass of the particulates in exhaust gases (CONCAWE, 1998). This is equally true for ambient air, in particular for nitroPAHs.

Just as PAHs are ubiquitous in the environment, so are nitroPAHs. This has been shown in particular for nitroPAHs that are formed not by combustion but by atmospheric transformation (e.g., 2-nitrofluoranthene and 2-nitropyrene), which have been found in different types of airsheds throughout the world (Ciccioli et al., 1995, 1996; see also chapters 3 and 5).

4.1.1.1 Distribution of nitroPAHs between fine and coarse fractions of inhalable atmospheric particulates

In recent monitoring investigations in downtown Rome, Italy, 1-nitropyrene, a marker indicative of direct emissions, was found not only in the coarse fraction (2.5–10 µm) of atmospheric particulates, but also in the fine fraction (0.01–2.5 µm [PM2.5]), whereas 2-nitrofluoranthene of photochemical origin was mostly found in the fine particulate fraction (Cecinato et al., 1999). 1-Nitropyrene and 1,3-, 1,6- and 1,8-dinitropyrene monitored in Kanazawa, Japan, were found almost exclusively in the particulate fraction <1.1 µm (Hayakawa et al., 1999b). It should be noted that particles with diameters below 2.1 µm can reach terminal bronchi and alveoli (Cecinato et al., 1999).

4.1.2 Distribution and transport in the hydrosphere

Owing to their low aqueous solubility or insolubility, nitroPAHs are not expected to accumulate in the hydrosphere. However, considering the low Henry’s law constants (see Table 2 in chapter 2), nitroPAHs present in the hydrosphere are not expected to be transferred significantly to the gas phase.

4.1.3 Adsorption onto soils and sediments

Although data are scarce, the sorption coefficients (log Koc) for nitroPAHs are high, indicating that nitroPAHs adsorb strongly to the organic fraction of soils and sediments. Leaching into groundwater is therefore thought to be negligible.

4.1.4 Bioaccumulation

The affinity of nitroPAHs for organic phases is much higher than that for water. The n-octanol/water partition coefficients (log Kow) range from 2.5 for 1-nitronaphthalene to 6.3 for 3-nitroperylene (see Table 2), indicating a potential for bioaccumulation. Bioaccumulation of 2-nitrofluorene by Daphnia magna was reported to follow first-order kinetics (Gang & Xiaobai, 1994). A bioconcentration factor of 170 was reported for daphnia exposed to a 2-nitrofluorene concentration of 0.124 mg/litre for up to 8 h.

4.1.5 Biomagnification

There were no data on biomagnification.

4.2 Transformation

4.2.1 Biotransformation

The diverse metabolic pathways for the microbial metabolism of nitroPAHs are summarized in Figure 4.

Less is known about the metabolism of nitroPAHs by aquatic and terrestrial microorganisms than for the parent PAHs. Although a wide variety of bacteria, fungi and algae have been shown to degrade the parent PAHs containing two to five rings, nitro-substituted PAHs are only slowly degraded by indigenous microorganisms and may persist in soils and sediments. The recalcitrance of high molecular weight nitroPAHs is due in part to the strong adsorption to soil organic matter, low solubility, large molecular size and the hydrophilic character of the nitro group (Cerniglia & Somerville, 1995).

4.2.1.1 Bacteria

The stability of 1-nitropyrene and 1,6-dinitropyrene was studied in four samples of water (sea, unpolluted river, polluted river and pond water) and filtrates of various soil suspensions with and without 0.1% peptone (Tahara et al., 1995). The mutagenicity decreased rapidly when 1-nitropyrene and 1,6-dinitropyrene were incubated at 30 °C, but not when the test solutions had been autoclaved. Mutagenicity attributed to 1-nitropyrene (3 µg/ml) decreased by 50% in 1.95–3.55 days for water samples and in 0.56–2.37 days for soil filtrate depending on the content of microflora in the test solutions. 1-Aminopyrene was detected as a degradation product of 1-nitropyrene. Mutagenicity attributed to 1,6-dinitropyrene (10 µg/ml) decreased by 50% in 0.53–2.15 days for water samples and in 0.50–0.61 days for soil filtrate.

Time course studies in microcosms showed that 1-nitropyrene was degraded slowly under aerobic and anaerobic conditions in estuarine sediments. Less than 1% had been converted to 14CO2 after 8 weeks of aerobic incubation. Addition of 1-nitropyrene to anaerobic sediments resulted in no 14CO2 evolution, but the 1-nitropyrene was reduced to 1-aminopyrene. The low mineralization of 1-nitropyrene compared with that of the parent compound pyrene could be due to the nitro substituent in the C1 position decreasing the enzymatic oxidation (Cerniglia & Somerville, 1995).

A bacterium isolated from sediments chronically exposed to petrogenic hydrocarbons mineralized 1-nitropyrene and 6-nitrochrysene only to a small extent (12.3% and 2%) compared with non-nitrated PAHs after 10 days of incubation (Heitkamp & Cerniglia, 1988). In pure culture, this bacterium, Mycobacterium sp. strain Pyr-1, was found to metabolize nitroPAHs by both oxidative and reductive pathways. In media with pyrene, the cells oxidized up to 20% of the added 1-nitropyrene to 1-nitro-cis-9,10- and 1-nitro-cis-4,5-dihydrodiols (Heitkamp et al., 1991). However, cells that had been grown in media without pyrene did not produce dihydrodiols, but reduced up to 70% of the 1-nitropyrene to aminopyrene. Further, extracts from cells that had been grown without pyrene activated 1-nitropyrene, 1,3- and 1,6-dinitropyrene and 6-nitrochrysene to DNA-damaging products, as shown in Salmonella typhimurium tester strains and by the umu test (Rafii et al., 1994).

Sphingomonas paucimobilis strain EPA 505 (a soil bacterium capable of utilizing fluoranthene as the sole source of carbon and energy) biodegraded 1-nitropyrene to 48.6% after 6 h (Ye et al., 1996).

4.2.1.2 Fungi

The filamentous fungus Cunninghamella elegans has been shown to metabolize a number of nitroPAHs via oxidation pathways to products that are, in general, less mutagenic than the nitroPAHs themselves. The nitroPAH is initially oxidized via a cytochrome P450 monooxygenase to arene oxides, which isomerize to form phenols (hydroxyl derivatives) or are enzymatically hydrated to form trans-dihydrodiols. The phenols can be subsequently conjugated with sulfate, glucose, xylose or glucuronic acid to form detoxified products (Cerniglia & Somerville, 1995) (Table 12 and Figure 4). The metabolites formed are similar to those oxidative metabolites found in rat microsomes and in vivo studies (see chapter 6); however, whereas the trans-dihydrodiol metabolites in rat liver microsomes are predominantly in the R,R configuration, metabolism by C. elegans produces trans-dihydrodiol metabolites in the S,S absolute configuration.

These studies with the fungus Cunninghamella elegans have been extended to include comparison of the biotransformation of nitroPAHs with that of their parent PAHs. For example, comparison of the metabolism pattern between 1-nitrobenzo[e]pyrene and its parent PAH, benzo[e]pyrene, indicates that the nitro group at the C1 position of benzo[e]pyrene drastically altered the regioselectivity of the metabolism (Pothuluri et al., 1999a).

The fungal biotransformation of a mixture of 2- and 3-nitrofluoranthenes was similar to that of the individual nitrofluoranthenes; however, the mammalian system (rat liver microsomes) showed differences in the regioselectivity of nitrofluoranthene at positions C4, C5, C8 and C9 (Pothuluri et al., 1998a).

4.2.1.3 Plants

A plant cell culture (100 mg/ml wet weight) derived from alligator weed (Alternanthera philoxeroides) detoxified 1-nitropyrene and 1,3-, 1,6- and 1,8-dinitropyrene, all direct-acting mutagens, when incubated with them, as shown by mutagenicity response in the Salmonella typhimurium TA98 assay (Shane et al., 1993).

Table 12. Biotransformation of nitroPAHs by the fungus Cunninghamella elegans (48-h incubation)

NitroPAH

Metabolites (major metabolites are in bold)

Reference

1-Nitropyrene

Glucoside conjugates of 1-nitropyren-6-ol and 1-nitropyren-8-ol

Cerniglia et al. (1985)

2-Nitrofluoranthene

Sulfate conjugates of 2-nitrofluoranthen-8-ol and 2-nitrofluoranthen-9-ol

Pothuluri et al. (1998a)

3-Nitrofluoranthene

Sulfate conjugates of 3-nitrofluoranthen-8-ol and 3-nitrofluoranthen-9-ol

Pothuluri et al. (1994)

6-Nitrochrysene

6-Nitrochrysene-1-sulfate
6-Nitrochrysene-2-sulfate

Pothuluri et al. (1998b)

1-Nitrobenzo[e]pyrene

Sulfate and glucoside conjugates of
1-nitrobenzo[e]pyren-6-ol

Pothuluri et al. (1999a)

6-Nitrobenzo[a]pyrene

Glucoside conjugates of
6-nitrobenzo[a]pyren-1-ol and
6-nitrobenzo[a]pyren-3-ol
Sulfate conjugate of 6-nitrobenzo[a]pyren-1-ol

Millner et al. (1986); Cerniglia & Somerville (1995)

2-Nitrofluorene

2-nitrofluoren-9-ol, 2-nitro-9-fluorenone,
2-nitrofluoren-6-ol
Sulfate conjugates of 2-nitro-9-fluorenon-7-ol, 2-nitrofluoren-7-ol

Pothuluri et al. (1996)

9-Nitroanthracene

Phenol and dihydrodiol derivatives

Pothuluri et al. (1999b)

4.2.1.4 Aquatic animals

The biotransformation of aquatic species is discussed in chapter 9.

4.2.2 Abiotic degradation

4.2.2.1 Direct photolysis

The photolysis of nitroPAHs has been studied under varied conditions of irradiation (see Table 13). The rate of photolysis depends not only on the conditions of irradiation but also on whether the nitroPAH is in the gaseous stage (e.g., 1-and 2-nitronaphthalene), in solution (type of solvent) or bound to solids or particles. In the latter case, the type and age of the particle seem to influence the photochemistry of the respective nitroPAH. The rate of photodecomposition, identification of photolytic products and the resulting loss or gain of mutagenic activity as determined by the Salmonella typhimurium assay have been the main end-points studied. Decomposition products include quinones, hydroxy-nitroPAHs and hydroxyPAHs (see Table 13). Although most studies show that the mutagenic activity of decomposition products was less than that of the original nitroPAH, some results show an increase.

Table 13. Rates of photolysis of nitroPAHs in different media and comparison of the mutagenic activity of the products

NitroPAH

Media

Source of light

Major product

Mutagenic activitya

Rate of photolysis

Reference

1-Nitronaphthalene

Gaseous

Sunlight

1,4-Naphthoquinone

 

Atmospheric lifetime = 1.7 h

Atkinson et al. (1989)

1- and 2-nitro-naphthalenes

Gaseous

Sunlight

   

0.5 h and 11 h, respectively

Feilberg et al. (1999)

1- and 2-nitro-naphthalenes

2-Propanol

320–418 nm for 2 h

   

No degradation

Stärk et al. (1985)

3-Nitrofluoranthene

DMSO

   

Less

12.5 days

Holloway et al. (1987)

2-Nitrofluorene

DMSO

Cool-white light or artificial sunlight; up to 5 h

 

More

 

White et al. (1985)

9-Nitroanthracene

Acetone and silica gel

 

Mainly 9,10-anthraquinone (via iminoxyl radical)

   

Chapman et al. (1966); Pitts et al. (1978)

9-Nitroanthracene

Hexane

 

Anthraquinone

 

Transformed to 40% after a 1-day exposure to light

Schlemitz & Pfannhauser (1997)

1-Nitropyrene

Solid

Sunlight

Pyrenol, pyrene quinone, pyrenediol

Less

Biphasic; half-times = 14 h and 533 h

Benson et al. (1985)

1-Nitropyrene

Methanol

>300 nm in presence of oxygen

Pyren-1-ol (88%) and 2-nitropyren-1-ol (7%)

 

95% transformation after 2.5 h

van den Braken-van Leersum et al. (1987)

1-Nitropyrene

Benzene

Sunlight

Nitropyren-9-ol

Less

Rapid, 10% after 1 h; 53% after 10 h

Koizumi et al. (1994)

1-Nitropyrene

Soot

Sunlight

Nitropyren-9-ol

Less

Slow, 42% in 40 days

Koizumi et al. (1994)

1-Nitropyrene

DMSO

Light
(>310 nm)

 

Less

1.2 days

Holloway et al. (1987)

1-Nitropyrene

Silica

Light
(>310 nm)

 

Less

6 days

Holloway et al. (1987)

1-Nitropyrene

2-Propanol

320–418 nm for 90 min

 

Almost total loss

Rapid

Stärk et al. (1985)

1-Nitropyrene

DMSO

Fluorescent sunlamps; 4 and 24 h

Pyrene quinone, pyrenol, 1-nitropyrenol

Less

 

Yang et al. (1994)

2-Nitropyrene

Methanol

>300 nm in presence of oxygen

Very stable

 

Only 19% conversion after 7.5 h

van den Braken-van Leersum et al. (1987)

4-Nitropyrene

Methanol

>300 nm in presence of oxygen

Pyrene (9%) and traces of other products

   

van den Braken-van Leersum et al. (1987)

1,8-Dinitropyrene

DMSO

Light
(>310 nm)

1-Nitropyren-8-ol

 

0.7 days

Holloway et al. (1987)

1,8-Dinitropyrene

Silica

Light
(>310 nm)

1-Nitropyren-8-ol

 

5.7 days

Holloway et al. (1987)

1,8-Dinitropyrene

Silica

   

Less

>20 days

Holloway et al. (1987)

7-Nitro-benz[a]-anthracene

DMSO

Cool-white light or artificial sunlight; up to 5 h

 

More

 

White et al. (1985)

7-Nitrodibenzo[a,h]-anthracene

DMSO

Fluorescent sunlamps; 4, 24 and 48 h

 

More at 4 h, then decreasing

 

Yang et al. (1994)

9-Nitrodibenzo[a,c]-anthracene

DMSO

Fluorescent sunlamps; 4, 24 and 48 h

 

More at 4 h, then decreasingb

 

Yang et al. (1994)

1-Nitrobenzo[a]pyrene

DMSO

Fluorescent sunlamps; 4, 24 and 48 h

 

Less

 

Yang et al. (1994)

6-Nitrobenzo[a]pyrene

Silica gel

 

Benzo[a]pyrene quinones: 1,6-, 3,6- and 6,12- isomers

   

Pitts (1983)

6-Nitrobenzo[a]pyrene

DMSO

Cool-white light or artificial sunlight; up to 5 h

 

More

 

White et al. (1985)

6-Nitrobenzo[a]pyrene

In solution

UV light

Benzo[a]pyrene-3,6-quinone via benzo[a]pyren-6-oxyl radical

   

Ioki (1977)

3,6-Dinitrobenzo[a]-pyrene

 

UV light at 312 nm

3-Nitrobenzo[a]-pyrene-6-quinone

   

Sera et al. (1991)

3-Nitrobenzo[e]pyrene

DMSO

Cool-white light or artificial sunlight; up to 5 h

 

More

 

White et al. (1985)

a Compared with original nitroPAH.

b Also increasing cytotoxicity with length of exposure time.

1) Photolysis of gaseous nitroPAHs

Feilberg et al. (1999) showed that 1-nitronaphthalene photolyses faster than 2-nitronaphthalene. Using a large outdoor smog chamber facility, the gas-phase photolysis rates were determined to be 0.07 × kNO2 and 0.005 × kNO2 for 1- and 2-nitronaphthalene, respectively (Feilberg et al., 1999). Using an average kNO2 of 5.2 × 10–3/s, the lifetimes of these two nitroPAHs with respect to photolysis were calculated to be 0.5 and 11 h, respectively. Therefore, gas-phase photolysis is the major degradation pathway for 1-nitronaphthalene; for 2-nitronaphthalene, other pathways (such as reaction with hydroxyl radicals; see Tables 14 and 15) may be important (Feilberg et al., 1999).

Table 14. Room temperature rate constants, k, for the gas-phase reactions of hydroxyl radicals, nitrate radicals and ozone with nitronaphthalenesa

Nitronaphthalene

k (cm3/molecule per second) for reaction with

Hydroxyl (OH)b

Nitrate
(NO3)

Ozone
(O3)b

1-Nitronaphthalene

5.4 x 10–12

3.0 x 10–29 [NO2]b

<6 x 10–19

2-Nitronaphthalene

5.6 x 10–12

2.7 x 10–29 [NO2]b

<6 x 10–19

a Adapted from Atkinson & Arey (1994).

b Atkinson (1991).

Table 15. Calculated atmospheric lifetimes of nitronaphthalenes due to photolysis and gas-phase reactions with hydroxyl and nitrate radicals and with ozone

Nitronaphthalenes

Atmospheric lifetimes due to reaction with

Hydroxyl (OH)

Nitrate (NO3)

Ozone
(O3)

Photolysis
(solar radiation)

1-Nitronaphthalene

2.7 daysa

18 yearsb

>28 daysc

0.5 hd

2-Nitronaphthalene

2.6 daysa

20 yearsb

>28 daysc

11 hd

a

From Atkinson & Arey (1994) for a 12-h daytime average hydroxyl radical concentration of 1.6 x 106 molecules/cm3 (Prinn et al., 1992).

b

From Atkinson & Arey (1994) for a 12-h average nighttime nitrate radical concentration of 5 x 108 molecules/cm3 (Atkinson, 1991) and nitrogen dioxide concentration of 2.4 x 1011 molecules/cm3.

c

From Atkinson & Arey (1994) for a 24-h average ozone concentration of 7 x 1011 molecules/cm3 (Logan, 1985).

d

From Feilberg et al. (1999) using an average 12-h daytime nitrogen dioxide photolysis rate kNO2 = 5.2 x 10–3/s.

2) Photolysis of PAHs in solution or on solids

Studies comparing the photodecomposition of nitroPAHs on solids or in solution show that decomposition rates or times are much longer on solids (see Table 13; e.g., with 1-nitropyrene [Koizumi et al., 1994]; 1-nitropyrene, 1,8-dinitropyrene, 3-nitrofluoranthene [Holloway et al., 1987]; see also section 3) below on photostability of nitroPAHs on particles).

Several authors have studied the photolysis of isomeric or other groups of nitroPAHs and compared their rates of decomposition and their mutagenicities with their differences in chemical structure (see Table 13).

Some nitroPAHs can be readily decomposed when exposed to light both in solution or on particles to form quinones and possibly phenolic derivatives (Pitts, 1983). The reactions are complex and depend on the presence or absence of air, the type of solvent and the wavelength of light used (Chapman et al., 1966). A mechanistic theory was suggested using the example of 9-nitroanthracene. Illumination in acetone resulted in rearrangement into a nitrite, followed by dissociation into nitric oxide and a phenoxy-type radical and ultimately anthraquinone (Chapman et al., 1966). 9-Nitroanthracene forms 9,10-anthraquinone on irradiation both in solution and on silica gel (Pitts et al., 1978).

UV light-induced oxidation of 6-nitrobenzo[a]pyrene resulted in the formation of benzo[a]pyrene-3,6-quinone (Ioki, 1977). 3,6-Dinitrobenzo[a]pyrene was readily decomposed by UV radiation at 312 nm to 3-nitrobenzo[a]pyrene-6-quinone (Sera et al., 1991).

6-Nitrobenzo[a]pyrene on silica gel photolyses rapidly to BaP quinones (1,6-, 3,6- and 6,12- isomers); the 1- and 3- isomers are more stable (Pitts, 1983). (According to Chapman’s hypothesis, the 6-nitro- isomer with two peri-hydrogens should be less stable than the 1- and 3- isomers with only one peri-hydrogen.)

A 1-day exposure of 9-nitroanthracene to light resulted in 40% conversion to anthraquinone (Schlemitz & Pfannhauser, 1997).

The influence of the nitro group on the aromatic B -system of pyrene has been studied by comparing the spectroscopic and photochemical properties of 1-, 2- and 4-nitropyrene (van den Braken-van Leersum et al., 1987). Whereas the UV and mass spectra of 1- and 4-nitropyrene show an interaction normal for nitro-aromatic compounds, 2-nitropyrene shows a lack of interaction, reflected in a UV spectrum very similar to that of pyrene and a mass spectrum with a low abundance of [M–NO]. The photochemical behaviour of the three compounds is governed by the degree of interaction. In the presence of oxygen, 1-nitropyrene shows the nitro-nitrite rearrangement, leading to 1-hydroxypyrene (pyren-1-ol) (88%) and 1-hydroxy-2-nitropyrene (2-nitropyren-1-ol) (7%); under anaerobic conditions, equimolar amounts of 1-nitrosopyrene and 2-nitropyren-1-ol are formed. The photochemical products of 4-nitropyrene in air are pyrene (9%) and unstable products that react with the solvent. 1-Nitropyrene was more reactive than 4-nitropyrene. 2-Nitropyrene is very stable under photochemical conditions due to lack of interaction. After irradiation of a solution of 2-nitropyrene 3 times longer than needed for 95% conversion of 1-nitropyrene, 81% of the starting material was recovered.

Yang et al. (1994) studied the photodecomposition of four sets of isomeric nitroPAHs (total of 10 nitroPAHs) in DMSO for 4, 24 and 48 h under fluorescent sunlamps. Decomposition products were multiple — e.g., quinones and/or hydroxy-nitroPAHs. The order of ease of photodecomposition for the different isomeric groups was as follows (see also Table 16):

6-nitro-BaP > 1-nitro-BaP $ 3-nitro-BaP

1-nitropyrene > 4-nitropyrene > 2-nitropyrene

9-nitroanthracene > 2-nitroanthracene

7-nitrodibenzo[a,h]anthracene = 9-nitrodibenzo[a,c]anthracene

Table 16. Comparison of the decomposition rates of the isomeric nitroPAHs after exposure in DMSO to fluorescent sunlampsa

Compound

Nitro orientation

Degree of decomposition

6-Nitrobenzo[a]pyrene

Perpendicular

+ + + + + +

1-Nitrobenzo[a]pyrene

Parallel

+ + +

3-Nitrobenzo[a]pyrene

Parallel

+ + +

1-Nitropyrene

Parallel

+ + +

4-Nitropyrene

Parallel

+ +

2-Nitropyrene

Parallel

+

9-Nitroanthracene

Perpendicular

+ + + +

2-Nitroanthracene

Parallel

+ +

7-Nitrodibenzo[a,h]anthracene

Perpendicular

+ + + +

9-Nitrodibenzo[a,c]anthracene

Perpendicular

+ + + +

a From Yang et al. (1994).

It appears that those nitroPAHs having a perpendicular orientation decompose faster than those with a parallel orientation (see Table 16).

1-Nitropyrene, 2-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 9-nitroanthracene and 6-nitrobenzo[a]pyrene were irradiated in cyclohexane solutions (Feilberg & Nielsen, 2000). In the absence of co-solutes, the photodegradation of nitroPAHs is strongly dependent on the orientation of the nitro group. In 9-nitroanthracene and 6-nitrobenzo[a]pyrene, the nitro group adapts an approximately perpendicular orientation relative to the aromatic plane, and, as expected, both compounds decayed very quickly, with less than 1% remaining after 15 min. 1-Nitropyrene decayed moderately, whereas 2-nitrofluorene, 2-nitrofluoranthene and 3-nitrofluoranthene were stable towards photolysis.

The mutagenicity of the photodecomposition products of 1-nitropyrene (a moderate direct-acting mutagen), 1-nitrobenzo[a]pyrene (a potent direct-acting mutagen), 7-nitrodibenzo[a,h]anthracene and 9-nitrodibenzo[a,c]anthracene (both non-direct-acting mutagens) were tested using the Salmonella typhimurium microsome assay with TA98 without metabolic activation (Yang et al., 1994). Most studies show that the mutagenic activity of decomposition products was less than that of the original nitroPAHs (Benson et al., 1985; Koizumi et al., 1994; Yang et al., 1994) (see Table 13). In contrast, following 3- or 5-h exposures in DMSO, 6-nitrobenzo[a]pyrene, 7-nitrobenz[a]anthracene, 3-nitrobenzo[e]pyrene and 2-nitrofluorene were found to have elevated mutagenicities in the S. typhimurium microsome assay when exposed to sunlight (White et al., 1985). Irradiated 6-nitrobenzo[a]pyrene was about 15 times more mutagenic than 7-nitrobenz[a]anthracene.

3) Photostability of nitroPAHs on particles

NitroPAHs that are particle-associated under atmospheric conditions may be fully or partially protected from photolysis (Atkinson & Arey, 1994).

In a chamber study, the stability of nitroPAHs (1-nitropyrene, 7-nitrobenz[a]anthracene and 6-nitrobenzo[a]pyrene) on airborne, fresh diesel soot particles was assessed (Kamens et al., 1994). They degraded very rapidly in the sunlight; the half-lives for the nitropyrenes were around 2.5 h. 6-Nitrobenzo[a]pyrene degraded more rapidly, with a half-life of 30 min. However, nitroPAHs associated with aerosolized diesel soot (SRM 1650) did not decay when exposed to sunlight (no further details given). 1-Nitropyrene adsorbed to coal fly ash was resistant to photodecomposition (Holder et al., 1994).

NitroPAHs, in spite of structural differences, all showed similar decay rates on diesel soot particles or wood smoke, indicating that other chemicals associated with the diesel particles somehow affect the photooxidation of nitroPAHs in sunlight and that the effect of the structure is not a dominant factor, as it is with individual nitroPAHs studied (Fan et al., 1996a; Feilberg & Nielsen, 2000, 2001).

4.2.2.2 Other atmospheric transformations

1) Gaseous nitroPAHs

Transformations of 1- and 2-nitronaphthalene with hydroxyl and nitrate radicals and ozone were studied, and the rate constants for the gas-phase reactions are given in Table 14.

The reactions of nitrate radicals and ozone with gaseous nitroPAHs (see Table 15) appear to be of negligible importance as atmospheric loss processes (Atkinson et al., 1989). Reactions of gaseous nitroPAHs with hydroxyl radicals are probably of secondary importance to photolysis (e.g., for 1-nitronaphthalene), although recent studies with 2-nitronaphthalene suggest that this loss process may be as important as photolysis (Feilberg et al., 1999).

2) NitroPAHs on particles

No physical or chemical loss processes were found when 1-nitropyrene was coated on glass and Teflon filters exposed to 100 ppb of nitric acid-free nitrogen dioxide, ozone and sulfur dioxide (Grosjean et al., 1983). In contrast, 1,3-, 1,6- and 1,8-dinitropyrene were detected when 1-nitropyrene-coated particles on a filter surface were exposed to 260 mg/m3 of nitrogen dioxide (Lee et al., 1989).

Oxidation of 1-nitropyrene adsorbed on silica gel with dimethyldioxirane led to the formation of 1-nitropyrene-4,5-oxide and 1-nitropyrene-9,10-oxide, in a ratio 74:26. When the adsorbed 1-nitropyrene was exposed to the gas-phase ozonolysis of tetramethylethylene, the same oxides were produced in the ratio 72:28. Reaction of 1-nitropyrene with ozone alone did not lead to oxide formation (Murray & Singh, 1998).

Degradation of nitroPAHs was observed in the presence of both ozone and nitrogen dioxide plus ozone on real soot particles studied in an outdoor smog chamber in both cool and warm temperatures (between 2 and 20 °C) (Fan et al., 1996b) (see Table 17). The degradation rate constants of 1- and 2-nitropyrene and 2-, 3- and 8-nitrofluoranthene with ozone ranged from 0.0015 to 0.0025/ppm per minute.

Table 17. Particle nitroPAH atmospheric half-lives of 2-nitrofluoranthene and 1-nitropyrene from photolysis and dark heterogeneous reactions with ozonea,b

Compound

Photolysis rate constant (min–1)

O3 rate constant
(ppm–1 min–1)

Half-life in sunlight
(h)

Half-life with 0.2 ppm O3 (h)

2-Nitrofluoranthene

0.013

0.002

1

29

1-Nitropyrene

0.020

0.002

0.6

29

a

Data from Fan et al. (1995, 1996b).

b

NitroPAH photolysis rate constants are derived from an average nitrogen dioxide photolysis rate of kNO2 = 0.5/min; an ozone concentration of 0.2 ppm (1 ppm = 1.96 mg/m3) is assumed, which represents a moderately to highly polluted atmosphere.

The rate constant of particle nitroPAHs with nitrogen dioxide–nitrate–nitrogen pentoxide was less than 0.001/ppm per minute. Particle oxidation of nitroPAHs by ozone does not appear to be as important as photodegradation of nitroPAHs in daytime, but it may be the main loss process at night.

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

5.1 Environmental levels

5.1.1 Air

5.1.1.1 Ambient air

NitroPAHs can be formed as direct or indirect products of incomplete combustion. Formation of nitroPAHs from diesel and gasoline exhaust is discussed in chapter 3. NitroPAHs can be expected in urban air as a result of traffic and domestic heating. However, nitroPAHs can also be formed indirectly from nitration of PAHs transported in ambient air to other sites (see chapter 3).

Some studies have monitored one sample to identify as many nitroPAHs as possible (Table 18). NitroPAHs that have been detected in ambient air include 1- and 2-nitronaphthalene, all 14 methylnitronaphthalene isomers (predominantly in the vapour phase), 2-nitrofluorene, 9-nitroanthracene, 9-nitrophenanthrene, 2-, 3- and 8-nitrofluoranthene, 1- and 2-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 6-nitrochrysene, 7-nitrobenz[a]anthracene and 1- and 2-nitrotriphenylene (Ishii et al., 2000, 2001). Also observed in low concentrations (relative to 2-nitrofluoranthene) were 7-nitrofluoranthene, 4-nitropyrene and nitroacephenanthrylene isomers. Other nitroPAHs have been cited in trace quantities by one author only (see Table 18). However, the importance of the nitroPAHs depends not only on their relative concentrations in air but also on their mutagenicity; dinitropyrenes and 3,6-dinitrobenzo[a]pyrene, for example, are present in relatively low concentrations but have a high mutagenicity (see below and chapter 7).

Ambient air monitoring of nitroPAHs in many parts of the world has revealed the unexpected presence in airborne particles of 2-nitrofluoranthene and 2-nitropyrene at levels equal to or higher than those found for 1-nitropyrene (see Table 19). 2-Nitrofluoranthene and 2-nitropyrene are not usually found as a result of combustion processes, but are formed by atmospheric reactions (see chapter 3).

Table 18. NitroPAHs detected in ambient aira

Parent PAH; nitro derivative

Concentration (ng/m3)b,c

a)

b)

c)

d)

e)

f)

g)

h)

i)

j)

k)

l)

m)

n)

P

P

V+P

V+P

V+P

V

P

P

V+P

P

P

 

V+P

P

Naphthalene

                           

1-Nitro-

m

 

2.7

5.7

0.10

4.7

   

0.39

 

0.90

 

0.352

0.39

2-Nitro-

   

2.6

3.1

0.17

2.3

   

0.55

 

0.55

 

0.065

0.21

1,3-Dinitro-

             

0.12

         

0.23

1,5-Dinitro-

             

0.06

         

0.029

1,8-Dinitro-

                         

0.017

Epsilon-Methylnitro-

   

X

X

X

6.0

 

0.005

           

Acenaphthene

                           

5-Nitro-

             

0.11

           

Fluorene

                           

2-Nitro-

             

0.05

   

0.21

   

0.37

2,5-Dinitro-

             

0.19

           

2,7-Dinitro-

             

1.5

         

0.095

Anthracene

                           

9-Nitro-

X

 

0.11

0.25

0.11

 

0.03

 

0.04

 

0.17

 

0.029

 

Phenanthrene

                           

9-Nitro-

               

0.29

     

0.105

 

Fluoranthene

                           

2-Nitro-

X

X

0.35

2.0

0.14

 

X

 

0.15

 

0.16

0.017

0.039

0.065

3-Nitro-

   

np

np

np

       

0.2–0.8

0.13

0.013

   

7-Nitro-

 

X

                       

8-Nitro-

 

X

0.003

0.004

0.003

 

X

             

3,7-Dinitro-

                   

0.005

     

3,9-Dinitro-

                   

0.004

     

Pyrene

                           

1-Nitro-

X

X

0.015

0.015

0.026

 

0.009

0.14

0.04

1.7–1.9

0.068

0.004

0.009

0.13

2-Nitro-

 

X

0.014

0.032

0.030

 

X

       

0.008

   

4-Nitro-

 

X

                       

1,3-Dinitro-

                   

0.01

     

1,6-Dinitro

                   

0.008

     

Benz[a]anthracene

                       

7-Nitro-

       

0.014

                 

10-Nitro-

           

0.014

             

Chrysene

                           

6-Nitro-

             

0.27

 

0.8–1.5

   

0.0002

 

Benzo[a]pyrene

                           

6-Nitro-

     

t

   

X

0.27

 

np

       

Total nitroPAHs

5.8

11.1

0.59

13.0

   

1.4

 

2.22

 

0.60

1.5

a

See also Tables 19 and 20 and Figures 5 and 6.

b

P = particulate; V = vapour; t = trace; m = monoisomers (specific isomers not identified); np = not present in detectable amounts; X = detected (no quantification). It should be noted that there is some historical misidentification of certain isomers due to lack of standards (e.g., nitrochrysenes and nitrofluoranthenes).

c

Samples were as follows:

 

a)

Urban air particles from St. Louis, Missouri, USA, NIST SRM (Ramdahl et al., 1982); 2-nitrofluoranthene originally reported as 3-nitrofluoranthene.

 

b)

Torrance, California, USA. Analysis by GC and negative ion atmospheric pressure ionization MS, by which means small peaks of 4-nitropyrene and 7-nitrofluoranthene could be detected (Korfmacher et al., 1987).

 

c)

Glendora, California, USA, daytime composite sample (Atkinson et al., 1988).

 

d)

Glendora, California, USA, nighttime composite sample (Atkinson et al., 1988).

 

e)

Concord, California, USA, daytime composite sample (Atkinson et al., 1988).

 

f)

Redlands, California, USA, nighttime sample (Gupta, 1995).

 

g)

ng/m3; mean of 30 samples of airborne particulate matter from rural area 30 km west of Copenhagen, Denmark, February to April 1982 (Nielsen et al., 1984). Original publication gives 3-nitrofluoranthene, but this was reanalysed and given as 2-nitrofluoranthene (Ramdahl et al., 1986).

 

h)

Tokyo air (Matsushita & Iida, 1986); only methylnitronaphthalene reported was 2-methyl-1-nitronaphthalene.

 

i)

Fresno, California, USA, composite daytime sample; five samples, December 1990 – January 1991 (Hunt & Maisel, 1995); see Figure 6.

 

j)

Suburban, Bayreuth, Germany, in 1983 (Garner et al., 1986). 3-Nitrofluoranthene reported as present, likely 2- + 3-nitrofluoranthene. No nitroPAHs in snow sample near motorway.

 

k)

Air sample from Sapporo, Japan, in 1989 (Tokiwa et al., 1990a).

 

l)

Aerosol from Amazon forest (Alta Floresta) near biomass burning (Vasconcellos et al., 1998; see also Figure 6).

 

m)

ng/m3; vapour + particulate phase; Houston, Texas, USA, annual average; see also seasonal pattern (Wilson et al., 1995).

 

n)

Assay of 19 independent 48-h air samplings from three sampling sites in downtown urban environment (Florence, Italy), January–February 1993; 3-nitrofluoranthene reported as present, likely 2- + 3-nitrofluoranthene (Berlincioni et al., 1995).

Table 19. Average concentrations of MW 247 nitroPAHs as represented by 2-nitrofluoranthene (2-NFL), 1-nitropyrene (1-NP)
and 2-nitropyrene (2-NP) detected in tropospheric samplesa,b

Source

Site

Year

Season

Concentration (pg/m3)

Reference

Total PAHs

BaP

2-NFL

1-NP

2-NP

2-NFL/
1-NP

Urban

Milan (Italy)

1990

Winter

   

240

30

4

8

Ciccioli et al. (1993)

Urban

Milan (Italy)

1990

Summer

   

170

20

nr

8.5

Ciccioli et al. (1993)

Urban

Barcelona (Spain)

1989–1990

Year

50 000

3500

120

30

20

4

Bayona et al. (1994)

Urban

Hamilton (Canada)

1990–1991

Spring/ summer

 

2000

6

12

4

0.5

Legzdins et al. (1995)

Downtown

Kanazawa (Japan), Site 1

1994

Summer

nr

nr

59

32

4

1.8

Murahashi & Hayakawa (1997)

Suburban

Kanazawa (Japan), Site 2

1994

Summer

nr

nr

16

10

2

1.6

Murahashi & Hayakawa (1997)

Urban

Damascus (Syria)

1994

Winter

 

4500

230

120

nr

1.9

Dimashki et al. (1996)

Urban

Milan, Viale Marche (Italy)

1991

Winter

48 600

2850

960

140

nr

6.8

Cecinato et al. (1998)

Urban

Milan, Brera Tower (Italy)

1993

Winter

70 200

1350

1730

590

nr

2.9

Cecinato et al. (1998)

Urban

Milan (Italy)

1991–1993

Winter

nr

nr

1140

220

300

5.2

Ciccioli et al. (1995)

Urban

Rome (Italy)

1991–1993

Winter

36 000

1600

250

80

nr

3.1

Cecinato et al. (1998)

Urban

Rome (Italy)

1991–1993

Summer

nr

nr

470

70

70

6.7

Ciccioli et al. (1995)

Urban

Sao Paulo (Brazil)

1991–1993

Winter

nr

nr

139

16

42

8.7

Ciccioli et al. (1995)

Urban

Athens (Greece)

1996

Year

23 900

610

90

40

60

2.3

Marino et al. (2000)

Urban

St. Louis (USA)

 

Year

   

560c

160c

60c

3.5

Ramdahl et al. (1986)

Remote

Svalbard Island (Norway)

1998–1999

Year

 

36

2

13

8

0.16

Cecinato et al. (2000)

Urban industrial

Bab Ezzouar (Algeria)

1999

Summer

 

2000

1500

0

n.ev.

Yassaa et al. (2001a)

Landfill

Oued Smar (Algeria)

1998–1999

Summer

 

3900

310

80

10

37

Yassaa et al. (2001b)

Urban

Algiers (Algeria)

1999

Winter

 

2300

450

140

20

3.2

Yassaa et al. (2001b)

Urban

Glendora (USA)

1986

Summer

 

240

630

16

19

~40

Atkinson et al. (1988)

Biomass burning

Yuba City (USA)

1986

Autumn

 

200

130

8

8

16

Atkinson et al. (1988)

Industrial

Concord (USA)

1986–1987

Winter

 

4400

290

30

50

10

Atkinson et al. (1988)

Wood smoke

Mammoth Lakes (USA)

1987

Winter

 

6200

29

8

3

3.6

Atkinson et al. (1988)

Oil production

Oildale (USA)

1987

Summer

 

490

28

7

1

4

Atkinson et al. (1988)

Urban

Reseda (USA)

1987

Summer

 

290

150

8

13

~19

Atkinson et al. (1988)

Remote

Pt. Arguello (USA)

1987

Summer

 

nd

5

0.5

0.3

10

Atkinson et al. (1988)

Remote

San Nicolas Island (USA)

1987

Summer

 

nd

2

3

nd

0.67

Atkinson et al. (1988)

Urban

Washington (USA)

 

Year

   

600c

200c

50c

3

Ramdahl et al. (1986)

Urban

Riversided (USA)

 

Autumn

nr

nr

193

22

8

8.8

Pitts et al. (1985d)

Suburban

Montelibretti (Italy)

1991–1993

Year

nr

nr

87

12

16

7.3

Ciccioli et al. (1995)

Suburban

Madrid (Spain)

1991–1993

Autumn

nr

nr

70

10

20

7

Ciccioli et al. (1995)

Suburban

Torranced (USA)

 

Winter

nr

nr

310

32

30

9.7

Arey et al. (1987)

Suburban

Glendorad (USA)

   

nr

nr

270

nr

12

 

Arey et al. (1988b)

Suburban

Claremontd (USA)

 

Autumn

nr

nr

408

16

3

25.5

Zielinska et al. (1989a)

Suburban

Claremont (USA)

1985

Autumn

nr

nr

2800c

360c

80c

7

Ramdahl et al. (1986)

Rural residential

Aurskog (Norway)

1984

Winter

nr

nr

560c

150c

170c

3.7

Ramdahl et al. (1986)

Forest areas

Castelporziano (Italy)

1992

Winter

1560

140

70

3

14

23

Ciccioli et al. (1995)

Forest areas

Castelporziano (Italy)

1993

Summer

600

10

25

nd

5

>25

Ciccioli et al. (1995)

Forest areas

Alta Floresta (Brazil)

1993

Winter, spring

2550

160

15

2

80

7.5

Ciccioli et al. (1995)

Forest areas

Storkow (Germany)

1991

Summer

430

10

3

nd

nd

>3

Ciccioli et al. (1995)

Remote

CNR Pyramid (Nepal)

1991

Autumn

130

3

3

nd

nd

>3

Ciccioli et al. (1995)

Remote

Terra Nova Bay (Antarctica)

1991

Summer

3

nd

nd

nd

nd

 

Ciccioli et al. (1995)

a

Adapted from Ciccioli et al. (1995, 1996).

b

nr = not reported; nd = not detected; Year (under "Season") = mean for year; n.ev. = 2-NP could not be evaluated since an interference co-eluted.

c

Units of ng/g; this is a NIST SRM of ambient particles.

d

California, USA.

Table 20. Concentrations of benzo[a]pyrene and selected nitroPAHs in urban air from Teplice and Prachatice, Czech Republica

Compound

Concentration (ng/m3)

Teplice,
October 1993 – March 1994

Teplice
April–September 1994

Teplice
October 1996 – March 1997

Prachatice
October 1993 –
March 1994

Prachatice
April–September 1994

BaP

6.05

0.57

4.76

3.68

0.41

9-Nitroanthracene

0.018

0.002

0.033

0.005

0.002

3-Nitrofluorantheneb

0.090

0.009

0.139

0.013

0.003

1-Nitropyrene

0.055

0.008

0.048

not given

0.005

a From Lenicek et al. (2001).

b Probably 2- plus 3-nitrofluoranthene.

From the results of these broad monitoring studies, other investigators have concentrated on the nitroPAHs that seem to be of quantitative/environmental (e.g., nitroPAHs of relative molecular mass 247 [MW 247]: 1-nitropyrene, 2-nitropyrene, 2-nitrofluoranthene; Table 19; Figures 5 and 6), 1-nitropyrene, 9-nitroanthracene, 3-nitrofluoranthracene (together with many PAHs in the Teplice, Czech Republic, program, Table 20) or carcinogenic (e.g., 1-nitropyrene, dinitropyrenes; Table 21) importance. As noted in Table 20, in many studies the report of only the 3- isomer of fluoranthene may in fact reflect a single GC peak in which 2- and 3-nitrofluoranthene co-eluted. Where both isomers have not been discussed, caution must be used in interpreting the data, for example, regarding the sources of the nitrofluoranthene isomers.

Table 21. Monitoring studies on dinitropyrenesa

Location

 

Date

BaP

1-Nitropyrene

1,3-Dinitropyrene

1,6-Dinitropyrene

1,8-Dinitropyrene

Reference

Shopping mall, Fukuoka, Japan

 

Winter 1992

   

15 pg/m3

9.5 pg/m3

7.5 pg/m3

Maeda et al. (1994)

Kanazawa, Japan

 

1989–1992

 

173 pg/m3

0.64 pg/m3

1.17 pg/m3

1.05 pg/m3

Hayakawa et al. (1995a)

Kanazawa, Japan

Downtown

July 1993–February 1994

2495±1260 pg/m3

222±101 pg/m3

1.3±0.8 pg/m3

1.0±0.6 pg/m3

1.4±0.7 pg/m3

Murahashi et al. (1995)

Location not given

     

15–134 pg/m3

nd–4.7 pg/m3

0.3–8.7 pg/m3

nd–6.6 pg/m3

Tanabe et al. (1986)

Location not given

     

190–1600 ng/g

nd–56 ng/g

5–105 ng/g

nd–79 ng/g

Tanabe et al. (1986)

Fukuoka, Japan

     

140 ng/g

12 ng/g

10 ng/g

25 ng/g

Sera et al. (1994)

Bermuda

Remote

August 1992

280 ng/g particulate

520 ng/g

 

8.1 ng/g

3.5 ng/g

Gibson (1986)

Bermuda

Remote

January/ February 1983

320 ng/g

720 ng/g

 

8.3 ng/g

4.4 ng/g

Gibson (1986)

Delaware (USA)

Rural

July/August 1982

570 ng/g

540 ng/g

 

4.9 ng/g

2.4 ng/g

Gibson (1986)

Warren (USA)

Suburban

December 1982

6500 ng/g

360 ng/g

 

<6 ng/g

<6 ng/g

Gibson (1986)

Warren (USA)

Suburban

June 1984

4100 ng/g

350 ng/g

 

4.6 ng/g

2.1 ng/g

Gibson (1986)

Detroit (USA)

Urban

June–August 1981

7800 ng/g

220 ng/g

 

3.6 ng/g

2.5 ng/g

Gibson (1986)

River Rouge (USA)

Industrial

July 1982

4900 ng/g

590 ng/g

 

46 ng/g

13.1 ng/g

Gibson (1986)

Dearborn (USA)

Industrial

June–August 1980

66 000 ng/g

150 ng/g

 

41 ng/g

20 ng/g

Gibson (1986)

   

February–April 1983

14 000 ng/g

       

Gibson (1986)

a nd = not detected.

Figure 5

Figure 5

Fig. 5. Seasonal studies of selected nitroPAHs in ambient air. 2-NFL = 2-nitrofluoranthene; 1-NP = 1-nitropyrene; 2-NP = 2-nitropyrene; 1,3-DNP = 1,3-dinitropyrene; 1,6-DNP = 1,6-dinitropyrene; 1,8-DNP = 1,8-dinitropyrene; 9-NPh = 9-nitrophenanthrene; 9-NA = 9-nitroanthracene; 1-NN = 1-nitronaphthalene; 2-NN = 2-nitronaphthalene; 6-NC = 6-nitrochrysene.

Figure 6

Figure 6

Fig. 6. Diurnal differences in MW 247 compounds. 2-NFL = 2-nitrofluoranthene; 3-NFL = 3-nitrofluoranthene; 8-NFL = 8-nitrofluoranthene; 1-NP = 1-nitropyrene; 2-NP = 2-nitropyrene; 1-NN = 1-nitronaphthalene; 2-NN = 2-nitronaphthalene; 9-NPh = 9-nitrophenanthrene; 9-NA = 9-nitroanthracene; 6-NC = 6-nitrochrysene; 1,6-DNP = 1,6-dinitropyrene.

3,6-Dinitrobenzo[a]pyrene was detected in airborne particulates in Santiago, Chile, in the winter of 1988–1989 at concentrations of 10 ng/g of total particulate (0.002 ng/m3 of air) (Sera et al., 1991). Although the concentration was usually low, the mutagenicity of the chemicals accounted for about 40% of the total activity of the crude extracts.

The nitroarene fraction of MW 247 is of interest because in diesel exhaust, about 10% of direct mutagenicity is associated with this fraction, comprising nitrofluoranthene and nitropyrene isomers (in particular 1-nitropyrene and 3- and 8-nitrofluoranthene in diesel exhaust) (Salmeen et al., 1984). Furthermore, 2-nitrofluoranthene and 2-nitropyrene, which are formed from atmospheric reactions, also have a relative molecular mass of 247.

Table 19 also compares the concentrations of total PAHs, BaP and nitroPAHs of MW 247. From the data available, it can be seen that the concentration of BaP always exceeds that of 1-nitropyrene (ranging from 2 to 100 times more). 2-Nitrofluoranthene concentrations usually exceed those of 1-nitropyrene (by 1–40 times). Because 2-nitrofluoranthene is formed from gas-phase radical-initiated reactions of fluoranthene and 1-nitropyrene is emitted in combustion sources, the high 2-nitrofluoranthene/1-nitropyrene ratio suggests the importance of atmospheric nitroPAH formation. Additionally, the very high nitronaphthalene and methylnitronaphthalene concentrations in Table 18 when vapour and particles have been measured also reflect the significance of the atmospheric formation of nitroPAHs (Atkinson et al., 1988; Gupta et al., 1996; Arey, 1998).

1) Abundance of volatile and particulate-bound nitroPAHs in ambient air

2-Nitrofluoranthene is usually the most abundant MW 247 nitroPAH observed in ambient particulate organic matter extracts; however, in the ambient air samples where vapour-phase nitroPAHs have been measured (see Table 19), the more volatile nitronaphthalenes and methylnitronaphthalenes (two-ring PAHs) were most abundant (Arey et al., 1987). From seasonal studies at Houston, Texas, USA, it was found that 1-nitronaphthalene was always the most prevalent nitroPAH, followed by 9-nitrophenanthrene (three-ring PAH), 2-nitronaphthalene and 2-nitrofluoranthene (four-ring PAH) and 9-nitroanthracene (three-ring PAH) (Wilson et al., 1995; see Figure 5). The concentrations of all these compounds were several times higher in summer and autumn than in winter and spring. In comparison, 1-nitropyrene (four-ring PAH) was present in comparatively small amounts, but more in autumn and winter. Although 1- and 2-nitronaphthalene are expected to be found dominantly in the gas phase, the other semivolatile nitroPAHs will be distributed between the particle and gas phases, depending upon the ambient temperature. Thus, many ambient measurements will underestimate the total nitroPAHs present unless both gas- and particle-associated species have been measured. Hunt & Maisel (1995) also reported 1-nitronaphthalene, 2-nitronaphthalene and 9-nitrophenanthrene as the most abundant nitroPAHs (see Figure 6).

At seven sites throughout California, USA (Atkinson et al., 1988), the nitronaphthalenes were the most abundant nitroPAHs; at Glendora (traffic impacted), Yuba City (biomass burning and traffic), Oildale (oil production) and Reseda (traffic), the nitronaphthalene concentrations exceeded those of BaP.

2) Studies on ambient particulate matter: worldwide surveys

Remote sites

There are some reports of the determination of nitroPAHs at remote and forest sites: Storkow (Germany), CNR Pyramid (Nepal), Terra Nova Bay (Antarctica) and Alta Floresta (Brazil) (see Table 19; Ciccioli et al., 1995, 1996). Recently, nitronaphthalenes were reported to be the most abundant nitroPAHs in Antarctica (1–200 fg/m3 range) (Vincenti et al., 2001).

NitroPAHs were either not detected or in the low picogram per cubic metre range (e.g., 2-nitrofluoranthene, 17 pg/m3; 1-nitropyrene, 4 pg/m3). NitroPAHs were determined in the aerosol from the Amazon forest (Alta Floresta), but the author notes that it was near biomass burning (Vasconcellos et al., 1998).

Urban sites

Reports of urban and suburban concentrations of nitroPAHs are given in Tables 18 and 19. The levels depend, for example, on the climatic conditions, the number and regulation of traffic vehicles and the type of heating used. For example, concentrations of nitroPAHs (9-nitroanthracene, 2-nitrofluoranthene and 1-nitropyrene) taken during the same time period (January to March 1994) were 2–4 times higher in Damascus, Syria, than in Birmingham, United Kingdom, possibly due to insufficient burning of fuel oil employed for domestic heating and older cars in the Syrian capital (Dimashki et al., 1996).

Even in urban areas, the concentrations of 2-nitrofluoranthene have been found to be greater than those of 1-nitropyrene (see Table 19), with some values exceeding 1 ng/m3. Maximum values of 2-nitrofluoranthene and 1-nitropyrene were reported in Milan (4540 and 1070 pg/m3, respectively). This is in the range of reported concentrations of BaP in particulate matter. The concentration of total PAHs is 2 orders of magnitude higher. In Glendora, California, USA, 2-nitrofluoranthene reached 2000 pg/m3 when the corresponding BaP concentration was 320 pg/m3 (Atkinson et al., 1988).

2-Nitrofluoranthene and 1-nitropyrene concentrations were monitored daily for 4 weeks (February/March 1991) in Milan and Rome, Italy, and compared with BaP and "total" PAH levels (Cecinato et al., 1998). Concentrations were, respectively, 0.95 and 0.25 ng/m3 in Milan compared with 0.25 and 0.08 ng/m3 in Rome. Together with the results of previous studies, it was shown that mean winter levels were higher than summer levels. In Milan, the concentrations of these two nitroPAHs increased from 1990 to 1993.

The outdoor background concentration of 1-nitropyrene measured in and around Southampton, United Kingdom, was usually below 10 pg/m3, but occasionally a higher value was observed (Scheepers et al., 1999).

Some studies on urban atmospheric particulates have concentrated on monitoring 2-nitrofluorene, which, together with 1-nitropyrene, is associated with diesel emissions. Concentrations of nitroPAHs in particulate samples in the summer in six different sites in the industrially developed region of Upper Silesia in Poland were 90–290 pg/m3 for 2-nitrofluorene and up to 350 pg/m3 for 1-nitropyrene (and/or 3-nitrofluoranthene) (Warzecha, 1993). In studies on levels of 2-nitrofluorene in various cities, the following concentrations were measured: 24–50 pg/m3 (Tokyo, Japan), 50–700 pg/m3 (Beijing, China) and 170–4190 pg/m3 (Berlin, Germany) (Moriske et al., 1984; Beije & Möller, 1988a).

3) Seasonal differences

Seasonal studies on certain selected nitroPAHs (Bayona et al., 1994; Hayakawa et al., 1995a; Wilson et al., 1995; Murahashi et al., 1999; Marino et al., 2000) (see Figure 5) show that the concentrations of 1-nitropyrene and dinitropyrenes (from combustion sources) in ambient air particulates are usually higher in winter than in the other months. In contrast, in most studies, levels of 2-nitrofluoranthene and 2-nitropyrene (atmospheric transformation) are less in winter months than in the warmer seasons. This is also the case for the more volatile nitroPAHs (e.g., 1-nitronaphthalene, 2-nitronaphthalene, 9-nitrophenanthrene and 9-nitroanthracene) (Wilson et al., 1995; see Figure 5). The concentrations of all these compounds were several times higher in summer and autumn than in winter and spring.

Table 20 shows the concentrations of BaP and some nitroPAHs measured in a large study of semivolatile and volatile compounds at two sites in the Czech Republic during the years 1993–1994 and 1996–1997. Higher concentrations were found in winter than in summer due to the higher fuel consumption in winter. Although coal combustion was replaced in this region by gas for heating purposes in the years 1994–1995, it was found that similar distribution profiles and concentrations of PAHs were in the air in Teplice in winter 1993 and 1996 (Lenicek et al., 2001).

4) Diurnal differences

Daytime and nighttime studies on MW 247 nitroPAHs in ambient air collected at three sites (Claremont, Torrance and Glendora) situated in the Los Angeles basin in California, USA (Arey et al., 1987, 1988b; Zielinska et al., 1989b), showed increases of certain nitroPAHs during the nighttime (Figure 6; Table 18). From studies of PAHs (and nitroPAHs) in simulated atmospheres, it has been proposed that PAHs react with gaseous co-pollutants in the atmosphere to produce these nitroPAHs. Whereas the nitration of particle-bound PAHs with nitrogen dioxide/nitric acid and nitrogen pentoxide is probably not significant under most atmospheric conditions, this may not be the case in airsheds that have high nitrogen dioxide and nighttime nitrogen pentoxide concentrations. Daytime hydroxyl radical-initiated reactions of PAHs lead to the formation of nitroPAHs in low yields. For a discussion of the atmospheric formation of nitroPAHs, see chapter 3. The high nighttime concentrations of 2-nitrofluoranthene (and 1- and 2-nitronaphthalene and certain methylnitronaphthalenes) are attributed to nighttime gas-phase reactions of the parent PAH with the nitrate radical.

Composite samples (vapour-phase and particulate portions) of a number of chemical compounds, including nitroPAHs, were monitored in Fresno, California, USA, during the winter months. Elevated retene (1-methyl-7-isopropylphenanthrene) and enhanced nocturnal PAH (in particular naphthalene) concentrations suggested the strong influence of residential wood burning. Photochemical reactions involving these parent compounds and hydroxyl radicals resulted in elevated concentrations of 1- and 2-naphthalene (Hunt & Maisel, 1995; see Figure 6).

In a study on the chemical composition of aerosol in the Amazon, it could be seen from diurnal studies that the nitroPAHs 2-nitrofluoranthene and 2-nitropyrene were formed from nighttime photochemical reactions. The comparatively high levels of daytime 3- and 8-nitrofluoranthene were thought to be due to direct emissions (flaming and smouldering) from forest fires (Vasconcellos et al., 1998; see Figure 6).

Diurnal studies in which 1-nitropyrene and dinitropyrene levels in ambient air in downtown Kanazawa, Japan (sampling period July 1993 to February 1994), were compared with BaP concentrations and traffic volume found the nitroPAH levels to be high in the morning and evening and low from midnight to early morning, indicating that the major contributor to the dinitropyrenes and 1-nitropyrene was traffic emission (Hayakawa et al., 1995a, 1999b; Murahashi et al., 1995; see Figure 7). Similar studies monitoring 2-nitrofluoranthene, 1-, 2- and 4-nitropyrene and 6-nitrochrysene (4–7 January 1995) showed the highest concentrations of 4-nitropyrene and 6-nitrochrysene around 10 a.m. to 2 p.m. (comparable to 1-nitropyrene); however, the highest concentrations of 2-nitrofluoranthene and 2-nitropyrene were observed about 6 h later, around 6 p.m. to 8 p.m. (Murahashi et al., 1999).

Diurnal and seasonal (summer/winter) atmospheric concentrations of 1-nitropyrene and dinitropyrenes were recorded in three cities in Japan: Kanazawa, Sapporo and Tokyo (Kakimoto et al., 2000, 2001; see Table 22 and also Table 21). The concentrations tended to be higher in winter than in summer and higher in daytime than at night.

Table 22. Mean atmospheric concentrations of 1-nitropyrene and dinitropyrenes in three Japanese cities in 1995a

City

Season

Day/night

1-Nitropyrene
(pg/m3)

Dinitropyrenesb
(pg/m3)

Kanazawa

Winter

Day

59.2

0.91

 

Night

38.3

0.62

Summer

Day

26.7

0.38

 

Night

11.3

0.28

Sapporo

Winter

Day

413

5.7

 

Night

197

2.6

Summer

Day

206

2.4

 

Night

149

2.3

Tokyo

Winter

Day

163

2.0

 

Night

120

2.2

Summer

Day

130

2.3

 

Night

75.3

2.9

a From Kakimoto et al. (2000, 2001).

b Dinitropyrenes = 1,6-dinitropyrene + 1,8-dinitropyrene +1,3-dinitropyrene.

Daytime and nighttime variations in concentrations of 2-nitrofluoranthene, 1-nitropyrene and 2-nitropyrene, together with aerosols, ozone and nitrated species, were recorded in downtown Milan, Italy, in September 1990 and February to March 1991 (Ciccioli et al., 1993).

Figure 7

Fig. 7. Diurnal studies of 1-nitropyrene (1-NP) and 1,3-, 1,6- and 1,8-dinitropyrene (1,3-, 1,6- and 1,8-DNP) in downtown Kanazawa, Japan, July to February 1994 (from Hayakawa et al., 1999b), compared with traffic volume, indicating that the major contributor to the dinitropyrenes and 1-nitropyrene was traffic emissions.

Hydroxyl radical-initiated reactions were assumed to be responsible for the formation of 2-nitrofluoranthene and 2-nitropyrene. Since 2-nitrofluoranthene in nighttime hours was at concentrations much higher than those of 2-nitropyrene, it was supposed that reaction with nitrate also occurred.

Thus, combustion emissions, the occurrence of atmospheric formation reactions (daytime hydroxyl radical reactions and nighttime nitrate radical reactions) and meteorological and atmospheric dynamics will all influence the measured concentrations of nitroPAHs.

5) Nitro-oxyPAHs

2- and 4-nitrodibenzopyranones were observed in an ambient particulate extract collected from Riverside, California, USA, and accounted for ~20% of the total mutagenic activity (Helmig et al., 1992a). It should be noted that the microsuspension modification of the Ames assay was used. 2-Nitrodibenzopyranone has an unusually high response in this assay. 2- and 4-nitrodibenzopyranone were also detected in other ambient air samples from California (0.05–1 and <0.02–0.21 ng/m3 for the 2- and 4- isomers, respectively) and in an urban dust sample (NIST SRM 1649) from Washington, D.C. (0.82 and 0.52 µg/g, respectively; see Table 23), but with much lower concentrations in diesel particulate material (~0.2 and <0.1 µg/g, respectively), suggesting that nitrodibenzopyranones are formed in the atmosphere (Helmig et al., 1992b). 3-Nitrodibenzopyranone has not been reported in ambient air samples.

Table 23. 2- and 4-nitrodibenzopyranone concentrations in atmospheric samples from Los Angeles, California, USA, in urban dust (SRM 1649) and diesel exhaust particles (SRM 1650) compared with MW 247 nitroPAHsa

Compound

Concentrations in the Los Angeles basin (ng/m3)

Concentration (µg/g particles)

SRM 1649
Urban dust

SRM 1650
Diesel exhaust particles

2-Nitrodibenzopyranone

0.05–1

0.82 ± 0.15

~0.2

4-Nitrodibenzopyranone

<0.02–0.21

0.52 ±0.09

<0.1

2-Nitrofluoranthene

0.03–2.0b

0.60c

 

1-Nitropyrene

0.003–0.06b

0.06d

19 ± 2d

2-Nitropyrene

0.001–0.06b

 

0.27

9-Nitroanthracene

 

0.2

10

7-Nitrobenz[a]anthracene

   

3

6-Nitrobenzo[a]pyrene

   

1.6

a Adapted from Helmig et al. (1992b).

b From Arey et al. (1987); Zielinska et al. (1989a).

c From Ramdahl et al. (1986).

d From May et al. (1992).

5.1.1.2 Indoor air

NitroPAHs have been detected in the emissions of kerosene heaters, fuel gas and LPG burners (city gas and heavy oil) used for heating and cooking at home, as well as in the fumes of cooking oils (see chapter 3). In poorly ventilated conditions, there is therefore a potential for indoor exposure to nitroPAHs.

Concentrations of polyaromatic compounds, including nitroPAHs, were measured in a study of indoor and outdoor air levels associated with 33 homes located in two US cities, Columbus, Ohio, and Azusa, California (Wilson et al., 1991). The overall levels were much higher in homes occupied by smokers, but the use of natural gas heating and cooking appliances also appeared to increase the nitroPAH levels slightly (Table 24). Concentrations were up to 0.28 ng/m3 for 1-nitropyrene and from 0.006 to 0.20 ng/m3 for 2-nitrofluoranthene. The average outdoor level for 2-nitrofluoranthene was 0.06 ng/m3. Concentrations of 1-nitropyrene were detected at <1.0–8.6 pg/m3 in indoor air in Southampton, United Kingdom (Scheepers et al., 1999).

Table 24. Typical nitroPAH concentrations in homes averaged over three locations: living room, bedroom and kitchena

NitroPAH

Concentrations (ng/m3)

Homes with smokers

Homes with non-smokers

with gas heating and gas stove

with electric heating and electric stove

with gas heating and gas stove

1-Nitropyrene

0.044

0.020

0.021

2-Nitrofluoranthene

0.052

0.012

0.022

a From Wilson et al. (1991).

5.1.2 Water

1-Nitropyrene was detected in water samples from the Yodo River, Kyoto, Japan, which is used as a source of drinking-water. No quantitative data were given (Ohe, 1996).

1- and 2-nitronaphthalene and 1,3- and 1,5-dinitronaphthalene were detected in river water in Japan at concentrations of 1.3, 11.7, 1.7 and 3.2 ng/litre, respectively (Takahashi et al., 1995).

The following nitroPAHs were not detected in the Nagura River in Japan: 1-nitropyrene (<20 ng/litre), 3-nitrofluoranthene (<20 ng/litre), 1,3-, 1,6- and 1,8-dinitropyrene (<200 ng/litre) and 2,7-dinitrofluorene (<200 ng/litre) (Nagai et al., 1999).

1-Nitropyrene (4.2–25 600 ng/litre) was detected in 36 of 55 samples of wastewater from oil/water separating tanks of gasoline stations and in used crankcase oil (Manabe et al., 1984). The 1-nitropyrene accounted for 0.3–58.5% of the total mutagenicity of the neutral fractions. Nineteen samples did not contain any 1-nitropyrene.

5.1.3 Soil, sewage sludge, sediment and incinerator ash

1-Nitropyrene and nitrofluoranthene isomers as well as nitronaphthalene, nitrofluorene and methylated derivatives were detected together with other PAHs in samples of sewage sludge in Upper Silesia, Poland (Bodzek & Janoszka, 1995; Bodzek et al., 1997) (levels not given). 1-Nitropyrene was identified in sewage sludge at 0.68 µg/kg dry weight (Fernández et al., 1992).

In samples of surface soil from the city of Basel, Switzerland, nitroPAHs (mainly 3-nitrofluoranthene and 1-nitropyrene) were found in concentrations between 0.03 and 0.8 µg/kg dry weight. Levels of oxyPAHs and parent PAHs were 102- to 104-fold higher (Niederer, 1998).

1,3-, 1,6- and 1,8-dinitropyrene (0.012–3.27, 0.014–5.59 and 0.013–6.80 µg/kg dry weight, respectively) were detected in all 110 samples of non-agricultural soil collected from five geographically different areas in Japan between November 1996 and March 1997 (Watanabe et al., 2000).

2-Nitrofluorene, 2,7-dinitrofluorene and 1-nitropyrene (1.5, 3.8 and 25.2 µg/kg sediment, respectively) were detected in sediments in the bed of the Suimon River. The direct-acting mutagenic activity that had been observed in sediment samples from this river was consistent with the mutagenicity of these nitroPAHs, in particular 1-nitropyrene (Sato et al., 1985).

Coastal sediments collected offshore from Barcelona, Spain, were fractionated and characterized using bioassay-directed chemical analysis (Fernández et al., 1992). 6-Nitrochrysene, 6-nitrobenzo[a]pyrene and nitrobenzofluoranthenes were identified at concentrations of 0.52, 0.34 and 0.26 µg/kg dry weight, respectively, in sediment from urban littoral stations.

Librando et al. (1993) reported concentrations of selected nitroPAHs of up to 5000 µg/kg in incinerator ash. The highest values were given by 1-nitronaphthalene (1.59–2.86 mg/kg), 2-nitronaphthalene (1.06–3.46 mg/kg) and 9-nitroanthracene (0.96–4.84 mg/kg). Values for 1-nitropyrene were <0.01–0.89 mg/kg.

5.1.4 Food and beverages

5.1.4.1 Food

Foodstuffs were monitored for the presence of 9-nitroanthracene and 1-nitropyrene in the United Kingdom (Dennis et al., 1984). Of the 28 samples analysed, 25 contained no detectable levels of these nitroPAHs. 9-Nitroanthracene was tentatively identified in peated malt at 0.9 µg/kg and 1-nitropyrene in two samples of tea leaf at 1.7 and 0.17 µg/kg.

1-Nitropyrene was detected in grilled corn, mackerel and (in considerable amounts) pork and yakitori (grilled chicken) grilled with sauce (up to 43 ng/g) (Ohnishi et al., 1986). The authors concluded that the formation of 1-nitropyrene is due to pyrene produced by the incomplete combustion of fat in the chicken, its nitration at acidic pH by nitrogen dioxide emitted by the burning of cooking gas and some components of the marinating sauce.

Various foods were analysed for the presence of 2-nitrofluorene, 1-nitropyrene and 2-nitronaphthalene (Schlemitz & Pfannhauser, 1996a; Table 25). These nitroPAHs were detected in most samples, the highest concentrations being found in spices and smoked food, but also in vegetables and fruits, probably due to atmospheric pollution. In a parallel study using another analytical method, the presence of nitroPAHs was monitored in cheese as well as in meats and fish prepared by grilling or roasting (Schlemitz & Pfannhauser, 1996b; see Table 25).

Table 25. Occurrence of nitroPAHs in food samples

Sample

Measurea

Concentrationsb (µg/kg)

Referencec

1-Nitronaphthalene

2-Nitronaphthalene

2-Nitrofluorene

1-Nitropyrene

Vegetable, fruits, nuts

         

Lettuce

n. sp.

<0.2

1.6

<0.2

Schlemitz & Pfannhauser (1996a)

Parsley

n. sp.

<0.2

0.8

1.7

Schlemitz & Pfannhauser (1996a)

Carrot

n. sp.

nd (0.2)

0.9

0.4

Schlemitz & Pfannhauser (1996a)

Apple

n. sp.

nd (0.2)

0.8

0.2

Schlemitz & Pfannhauser (1996a)

Peanuts

n. sp.

nd (0.2)

16.6

<0.5

Schlemitz & Pfannhauser (1996a)

Spices

           

Paprika

n. sp.

7.8

26.4

9.3

Schlemitz & Pfannhauser (1996a)

Marjoram

n. sp.

3.6

23.1

14.1

Schlemitz & Pfannhauser (1996a)

Caraway

n. sp.

3.1

350.7

10.9

Schlemitz & Pfannhauser (1996a)

Oils

           

Olive oil

n. sp.

<0.2

0.8

0.6

Schlemitz & Pfannhauser (1996a)

Milk products

           

Alp-cheese I

mean (3)

nd

1.1

nd

Schlemitz & Pfannhauser (1996b)

Alp-cheese II

mean (3)

nd

0.3

nd

Schlemitz & Pfannhauser (1996b)

Smoked cheese

mean (3)

0.6

0.5

nd

Schlemitz & Pfannhauser (1996b)

Fish

           

Grilled fish

mean (3)

0.2

0.3

nd

Schlemitz & Pfannhauser (1996b)

Grilled mackerel

       

0.45

Ohnishi et al. (1986)

Smoked fish

mean (3)

0.3

0.2

nd

Schlemitz & Pfannhauser (1996b)

Meat

           

Grilled meat (pork)

n. sp.

0.1

2.0

1.0

Schlemitz & Pfannhauser (1996a)

Grilled sausages

n. sp.

 

<0.2

0.2

0.8

Schlemitz & Pfannhauser (1996a)

mean (3)

nd

 

0.1

 

Schlemitz & Pfannhauser (1996b)

Smoked meat

n. sp.

10.2

2.0

2.2

Schlemitz & Pfannhauser (1996a)

Smoked sausages

n. sp.

8.4

19.6

4.2

Schlemitz & Pfannhauser (1996a)

Roasted meat (pork)

mean (3)

nd

0.5

0.3

Schlemitz & Pfannhauser (1996b)

Roasted turkey

mean (3)

nd

0.3

nd

Schlemitz & Pfannhauser (1996b)

Grilled chicken

range (10)

0.4–11

Kinouchi et al. (1986a)

Grilled chicken

       

up to 43

Ohnishi et al. (1986)

a

In parentheses: number of samples; n.sp.= not specified.

b

nd = not detected (detection limit in parentheses, if given); – = no data available.

c

Schlemitz & Pfannhauser (1996a, 1996b) studies conducted in Austria; Kinouchi et al. (1986a) and Ohnishi et al. (1986) studies conducted in Japan.

In another study (Ziegler et al., 1999), concentrations of 2-nitrofluorene and 1- and 2-nitronaphthalene in a variety of fruits and vegetables (apple, grapes, red pepper, broccoli, kohlrabi and cauliflower) were below the detection limit (5 µg/kg).

To study the effect of cooking on nitroPAHs, cauliflower and broccoli samples were artificially contaminated with a solution of 2-nitrofluorene and 1- and 2-nitronaphthalene and cooked for 20 min. No more than 4% of the nitroPAHs transferred into the boiling water. The nitronaphthalenes volatilized to a great extent, but 2-nitrofluorene remained almost quantitatively on the solid parts.

The same solution of three nitroPAHs was dropped onto the peel of apples. After 18 h, it was found that almost the whole content remained in the peel fraction, only a small amount of the nitronaphthalenes being found in the edible interior part. Washing the contaminated apples in hot water did not remove any nitroPAHs from the peel. Similar results were found with kohlrabi. Therefore, nitroPAHs remain on the surface of fruits and vegetables. In the environment, however, the nitroPAHs are attached to particles and are not applied directly to the surface of plants, so washing the fruit with hot water should remove the particulate matter and the nitroPAHs; peeling fruit is the best way to reduce potential exposure to nitroPAHs (Ziegler et al., 1999).

NitroPAHs (2-nitrofluorene, 1-nitropyrene and 1- and 2-nitronaphthalene) were found in smoked foods from the market (n = 92), such as fish (n = 69) and meat products (n = 14). No traces of such compounds could be detected in smoked cheese (n = 9) after discarding the cheese rind (Dafflon et al., 2000).

5.1.4.2 Beverages

Surveys of nitroPAH levels in various foods showed that substantial concentrations of nitroPAHs were present in tea and coffee (Schlemitz & Pfannhauser, 1996a). In a further study (using supercritical fluid extraction), samples of a variety of teas were investigated for their nitroPAH and PAH content (Schlemitz & Pfannhauser, 1997; see Table 26). Six nitroPAHs were measured. Very high concentrations of nitroPAHs (128 µg/kg) and PAHs (7536 µg/kg) were found in Mate tea, which is roasted with combustion fumes to gain its unique aroma. Other teas had concentrations of around 20 µg/kg for the six nitroPAHs, with 2-nitrofluorene and 9-nitroanthracene having the highest levels. Although nitroPAHs are usually not very soluble in water (see chapter 2), the authors found that up to 25% of the nitroPAH concentration could be measured in the tea water. It is possible that other components of the tea act as co-solvents, thereby increasing the solubility of nitroPAHs in the tea water. In all tea samples, the PAH content was always much higher than that of nitroPAHs. The presence of nitroPAHs and PAHs in tea probably originates from several sources, including technological processes used during the preparation of tea, such as roasting and drying, and atmospheric pollution.

Table 26. Occurrence of nitroPAHs in tea leaves and coffee beansa

Sample (n = 3)

Concentrationb (µg/kg)

1-NN

2-NN

2-NF

1-NP

9-NA

3-NFL

Total nitroPAHs

Total PAHs

Assam tea

nd

0.58

6.45

2.32

5.21

4.36

18.92

28.56

Earl Grey tea

0.90

1.38

9.77

7.75

4.17

1.38

25.35

313.77

Ceylon tea

0.39

0.67

0.61

1.54

16.64

1.73

21.58

79.7

Darjeeling tea

13.07

1.43

nd

4.00

2.5

0.98

21.98

775.67

Mate tea (roasted)

0.47

4.09

20.04

37.89

22.19

43.04

127.72

7536.33

Mate tea (green)

nd

1.52

5.32

0.80

1.81

1.21

10.66

6475.9

Formosa Sencha (green tea)

nd

1.05

9.27

3.10

3.06

3.66

20.14

549.76

Nettle leaf tea

0.25

0.18

10.02

1.96

12.6

3.71

28.72

97.77

Peppermint tea

0.46

0.85

16.41

3.79

3.94

1.85

27.30

140.39

Fennel tea (instant)

nd

0.41

0.09

nd

0.51

nd

1.01

13.41

Fruit tea (instant)

0.82

0.22

0.56

0.55

0.60

0.28

3.03

17.53

Teac

1.5

5.3

nd

         

Coffeec

4.0

30.1

2.4

         

a

From Schlemitz & Pfannhauser (1997).

b

1-NN = 1-nitronaphthalene; 2-NN = 2-nitronaphthalene; 2-NF = 2-nitrofluorene; 1-NP = 1-nitropyrene; 9-NA = 9-nitroanthracene; 3-NFL = 3-nitrofluoranthene; nd = not detected.

c

From Schlemitz & Pfannhauser (1996b); in this study, only three nitroPAHs were determined.

NitroPAHs are found in the ground coffee bean, so filtered coffee would probably not contain nitroPAHs at such levels. Drinking instant coffee would lead to the direct ingestion of the nitroPAHs (Schlemitz & Pfannhauser, 1996a).

5.1.5 Other sources

5.1.5.1 Carbon black and toners

Extracts of selected xerographic toners and paper photocopies were found to be mutagenic (Löfroth et al., 1980). The fraction of the carbon black B responsible for 80% of the mutagenicity contained 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene. As a result of this finding, the manufacturers modified the production of carbon black B, substantially reducing the levels of nitropyrenes (Rosenkranz et al., 1980).

5.1.5.2 Cigarette smoke

Although "parent" PAHs have been detected in tobacco smoke (IARC, 1986), there are no reports of nitroPAHs in cigarette smoke condensate. 1-Nitronaphthalene (<10 ng/cigarette), 1-nitropyrene (<10 ng/cigarette) and 6-nitrochrysene (<1 ng/cigarette) were not detected in the mainstream cigarette smoke of three different types of cigarette (El-Bayoumy et al., 1985). No 1-nitropyrene was detected in the tar extracts of five commercial brands of cigarettes at a limit of determination of 30 pg/cigarette (Scheepers et al., 2001).

5.2 General population exposure

NitroPAHs have been found to be ubiquitous in ambient air and in particular in air polluted by vehicular traffic. Indoor air exposure from kerosene heaters and from cooking oils has been described. NitroPAHs are found in certain foods, in particular grilled foods (see section 5.1.4.1). Drinking-water is not likely to contain nitroPAHs, as they are sparingly soluble or insoluble in water.

NitroPAHs have been detected in samples of resected lung tissue from patients in Japan (Tokiwa et al., 1993a, 1998a,b; Sera, 1998; Tokiwa & Sera, 2000) (see Table 27; section 8.1). The lung specimens were surgically resected from 293 patients with carcinoma and 63 patients with tuberculosis without carcinoma as controls during the periods 1961–1962 (time of heavy air pollution in Japan) and 1991–1996 (time of regulated air pollution in Japan). The ages of the patients ranged from 28 to 68 years. Lung specimens collected in this study originated from patients, including smokers and non-smokers, who had lived in the Fukuoka prefecture in Japan for over 20 years.

Table 27. Concentrations of PAHs and nitroPAHs in lung tissuesa

Compound

Concentration of compound (pg/g of dry weight)

With carcinomab
(n = 37)

Controlb
(n = 37)

With carcinomac
(n = 256)

Controlc
(n = 26)

1-Nitropyrene

30.8 ± 11.4

27 ± 14.8

19.7 ± 10.5*

18.7 ± 10.2

1,3-Dinitropyrene

4 ± 0.7

4.2 ± 0.35

3.5 ± 0.14

2.6 ± 0.17

1,6-Dinitropyrene

5.1 ± 2.3

5 ± 2.8

4.82 ± 1.69

4.6 ± 1.8

1,8-Dinitropyrene

6.7 ± 2.6

6.2 ± 2.7

6.26 ± 1.76

6.1 ± 2.1

2-Nitrofluoranthene

43.2 ± 19.7

43.5 ± 2.5

38.6 ± 17.2

34.7 ± 15.4

3-Nitrofluoranthene

32.6 ± 12.8

33.6 ± 15.2

39.1 ± 14.2

36.5 ± 13.8

Benzo[a]pyrene

341 ± 210

330 ± 107

196 ± 114*

178 ± 118

Pyrene

583 ± 220

539 ± 356

510 ± 286

462 ± 115

Fluoranthene

701 ± 356

673 ± 421

605 ± 370*

586 ± 136

a From Tokiwa et al. (1998a); * P < 0.05.

b Periods 1961–1962 (time of heavy air pollution in Japan); samples from patients with lung carcinoma and, as control, from patients with tuberculosis but without carcinoma.

c Periods 1991–1996 (time of regulated air pollution in Japan); samples from patients with lung carcinoma and, as control, from patients with tuberculosis but without carcinoma.

5.3 Occupational exposure

Occupational exposure to nitroPAHs is expected in workplaces associated with the use of diesel engines. Underground workers (drivers of diesel-powered excavators) at an oil shale mine in Estonia were subjects in a study for the evaluation of 1-nitropyrene as a biomarker of exposure. The concentrations of 1-nitropyrene associated with respirable particles as determined in the breathing zones of the workers ranged up to 42 ng/m3 (mean 2.5 ng/m3) (Scheepers et al., 2002; see Table 28).

Table 28. Concentration of particle-associated 1-nitropyrene (pg/m3) in the breathing zones of drivers of diesel-powered excavators
(main study and pilot study)a

 

Surface

Underground

 

No. of measurements

Geometric mean (pg/m3)

Median (pg/m3)

Range (pg/m3)

No. of measurements

Geometric mean (pg/m3)

Median (pg/m3)

Range (pg/m3)

Main study

42b

85

83

4–2166

50

637

694

29–5031

Pilot study (first shift)

9

9

6

3–50

10

2483

2021

602–42 190

Pilot study (second shift)

9

45

34

12–686

10

984

1179

134–3455

a

From Scheepers et al. (2002).

b

1-Nitropyrene could not be quantified in seven workers because the internal standard peak area was too low. Air sampling failed in one worker.

Air concentrations of 1-nitropyrene were measured in various workplaces associated with the use of diesel engines (Scheepers et al., 1994a; see Table 29). The highest mean level (1 ng/m3) was reported for forklift truck drivers at an aluminium rolling factory.

Table 29. Air concentrations of 1-nitropyrene as determined from extracts of total suspended particulate matter collected at workplaces associated with the use of diesel enginesa

Facility

Sources of diesel exhaust

No. of samples (n)

1-Nitropyrene (mean)b (ng/m3)

River vessel

Ship’s engine

3

0.05

Repair shop for trains

Train engines

4

0.32

Army driving lessons

Armoured cars

2

0.01

Flower auction

Trucks

2

0.08

Farming

Tractor

1

nd

Gardening

Passing traffic

1

0.04

Airport platform

Platform vehiclesc

3

0.04

Concrete manufacturing

Forklift trucks

2

0.66

Chemical plant

Forklift trucks

4

0.27

Aluminium rolling

Forklift trucks

4

1.06

Galvanization workshop

Forklift trucks

4

0.10

Grass verge maintenance

Lawn mowers

1

0.007

River vessel

Ship’s aggregate

1

0.79

a From Scheepers (1994); Scheepers et al. (1994a); sample volumes 135–600 m3.

b Using gas chromatography–high-resolution mass spectrometry detection; nd = not detected.

c Lift platforms, power supplies, trucks, pull-offs.

In a more detailed report, 1-nitropyrene levels were determined in stationary samples of total suspended particulate matter collected on two consecutive workdays in January in a repair shop for train engines (Scheepers et al., 1994b). Air concentrations of particulate-associated 1-nitropyrene ranged from non-detectable to 5.6 ng/m3. In spot urine samples, urinary metabolites of PAHs and nitroPAHs were determined using enzyme-linked immunosorbent assay (ELISA), based on an antibody with high affinity to 1-aminopyrene. In urine samples of three non-smoking diesel mechanics, both cumulative and average excretion of urinary metabolites over 48 and 72 h were significantly enhanced (P < 0.05) compared with the excreted levels in urine samples from two office clerks. However, in a further study in May, this could not be confirmed (Scheepers et al., 1995a). Spot urine samples were collected during two consecutive workdays in January and May from a group of 30 railroad workers. Three job categories were involved: a) diesel mechanics exposed indoors to exhaust derived from running diesel engines in a repair shop for diesel trains; b) electrical engineers working outdoors and exposed to low levels of emissions derived from remote sources of incomplete combustion; and c) office workers. Levels of 1-aminopyrene equivalents were very similar in subjects working indoors and outdoors, regardless of whether they were smokers or non-smokers. It is possible that the significant effect found in the January study was due to the higher number of diesel engines being repaired or a lack of ventilation compared with the study in May, when windows, for example, were left open (Scheepers, 1994; Scheepers et al., 1995a,b, 1999, 2001).

For further studies on biomonitoring, see chapter 8.

6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS

6.1 Overview of the metabolism of nitroPAHs

The metabolism of nitroPAHs in vivo, with the exception of 1-nitropyrene and 2-nitrofluorene, has not been well studied. However, from the results on these two compounds, it can be expected that, in general, nitroPAHs administered by various routes are rapidly absorbed and metabolized, mainly by ring oxidation and nitroreduction, followed by conjugation and excretion of the metabolites formed, mainly in faeces and urine. Radiolabelled 1-nitropyrene was found to be widely distributed in the body of rats and mice after administration by all routes. For recent reviews on the metabolism and activation of nitroPAHs, see Fu (1990), Beland & Marques (1994), Fu & Herrenos-Saenz (1999) and Purohit & Basu (2000).

NitroPAHs constitute a complex group of chemicals showing different metabolisms. For a particular nitroPAH, there may be several metabolic pathways, often depending on the route of administration. Intestinal microflora seem to play a role in metabolism through nitroreduction and/or deconjugation. Deconjugation can enhance enterohepatic circulation. For most of the compounds studied, the metabolic pathways are not clearly understood.

While metabolism of PAHs involves oxidation and subsequent hydrolysis and/or conjugation reactions (IPCS, 1998), the metabolism of nitroPAHs is even more complex. It seems that there are at least five metabolic activation pathways through which mutations can be induced by nitroPAHs in bacterial and mammalian systems and/or through which DNA binding occurs. These are:

Figure 8

Fig. 8. Metabolic activation pathways of nitroPAHs leading to mutation (Fu, 1990).

In bacteria, nitroreduction seems to be the major metabolic pathway, whereas the fungus Cunninghamella elegans is an example of a species in which nitroPAHs are metabolized by ring oxidation (see chapter 4).

Studies into the enzymes involved in metabolic pathways — in particular those of cytochrome P450 — have shown that different P450 enzymes may be involved in metabolism of a specific nitroPAH (Chae et al., 1993); that these may differ in the related isomers, resulting in possibly different kinetics and pathways (Chae et al., 1999a); and that P450 enzymes responsible for the metabolism of nitroPAHs may vary from species to species and in different target organs (Howard et al., 1988, 1990; Silvers et al., 1992, 1994; Guengerich et al., 1999).

The nitroreduction of a nitroPAH may involve one- and/or two-electron transfers and result in the formation of the corresponding nitrosoPAH, further reduction to the N-hydroxyaminoPAH and final reduction to the aminoPAH. Both bacterial and mammalian enzymatic systems are capable of reductively metabolizing nitroPAHs under anaerobic or hypoxic incubation conditions (see also chapter 4). Nitroreduction of nitroPAHs in vivo probably occurs mainly by bacteria in the intestinal tract. In mammalian cells, nitroreduction is catalysed by a variety of enzymes, including cytosolic aldehyde oxidase (Tatsumi et al., 1986), xanthine oxidase (Howard & Beland, 1982; Bauer & Howard, 1990, 1991), DT-diaphorase or microsomal NADPH cytochrome P450 reductase (Djuric et al., 1986a). These enzymes are found in a variety of tissues; for example, xanthine oxidase occurs in liver, intestinal mucosa and mammary glands and is also present in the milk and colostrum of most mammals (Howard et al., 1995).

In oxidative metabolism, the first step is transformation to phase I primary metabolites, such as epoxides, phenols and dihydrodiols, and then to secondary metabolites, such as diol epoxides, tetrahydrotetrols and phenol epoxides. In mammalian systems, the phase I metabolites are then conjugated with glutathione, sulfate or glucuronic acid to form phase II metabolites, which are more polar and water soluble than the parent hydrocarbons. On reaching the intestine, the conjugated metabolites can be deconjugated by the intestinal microflora and absorbed, entering the enterohepatic recirculation. Nitroreduction and N-acetylation can occur, resulting in the excretion in urine and faeces of metabolites such as, for example, acetylaminopyrenols after 1-nitropyrene administration.

Acetylation is an important conjugation reaction whereby water solubility is reduced. N-Acetyltransferases are found in the liver and in the intestine. Arylhydroxyamines can be acetylated at either the -NH or -OH of the -NHOH moiety, resulting in either an arylhydroxyamic acid or an acetoxyarylamine.

NitroPAHs do not follow the same activation pathways. Mutagenic activation can occur by deacetylation (to arylhydroxyamines) or O-acetylation (e.g., 1,8- and 1,6-dinitropyrene) and intramolecular acyl transfer to acetoxyarylamines with the production of electrophilic nitrenium ion (-N+), which binds to DNA. Some nitroPAHs (e.g., 6-nitrobenzo[a]pyrene) may be mutagenic only after activation by oxidation to reactive epoxides or dihydrodiol epoxides, similarly to BaP.

The main DNA adducts detected with nitroPAHs in vivo and in vitro are N-(deoxyguanosin-8-yl) (C8-substituted deoxyguanosine [dG]) derivatives, but N2-substituted dG and C8-substituted deoxyadenosine (dA) derivatives have also been detected and may predominate in certain nitroPAHs with greater hydrocarbon character (e.g., 3-nitrobenzo[a]pyrene and 6-nitrochrysene). DNA adducts of dinitropyrenes are formed only via nitroreduction, presumably owing to the high electron deficiency in the aromatic rings caused by the presence of two nitro groups. The DNA adducts resulting from the nitroreduction of nitroPAHs are better characterized than those arising from oxidative metabolism, although the latter may be of more importance in mammalian metabolism. There is recent evidence for oxidative metabolism, e.g., in the genotoxicity of the atmospheric reaction product 2-nitronaphthalene in human lymphoblastoid cell lines (Grosovsky et al., 1999; Sasaki et al., 1999).

In this document, it is not possible to present the results of all the studies on the metabolism of nitroPAHs because of the amount of data and the number of different compounds. Most studies on the metabolism of nitroPAHs have concentrated on 1-nitropyrene and, to a lesser extent, 2-nitrofluorene, as they are the most abundant nitroPAHs in diesel exhaust. These are given in sections 6.2 and 6.4, respectively. An overview of the metabolism, metabolic activation and DNA binding of isomeric and some individual nitroPAHs (sections 6.3, 6.5–6.9) is then given to illustrate how the three-dimensional structure of individual nitroPAHs, in particular the orientation of the nitro functional group, seems to determine the biological activity of individual nitroPAHs (reviewed by Fu et al., 1988a; Hart et al., 1988; Fu, 1990). For recent reviews on the metabolism and activation of nitroPAHs, refer to the first paragraph of this chapter.

6.2 1-Nitropyrene metabolism in mammals

6.2.1 Absorption

Early peak levels of radioactivity observed in blood and tissue homogenates indicated a rapid (within 3 h) absorption from the gastrointestinal tract after intragastric administration of [14C]1-nitropyrene (single dose, 27.6 µCi, 750 mg/kg bw) to male Cpb:WU (Wistar) rats (van Bekkum et al., 1999).

After gavage administration of radiolabelled 1-nitropyrene to rats, rapid absorption was found after 30 min (Sun et al., 1983) and 1 h (Bond et al., 1986). Binding of 1-nitropyrene to particles reduced absorption significantly (see section 6.2.2).

No data were available on dermal absorption.

6.2.2 Distribution

Radioactivity was found to be widely distributed in tissues of rats after intragastric administration of 14C- or 3H-labelled 1-nitropyrene, being found mainly in the liver and kidney, but also in the gastrointestinal tract, bladder, adipose tissue, brain, lung and heart (Kinouchi et al., 1986b).

Carbon-14 was widely distributed in tissues of male F344/Crl rats 1 h after nose-only exposure to 50 and 490 ng [14C]1-nitropyrene/litre air or [14C]1-nitropyrene (650 ng/litre) coated on diesel exhaust particles. Highest concentrations of 14C were found in the respiratory tract (nasal turbinates, trachea, lung), kidneys, liver and urinary bladder. Lungs of rats exposed to the diesel particles contained nearly 5 times (148 versus 29 pmol/g lung) more 14C than lungs exposed to aerosols without particles 1 h after exposure and 80-fold (80 versus 1 pmol/g lung) 94 h after exposure, demonstrating that particle association of nitropyrene significantly alters the biological fate of inhaled nitropyrene (Bond et al., 1986). Concentrations of diesel soot in the exposure chamber ranged from 3.7 to 6.1 µg/litre air. The activity median diffusion equivalent diameter of aerosols of [14C]1-nitropyrene coated on diesel exhaust particles was 0.22 µm.

6.2.3 Metabolism

6.2.3.1 Introduction

Once 1-nitropyrene becomes available in the bloodstream, it is rapidly metabolized, followed by conjugation and rapid elimination of the metabolites formed. Evidence for pathways of metabolism has come from in vivo studies on these metabolites in rodents, including studies with germ-free animals (see section 6.2.3.5 and Table 30) and studies on bile-cannulated rats (see Tables 30 and 31), as well as from in vitro studies (El-Bayoumy & Hecht, 1983; Ball & King, 1985; Howard et al., 1985; King & Lewtas, 1993).

Table 30. Metabolites formed in in vivo studies (either unconjugated 1-nitropyrene metabolites or aglycons, after incubation with,
for example, aryl sulfatase or β-glucuronidase)a,b

Metabolite

I

II

IIIa germ-free

IIIb

IIIc

IV

Va

Vb

Vc

VI

VII

i.p.

i.v.

oral

oral

oral

inhal.

oral

oral

oral

oral

oral

1-Nitropyren-3-olc

U, F

Bil

F,U

 

Bil

 

B

 

nd

U

P, Li, Ki, Lu

1-Nitropyren-6- and/or -8-ol

U, F

Bil

F,U

 

Bil

U

B

x

x

U

P, Li, Ki, Lu

Aminopyrenols

       

Bil

           

N-Acetyl-1-aminopyren-3-ol

U, F

   

F

Bil

       

nd

 

N-Acetyl-1-aminopyren-6-ol

U ,F

   

U, F

Bil

U, F

     

U

P, Li, Ki,

N-Acetyl-1-aminopyren-8-ol

U, F

   

U, F

Bil

U, F

     

U

P Li, Ki,

trans-4,5-Dihydro-4,5-dihydroxy-1-nitropyrene (1-nitropyrene-4,5-dihydrodiol)

U, F

Bil

F,U

 

Bil

 

B

 

x

 

P Li, Ki,

N-Acetyl-1-aminopyrene

U, F

       

F

nd

x

nd

   

1-Aminopyrene

U, F

   

F

 

F

B

x

x

U

P Li, Ki, Lu

1-Nitropyrene

U, F

       

F

     

nd

P, Li, Ki, Lu

a

i.p. = intraperitoneal; i.v. = intravenous; inhal. = inhalation; P = in plasma; B = in blood; U = in urine; F = in faeces; Bil = biliary glucuronide conjugates; Li = liver, Ki = kidney; Lu = lung ; nd = not detected; x = whole tissue; bold type, main metabolites.

b

Studies were as follows:

 

I)

Intraperitoneal administration of [14C]1-nitropyrene to CD rats (Ball et al., 1984a).

 

II)

Intravenous administration of [4,5,9,10-3H]1-nitropyrene to bile-cannulated rats resulted in rapid appearance of radioactivity in the bile with maximum concentration of radioactivity being observed between 0 and 1 h. Thirty per cent was excreted in 4 h. By comparison, following oral administration of [4,5,9,10-3H]1-nitropyrene, the highest concentration of radioactivity in the bile was detected between 2 and 4 h, and only 10% of the dose was found in the bile within 12 h. Nearly all the radioactivity in the bile was due to conjugated 1-nitropyrene metabolites (Howard et al., 1985).

 

IIIa)

Major faecal metabolites in germ-free rats following oral administration of [3H]1-nitropyrene (48 h). The same metabolites were found in the urine as their glucuronide conjugates. 1-Nitropyrene was found in the faeces (Kinouchi et al., 1986b). Similar results were found using [4,5,9,10-14C ]1-nitropyrene (120 h) (El-Bayoumy et al., 1984a).

 

IIIb)

Major faecal metabolites in conventional rats following oral administration of [3H]1-nitropyrene (48 h). Substantial nitroreduction and N-acetylation occurred (Kinouchi et al., 1986b). Similar results were found using [4,5,9,10-14C]1-nitropyrene (120 h) (El-Bayoumy et al., 1984a).

 

IIIc)

As above using bile-cannulated rats (Kinouchi et al., 1986b). Nearly all the metabolites in the bile were conjugated. With time (24–48 h), the proportion of nitrated metabolites decreased, while the proportion of aminopyrenols and acetylaminopyrenols increased.

 

IV)

Rats exposed to 50 and 490 ng [14C]1-nitropyrene/litre air, or [14C]1-nitropyrene (650 ng/litre) coated on diesel exhaust particles (Bond et al., 1986). Large quantities of unmetabolized 1-nitropyrene (presumably cleared from upper respiratory tract and cleared unmetabolized and unabsorbed via gastrointestinal tract). Lungs: unmetabolized nitropyrene, unconjugated acetylaminopyrenols, small quantities of polar metabolites. Liver and kidneys: polar nitropyrene metabolites.

 

Va)

HPLC analysis of organic extractable metabolites (24 h) from blood samples of female C57B1/6N mice administered [4,5,9,10-3H]1-nitropyrene by gavage (Howard et al., 1995).

 

Vb)

C57B1/6N mouse fetal homogenates. 1-Aminopyrene was detected in the amniotic fluid, and 0.7% of the administered dose passed the placenta (Howard et al., 1995).

 

Vc)

Neonatal homogenate from nursing mice 12 h after mothers administered 1-nitropyrene by gavage. Each neonate received about 0.1% of the administered dose (Howard et al., 1995).

 

VI)

Single oral dose of 20 mg of diesel exhaust particle sample to rats. Approximately 13% of the 1-nitropyrene present on the diesel exhaust particle sample was excreted in urine and identified as 1-nitropyrene metabolites (van Bekkum et al., 1998).

 

VII)

Metabolites present following intragastric administration of [14C]1-nitropyrene to male Wistar rats (van Bekkum et al., 1999).

c

Also called 3-hydroxy-1-nitropyrene.

1-Nitropyrene undergoes extensive metabolism both on the pyrene moiety and on the nitro function of the molecule (see Figure 9). The metabolism of 1-nitropyrene occurs 1) through cytochrome P450-mediated ring C-oxidation to epoxides, with subsequent rearrangement to nitropyrenols and conjugation or hydration to dihydrodiols, or 2) through nitroreduction in one- or two-electron steps to form 1-nitrosopyrene, N-hydroxy-1-aminopyrene or 1-aminopyrene with or without subsequent acetylation, or 3) a combination of ring oxidation and nitroreduction followed by acetylation. The complex biotransformation is reflected in the variety of metabolites present in plasma and tissue homogenate (see Figures 10–12) and in the variety of adducts formed with blood proteins (van Bekkum et al., 1999) and with DNA (El-Bayoumy et al., 1994a) (see section 6.2.5).

6.2.3.2 Identification of metabolites

The metabolites of 1-nitropyrene are highly conjugated in vivo. After an i.p. administration of [14C]1-nitropyrene to CD rats, >95% of the urinary 14C eluted with the solvent front (Ball et al., 1984a). Incubation of excreted metabolites in the urine with specific deconjugating enzymes β-glucuronidase and sulfatase reduced this (see Figure 10), but still over 50% of the radioactivity was to be detected in the solvent front, so that many metabolites have not yet been identified.

Urinary metabolites that in general have been observed in in vivo studies with rats and mice (following hydrolysis of conjugates) are 1-nitropyren-3-ol, 1-nitropyren-6-ol and 1-nitropyren-8-ol (also referred to as 3-, 6- and 8-hydroxy-1-nitropyrene), N-acetyl-1-aminopyren-3-ol, N-acetyl-1-aminopyren-6-ol and N-acetyl-1-aminopyren-8-ol (also referred to as 3-, 6- and 8-hydroxy-N-acetylaminopyrene), 1-nitropyrene-trans-dihydrodiols (mainly trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene, also called 1-nitropyrene-4,5-dihydrodiol), N-acetyl-1-aminopyrene and 1-aminopyrene, and in some studies unmetabolized 1-nitropyrene (Ball et al., 1984a; El-Bayoumy & Hecht, 1984a; Howard et al., 1985, 1995; Kinouchi et al., 1986b; van Bekkum et al., 1999). In all studies, the main metabolite identified (total up to 48 h) in urine of rats administered 1-nitropyrene was N-acetyl-1-aminopyren-6-ol, which (e.g., in the study by Ball et al., 1984a) accounted for about 20% of the total radioactivity observed in the urine and is the predominant source of mutagenic activity from this source in the urine. 1-Aminopyrene is hardly measurable in the urine but is found as the major metabolite in faeces (Ball et al., 1984a; Kinouchi et al., 1986b; see Figures 10 and 11). The major metabolites in the bile from bile-cannulated rats were 1-nitropyren-3-ol, 1-nitropyren-6/8-ol and trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene (Kinouchi et al., 1986b; Figure 11). An overview of the studies into the metabolites formed during the metabolism of 1-nitropyrene is given in Table 30.

The nature of the metabolites (biliary, urinary and faecal) changes with time. The early metabolites are predominantly primary oxidative products — phenols and dihydrodiol(s). Ball et al. (1984a) measured metabolites after i.p. injection after 4 h and found that 1-nitropyren-3-ol, 1-nitropyren-6/8-ol and trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene were the major metabolites (Figure 10). The major metabolites in plasma, liver and kidneys after 3 and 6 h were likewise 1-nitropyren-6/8-ol (van Bekkum et al., 1999; see Figure 12). At later time points, the major products (N-acetyl-1-aminopyren-6-ol, N-acetyl-1-aminopyren-8-ol and N-acetyl-1-aminopyren-3-ol) have undergone ring oxidation, nitroreduction and also N-acetylation (see Figures 9–12).

Figure 9

Fig. 9. Scheme of metabolism of 1-nitropyrene in vivo (from Howard et al., 1995).

Figure 10

Fig. 10. The metabolites of [14C]1-nitropyrene in the urine and faeces of rats at specified times after i.p. administration (Ball et al., 1984a). Deconjugating enzymes: β-glucuronidase and sulfatase; % of urinary [14C] on solvent front = 52, 72, 64, 58 and 79% for the given time points; 3-OH-NP = 1-nitropyren-3-ol; 8-OH-NP = 1-nitropyren-8-ol; 6-OH-NAAP = N-acetyl-1-aminopyren-6-ol; 8-OH-NAAP = N-acetyl-1-aminopyren-8-ol; 3-OH-NAAP = N-acetyl-1-aminopyren-3-ol; NAAP = N-acetyl-1-aminopyrene.

Figure 11

Fig. 11. Metabolites excreted from rats at given times after oral exposure to [łH]1-nitropyrene (percentage of diethylether-soluble material after β-glucuronidase treatment) (Kinouchi et al., 1986b). 1-NP = 1-nitropyrene; 3-OH-NP = 1-nitropyren-3-ol; 6/8-OH-NP = 1-nitropyren-6/8-ol; 1-NP-4,5-DHD = trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene; 1-AP = 1-aminopyrene; 6-OH-NAAP = N-acetyl-1-aminopyren-6-ol; 8-OH-NAAP = N-acetyl-1-aminopyren-8-ol; 6-OH-AP = 1-aminopyren-6-ol; 8-OH-AP = 1-aminopyren-8-ol.

Figure 12

Figure 12

Fig. 12. Metabolites excreted after oral application to Wistar rats after given times; single dose of [14C]1-nitropyrene; 27.6 µCi, 750 mg/kg bw (van Bekkum et al., 1999). 1-NP = 1-nitropyrene; 3-OH-NP = 1-nitropyren-3-ol; 6/8-OH-NP = 1-nitropyren-6/8-ol; 1-NP-4,5-DHD = trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene; 1-AP = 1-aminopyrene; 6-OH-NAAP = N-acetyl-1-aminopyren-6-ol; 8-OH-NAAP = N-acetyl-1-aminopyren-8-ol.

6.2.3.3 Cytochrome P450-mediated ring C-oxidative pathway

The C-oxidative metabolism of 1-nitropyrene in different species is catalysed by different cytochrome P450 isoenzymes: in rabbit by CYP2C3 (Howard et al., 1988) and in rat by CYP2B1 and CYP2C (Silvers et al., 1994). The CYP3A subfamily (in particular CYP3A3 and CYP3A4) seems to be the enzymes involved in human metabolism of 1-nitropyrene (Howard et al., 1990; Silvers et al., 1992; Chae et al., 1999a).

Cytochrome P450 can catalyse the direct formation of three phenols (1-nitropyren-3-ol, 1-nitropyren-6-ol and 1-nitropyren-8-ol). These phenols are further conjugated to sulfate and glucuronide derivatives. However, in vitro studies showed that human liver enzymes preferentially produce metabolites with the -OH function situated at the 3- position, whereas 6- and 8- isomers are the main metabolites produced by rat liver enzymes (Howard et al., 1990; Silvers et al., 1992; Chae et al., 1999a). 1-Nitropyren-3-ol is considerably more mutagenic in Salmonella typhimurium than 1-nitropyren-6-ol and 1-nitropyren-8-ol, the mutagenicity of the phenols presumably resulting from nitroreduction to hydroxyamine derivatives (Ball et al., 1984a; Consolo et al., 1989).

Alternatively, the C-oxidation of 1-nitropyrene by cytochrome P450 can result in the formation of two arene K-region oxides, 1-nitropyrene-4,5-oxide and 1-nitropyrene-9,10-oxide. The K-region oxides are mutagenic in S. typhimurium either with or without exogeneous activating enzymes (Smith et al., 1990a; Beland, 1991). The K-region oxides can be hydrolysed by epoxide hydrolase to the corresponding K-region trans-dihydrodiols or can rearrange to form four K-region phenols (1-nitropyren-4-ol, 1-nitropyren-5-ol, 1-nitropyren-9-ol or 1-nitropyren-10-ol). Mutagenicity of K-region derivatives of 1-nitropyrene was reported (El-Bayoumy & Hecht, 1986).

Species differences were found in both activation of 1-nitropyrene to 1-nitropyrene oxides and inactivation of 1-nitropyrene oxides by epoxide hydration and glutathione conjugation in hepatic subcellular fractions. For example, 1-nitropyrene-4,5-oxide-producing activity was highest in guinea-pig and dog, followed in order by hamster, rat, human and mouse; 1-nitropyrene-9,10-oxide-producing activity was highest in hamster, followed in order by guinea-pig, rat, dog, mouse and human; glutathione conjugation of 1-nitropyrene oxides was higher in rodents than in human and dog. There was also a wide degree of interindividual variations in these activities (Kataoka et al., 1991).

6.2.3.4 Nitroreduction pathway

In S. typhimurium, 1-nitropyrene is mutagenic through nitroreduction to the corresponding nitroso derivative, then to the hydroxyamino derivative, which has been shown to form a C8-guanyl adduct (Howard et al., 1983a). This adduct is also responsible for the mutagenicity of 1-nitropyrene in Chinese hamster ovary (CHO) cells (Heflich et al., 1986a) and cultured human diploid fibroblasts (Howard et al., 1983b; Beland et al., 1986). The reductive metabolism of 1-nitropyrene by rat liver microsomes gave 1-aminopyrene as the sole metabolite (Saito et al., 1984a).

Human metabolism of 1-nitropyrene has been studied in vitro. Metabolites derived from nitroreduction (1-aminopyrene) as well as ring oxidation (trans-dihydriols and phenols) were detected in the human hepatoma cell line HepG2 treated with 1-nitropyrene (Eddy et al., 1987; Silvers et al., 1994, 1997). In contrast, human hepatic microsomal incubations of 1-nitropyrene yielded only oxidized products (Silvers et al., 1992; Chae et al., 1999a).

Studies have shown that human red blood cells may also possess the metabolic competence to reduce 1-nitropyrene (Belisario et al., 1996).

6.2.3.5 Human and rodent intestinal microflora

Metabolism of nitroPAHs by intestinal microflora has been demonstrated both in vitro and in vivo (Cerniglia & Somerville, 1995). Intestinal microflora have been shown to be important in hydrolysing conjugated metabolites (e.g., via β-glucuronidase activity) and thereby facilitating enterohepatic recirculation of aglycones as well as playing a role in nitroreduction (Morotomi et al., 1985; Cerniglia & Somerville, 1995).

Studies using germ-free animals (intestinal microflora were absent) treated with 1-nitropyrene showed that the predominant metabolites formed were primarily ring-hydroxylated derivatives of 1-nitropyrene, whereas conventional animals used both oxidative and reductive pathways to form aminopyrene and acetylated and ring-hydroxylated metabolites. Further, the excreta collected from germ-free animals were less mutagenic than those from conventional animals. Following oral administration, the radioactivity passed through the conventional rats much more rapidly than through the germ-free rats (El-Bayoumy et al., 1983, 1984a; Kinouchi et al., 1986b). In studies on the major end metabolite N-acetyl-1-aminopyren-6-ol, which is excreted mainly as its β-glucuronidase conjugate, it was shown using germ-free animals that intestinal microflora are vital not only for nitroreduction but also for the hydrolysis of glucuronides released for enterohepatic recirculation (Ball et al., 1991). Scheepers et al. (1994d; ref #2 from Bos) studied haemoglobin (Hb) adduct formation after intragastric administration of 2-nitrofluorene (and 2-aminofluorene) in germ-free rats compared with rats equipped with a bacterial flora derived from human or rat faeces. After administration of 2-nitrofluorene, no Hb adducts could be detected in germ-free rats. Rats with a rat or human microflora showed low levels of Hb adducts, less than 1% of the Hb adduct levels obtained after administration of the same dose of 2-aminofluorene.

These results, together with those on pure and mixed cultures of intestinal flora from humans and rodents (see Cerniglia & Somerville, 1995), which reduced 1-nitropyrene to 1-aminopyrene, indicate the important role of intestinal flora in the bioactivation of 1-nitropyrene (as a model for nitroPAHs in general).

However, the intestinal microflora are not separate from the host metabolic processes, and the synergistic metabolic interactions between enzymes of the gut mucosa, hepatic tissue and microflora are important for the metabolic activation of nitroPAHs. The extent of nitroreduction of nitroPAHs to the corresponding aryl amines by cultures of intestinal microflora depends upon the geometric structure of the nitroPAH, the position of the nitro substituent, the source of intestinal microflora and diet. Generally, the nitroreductive capacities of mixed populations of intestinal microflora are greater than those observed in pure culture (Cerniglia & Somerville, 1995).

It has been suggested that in vivo, intestinal microflora are involved in the biotransformation of 1-nitropyrene to 1-aminopyrene (El-Bayoumy et al., 1983; Howard et al., 1983c; Ayres et al., 1985). The upper intestinal tract has, however, been shown to contain a low capacity of nitroreductase activity compared with the lower intestinal tract (Kinouchi et al., 1993). Data from a recent study in rats after intragastric administration of 1-nitropyrene (van Bekkum et al., 1999) imply that 1-nitropyrene is hardly reduced by the intestinal microflora in the rat prior to absorption from the upper intestinal tract and transportation to the liver. Ring hydroxylation in the liver initially seems to be the predominant route of biotransformation, whereas nitroreduction in the liver occurs to a small extent. v Nitroreduction seems to play a significant role in the enterohepatic recirculation only following biliary excretion and deconjugation, explaining the presence of reduced and acetylated metabolites in plasma, urine and tissue homogenates at later time points (van Bekkum et al., 1999).

6.2.3.6 Suggested metabolic pathway

From the profile of the metabolites, it seems that the main metabolic pathway for 1-nitropyrene is absorption, hydroxylation of the aromatic moiety and conjugation in the liver. After passing via the bile to the intestine, it is deconjugated, reduced (intestinal microflora) and reabsorbed. N-Acetylation of amino derivatives of 1-nitropyrene in vivo appears to occur primarily in the liver (Kinouchi et al., 1986b). Excretion of metabolites is via urine, but the unabsorbed metabolites are reduced to 1-aminopyrene and excreted in the faeces.

6.2.4 Elimination and excretion

6.2.4.1 Elimination

After oral or i.p. administration of [4,5,9,10-3H]1-nitropyrene to female C57B1/6N mice, rapid distribution and elimination of the 1-nitropyrene and its metabolites were observed. In both routes, the radioactivity in the serum rose to maximum levels between 6 and 12 h. The elimination kinetics was described as a two-component curve with half-lives for the rapid component and for the slower phase, respectively, of 0.5 and 3 days for the i.p. route and 0.3 and 1.8 days for animals treated orally (Howard et al., 1995).

Radioactivity (14C) was cleared rapidly from all tissues of male F344/Crl rats in a biphasic manner after inhalation exposure to 50 and 490 ng [14C]1-nitropyrene/litre (0.05 and 0.49 mg/m3) air or [14C]1-nitropyrene (650 ng/litre [0.65 mg/m3] air) coated on diesel exhaust particles. The short-term half-life of 14C in the lungs, liver and kidneys was 1, 3 and 0.5 h, respectively, and the long-term half-life was 40, 35 and 120 h, respectively (Bond et al., 1986).

1-Nitro[4,5,9,10-14C]pyrene, alone, co-administered with or vapour-coated onto aerosolized diesel particles, was administered to Sprague-Dawley rats by intratracheal instillation (Ball et al., 1986). After 24 h, the pattern of excretion was similar in all three regimens, the majority of the radioactivity being in the gastrointestinal tract. The pure compound resulted in the most extensive clearance of 14C from the lung and faster movement of 14C along the gut. The urines analysed by HPLC were similar to each other (and to urines from rats injected i.p.) in terms of the distribution of metabolites.

In a nose-only inhalation study, the elimination half-life of 1-nitropyrene in the lungs was about 1 h for rats exposed to 8 mg/m3 and 6 h for rats exposed to 50 mg/m3 (NTP, 1996). Lung burdens of 1-nitropyrene in rats exposed to 8 mg/m3 remained the same for the 13-week duration; however, lung burdens in rats exposed to 50 mg/m3 increased with time, indicating that the rats were unable to clear the 1-nitropyrene between exposures. The half-life of 1-nitropyrene in the plasma of rats was about 1 h (NTP, 1996).

6.2.4.2 Excretion

Following intragastric and i.p. administration and following inhalation of 1-nitropyrene or 1-nitropyrene coated on diesel exhaust particles, the majority (50–60%) of the administered dose has been shown to be excreted in the faeces, whereas urine contained about 15–20% of the dose (see Table 31).

Table 31. Excretion pattern for 1-nitropyrene in vivo

Route of administration

Radiolabel

Dose

Rats

Time
(h)

% of dose in faeces

% of dose in urine

% of dose in bile

Reference

Oral

3H

n.g.

Female Fischer-344

96

46a

18a

 

Dutcher & Sun (1983)

i.p.

14C

10 mg/kg bw

Female Fischer-344

24
120

15
20

30
60

 

Dutcher et al. (1985)

Oral

14C

10 mg/kg bw

Female Fischer-344

48

35

55

 

Dutcher et al. (1985)

i.p.

14C

10 mg/kg bw

Not given

24

40

15

 

Ball et al. (1984a)

i.p.

14C

6 µmol

Male Sprague-Dawley

24

40–60

20–30

 

Ball & King (1985)

Oral; coated on diesel particles

14C

380 µg/g; 5 mg/rat

Sprague-Dawley

24

40–60

20–30

 

Ball & King (1985)

Intratracheal instillation; coated on diesel particles

14C

380 µg/g; 5 mg/rat

 

24

40–60

20–30

 

Ball & King (1985)

Gavage

14C

100 mg/kg bw

Male F344

48
120

41
51

16
19

 

El-Bayoumy & Hecht (1984a)

Gavageb

14C

100 mg/kg bw

Male F344b

72

 

6

37

Oral

14C

10 µg/kg bw

Male F344/Crl

c

     

Bond et al. (1986)

i.v.

14C

10 µg/kg bw

 

c

     

Oral; coated on diesel particles

14C

10 µg/kg bw

 

c

     

Inhalation (nose only)

14C

70–1000 ng/litre

 

c

     

Inhalation; coated on diesel particles

14C

50–1100 ng/litre

 

c

     
                 

Oral (gavage)

3H

30 µmol (700 µg)

Male Wistar

24
48

52
62

18
22

 

Kinouchi et al. (1986b)

Oral (gavage)

3H

30 µmol (700 µg)

Male Wistar (germ-free)

24
48

9
45

4
8

 

i.v.

3H

0.3, 1.2 µmol (71, 286 µg)

Male F344

24

3, 12

17, 7

 

Medinsky et al. (1985)

i.v.b

3H

0.3, 1.2 µmol

Male F344b

24

 

8

80, 60

Intragastric

14C

750 mg/kg bw

Male Cpb:WU

 

50–60

15–20

 

van Bekkum et al. (1999)

i.p.

3H

24 mg/kg bw

Female CD

24

3.2

6.5

 

Chae et al. (1997)

a

Greater than 36% was converted into the volatile form, presumably 3H2O.

b

Bile duct-cannulated rats.

c

Excretion in faeces was the dominant route of elimination. In general, quantities of 14C excreted were approximately 1.5–2 times greater than those measured in urine. Elimination half-lives for 14C in urine for different concentrations and different exposure modes ranged from 13 to 20 h. For faeces, elimination half-lives for 14C ranged from 15 to 21 h (Bond et al., 1986).

Elimination half-lives for 14C in urine for different concentrations and different exposure modes ranged from 13 to 20 h. For faeces, elimination half-lives for 14C ranged from 15 to 21 h (Bond et al., 1986).

6.2.4.3 Biliary excretion and enterohepatic circulation

In biliary excretion, metabolites are excreted directly from the liver into the bile and thus into the small intestine without first entering the bloodstream for excretion by the kidneys. In the small intestine, if conditions are favourable, reabsorption can occur, resulting in an enterohepatic recirculation.

In studies with 1-nitropyrene using bile duct-cannulated rats, biliary excretion accounted for 37% of an intragastric dose after 72 h in one study (El-Bayoumy & Hecht, 1984a) and for 60–80% after 24 h in an intravenous (i.v.) study (Medinsky et al., 1985) (see Table 31). It was shown using germ-free animals that intestinal microflora are vital for the hydrolysis of glucuronides, enabling enterohepatic recirculation (Ball et al., 1991). Enterohepatic recirculation following biliary excretion seems to be an important route of excretion, at least in the rat (Howard et al., 1985; Medinsky et al., 1985; Kinouchi et al., 1990).

6.2.5 Reaction with body components

Reactive metabolites have been shown to bind to macromolecules, such as DNA and blood proteins, in in vitro and in vivo studies (Howard & Beland, 1982; Howard et al., 1983a; Djuric et al., 1986b; Roy et al., 1989; Smith et al., 1990b; El-Bayoumy et al., 1994a,b,c).

6.2.5.1 Protein binding

Hb binding to 1-nitropyrene, 2-nitronaphthalene and 2-nitrofluorene administered orally to male SD rats was found to be significantly lower than Hb binding to the corresponding amines. Reactive metabolites of 1-nitropyrene bound abundantly to the plasma proteins (Suzuki et al., 1989).

Reactive metabolites of 1-nitropyrene were reported to bind to Hb of both male and female F344 rats at a level of 0.08% of the dose in a dose-related manner. 1-Nitropyrene–Hb adducts appeared to be stable and accumulated with long-term exposure. After administration of a single dose of 1-nitropyrene, the half-life for clearance of Hb-associated radioactivity was 1.6 days. It seemed that reactive metabolites of 1-nitropyrene bind to the haem rather than to the globin moiety (El-Bayoumy et al., 1994a,c).

Van Bekkum et al. (1997) developed a method for determining Hb adducts following oral administration to rats. Low levels of Hb adducts were found 24 h after administration of a single dose of 1-nitropyrene. This method was used for biomonitoring studies (see below).

Van Bekkum et al. (1999) found that with intragastric administration of [14C]1-nitropyrene, levels of radioactivity associated with plasma proteins were approximately 4 times higher than the radioactivity associated with Hb (401.0 and 84.1 pmol/g protein per µmol 1-nitropyrene per kg bw, respectively, at 24 h). There were no indications of reactive metabolites of 1-nitropyrene binding to the haem of the Hb.

6.2.5.2 DNA adducts

Aryl nitrenium ions generated by nitro reduction or K-region nitropyrene epoxides generated by ring oxidation can react with DNA, forming adducts (for review on DNA adducts from nitroPAHs, see Beland & Marques, 1994). In bacteria and in in vitro studies, the predominant DNA adduct derived from 1-nitropyrene is N-(deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP), formed via the nitroreduction pathway. In vivo experiments with rats and mice have shown the presence of dG-C8-AP and, in smaller amounts, further adducts that have not yet been identified, formed via oxidative metabolic pathways (El-Bayoumy et al., 1994a,c; see Table 32 and also Tables 33 and 34 for some other nitroPAHs).

Table 32. DNA adducts in vivo after exposure to 1-, 2- and 4-nitropyrene (or their metabolites)a

NitroPAH

Species; strain; sex

Route

Organ

N or O

Adducts identified

Reference

1-Nitropyrene

Rat; Wistar; m

i.p.

Liver

N

dG-C8-AP

Hashimoto & Shudo (1985)

1-Nitropyrene

Rat; Wistar; f

i.p.

Liver, kidney, mammary gland

N

dG-C8-AP

Stanton et al. (1985)

1-Nitropyrene

Mouse (newborn)

i.p.

Liver, lung

N

dG-C8-AP

El-Bayoumy et al. (1988a)

1-Nitropyrene

Mouse; B6C3F1; m

i.t.

Lung, liver, kidney

N

dG-C8-AP, major adduct; further unidentified adducts

Mitchell (1988a)

1-Nitropyrene

Rat; Sprague-Dawley; f

Gavage

Liver, mammary fat pads

N/O

dG-C8-AP, a minor adduct; further unidentified adducts

Roy et al. (1989)

1-Nitropyrene

Mouse; B6C3F1; m

Inhalation

Lung

N

dG-C8-AP

Bond et al. (1990)

1-Nitropyrene

Rat; CD; f
(newborn)

s.c.

Injection site

N

dG-C8-AP
N-(deoxyguanosin-8-yl)-1-amino-3-, 6- or 8-nitropyrene

Smith et al. (1990b)

Mammary gland

dG-C8-AP
N-(deoxyguanosin-8-yl)-1-amino-3-, 6- or 8-nitropyrene
unknown adduct

1-Nitropyrene

Mouse; CD-1; m (newborn)

s.c.

Liver

N

dG-C8-AP
N-(deoxyguanosin-8-yl)-1-amino-3-, 6- or 8-nitropyrene

Smith et al. (1990b)

1-Nitropyrene

Mouse; A/J; m
(8 weeks)

i.p.

Lung

N

dG-C8-AP
N-(deoxyguanosin-8-yl)-1-amino-3-, 6- or 8-nitropyrene

Smith et al. (1990b)

1-Nitropyrene

Rat; Sprague-Dawley; f

 

Mammary gland

N

dG-C8-AP
6-(deoxyguanosin-N2-yl)-1-aminopyrene
8-(deoxyguanosin-N2-yl)-1-aminopyrene

Herreno-Saenz et al. (1995)

1-Nitropyrene

Rat; Sprague-Dawley; f

Gavage

Liver

N

dG-C8-AP
(minor adduct)

El-Bayoumy et al. (1994c)

1-Nitropyrene-4,5-oxide

Mouse; ICR; m

Gavage

Lower intestinal mucosa

O

Three different adducts, unidentified

Kinouchi et al. (1993)

1-Nitropyrene-9,10-oxide

Mouse; ICR; m

Gavage

Lower intestinal mucosa

O

Three different adducts, unidentified

Kinouchi et al. (1993)

[3H]2-Nitropyrene

Rat; Sprague-Dawley; f

Gavage

Liver, kidney, mammary gland

 

Multiple adducts; minor: dG-C8-2-AP and dA-C8-2-AP

Upadhyaya et al. (1992)

[3H]2-Nitropyrene

Rat; CD; f

i.p.

Liver, mammary gland

 

No adducts found

Chae et al. (1997)

[3H]4-Nitropyrene

Rat; CD; f

i.p.

Liver, mammary gland

N

Four radioactive peaks: two unstable adducts decomposed to pyrene-4,5-dione, a tentative identification of dI adduct

Chae et al. (1997, 1999b)

a

m = male; f = female; i.p. = intraperitoneal; i.t. = intratracheal instillation; s.c. = subcutaneous; N = nitroreduction pathway; O = oxidation pathway; major adduct given in bold type; dG-C8-AP = N-(deoxyguanosin-8-yl)-1-aminopyrene; dG-C8-2-AP = N-(deoxyguanosin-8-yl)-2-aminopyrene; dA-C8-2-AP = N-(deoxyadenosin-8-yl)-2-aminopyrene.

Table 33. DNA adducts from 2-nitrofluorenea

Animal

Route

 

Organ

Main adduct

Others

Reference

Rat, Wistar; m

Oral

 

Liver

Unidentified (80%)

dG-C8-AF (15%)
dG-C8-AAF (4%)
dG-N2-AAF (2%)

Mulder et al. (1990)

Rat, Wistar; m

Oral

 

Liver

Unidentified

dG-C8-AF

Wierckx et al. (1991)

Rat, Wistar; m

i.v.

With catheterized bile ducts

 

Same unidentified as above

 

Wierckx et al. (1991)

Rat, Wistar; m

i.p.

 

Liver

dG-C8-AF

dG-C8-AAF

Möller et al. (1993b)

Rat, Wistar; m

Gavage

 

Liver

dG-C8-AF

dG-C8-AAF

Möller & Zeisig (1993)

Rat, AGUS; f

Gavage

 

Liver (kidney, lung, heart)

dG-C8-AF (95%)

 

Möller et al. (1994)

Rat, AGUS; f

Gavage

Germ-free

Liver

Unidentified

 

Möller et al. (1994)

Rat, Wistar; f

Gavage

Germ-free

Liver

Unidentified

dG-C8-AF

Scheepers et al. (1994c)

Rat, Wistar; f

Gavage

Germ-free + rat microflora

Liver

Unidentified

dG-C8-AF

Scheepers et al. (1994c)

Rat, Wistar; m

Diet

 

Forestomach (liver, kidney)

dG-C8-AF

2–4 unidentified

Cui et al. (1995)

Rat, Wistar; m

Diet

 

Forestomach, liver, kidney (spleen)

dG-C8-AF, dG-C8-AAF, 2 unidentified

 

Cui et al. (1999)

a

m = male; f = female; i.v. = intravenous; i.p. = intraperitoneal; dG-C8-AF = N-(deoxyguanosin-8-yl)-2-aminofluorene; dG-C8-AAF = N-(deoxyguanosin-8-yl)-2-acetylaminofluorene; dG-N2-AAF = C3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene.

Table 34. DNA adducts of nitroPAHs other than 1-nitropyrene and 2-nitrofluorenea

NitroPAH

Animal

Route

Organ

N or O

Major DNA adducts

Reference

2-Nitrofluoranthene

Neonatal B6C3F1 mice

   

N

dG-C8-2-aminofluoranthene

Herreno-Saenz et al. (1994)

3-Nitrofluoranthene

In vitro

   

N

dG-C8-3-aminofluoranthene

Dietrich et al. (1988)

1-Nitrobenzo[a]pyrene

In vitro

   

O

dG-1-nitroBaP trans-7,8-diol anti-9,10-epoxide

Fu et al. (1997)

1-Nitrobenzo[a]pyrene

In vitro

 

 

N

6-dG-N2-1-aminoBaP

Fu et al. (1997)

3-Nitrobenzo[a]pyrene

In vitro

   

O

dG-3-nitroBaP trans-7,8-diol anti-9,10-epoxide

 

3-Nitrobenzo[a]pyrene

In vitro

   

N

6-dG-N2-3-aminoBaP

Herreno-Saenz et al. (1993)

1,6-Dinitropyrene

Male CD-1 preweanling mice

i.p.

Liver

N

dG-C8-1-amino-6-NP

Delclos et al. (1987b)

1,6-Dinitropyrene

Male Sprague-Dawley rats

i.p.

Bladder, liver, mammary gland, kidney, lung

N

dG-C8-1-amino-6-NP

Djuric et al. (1988)

1,6-Dinitropyrene

Rats

 

Lung

N

dG-C8-1-amino-6-NP

Smith et al. (1993); Beland et al. (1994)

1,6-Dinitropyrene

Male F344 rats

 

Lung, liver

N

dG-C8-1-amino-6-NP

Beland et al. (1994)

1,6-Dinitropyrene

Male B6C3F1 mice

 

Liver

N

dG-C8-1-amino-6-NP

Howard & Beland (1994)

1,8-Dinitropyrene

Rat

 

Mesentery, mammary glands

N

dG-C8-1-amino-8-NP

Heflich et al. (1986b)

6-Nitrochrysene

In vitro and in bacterial systems

   

N

N-(dG-8-yl)-6-aminochrysene; N-(dI-8-yl)-6-aminochrysene; 5-(dG-N2-yl)-6-aminochrysene

Delclos et al. (1987b)

6-Nitrochrysene

Preweanling CD-1 mice

i.p.

Lung

O/N

dG with 1,2-DHD-6-aminochrysene-3,4-epoxide

Delclos et al. (1988); Li et al. (1993, 1994)

6-Nitrochrysene

Female CD rat

i.p.

Lung, liver, mammary gland and colon

O/N

dG with 1,2-DHD-6-aminochrysene-3,4-epoxide

Chae et al. (1996)

N

5-(dG-N2-yl)-6-aminochrysene

a

i.p. = intraperitoneal; N = nitroreductive pathway; O = oxidative pathway; O/N = oxidative or reductive; dG-C8-2-aminofluoranthene = N-(deoxyguanosin-8-yl)-2-aminofluoranthene; dG-C8-3-aminofluoranthene = N-(deoxyguanosin-8-yl)-3-aminofluoranthene; dG-1-nitroBaP trans-7,8-diol anti-9,10-epoxide = deoxyguanosine-1-nitrobenzo[a]pyrene trans-7,8-diol anti-9,10-epoxide; dG-3-nitroBaP trans-7,8-diol anti-9,10-epoxide = deoxyguanosine-3-nitrobenzo[a]pyrene trans-7,8-diol anti-9,10-epoxide; 6-dG-N2-1-aminoBaP = 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene; 6-dG-N2-3-aminoBaP = 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene; dG-C8-1-amino-6-NP = N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene; dG-C8-1-amino-8-NP = N-(deoxyguanosin-8-yl)-1-amino-8-nitropyrene; N-(dG-8-yl)-6-aminochrysene = N-(deoxyguanosin-8-yl)-6-aminochrysene; N-(dI-8-yl)-6-aminochrysene = N-(deoxyinosin-8-yl)-6-aminochrysene; 5-(dG-N2-yl)-6-aminochrysene = 5-(deoxyguanosin-N2-yl)-6-aminochrysene; 1,2-DHD-6-aminochrysene-3,4-epoxide = trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene-3,4-epoxide.

1) Reduction pathway of 1-nitropyrene and formation of dG-C8-AP in in vitro studies

Early in vitro studies, such as the detection of reduced metabolites (e.g., 1-aminopyrene and N-acetyl-1-aminopyrene; Messier et al., 1981) and decreased mutagenic activity in nitroreductase-deficient strains of S. typhimurium (Mermelstein et al., 1981; Rosenkranz et al., 1981), showed that the reduction of 1-nitropyrene to N-hydroxy-1-aminopyrene was associated with the induction of mutations. N-Hydroxy-1-aminopyrene has been shown to undergo an acid-catalysed reaction with DNA to yield the C8-substituted dG adduct dG-C8-AP (see Figure 13) (Howard et al., 1983a). This adduct is the predominant product formed from 1-nitropyrene and from 1-nitrosopyrene, its reduced derivative, in S. typhimurium TA1538 (Howard et al., 1983a; Heflich et al., 1985a,b), CHO cells (Heflich et al., 1985a, 1986a; Thornton-Manning et al., 1991a,b), Chinese hamster lung fibroblasts (Edwards et al., 1986a) and human diploid fibroblasts (Beland et al., 1986; Patton et al., 1986).

Evidence that dG-C8-AP is probably the premutagenic lesion comes from the observation that mutations are induced primarily at G:C base pairs in the lambda cI gene of an Escherichia coli uvr lysogen (Stanton et al., 1988), in pBR322 introduced into E. coli (Melchior et al., 1990), in pZ189 replicating in human embryonic kidney cells (Yang et al., 1988; Maher et al., 1990) and in the hprt gene of CHO cells (Newton et al., 1992). dG-C8-AP causes a –2 deletion of a G:C or C:G pair within a CGCGCGCG hot-spot sequence upstream of the hisD3052 mutation in S. typhimurium strain TA98 (Bell et al., 1991; Malia et al., 1996). It could be shown in a comparative study using S. typhimurium strain TA98NR (which does not produce a nitroreductase), strain TA98 and strain YG1021 (which contains a plasmid carrying the nitroreductase-encoding gene) that nitroreductase activity, DNA adduct level and mutagenicity were strongly correlated with each other and that the higher the adduct level, the higher the level of mutagenicity (Arimochi et al., 1998).

Ring oxidation, which is a major metabolic pathway for 1-nitropyrene in vivo, results in the formation of metabolites such as phenols, dihydrodiols and K-region epoxides, which are more mutagenic than 1-nitropyrene (El-Bayoumy & Hecht, 1986). In vitro studies reacting calf thymus DNA with 1-nitropyrene-4,5-epoxide yielded three major adducts, identified as diastereomers, two with a trans configuration at the C4–C5 bond and one cis, resulting from the addition of the exocyclic N2 of dG to the C5 benzylic position of the epoxide (Roy et al., 1991; see Figure 13). These were probably the same adducts as those described in a similar experiment (Smith et al., 1990b). DNA adducts with characteristics of ring oxidation have also been detected in CHO cells incubated with 1-nitropyrene in the presence of an exogeneous microsomal preparation from liver (S9) (Thornton-Manning et al., 1991a).

The role of nitroreduction and C-oxidation in DNA adduct formation was investigated in HepG2 cells (Silvers et al., 1997). Cytochrome P450s were induced with differing concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). As the P450s were induced, the metabolism of 1-nitropyrene in the HepG2 switched from nitroreduction (i.e., formation of 1-aminopyrene) to C-oxidation (i.e., formation of pyrenols). The metabolic switch from nitroreduction to C-oxidation was accompanied by a decrease in DNA adduct formation, suggesting that generation of C-oxidized metabolites in HepG2 does not result in DNA adduct formation.

2) DNA adducts in in vivo studies

Metabolites indicative of ring oxidation and nitroreduction have also been detected in vivo in experimental animals. In mammalian systems, nitroreductase enzymes typically exhibit low levels of activity (Peterson et al., 1979). dG-C8-AP has also been detected in vivo in mice and rats treated with 1-nitropyrene or 1-nitrosopyrene (see Table 32), but it represents only a small proportion of the total molecular binding. Additional adducts were found in most cases, but none of these has been identified fully. For example, Kinouchi et al. (1992) studied DNA formation in the liver of B6C3F1 mice after administration of 1-nitropyrene and found that the major adduct was not dG-C8-AP; however, the major 32P-labelled spots migrated to the same position as the in vitro DNA adduct spots of K-region epoxides of 1-nitropyrene. In a separate investigation, similar findings were reported (El-Bayoumy et al., 1994a,c).

Further activation of oxidized metabolites of 1-nitropyrene has been suggested (Ball et al., 1996). 1-Nitropyren-6-ol can be activated to K-region epoxidation. 1-Acetamidopyren-6-ol undergoes several steps, possibly including both deacetylation and N-oxidation, yielding an arylnitrenium ion, as well as K-region oxidation, yielding an epoxide as active intermediate. DNA adducts derived from these activated compounds may be formed in tissues susceptible to 1-nitropyrene.

6.2.6 Biomonitoring studies

For approaches to biomonitoring of persons exposed to nitroPAHs, in particular 1-nitropyrene as a marker for diesel exhaust exposure, see chapter 8.

6.3 Mononitropyrenes (1-, 2- and 4-nitropyrene) — a comparison

4-Nitropyrene seems to differ from 1- and 2-nitropyrene concerning elimination and excretion patterns, metabolites and DNA adducts (see below). This may account for 4-nitropyrene being the most carcinogenic of the nitropyrenes in rat mammary gland.

6.3.1 Faecal and urinary excretion

The data on faecal and urinary excretion of 2- and 4-nitropyrene after intragastric gavage suggest differences between the excretion patterns of 4-nitropyrene compared with 1- and 2-nitropyrene (Upadhyaya et al., 1992, 1994; see Table 35). The results of comparative studies via the i.p. route are inconclusive owing to the low excretion rate reported (Chae et al., 1997).

Table 35. Excretion pattern for 2- and 4-nitropyrene compared with that of 1-nitropyrene

NitroPAH

Route

Radio-label

Rats

Time (h)

Faeces (% of dose)

Urine (% of dose)

Main metabolites in urine and faeces

Reference

1-Nitropyrene

All

   

24

50–60

15–20

See Table 30

See Table 30

2-Nitropyrene

Intragastric

14C

Male F344

48

60

10

N-Acetyl-2-aminopyren-6-ol

Upadhyaya et al. (1992)

4-Nitropyrene

Intragastric

3H

Female Sprague-Dawley

48 and
168

30

40

4-Aminopyrene, N-acetyl-4-aminopyren-9(10)-ol + many unknown metabolites

Upadhyaya et al. (1994)

1-Nitropyrene

i.p.

3H

Female CD

24

3.2

6.5

 

Chae et al. (1997)

2-Nitropyrene

i.p.

3H

Female CD

24

2.2

6.2

 

Chae et al. (1997)

4-Nitropyrene

i.p.

3H

Female CD

24

0.8

2.1

 

Chae et al. (1997)

6.3.2 Metabolism

Analogous to 1-nitropyrene, N-acetyl-2-aminopyren-6-ol was the main metabolite detected in urine and faeces (2% and 20% of the dose, respectively) after intragastric gavage of 2-nitropyrene in rats (Upadhyaya et al., 1992). In contrast, after 4-nitropyrene administration, 4-aminopyrene and N-acetyl-4-aminopyren-9(10)-ol were the major metabolites, but at much lower yields. There were many unknown metabolites, including possibly 4-nitropyrene-9,10-dione (Upadhyaya et al., 1994). In a comparative study on 1-, 2- and 4- nitropyrene administered i.p., metabolites derived from nitroreduction and ring oxidation pathways (acetylaminopyrenes and their phenolic derivatives) were found in all three cases, although a quantitative analysis was not possible as a result of the low levels detected (Chae et al., 1997).

A comparative study on 1-, 2- and 4-nitropyrene in human hepatic and pulmonary microsomes showed some differences in their metabolism (Chae et al., 1999a). Human hepatic samples were competent in metabolizing 1-, 2- and 4-nitropyrene. With human pulmonary microsomal samples, similar patterns were obtained, but at much lower levels. Ring-oxidized metabolites (phenols and trans-dihydrodiols) were produced from all three isomers. However, the reductive metabolism leading to the formation of aminopyrene was evident only with 4-nitropyrene, which is the most potent carcinogen of the three.

Whereas most of the hepatic microsomal metabolism of 1- and 4-nitropyrene could be attributed to CYP3A4, none of the P450 enzymes tested seemed to be involved in the human hepatic microsomal metabolism of 2-nitropyrene (Chae et al., 1999a). Thus, the role of specific human P450 enzymes depends upon the position of the nitro group.

6.3.3 DNA adducts

The major DNA adduct found in S. typhimurium (Howard et al., 1983a), cells in culture (Heflich et al., 1986a; Silvers et al., 1997) and rodents treated with 1-nitropyrene (see Table 32) is dG-C8-AP. With 2-nitropyrene, both dG and, to a lesser extent, dA adducts derived from nitroreduction [N-(deoxyguanosin-8-yl)-2-aminopyrene (dG-C8-2-AP) and N-(deoxyadenosin-8-yl)-2-aminopyrene (dA-C8-2-AP)] were formed in S. typhimurium (Yu et al., 1991) and upon incubation of 2-nitropyrene with DNA in the presence of rat hepatic microsomes (Fu et al., 1991).

DNA binding with 2-nitropyrene has been studied in vivo in female rats administered a single dose by gavage (Upadhyaya et al., 1992). The two adducts, dG-C8-2-AP and dA-C8-2-AP, formed by nitroreduction were detected in liver, mammary gland and kidney DNA, but were only a small percentage of the total DNA hydrolysate products (see Table 32).

In a comparative study, after i.p. treatment with [3H]1-, 2- or 4-nitropyrene, binding to rat mammary gland DNA was 0.6, 0.3 and 2.1 pmol/mg DNA, respectively. Only 4-nitropyrene yielded multiple putative DNA adducts (Chae et al., 1997). HPLC analysis yielded four radioactive peaks, which were found to co-elute with standards derived from the nitroreduction of 4-nitropyrene. One peak was identified as pyrene-4,5-dione and was formed from decomposition of two putative DNA–4-nitropyrene adducts. Another was tentatively identified as a deoxyinosine (dI) adduct. None of the peaks co-eluted with the major DNA adducts derived from 4-nitropyrene-9,10-epoxide, a ring-oxidized metabolite, or with the adduct standard N-(deoxyguanosin-8-yl)-4-aminopyrene (dG-C8-4-AP) (Chae et al., 1999b).

6.4 2-Nitrofluorene

Although earlier studies inferred that the metabolism and excretion patterns of 2-nitrofluorene differed greatly from those of 1-nitropyrene, it seems with increasing data that there are many similarities. Whereas it was postulated that 2-nitrofluorene is metabolized in vivo via reduction to 2-aminofluorene (via reductase) in the intestine (and liver) followed by acetylation to N-acetyl-2-aminofluorene and then hydroxylation (Möller et al., 1985), this now appears to be only the case for one pathway after oral administration. Other studies show that the metabolic pathway via hydroxylated nitrofluorenes is similar to that of 1-nitropyrene (see Figure 14). Hydroxylated nitrofluorenes are more mutagenic in Salmonella than 2-nitrofluorene alone (Möller et al., 1988).

Figure 13

Fig. 13. Formation of DNA adduct from 1-nitropyrene; oxidative pathways and DNA adduct formation in vitro
(adapted from Beland & Marques, 1994).

Figure 14

Fig. 14. Metabolism of 2-nitrofluorene in vivo
(adapted from Möller et al., 1985, 1987a,b, 1994).

6.4.1 Absorption, distribution and elimination

2-Nitrofluorene is readily absorbed, distributed and excreted after administration by all routes. Data on excretion studies are given in Table 36. The data are contradictory, with some studies showing a higher percentage of the dose in urine than in faeces and vice versa. Some studies show that, similar to 1-nitropyrene, about 20–30% of the dose is excreted in the urine after 24 h. Excretion of metabolites was accompanied by an excretion of mutagenicity. (In both urine and faeces, the direct-acting mutagenicity [–S9] dominated over mutagenicity with S9 [Möller et al., 1987a, 1988, 1994]. Forty-three per cent of the total mutagenicity [and ~75% of the direct-acting mutagenicity] in the urine of rats after an oral dose of 2-nitro[9-14C]fluorene is due to free [unconjugated] metabolites: mostly N-acetylaminofluorene and some nitrofluorenols [Möller et al., 1987a].)

Table 36. Excretion pattern and metabolites for 2-nitrofluorene in vivoa

Route

Radio-label

Dose

Rats

Time (h)

Faeces
(% of dose)

Urine
(% of dose)

Bile
(% of dose)

Main metabolites in the urine and bile

Reference

Oral, intragastric

14C

Single dose, 5 mg/rat

Sprague-Dawley

24
144

2–9
31

31–35
57–60

 

7- and 5-OH-AAF; minor: 1-, 3-, 8-, 9-OH-AAF; 2-NF not observed

Möller et al. (1985, 1987a)

Oral, intragastric

14C

Single dose, 5 mg/rat

AGUS conventional rats

24
144

17
48

22
31

 

7- and 5-OH-AAF

Möller et al. (1988)

Oral, intragastric

 

Single dose, 5 mg/rat

Germ-free

24
144

8
50

18
21

 

9-OH-NF and 2-NF;
no OH-AAF metabolites

Möller et al. (1988)

Oral

14C

Not given

Male Wistar

24

Not given

20

 

OH-NF and 2-AAF

Mulder et al. (1990)

i.v.

3H and 14C

6 µmol/kg bw

Male Wistar

2

   

40

In bile, glucuronide conjugate of 9-OH-NF

Mulder et al. (1990)

i.p.

None

1 mmol/kg bw

Male Sprague-Dawley

Up to 72; maximum at 24

     

Five hydroxylated 2-nitrofluorenes

Castaneda-Acosta et al. (1997)

a

i.v. = intravenous; i.p. = intraperitoneal; 2-NF = 2-nitrofluorene; 2-AAF = 2-acetylaminofluorene; x-OH-AAFs = N-acetylaminofluoren-x-ols; x-OH-NF = nitrofluoren-x-ols.

The pharmacokinetics of 2-nitrofluorene elimination from the blood after i.v. administration was studied in rats with cannulated bile ducts. 2-Nitrofluorene was found to be removed from the blood in a biphasic manner, with t˝ values of 2.5 min and 2.5 h. Biliary excretion was likewise biphasic, with t˝ values of 9 min and 1 h. After 2 h, about 40% of the dose had been excreted in the bile. The major metabolite was the glucuronide conjugate of 2-nitrofluoren-9-ol. The great difference between the high biliary excretion of 2-nitrofluorene metabolites (40% in 2 h) and the rather low excretion of the compound and its metabolites in urine (20% in 24 h) is possibly due to reabsorption of 2-nitrofluorene or its metabolites from the gut and subsequent enterohepatic recirculation (Mulder et al., 1990).

6.4.2 Metabolism/mechanism of action

6.4.2.1 Metabolites (see also Table 36)

In contrast to former studies using oral administration (see below), the time-dependent metabolism of 2-nitrofluorene after i.p. administration to rats (Castaneda-Acosta et al., 1997) showed that N-acetylaminofluorenols, 2-acetylaminofluorene and 2-aminofluorene were not detected in the urine using HPLC or 1H-nuclear magnetic resonance (NMR); radioactive labelling was not used. After hydrolysis with β-glucuronidase/arylsulfatase, five 2-nitrofluorenols were(isolated by HPLC and identified by a combination of data from NMR, UV, and MS but not radioactive labelling) identified: trans-6,9-dihydro-6,9-dihydroxy-2-nitrofluorene [trans-6,9-dihydro-2-nitrofluoren-6,9-diol], 2-nitrofluoren-6-ol, 2-nitrofluoren-7-ol, 2-nitrofluoren-8-ol and 2-nitrofluoren-9-ol. Two conjugated metabolites were identified as 6- and 7-[(2-nitrofluoren-6-ol and 2-nitrofluoren-7-ol sulfate esters)oxy]-2-nitrofluorene. These latter conjugates were present as a mixture and were probably sulfates. They were by far the main metabolites and had a maximum at 12 h (the first time point given).

After an oral dose to rats, 2-nitrofluorene was metabolized to unconjugated hydroxy metabolites of 2-acetylaminofluorene compounds, mainly the 7- and 5-hydroxyl metabolites and, to a lesser extent, the 9-, 8-, 3- and 1-hydroxy metabolites and N-acetyl-2-amino(x)fluorenol (Möller et al., 1985). In another study, N-acetyl-2-aminofluoren-9-ol and, to a lesser extent, N-acetyl-2-aminofluoren-7-ol were observed as abundant urinary metabolites after 2-nitrofluorene intragastric administration. 2-Nitrofluorene phenols were also detected (Scheepers et al., 1994c). In contrast, in germ-free animals, 2-acetylaminofluorenols are hardly detected in urine or faeces after a single oral dose of 2-nitrofluorene; instead, 2-nitrofluorene phenols are mainly detected (Scheepers et al., 1994c).

After administration of a single oral dose of 2-nitrofluorene to germ-free and conventional rats, radioactivity with associated mutagenic activity was rapidly excreted in both urine and faeces. The mutagenicity in the excreta from germ-free animals exceeded that from conventional animals (in contrast to findings with 1-nitropyrene). Nitrofluorenols were associated with this high direct-acting mutagenicity (Möller et al., 1988). It is suggested that the microflora are responsible for reducing the nitrofluorenols (or 2-nitrofluorene). After oral administration to conventional rats, another metabolic route (similar to that for 1-nitropyrene) results in the formation of hydroxylated nitrofluorenes (2-nitrofluorenols), which could play a role in the carcinogenicity of 2-nitrofluorene, since the tumour patterns for 2-nitrofluorene differ from those for N-acetylaminofluorene (Miller et al., 1955; Cui et al., 1995; see chapter 7). 2-Nitrofluoren-7-ol and 2-nitrofluoren-9-ol (but not 2-nitrofluoren-5-ol) were shown to induce similar DNA adducts and preneoplastic liver lesions, but in smaller quantities than with 2-nitrofluorene (Cui et al., 1996).

The formation of 2-nitrofluorenols has also been shown in inhalation studies (in isolated, perfused lung and liver; Möller et al., 1987b) as well as in germ-free animals (Möller et al., 1988) and after induction of the cytochrome P450 system in vivo with β-naphthoflavone, resulting in an increase in the mutagenicity of urine and faeces in induced animals (Mφller et al., 1987a). A β-naphthoflavone-inducible microsomal enzyme, most likely CYP1A1, catalyses the hydroxylation of nitrofluorene both in the 9- position and in other positions (Törnquist et al., 1990). It seems that inhaled 2-nitrofluorene is metabolized by the lung to 2-nitrofluorenols or transported to the liver as nitrofluorene and then ring hydroxylated. The liver conjugates the 2-nitrofluorenols and excretes them as glucuronides via the bile. In the intestine, however, the 2-nitrofluorenols may be liberated by the action of β-glucuronidase (Mφller et al., 1994).

The in vivo metabolism of 2-nitrofluorene, an environmental pollutant, and 2-aminofluorene and its alkylated derivatives, 2-formylaminofluorene and N-acetyl-2-aminofluorene, was examined in rat and dog (Ueda et al., 2001a). 2-Nitrofluoren-7-ol, 2-nitrofluoren-5-ol, 2-aminofluorene, N-acetyl-2-aminofluorene, N-formyl-2-aminofluorene, 2-aminofluoren-5-ol, 2-aminofluoren-7-ol, N-acetyl-2-aminofluoren-5-ol, N-acetyl-2-aminofluoren-7-ol, N-formyl-2-aminofluoren-5-ol and N-formyl-2-aminofluoren-7-ol were identified as urinary and faecal metabolites of nitrofluorene in rat and dog. Acetylaminofluorene and its hydroxylated derivatives were detected as major metabolites of nitrofluorene in rat, but formylaminofluorene and its hydroxylated derivatives were mainly excreted in dogs.

6.4.2.2 DNA adducts

DNA adduct formation was studied in the liver, kidney, spleen and stomach after oral administration of 2-nitrofluorene in rats (Cui et al., 1999). Four major DNA adducts were induced. DNA adduct D co-migrating with N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) had previously been identified after oral administration of 2-nitrofluorene (Wierckx et al., 1991; Möller & Zeisig, 1993; Cui et al., 1995). DNA adduct C co-migrated with C3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG-N2-AAF), whereas the other two (A and B) did not co-migrate with any of the adduct standards used and could not be identified. The four DNA–2-nitrofluorene adducts showed different kinetics of formation and persistence, which may play different roles in 2-nitrofluorene-induced tumour formation (Cui et al., 1999). In the forestomach, after 10 days, there was a high level of only adduct D (dG-C8-AF), but the amount of DNA adducts after 11 months could not be analysed due to multiple tumours. In the liver, the amount of dG-C8-AF decreased with time. The unidentified adducts A and B were the major adducts after 11 months of 2-nitrofluorene feeding. All adducts, in particular A and C, persisted long after the withdrawal of 2-nitrofluorene (Cui et al., 1999).

Other studies on DNA adducts after 2-nitrofluorene administration are summarized in Table 33. Almost all studies after oral administration show the presence of unidentified DNA adducts that do not elute with known DNA adducts found after oral administration of 2-aminofluorene or N-acetyl-2-aminofluorenol (i.e., dG-C8-AF, N-(deoxyguanosin-8-yl)-2-acetylaminofluorene [dG-C8-AAF], dG-N2-AAF; e.g., Scheepers et al., 1994c). The metabolites 2-nitrofluoren-7-ol and 2-nitrofluoren-9-ol (but not 2-nitrofluoren-5-ol) were shown to induce DNA adducts that were similar to those unidentified DNA adducts after 2-nitrofluorene administration (Cui et al., 1996), suggesting that these DNA adducts are formed via an oxidative pathway. Möller et al. (1987a) had previously shown that cytochrome P450 induction dramatically increases the excretion of hydroxylated nitrofluorenes, indicating the involvement of epoxides.

Germ-free animals administered 2-nitrofluorene by gavage had lower levels of DNA adducts than conventional animals, suggesting that the microflora plays an important role in the formation of DNA adducts. Although DNA adducts were detected in the liver, they have not been identified (Möller et al., 1994; Scheepers et al., 1994c).

6.4.2.3 Haemoglobin adducts

After oral administration of 2-nitrofluorene, Hb adduct levels were low, and no Hb adducts were detected in the blood in the absence of microflora (Scheepers et al., 1994c,d).

6.5 Dinitropyrenes (1,3-, 1,6- and 1,8-dinitropyrene)

Dinitropyrenes are detected at much lower concentrations than 1-nitropyrene; however, these compounds, in particular 1,6- and 1,8-dinitropyrene, are exceedingly potent bacterial mutagens and in most studies are more tumorigenic than 1-nitropyrene (see chapter 7).

The metabolic activation of dinitropyrenes occurs by reduction of one nitro group to yield N-hydroxy-1-amino-x-nitropyrene, where x is 3, 6 or 8, depending on the original compound. These N-hydroxyarylamine intermediates can undergo acid-catalysed DNA binding or, in contrast to 1-nitropyrene, which is only N-acetylated, can be converted into highly reactive O-acetyl metabolites by bacterial (Orr et al., 1985) and mammalian (Djuric et al., 1985) transacetylases (Beland & Marques, 1994). This activation pathway has been shown to be responsible for their extreme mutagenicity in Salmonella (Fu, 1990; Beland, 1991). In rat liver cytosolic incubations, 1-nitropyrene and 1,3-dinitropyrene were reduced to a much lesser extent than 1,6- or 1,8-dinitropyrene, which suggests that there may be fundamental differences in the reduction pathways between these nitroPAHs (Djuric et al., 1986a; Fu, 1990).

Following a single dose of 1,8-dinitropyrene, only one adduct, identified as N-(deoxyguanosin-8-yl)-1-amino-8-nitropyrene (dG-C8-1-amino-8-NP), was detected in mesentery and in mammary glands of rats (Heflich et al., 1986b) (see Table 34 and Figure 15).

Figure 15

Fig. 15. Metabolic activation pathway and DNA adduct formation of 1,8-dinitropyrene (from Beland & Marques, 1994).

Similarly, a single DNA adduct, N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene (dG-C8-1-amino-6-NP), has been found in the livers of newborn mice (Delclos et al., 1987a) and in the mammary glands (Djuric et al., 1988) and lungs of rats (Smith et al., 1993) treated with 1,6-dinitropyrene. After administration by gavage, measurable DNA adducts (probably dG-C8-1-amino-6-NP) were detected only in intestinal mucosa and urinary bladder; after i.p. injection, higher levels of the DNA adduct were found, mostly in bladder, white blood cells and lung, but only a lower adduct level was found in liver (Wolff et al., 1993). In male F344 rats after direct pulmonary instillation of 1,6-dinitropyrene, the major DNA adduct in target (lung) and surrogate (liver, white blood cells and spleen lymphocytes) tissues was dG-C8-1-amino-6-NP (Beland et al., 1994). Nitroreduction as well as factors such as O-acetylation seem to be important in determining the extent of DNA binding by 1,6- and 1,8-dinitropyrene in vivo (Beland, 1989).

The relationship between induction of DNA adducts and gene mutations was analysed in F344 rats given 1,6-dinitropyrene directly to the lungs by implantation. A dose-dependent increase of adducts was found in spleen lymphocytes but not in the lung. In parallel, a significant increase of gene mutation frequency (hprt locus) was detected in spleen T-lymphocytes (Smith et al., 1993). Similar studies (Beland et al., 1994; Beland, 1995) compared the adducts in lung, liver and lymphocytes and gene mutations in spleen T-lymphocytes. These findings indicate that concentrations of 1,6-dinitropyrene that produce a dose-dependent induction of lung tumours also result in a dose-dependent formation of DNA adducts and induction of lymphocyte mutations, but that the dose–response curves for DNA binding and mutations are different. These results suggest that T-lymphocyte mutations may be a more sensitive and longer-lived biomarker than DNA adducts for assessing previous exposures to nitroPAHs.

Sequencing of DNA amplification products from 20 1,6-dinitropyrene-induced lung tumours identified five mutations in K-ras codon 12 (four GGT to TGT transversions and one GGT to GAT transition), but not K-ras codons 13 or 61; and mutations in p53 exons 5–8 (eight substitutions at G:C base pairs and one deletion) in 9 of 20 tumour samples. The mutations identified in the dinitropyrene-induced lung tumours and TGr T-lymphocytes are consistent with the formation of dG adducts by 1,6-dinitropyrene (Smith et al., 1997).

The i.p. administration of 100 nmol of 1,6-dinitropyrene to B6C3F1 mice resulted in the detection of 0.46 ± 0.05 fmol of DNA adducts per microgram of DNA. The co-administration of a 25-fold molar excess of 1-nitropyrene (but not 2.5-fold) increased the 1,6-dinitropyrene DNA adduct level to 0.59 ± 0.07 fmol/µg DNA. Conversely, co-administration of 25-fold molar excess of pyrene resulted in a significant decrease in 1,6-dinitropyrene DNA adducts to 0.34 ± 0.04 fmol/µg DNA. While there has been no follow-up on these observations, they do suggest that the metabolic activation of 1,6-dinitropyrene, when existing as part of a complex mixture (e.g., inhaled pollution or particulates), might be affected by the more highly abundant mononitroPAHs or PAHs (see also section 7.5.5 on complex mixtures).

6.6 Mononitrobenzo[a]pyrenes (1-, 3- and 6-nitrobenzo[a]pyrene)

The orientation of the nitro group may be the reason for the differences in mutagenicity and tumorigenicity of the mononitro derivatives of the potent carcinogenic BaP (see chapter 7). While 1- and 3-nitrobenzo[a]pyrene have their nitro group preferentially adopting a parallel (coplanar) orientation, 6-nitrobenzo[a]pyrene has its nitro group in a perpendicular orientation.

Very few data were found on in vivo studies on excretion patterns or metabolites after application of nitrobenzo[a]pyrene. Most studies available are in vitro studies.

As a comparison, BaP requires metabolic activation and is metabolized by a number of oxidative pathways; it is also activated via "bay-region" vicinal diol epoxides (e.g., 7,8-dihydrodiol-9,10-epoxide) to form DNA adducts (IPCS, 1998).

Isomeric nitrobenzo[a]pyrenes are activated to DNA-damaging and mutagenic derivatives by nitroreduction, ring oxidation or a combination of these two pathways. Comparison of metabolic patterns between BaP and 1-, 3- and 6-nitrobenzo[a]pyrene indicates that nitro orientation may affect the regioselectivity of the cytochrome P450 isozymes (Wang et al., 1988).

For 2-nitrobenzo[a]pyrene, the principal metabolic activation pathway using mouse or rat liver microsomes is similar to that of BaP, via ring oxidation to the corresponding 7,8- and 9,10-dihydrodiols. 2- Nitrobenzo[a]pyrene and both of its dihydrodiols are potent mutagens with and without S9 activation. 2-Nitrobenzo[a]pyrene, however, showed higher mutagenic activity in the presence of S9 than in the absence of S9. Collectively, these results suggest that ring oxidation of 2-nitrobenzo[a]pyrene to the bay-region diol epoxide, 2-nitrobenzo[a]pyrene trans-7,8-dihydro-9,10-epoxide, is the principal route of metabolic activation (Von Tungeln et al., 1994a).

Incubation of 3-nitrobenzo[a]pyrene with rat liver microsomes under aerobic conditions yielded 3-nitrobenzo[a]pyrene trans-7,8-dihydrodiol and 3-nitrobenzo[a]pyrene trans-9,10-dihydrodiol as major metabolites, which give 3-nitrobenzo[a]pyrene trans-7,8-diol anti-9,10-epoxide. In contrast, metabolism of 3-nitrobenzo[a]pyrene under hypoxic conditions yielded 3-aminobenzo[a]pyrene, presumably via 3-nitroso- and N-hydroxy-3-aminobenzo[a]pyrene (Chou et al., 1985; Wang et al., 1988). Analogous metabolites were found with 1-nitrobenzo[a]pyrene (Chou et al., 1986).

The S9-mediated mutagenicity of 1- and 3-nitrobenzo[a]pyrene seems to result from binding of 1- and 3-nitrobenzo[a]pyrene trans-7,8-diol anti-9,10-epoxide metabolites to DNA and the subsequent formation of 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene (dG-1-nitroBaP-DE) and 10-(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]pyrene (dG-3-nitroBaP-DE), respectively. In contrast, the direct-acting mutagenicity of 1- and 3-nitrobenzo[a]pyrene is due to the reaction of N-hydroxyamino derivatives with DNA, which produces the corresponding 6-(deoxyguanosin-N2-yl)-1-aminobenzo[a]pyrene (6-dG-N2-1-amino-BaP) and 6-(deoxyguanosin-N2-yl)-3-aminobenzo[a]pyrene (6-dG-N2-3-amino-BaP) adducts (Fu et al., 1997; Fu & Herreno-Saenz, 1999) (see Table 34).

The aerobic metabolism of 6-nitrobenzo[a]pyrene by rat liver microsomes generated 6-nitrobenzo[a]pyren-3-ol as the major metabolite and 6-nitrobenzo[a]pyren-6-ol and quinones as minor products (Fu et al., 1982).

6.7 The nitrofluoranthene family

Whereas 3- and 8-nitrofluoranthene and 3,7- and 3,9-dinitrofluoranthene have been detected in diesel exhaust, 2-nitrofluoranthene has been found to be one of the most abundant nitroPAHs in ambient particulate matter in most locations not associated with traffic pollution (see chapter 5). It is formed from the parent fluoranthene by gas-phase hydroxyl radical- or nitrate radical-initiated reactions (see chapter 3).

The oxidative metabolism of 3-nitrofluoranthene by liver microsomes and cytosols of several mammalian species has been reported to yield various phenolic derivatives, the major metabolites from microsomes isolated from Sprague-Dawley rat and C57B16 mouse being 3-nitrofluoranthen-8-ol and 3-nitrofluoranthen-9-ol (Howard et al., 1988). After i.p. administration of [14C]3-nitrofluoranthene, urine was found to contain 15–20% and faeces about 30% of the dose eliminated within the first 24 h after dosing. Analysis of the metabolic fractions after hydrolysis with β-glucuronidase and sulfatase indicated that positions 4, 8 and 9 were major sites of oxidation. Four major metabolites were tentatively identified as 3-acetamidofluoranthen-1-ol, 3-aminofluoranthene-4,9-quinone and 4-hydroxyfluoranthene-3,8-quinone (from the oxidation of 3-aminofluoranthen-4,9-diol and 3-aminofluoranthen-4-ol-8,9-quinone) and 3-aminofluoranthen-4-ol-8,9-quinone (Gold et al., 1996).

Studies using rat lung cytosol suggest that the mononitrofluoranthenes can be metabolized in the lung by both nitroreductive and oxidative pathways. The absence of any significant oxidative metabolism of 3,9-dinitrofluoranthene suggests that the major pathway for its activation may be nitroreduction followed by O-acetylation. Nitroreduction of 3,9-dinitrofluoranthene occurred at rates twice that of 3-nitrofluoranthene, while both 8- and 2-nitrofluoranthene were metabolized more slowly (Mitchell et al., 1993).

Rat hepatic enzymes catalyse both reductive and oxidative nitrofluoranthene metabolism in vitro. Under aerobic conditions, hydroxylation of the aromatic ring is the main pathway, whereas in the absence of oxygen, only reduction of the nitro group occurs. However, it seems that the isomeric position of the nitro group and the coplanar or perpendicular conformation of the nitro group with respect to the plane of aromatic rings have an influence on the biological activity of the nitrofluoranthenes. The isomers 1-, 7- and 8-nitrofluoranthene were better substrates of rat hepatic microsomal hydrolases than 3-nitrofluoranthene, which, along with 1-nitrofluoranthene, was the preferred substrate for microsomal and cytosolic rat liver nitroreductases. Further, 1- and 3-nitrofluoranthene isomer oxidative metabolism was mediated to a larger extent by phenobarbital-induced rat liver microsomes, whereas 7- and 8-nitrofluoranthene isomer activation was greater in liver microsomes from 3-methylcholanthrene-induced rats (Belisario et al., 1990).

Results from studies of 2-nitrofluoranthene with S. typhimurium TA98 suspension cultures and from neonatal mice administered 2-nitrofluoranthene suggest that 2-nitrofluoranthene is metabolically activated to N-hydroxy-2-aminofluoranthene via nitroreduction, and the resulting DNA adduct [N-(deoxyguanosin-8-yl)-2-aminofluoranthene] is responsible, at least in part, for the mutagenic properties of this compound (Herreno-Saenz et al., 1992, 1994). The major adduct formed in vitro from 3-nitrofluoranthene in the presence of xanthine oxidase and DNA is similarly N-(deoxyguanosin-8-yl)-3-aminofluoranthene. No evidence for other adducts was found (Dietrich et al., 1988; see Table 34).

6.8 2- and 9-nitroanthracene

2-Nitroanthracene contains a nitro group that is coplanar or nearly coplanar with the aromatic ring, whereas 9-nitroanthracene has a nitro group that is perpendicular or nearly so to the aromatic moiety, so that steric interactions with adjacent protons are minimized. This difference in structure is hypothesized to be the basis for differences in metabolism and biological activity of the two nitroPAHs (Fu, 1990).

2-Nitroanthracene was reduced to 2-aminoanthracene in vitro under hypoxic conditions (3% oxygen), but the 9- isomer was not nitro reduced, even with a lower oxygen concentration (0.2%). Aerobic metabolism of 2-nitroanthracene by liver microsomes of rats (after induction with 3-methylcholanthrene) produced 2-nitroanthracene trans-5,6-dihydrodiol and 2-nitroanthracene trans-7,8-dihydrodiol, which were further metabolized mainly to 2-nitroanthracene 5,6,7,8-tetrahydro-trans-diols (dihydrodiol ketones). Under similar conditions, aerobic metabolism of 9-nitroanthracene via 9-nitroanthracene trans-3,4-dihydrodiol yielded 9-nitroanthracene 1,2,3,4-tetrahydrotetrol as the principal metabolite (Fu et al., 1985a, 1986).

6.9 6-Nitrochrysene

Although 6-nitrochrysene is present in the environment in rather low levels, research into this nitroPAH has been based on its remarkable carcinogenic activity in the newborn mouse lung adenoma assay, being the most potent nitroPAH tested in this model (see chapter 7). Figure 16 describes some of the main pathways of 6-nitrochrysene metabolism.

Figure 16

Fig. 16. Metabolic activation pathways and DNA adducts of 6-nitrochrysene (Beland & Marques, 1994).

In rats injected with [3,4,9,10-3H]6-nitrochrysene i.p., after 24 h, only 1.3% of the dose was excreted in the urine and 23% in the faeces (compare with excretion of 1-nitropyrene in Table 31). The extent of metabolism in rats was extremely limited, with 6-nitrochrysene being the major component found in the faeces after 24 h, accounting for 98% of the radioactivity. There is evidence of both reductive and aromatic ring oxidation pathways. The major faecal and urinary metabolite was 6-aminochrysene. Other metabolites observed in the faeces were trans-1,2-dihydro-1,2-dihydroxy-6-nitrochrysene and chrysene-5,6-quinone; further urinary metabolites were trans-1,2-dihydro-1,2-dihydroxy-6-nitrochrysene and trans-9,10-dihydro-9,10-dihydroxy-6-nitrochrysene (Chae et al., 1996). Oral administration of 6-nitrochrysene to female CD rats was also performed, and similar results were obtained (Boyiri et al., 2000). Metabolites detected in extracts from whole animals (mice) after dosing with [3,4,9,10-3H]6-nitrochrysene i.p. were likewise 6-aminochrysene, trans-1,2-dihydro-1,2-dihydroxy-6-nitrochrysene and trans-9,10-dihydro-9,10-dihydroxy-6-nitrochrysene (Delclos et al., 1988).

Studies in mice and in vitro assays have indicated that 6-nitrochrysene can be activated by two major pathways (Figure 16; Li et al., 1994). The major pathway seems to proceed by a combination of ring oxidation and nitroreduction via the formation of the proximate tumorigen trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene to yield a single major DNA adduct, which has not been rigorously characterized but appears to involve the formation of trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene-3,4-epoxide with dG (Delclos et al., 1988; Li et al., 1993, 1994). This pathway seems to be the major contributor to the formation of DNA adducts in preweanling mice (Delclos et al., 1987a, 1988; El-Bayoumy et al., 1989a) and rats in vivo (Chae et al., 1996; see Table 34).

Adducts derived from simple nitroreduction of 6-nitrochrysene differ from those obtained from incubating trans-1,2-dihydro-1,2-dihydroxy-6-nitrochrysene in vitro with calf thymus DNA (Krzeminski et al., 2000).

Oxidation to trans-1,2-dihydro-1,2-dihydroxy-6-nitrochrysene is catalysed by CYP1A2 in human liver and CYP1A1 in human lung; reduction to aminochrysene is catalysed by CYP3A4 (Chae et al., 1993). Results of Chen et al. (2000) suggest that 6-nitrochrysene is an inducer of human CYP1A1, and the induction occurs at a transcriptional level in HepG2 cells.

A second pathway has been observed in isolated rat hepatocytes treated with 6-nitrochrysene, involving the formation of N-hydroxy-6-aminochrysene by simple nitroreduction, yielding three major adducts: N-(deoxyguanosin-8-yl)-6-aminochrysene, N-(deoxyinosin-8-yl)-6-aminochrysene, and 5-(deoxyguanosin-N2-yl)-6-aminochrysene (Delclos et al., 1987b). The dI adduct probably results from the oxidative deamination of the corresponding dA adduct.

6.10 K- and H-ras mutations in tumours produced by nitroPAHs

1-, 2- and 3-BaP, 1- and 3-nitrobenzo[e]pyrene, 2- and 3-nitrofluoranthene and 9-nitrodibenz[a,c]anthracene were tested for tumorigenicity in the neonatal male B6C3F1 mouse with 6-nitrochrysene as positive control (see chapter 7). K- and H-ras mutations were analysed in liver tumours of the treated mice and were found to occur mainly at the first base of K-ras codon 133, resulting in GGCright arrowCGC transversion (von Tungeln et al., 1999b). The results indicate that liver tumours from mice treated with nitroPAHs possess ras mutations typical of PAHs and their derivatives rather than H-ras mutations, which are typical for those produced by arylamines (nitroreduction pathway). This result is consistent with the theory that positive responses for nitroPAHs in the neonatal B6C3F1 mouse bioassay are due to metabolism via ring oxidation.

Sixty-four (88%) of 73 analysed lung adenomas and all 15 analysed adenocarcinomas from newborn CD-1 mice injected with 6-nitrochrysene i.p. had a K-ras mutation in codon 12, 13 or 61. All of the mutations in codons 12 and 13 involved a G:C base pair (Li et al., 1994).

6.11 Human enzymes expected to be involved in nitroPAH metabolism

The Ah receptor-regulated enzymes CYP1A+, CYP1A2 and CYP1B1 are especially important for the activation of nitroPAHs. The induction of these enzymes by smoking and other exposures will cause a high activating capacity in a fraction of exposed individuals. Many of the enzymes involved (cytochrome P450s, N-acetyltransferases, glutathione S-transferase µ1 [GSTµ1], etc.) show high interindividual variation in humans due to genetic polymorphism (for more information, see section 8.3.2.2).

7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

7.1 Acute toxicity

There are few studies on the acute toxicity of nitroPAHs (Table 37). Only studies on acute lung and liver toxicity of 1- and 2-nitronaphthalene after parenteral application have been reported in any detail.

Table 37. Acute toxicity of nitroPAHs

Substance

Species

Sex

Route

LD50 (mg/kg bw)

Referencea

1-Nitro-naphthalene

Sprague-Dawley
rats

Male

i.p.

86

Johnson et al. (1984)

2-Nitro-naphthalene

Swiss-Webster mice

Male

Oral

1300

Simmon et al. (1979)

2-Nitrofluorene

Swiss-Webster mice

Male

Oral

1600

Simmon et al. (1979)

1-Nitropyrene

F344 rats

Male or female

Gavage

>5000

Marshall et al. (1982)

a Cited studies not conducted according to current guidelines.

7.1.1 1-Nitronaphthalene

Johnson et al. (1984) reported respiratory distress in male Sprague-Dawley rats (n = 4–23 per group) within 24 h after a single i.p. injection of 25–200 mg 1-nitronaphthalene/kg bw (ED50 = 60 mg/kg bw). Rats showed laboured breathing and pronounced gasping (severe form). Histopathology revealed acute necrosis of non-ciliated bronchiolar epithelial cells and inflammation. Furthermore, authors reported centrilobular liver necrosis at 100 mg/kg bw, which was most pronounced 72 h after injection. At the time of sacrifice, the relative liver weight was also significantly elevated. The authors estimated an LD50 of 86 mg/kg bw after i.p. application. Further details on lung and liver toxicity are given in section 7.7.

7.1.2 2-Nitronaphthalene

Acute toxicity in male mice after oral application was determined by Simmon et al. (1979). The authors reported an LD50 of 1300 mg/kg bw at a post-exposure observation period of 24 h (no further data).

Details on lung and liver toxicity are given in section 7.7.

7.1.3 5-Nitroacenaphthene

Morita et al. (1997) reported an LD100 of approximately 1700 mg/kg bw in male and female rats after i.p. injection. Rats died 48–72 h after treatment (no further data available).

7.1.4 2-Nitrofluorene

An LD50 of 1600 mg/kg bw at a post-exposure observation period of 24 h was reported in male mice after oral application of 2-nitrofluorene (no further data) (Simmon et al., 1979).

7.1.5 3,9-Dinitrofluoranthene

Male rats receiving intrapulmonary implants at a dose of 50 µg per rat showed reduced body weight (Horikawa et al., 1991).

7.1.6 1-Nitropyrene

In a dose range-finding study, no observable toxic effects and no histopathological alterations were reported in female F344 rats (= 1 per dose) that were gavaged with 500–5000 mg/kg bw and sacrificed 4 days after treatment. Similar results were noted after 5000 mg/kg bw (two males and two females) and post-exposure observation periods up to 14 days (Marshall et al., 1982).

7.2 Short-term and long-term exposure (non-neoplastic effects)

There are very few (if any) reports on just the non-neoplastic effects of nitroPAHs, but details can be obtained from studies available on the carcinogenicity of nitroPAHs after repeated administration (see section 7.6). In most carcinogenicity studies, non-neoplastic effects such as increased mortality or reduced body weight are due to tumour formation. In this section, those studies are presented in which non-neoplastic toxic effects that are presumably independent of carcinogenic effects appeared or the specified dose resulted in neither carcinogenic effects nor other toxic effects. However, there seems to be no underlying pattern, and it is impossible to generalize about these effects. Details on experimental design and results are described in Tables 38 and 39. Studies with limited validity (see footnotes for Table 39) are not discussed in this section. The criteria that were considered include (a) toxic effects that were statistically significant, (b) dose–response and (c) clear positive results where statistics were not given.

The Task Group evaluated the strength of each of the in vivo studies based on the following criteria: (a) numbers of animals in each group, (b) duration of study, (c) adequate dosing (e.g., maximum tolerated dose [MTD] in negative studies) and (d) appropriateness of controls. Specific comments on the strengths and weaknesses of the studies are included in Table 39.

7.2.1 1-Nitronaphthalene

In chronic feeding studies on mice and rats, up to 160 or 90 mg/kg bw per day, respectively, for 78 weeks plus latency period resulted in no obvious toxic effects (NCI, 1978a).

7.2.2 5-Nitroacenaphthene

In a subchronic intermittent feeding study on rats, a dose of approximately 500 mg/kg bw per day resulted in reduced body weight and increased mortality in females but not in males (Takemura et al., 1974).

In male and female mice, lipidosis of the liver, liver tumour formation and calcification of the renal papilla were observed after exposure via the diet for 78 weeks at doses >40 mg/kg bw per day. The authors also noted reduced survival (NCI, 1978b).

7.2.3 2-Nitrofluorene

Cui et al. (1995) reported reduced body weight during the exposure period of 11 months in a feeding study on rats at a dose level of about 25 mg/kg bw per day. No effect was noted at <10 mg/kg bw per day.

7.2.4 1-Nitropyrene

In a nose-only inhalation study, F344 rats (40 males and 40 females per group) were exposed to 0 or 7.5 mg 1-nitropyrene/m3 for 2 h per day, 5 days per week, for 4 weeks. One day, 2 weeks, 6 months or 12 months after the exposure period, groups of three males and three females were sacrified for histopathology of lung and nasal cavity. There was no effect on body or lung weight, and histopathology revealed no significant lesion. In additional immunization experiments, no increase in the number of antibody-forming cells in the lung-associated lymph nodes was noted (Wolff et al., 1988).

In contrast to the study of Wolff et al. (1988), adverse effects at much lower concentrations were observed in a nose-only study on F344 rats after 13 weeks of exposure (NTP, 1996; for details, see Table 38). Even at the low dose of 0.51 mg/m3, squamous metaplasia of the epiglottis was reported (see also section 7.7).

Repeated administration to rats via gavage (3 times weekly for 4 weeks) at a dose of approximately 2.5 mg/kg bw showed no alteration in body weight and survival (King, 1988).

No effect on body or organ weight was reported in rats after a single intrapulmonary implantation of 1.5 mg per rat (Maeda et al., 1986) or i.p. injection of approximately 17 mg/kg bw, 3 times weekly for 4 weeks (Imaida et al., 1991b).

Table 38. Short-term studies on toxicity and preneoplastic lesions induced by nitroPAHsa

NitroPAH; purity

Species; strain; number per group, sex, ageb

Route and dose

Treatment duration; study duration

Type of lesion: incidence in male and female dose groups

Results

Non-neoplastic effects; remarks

Reference

2-Nitro-fluorene; n.g.

Rat; Wistar; n.g., 6 weeks

Gavage
0, 5, 20 mg/kg bw per day

Four applications on consecutive days and two 2 and 4 days after two-thirds hepatectomy on day 5; 7 weeks

gamma-Glutamyl transferase-positive foci (preneoplastic changes) volume in % of liver volume:
m 0.05, 0.1**, 0.19**

+

No data on toxicity
TS as promotor; initiation by 200 mg diethylnitrosamine/kg bw 2 weeks before TS treatment

Möller et al. (1989)


2-Nitro-fluorene; 98%

Rat; Wistar; 3 m, 6–8 weeks

Gavage
0, 50, 200 mg/kg bw, 18 h after two-thirds hepatectomy

Single application; 6 weeks

gamma -Glutamyl transferase-positive foci per cm3 in the liver:
m ~10, 200**, 400**; positive control ~1150**

+

No data on toxicity
TS as initiator; promotor: 0.02% acetylaminofluorene in the diet (weeks 2–4) and 2 ml carbon tetrachloride/kg bw p.o. once at week 3; positive control 50 mg diethylnitrosamine/kg bw

Möller et al. (1993b)

2-Nitro-fluorene; n.g.

Rat; Wistar; n.g., m, 6 weeks

i.p.
0, 20, 100, 200 mg/kg bw, 16 h after two-thirds hepatectomy

Single application; 6 weeks

gamma -Glutamyl transferase-positive foci per cm3 in the liver:
m 8, 12, 39, 98***; positive control 258***

+

No data on toxicity
TS as initiator; promotor: 0.02% acetylaminofluorene in the diet (weeks 2–4) and 2 ml carbon tetrachloride/kg bw p.o. once at week 3; positive control 20 mg diethylnitrosamine/kg bw

Möller et al. (1989)


2-Nitro-fluorene;
98%

Rat; Wistar; 3 m, 6–8 weeks

i.p.
0, 50, 200 mg/kg bw, 18 h after two-thirds hepatectomy

Single application; 6 weeks

gamma -Glutamyl transferase-positive foci per cm3 in the liver:
m ~20, 150, 350*; positive control ~1350**

+

No data on toxicity
TS as initiator; promotor: 0.02% acetylaminofluorene in the diet (weeks 2–4) and 2 ml carbon tetrachloride/kg bw p.o. once at week 3; positive control 50 mg diethylnitrosamine/kg bw

Möller et al. (1993b)

2-Nitrofluorene;
>98%

Rat; Wistar; 5–7 m, 6–8 weeks

i.p.
0, 50, 200 mg/kg bw, 18 h after two-thirds hepatectomy

Single application; 6 weeks

gamma -Glutamyl transferase-positive foci per cm3 in the liver:
m ~20, 100*, 200**; positive control ~1200

+

No data on toxicity
TS as initiator; promotor: 0.02% acetylaminofluorene in the diet (weeks 2–4) and 2 ml carbon tetrachloride/kg bw p.o. once at day 21; positive control 50 mg diethylnitrosamine/kg bw

Möller et al. (1993b)

2,7-Dinitro-fluorene;
97%

Rat; Wistar; 3 m, 6–8 weeks

Gavage
0, 50, 200 mg/kg bw, 18 h after two-thirds hepatectomy

Single application; 6 weeks

gamma -Glutamyl transferase-positive foci per cm3 in the liver:
m ~10, 250**, 950**;
positive control ~1150**

+

No data on toxicity
TS as initiator; promotor: 0.02% acetylaminofluorene in the diet (weeks 2–4) and 2 ml carbon tetrachloride/kg bw p.o. once at week 3; positive control 50 mg diethylnitrosamine/kg bw

Möller et al. (1993b)

2,7-Dinitro-fluorene;
97%

Rat; Wistar; 3 m, 6–8 weeks

i.p.
0, 50, 200 mg/kg bw, 18 h after two-thirds hepatectomy

Single application; 6 weeks

gamma -Glutamyl transferase-positive foci per cm3 in the liver:
m ~20, 100, 350*;
positive control ~1350**

+

No data on toxicity
TS as initiator; promotor: 0.02% acetylaminofluorene in the diet (weeks 2–4) and 2 ml carbon tetrachloride/kg bw p.o. once at week 3; positive control 50 mg diethylnitrosamine/kg bw

Möller et al. (1993b)

1-Nitropyrene;
purified, no di- or trinitropyrenes detected by HPLC

Rat; Fischer 344; 6–20 m,
6 weeks

Gavage
0, 100, 250, 500, 1000 mg/kg bw once a day for 6 days

6 days; 6 weeks

gamma -Glutamyl transferase-positive foci per cm3 in the liver:
18, 34***, 38***, 54***, 37***;
positive control 786***

+

No data on toxicity
TS as initiator; promotor: 0.02% acetylaminofluorene in the diet (weeks 4+5) and 1 ml carbon tetrachloride/kg bw p.o. once after week 4; positive control 200 mg BaP/kg bw; two-thirds hepatectomy after 4th application; no effect with a single dose of 1000 mg 1-nitropyrene/kg bw

Denda et al. (1989)

1-Nitropyrene;
>99%

Rat; F344/N; 7 weeks

Inhalation
0, 0.51, 1.99, 7.78, 19.9, 46.6 mg/m3 (mean analytical concentration), 6 h per day, 5 days per week

13 weeks; 13 weeks

Epiglottis squamous metaplasia #:
m: 0/10, 0/10, 5/10, 10/10, 10/10, 10/10;
f: 0/10, 4/10, 4/10, 10/10, 10/10, 10/10
Bronchus squamous metaplasia #:
m: 0/10, 0/10, 0/10, 1/10, 2/10, 5/10;
f: 0/10, 0/10, 0/10, 0/10, 2/10, 3/10

+

No effect on body weight except slight decrease in high-dose males, increased relative and absolute liver weights in m at >2 mg/m3; haematology and clinical chemistry revealed no treatment-related effects; only non-neoplastic effects but short observation period; nose-only exposure to aerosol; NOAEL for m 0.51 mg/m3; LOAEL for f 0.51 mg/m3

NTP (1996)

a

n.g. = not given; m = male; f = female; # = no data on statistical evaluation; TS = test substance; p.o. = per os; NOAEL = no-observed-adverse-effect level; LOAEL = lowest-observed-adverse-effect level; significant compared with control, * (P < 0.05), ** (P < 0.01), *** (P < 0.001).

b

Age of animals at start of the experiment.

Table 39. Long-term toxicity and carcinogenicity of nitroPAHsa

NitroPAH and purity

Species; strain; number per group per sex; ageb

Route and dose

Treatment duration; study duration

Type of lesion: incidence in male and female dose groups

Resultc

Non-neoplastic effects; remarks

References

1-Nitronaphthalene

           

Technical grade, impurity not specified

Mouse; B6C3F1;
50 m, 50 f (1 control group for each dosed group);
6 weeks

Diet, 0, 0.06, 0.12% (~0, 80, 160 mg/kg bw per day)

78 weeks; 96–98 weeks

No significant effects with any tumour type

(–)

No clinical abnormalities; no effect on survival, but body weight reduced in m and f (no statistics); increase in incidences of non-neoplastic lesions not compound related; complete histopathology at termination; statistical evaluation with all animals surviving 52 weeks; reduced body weight also in low-dose control compared with high-dose control; high dose presumably not the MTD

NCI (1978a)

Technical grade, impurity not specified

Rat; F344; 50 m, 50 f (separate high-dose control 25 m and 25 f);
6 weeks

Diet, 0, 0.05% for 12 weeks then 0.06% for 66 weeks, 0.18% (~0, 25/30, 90 mg/kg bw per day)

78 weeks; 107–109 weeks

No significant effects with any tumour type

(–)

No treatment-related clinical abnormalities; no effect on survival; body weight decreased in high-dose group compared with concurrent control but not with low-dose control (no statistics); no compound-related increase in non-neoplastic lesions; complete histopathology at termination; statistical evaluation with all animals surviving 52 weeks; high dose presumably not the MTD

NCI (1978a)

2-Nitronaphthalene

           

Purified

Rhesus monkey; n.g.; 1 f; n.g.

Oral gelatin capsules, 121 mg/kg bw twice daily, 6 days per week

54 months;
54 months

Papilloma in urinary bladder:
1/1

(+)

No data on toxicity; four papillomas observed; no further macroscopic lesions detected; no control

Conzelman et al. (1970)

n.g.

Mouse; n.g.;
control 82, 140, 72; treated 80, 76, 41; sex n.g.; n.g.

n.g.

Single implantation;
mice observed for up to 70 weeks

Bladder carcinomas:
control 1/82, 2/140; 4/72 versus 7/80, 2/76, 2/41 in treatment groups (not significant); carcinogenic effect of cholesterol pellet (no carcinoma in sham operated mice without pellet)

(–)

No effect on survival (no further data); cholesterol pellet containing the TS implanted into the bladder; only mice surviving longer than 175 days evaluated

Bryan et al. (1964)

5-Nitroacenaphthene

           

n.g.

Rat; Wistar;
30 f; weanling

Diet, 0, 1%
(~500 mg/kg bw per day)

4 months, 2 interruptions, each of 3 weeks after 1 and 2 months of exposure; 282–500 days

Rhabdomyosarcomas #:
f 0/29, 1/12;
ear duct carcinomas #:
f 0/29, 2/12;
mammary carcinomas #:
f 0/29, 5/12;
small intestine adenocarcinomas #:
f 0/29, 10/12

+

Reduced body weight and survival also without tumour formation; only 12 treated rats lived more than 200 days; 29 controls survived >500 days

Takemura et al. (1974)

n.g.

Rat; Wistar;
20 m; weanling

Diet, 1% (~500 mg/kg bw per day)

6 months;
500 days

No malignant tumours (no further data)

(–)

No effects on survival (>500 days) or on body weight; no control data

Takemura et al. (1974)

Fairly high purity (no further data)

Rat; F344;
50 m, 50 f;
6 weeks

Diet, 0 (two control groups), 0.12, 0.24% (~0, 60, 120 mg/kg bw per day)

78 weeks (high-dose m 70 weeks); control 108–110 weeks, low dose 100 weeks, high-dose m 70 weeks (f 87 weeks)

Lung adenomas/carcinomas:
m 0/48, 1/48, 7/38**, 3/45;
f 0/49, 1/50, 8/46**, 3/31;
ear canal/skin carcinomas:
m 1/48, 0/48, 21/42***, 20/47***; f 0/49, 0/50, 27/49***, 35/47***;
mammary adenocarcinoma:
f 0/49, 0/50, 5/46*, 5/31**;
clitoral gland carcinoma:
f 0/49, 0/50, 6/46*, 5/39*

+

Dose-dependent reduction in body weight in m and f (no statistics); dose-dependent increase in mortality in m and f (high tumour incidence); histopathology revealed no compound-related increase in non-neoplastic lesions; concurrent high-dose and low-dose control groups

NCI (1978b)

Fairly high purity (no further data)

Mouse; B6C3F1;
50 m, 50 f;
6 weeks

Diet, 0, 0.06 (51 weeks) and then 0.03, 0.12% (~0, 80/40, 160 mg/kg bw per day)

78 weeks;
96 weeks

Hepatocellular carcinomas:
m 12/50, 7/31, n.g.;
f 2/47, 23/47***, 18/19***;
ovarian tumours:
f 0/45, 4/41*, 7/37**

+

Dose-dependent depression in body weight in m but not in f; reduced survival (not tumour related) in m and f; treatment-related fatty metamorphosis of the liver (low- and high-dose m, high-dose f) and calcification of renal papilla (high-dose f, low- and high-dose m)

NCI (1978b)

n.g.

Syrian golden hamster; n.g.;
control 20 f but no m, treated 24 f and 10 m;
weanling

Diet, 0, 1%
(~0, 830 mg/kg bw per day)

6 months;
>270 days (no further data)

Cholangiomas #:
m no control, 0/7; f 0/20, 7/13

+

In f, reduced body weight compared with control (no further data); reduced survival, 13 f and 7 m survived 270 days (no data on control)

Takemura et al. (1974)

2-Nitrofluorene

           

n.g.

Rat; Holtzman;
control 18 m and 20 f, treated 9 m and 9 f; n.g.

Diet, 0, 342 mg/kg diet
(~17 mg/kg bw per day)

8 months;
10 months

Mammary gland tumours #:
m 0/18, 0/9; f 1/20, 4/9;
most treated rats (no further data) had forestomach tumours

(+)

No effect on survival (no data on body weight); metabolites 2-aminofluorene and 2-acetylaminofluorene were more active

Miller et al. (1955)

n.g.

Rat; Holtzman;
control 10 m, treated 20 m;
n.g.

Diet, 0, 342 mg/kg diet
(~17 mg/kg bw per day)

12 months;
12 months

Forestomach squamous cell carcinomas #:
m 0/10, 17/18;
liver tumours #:
m 0/10, 14/18;
ear duct tumours #:
m 0/10, 4/18;
small intestine tumours #:
m 0/10, 2/18

+

No effect on health or survival up to the time of tumour formation (no further data); glandular portion of the forestomach not involved in tumour formation

Miller et al. (1955)

n.g.

Rat; Minnesota;
control 3 m and 3 f, treated 6 m and 3 f; n.g.

Diet, 0, 0.05%
(~0, 25 mg/kg bw per day)

23 weeks;
44 weeks

Mammary adenocarcinomas #:
m 0/3, 0/6; f 0/3, 1/3;
squamous cell carcinomas of the ear canal #:
m 0/3, 0/6; f 0/3, 1/3

(–)

No effect on survival; reduced body weight; small number of animals

Morris et al. (1950)

95.3%

Rat; Wistar;
18–20 m; n.g.

Diet, 0, 0.24, 0.95, 2.37 mmol/kg diet
(~2.5, 10, 25 mg/kg bw per day)

11 months;
24 months

Liver hepatocellular carcinomas #:
m 0/20, 2/18, 15/19, 20/20;
forestomach carcinomas #:
m 0/20, 10/18, 16/19, 10/20;
kidney cortical carcinomas #:
m 0/20, 1/18, 15/19, 11/20

+

During exposure period, reduced body weight in the high-dose group; after exposure period, dose-dependent increase in mortality due to tumour formation in mid- and high-dose group

Cui et al. (1995)

98%

Mouse; Sencar; 20 f;
6–8 weeks

Dermal, 0, 50, 150 µg/mouse

Single application;
14 weeks

A few skin tumours arose, but effect was not significant concerning tumours per animal (no data on incidence)

(–)

No effect on body weight; initiation/promotion study; TS as initiator; vehicle acetone; 6 days after initiation was complete, 2 µg tetradecanoylphorbol acetate applied twice weekly for 13 weeks (promotion)

Möller et al. (1993c)

98%

Mouse; Sencar; 25 f;
6–8 weeks

Dermal, 0, 500, 1500 µg/mouse

Single application;
20 weeks

A few skin tumours arose, but effect was not significant concerning tumours per animal (no data on incidence)

(–)

Final body weight significantly decreased in both TS treatment groups; TS without tetradecanoylphorbol acetate treatment increased body weight; initiation/promotion study; TS as initiator; vehicle acetone; 5 days after initiation was complete, 5 µg tetradecanoylphorbol acetate applied twice weekly for 19 weeks (promotion)

Möller et al. (1993c)

n.g.

Rat; Minnesota;
see incidence;
n.g.

Dermal, 3 or, after 6 months, 6 drops of 0 or 2% thrice weekly

80 weeks;
80 weeks

Total tumours #:
m 0/3, 6/7; f 0/3, 0/3

(+)

Slightly reduced survival, no effect on body weight; no skin tumours induced; TS dissolved in acetone; total dose 69 mg/rat; small number of animals

Morris et al. (1950)

>98%

Rat; Sprague-Dawley; 15 f; 50 days

Intramammary, 0 or 2.04 µmol/injection, see remarks

Single application;
82 weeks

Mammary gland tumour (malignant tumours): 10(2)/15, 5(1)/15; total number of tumours (malignant tumours): 12(2)/15; 8(1)/15

(–)

No effect on body weight gain; vehicle DMSO; each of eight glands injected underneath the nipples; high incidence in vehicle control

Malejka-Giganti et al. (1999)

2,5-Dinitrofluorene

           

n.g.

Rat; n.g.;
control 12 m and 12 f, treated 8 f and 8 m; n.g.

Diet, 0 or 1.62 mmol/kg diet
(0 or 24 mg/kg bw per day)

8 months;
10 months

Mammary gland tumours #:
m 0/12, 0/8; f 0/12, 2/8

(+)

Survivors at termination: m 9/12, 6/8; f 11/12, 6/8 (no further data); autopsy included ear duct, mammary gland and abdominal and thoracic organs; only histopathology of tumours

Miller et al. (1962)

2,7-Dinitrofluorene

           

n.g.

Rat; n.g.;
control 12 m and 12 f, treated 8 f and 8 m; n.g.

Diet, 0 or 1.62 mmol/kg diet
(0 or 24 mg/kg bw per day)

8 months;
10 months

Malignant mammary gland tumours #:
m 0/12, 1/8; f 0/12, 8/8

(+)

Survivors at termination: m 9/12, 4/8; f 11/12, 0/8 (no further data); autopsy included ear duct, mammary gland and abdominal and thoracic organs; only histopathology of tumours

Miller et al. (1962)

>98%

Rat; Sprague-Dawley; 15 f; 50 days

Intramammary, 0 or 2.04 µmol/injection, see remarks

Single application;
82 weeks

Mammary gland tumour (malignant tumours): 10(2)/15, 14(14)/15; total number of tumours (malignant tumours): 12(2)/15; 33(27)/15***

(+)

No effect on body weight gain; vehicle DMSO; each of eight glands injected underneath the nipples; high incidence in vehicle control

Malejka-Giganti et al. (1999)

2-Nitrofluoranthene

           

>99%

Mouse; B6C3F1;
control 24 m, treated 23 m;
newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 2/24, 8/23; liver carcinomas: 1/24, 3/23; lung tumours: 0/24, 0/23

(–)

Mortality: 0%, 4%; no effects on body weight; solvent DMSO; only liver and lung tumours studied

von Tungeln et al. (1994b, 1999b)

3-Nitrofluoranthene

           

99.99%

Rat; F344/DuCrj;
20 m; 11 weeks

Intrapulmonary implants, 0, 1000 µg/rat

Single implantation;
100 weeks

Lung squamous cell carcinomas #:
m 0/20, 1/20

(–)

Reduced body weight (but not significant) in treated rats; survival not reduced; further lung lesions also in controls; TS in a mixture of beeswax–tricaprylin

Horikawa et al. (1991)

Purified (no further data)

Rat; n.g.; 20 m; n.g.

Intrapulmonary implants, 0, 1000 µg/rat

Single implantation;
70 weeks

Lung tumours #:
m 0/20, 2/20

(–)

No data on toxicity; TS in a mixture of beeswax–tricaprylin

Tokiwa et al. (1990b)

>99%

Rat; F344/DuCrj;
control 20 m, treated 10 m; 8 weeks

s.c., 0, 2 mg/rat twice weekly

7.5 weeks;
lifetime

Tumours at injection site:
m 0/20, 4/10* (2 malignant fibrous histiocytomas, 2 dermato-fibrosarcomas)

+

Reversible inflammation and ulcerations at the injection site after treatment with TS; decreased body weight gain; solvent DMSO

Ohgaki et al. (1982)

>99.9%

Mouse; BLU:Ha;
control 91 m and 101 f, treated 25–29 m and 24–27 f; newborn

i.p., total dose 0, 63, 315 µg/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected;
26 weeks

total no. of mice with lung tumours: 20/192, 15/53***, 16/52**;
no. of lung tumours/ mouse:
0.11, 0.30**, 0.63***

+

No data on toxicity; solvent DMSO; lung tumours studied; sexes not separately evaluated for statistical purposes

Busby et al. (1989)

>99%

Mouse; B6C3F1;
24 m; newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 2/24, 7/24; liver carcinomas: 1/24, 1/24; lung tumours: 0/24, 0/24

(–)

Mortality: 0%, 0%; no effects on body weight; solvent DMSO; only liver and lung tumours studied

Von Tungeln et al. (1994b, 1999b)

3,7-Dinitrofluoranthene

           

Purified by recrystallization

Rat; F344/DuCrj;
21 m; 6 weeks

s.c., 0, 50 µg/rat twice weekly

10 weeks;
50 weeks

Local subcutaneous tumours #:
m 0/21, 21/21 (20 malignant fibrous histiocytomas and 1 rhabdomyosarcoma)

+

<10% of treated rats alive at termination (control 100%); TS dissolved in DMSO

Tokiwa et al. (1987a)

99.90%

Rat; F344/DuCrj;
20–22 m; 11 weeks

Intrapulmonary implants, 0, 200 µg/rat

Single implantation;
100 weeks

Lung tumours #:
m 0/20, 12/22 (11 squamous cell carcinomas, 1 undifferentiated neoplasm)

+

Reduced body weight and survival in treated rats, presumably due to tumour formation; TS in a mixture of beeswax–tricaprylin

Tokiwa et al. (1990b); Horikawa et al. (1991)

3,9-Dinitrofluoranthene

           

Purified by recrystallization

Rat; F344/DuCrj;
control 21 m, treated 11 m; 6 weeks

s.c., 0, 50 µg/rat twice weekly

10 weeks;
50 weeks

Subcutaneous tumours #:
m 0/21, 10/11 (7 malignant fibrous histiocytomas and 3 rhabdomyosarcomas)

+

All treated rats died or were sacrificed moribund at weeks 15–36 (survival in control 100% at week 50) due to local tumour formation; TS dissolved in DMSO

Tokiwa et al. (1987a)

99.98%

Rat; F344/DuCrj;
10–21 m; 11 weeks

Intrapulmonary implants, 0, 50, 100, 200 µg/rat

Single implantation;
100 weeks

Lung tumours #:
m 0/20, 1/10, 7/10, 19/21 (mostly squamous cell carcinomas)

+

Reduced body weight in treated rats (dose related); reduced survival in high-dose group; effects in low-dose group presumably not related to tumour formation; TS in a mixture of beeswax–tricaprylin

Horikawa et al. (1991)

1-Nitropyrene

           

>99.9%

Rat; CD; 35–36 f; weanling

Gavage, 0, 10 µmol (2.5 mg)/kg bw, thrice weekly

4 weeks;
76–78 weeks

No significant carcinogenic effects

(–)

No effects on body weight and survival; solvent DMSO

King (1988); Imaida et al. (1991a)

>99.9%

Rat; CD; 30 f; 30 days

Gavage, 0, 50 µmol/rat (42–125 mg/kg bw), once weekly

8 weeks;
49 weeks

Mammary fibroadenomas:
f 0/30, 10/30**;
mammary desmoplastic adenomas: f 8/30, 15/30*

+

No effect on body weight and survival; vehicle trioctanoin

El-Bayoumy et al. (1995)

>99.9%, absence of dinitropyrenes

Rat; Sprague-Dawley; 22–36 m and 24–31 f;
newborn

Gavage, 0, 100, 250 µmol/kg bw (0, 25, 62 mg/kg bw), once weekly

16 weeks;
94 weeks

Mammary adenocarcinomas:
f 1/31, 14/33**, 15/24**;
pancreas islet cell adenomas:
m 0/22, 4/25*, 3/36;
f 0/31, 1/33, 1/24;
lung tumours: m 0/22, 1/25, 4/36;
f 0/31, 0/33, 3/24*

+

No effect on body weight, but reduced survival in both treatment groups, presumably due to tumour formation; vehicle trioctanoin; metabolites 1-nitrosopyrene and 1-aminopyrene were less carcinogenic

El-Bayoumy et al. (1988b)

>99%

Mouse; Crl/CD-1 (ICR)BR;
20 f; 50–55 days

Dermal, 0, 0.1 mg/mouse per day

10 days;
25 weeks

No significant effect on skin tumour formation

(–)

No data on toxicity; initiation/promotion study; TS as initiator; vehicle acetone; 10 days after initiation was complete, 2.5 µg tetradecanoylphorbol acetate applied thrice weekly for 25 weeks (promotion)

El-Bayoumy et al. (1982)

99.5%

Mouse;
SENCAR;
39–40 m and 39–40 f; 7 weeks

Dermal, 0, 0.03, 0.1, 0.3, 1, 3 mg/mouse

Single application except high dose (2 treatments, no further information);
30 weeks

No significant tumour initiating activity in contrast to the positive control BaP (0.05 mg/mouse)

(–)

No data on toxicity; initiation/promotion study; vehicle acetone; 1 week after initiation was complete, 2 µg tetradecanoylphorbol acetate applied twice weekly for 30 weeks (promotion)

Nesnow et al. (1984)

98% (0.008% 1,3-dinitropyrene, 0.6% 1,6- and 1,8-dinitropy-rene, 1.3% pyrene)

Syrian golden hamster; n.g.;
control 19 m, treated 34 m; 8 weeks

Intratracheal instillation, 0, 2 mg/hamster, once weekly

15 weeks;
663 days, control 684 days

No significant effects on lung or trachea tumour formation in contrast to the positive control BaP (same dose)

(–)

24 (control 16) hamsters alive after treatment period (pneumonia during exposure period), no effect on survival; vehicle phosphate buffer; animals observed during entire life span

Yamamoto et al. (1987)

99.9%

Rat; F344/DuCrj;
31–32 m; 10–11 weeks

Intrapulmonary implants, 0, 1.5 mg/rat

Single implantation;
72 weeks

No lung tumour induction in contrast to the positive control 0.5 mg 3-methylcholanthrene (19/19)

(–)

No effect on body or organ weight (no data on survival); TS in a mixture of beeswax–tricaprylin

Maeda et al. (1986)

99.9%

Mouse; BALB/c; 20 m; 6 weeks

s.c., 0, 0.1 mg/mouse (~3.3 mg/kg bw), once weekly

20 weeks;
60 weeks

No significant carcinogenic effect in contrast to 1,6-dinitropyrene (same dose; subcutaneous tumours)

(–)

Slightly reduced survival, presumably not treatment related; no data on body weight; solvent DMSO; low number of mice

Tokiwa et al. (1984, 1986)

>99.75%

Rat; F344/DuCrj;
control 20 m, high dose 20 m, low dose 10 m; 6 weeks

s.c., 0, 0.2, 2 mg/rat (0, 2, 20 mg/kg bw), twice weekly

10 weeks;
650 days

No tumours at injection site, no other carcinogenic effects

(–)

No effect on body weight and survival; solvent DMSO

Ohgaki et al. (1985)

Purified, contamination by dinitro-pyrenes <0.02%

Rat; Sprague-Dawley CD;
28–35 rats of each sex; newborn

s.c., 0, 50, 100 µmol (12.5 or 25 mg)/kg bw, once weekly

8 weeks;
62 weeks

Tumours at injection site: m 0/28, 2/29, 10/31***; f 0/31, 3/31, 9/32** (mostly malignant fibrous histiosarcomas); mammary tumours: f 2/31, 7/31, 15/32*** (mostly adenocarcinomas and fibroadenomas)

+

No significant effect on body weight or survival; no effect on liver and kidney weight, but spleen weight increased in m and f in high-dose group (marked haematopoiesis due to tumour formation); solvent DMSO

Hirose et al. (1984)

n.g.

Rat; CD; 29–30 f; weanling

s.c., 0, 100 µmol (25 mg)/kg bw, once weekly

4 weeks; 87–90 weeks

Mammary fibroadenomas:
f 8/30, 15/29*

(+)

No significant effect on body or organ weight (liver, kidney, spleen); no data on survival; solvent DMSO

Imaida et al. (1991b)

>99.9%

Rat; F344; 55 f; newborn

s.c., 0, 100 µmol (25 mg)/kg bw, once weekly

8 weeks;
86 weeks

Leukaemia:
f 0/55, 4/55*

+

No effects on survival; no data on body weight; solvent DMSO

King (1988); Imaida et al. (1995)

>99.9%

Rat; CD; 47–48 f; newborn

s.c., 0, 100 µmol (25 mg)/kg bw, once weekly

8 weeks;
86 weeks

Mammary adenocarcinomas:
f 3/47, 10/48*

+

No effects on survival; no data on body weight; solvent DMSO; no significant effects with the metabolites 1-nitropyren-3-ol, 1-nitropyren-6-ol and 1-nitropyren-8-ol

King (1988); Imaida et al. (1995)

>99.9%

Rat; CD; control 40 f, treated 49 f; newborn

s.c., 0, 2.5–10 µmol/kg bw (0.6–2.5 mg/kg bw) (see remarks), once weekly

8 weeks;
67 weeks

Mammary adenocarcinomas:
f 1/40, 10/49*

+

No effects on survival; solvent DMSO; 1st injection 2.5 µmol, 2nd and 3rd 5 µmol, 4th–8th 10 µmol/kg bw

King (1988); Imaida et al. (1995)

>99.99%, no dinitropyrenes detected

Mouse, A/J;
26 m; 6 weeks

i.p., 0 or 93 mg/kg bw, thrice weekly

6 weeks (total of 17 injections);
24 weeks

Incidence of lung tumours significantly increased (< 0.0001, control 1/23, no further data)

(+)

No data on toxicity; solvent DMSO; no definitive carcinogenicity test with this mouse strain

Bai et al. (1998)

>99%, absence of dinitropyrenes

Mouse, A/J; 15–16 m and 12–16 f; 6–8 weeks

i.p., not specified, but total dose 0, 175, 525, 1575 mg/kg bw, injections thrice weekly

6 weeks; 24 weeks

Lung tumours:
m 3/16, 4/10, 6/14, 14/16*;
f 4/16, 3/14, 5/13, 8/12*;
mean number of lung tumours per mouse: m 0.3, 0.6*, 0.6, 1.6***; f 0.3, 0.2, 0.4, 0.8*

(+)

No data on toxicity; vehicle trioctanoin; the strain used in this study is sensitive to lung tumour development and, on occasion, conflicts with standard 2-year studies

El-Bayoumy & Hecht (1983); El-Bayoumy et al. (1984b)

>99%

Mouse; CD-1;
28–45 m and 26–50 f; newborn

i.p., total dose 0, 700, 2800 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 12 months

Liver carcinomas:
m 0/28, 0/45 (2nd control), 3/34*, 5/29*; no liver tumours in female mice

+

54–85% survived to weaning (no further data on toxicity); vehicle DMSO; two vehicle-treated control groups; tumour incidence from weaning to month 12 recorded; metabolite 1-nitrosopyrene was more effective

Wislocki et al. (1986)

>99.9%

Mouse; BLU:Ha;
control 91 m and 101 f; treated 23–29 m and 23–30 f; newborn

i.p., total dose 0, 21, 105 µg/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; days 1–15; 26 weeks

No effects on lung tumour formation

(–)

No data on toxicity; solvent DMSO; only lung tumours studied

Busby et al. (1989)

99.5%

Mouse; SENCAR;
39–40 m, 39–40 f; 7 weeks

i.p., 0, 1, 2, 4, 6, 8 mg/mouse (~0, 33, 66, 132, 200, 264 mg/kg bw)

Single application;
30 weeks

No significant tumour initiating activity in contrast to the positive control BaP

(–)

No data on toxicity; initiation/promotion study; vehicle corn oil; 1 week after initiation, 2 µg tetradecanoylphorbol acetate applied dermally twice weekly for 30 weeks (promotion)

Nesnow et al. (1984)

>99.9%

Rat; CD; 36 f; weanling

i.p., 0, 10 µmol/kg bw (2.5 mg/kg bw), thrice weekly

4 weeks;
76–78 weeks

Mammary tumours:
f 7/31, 25/36*** (14 rats with adenocarcinoma [control 3] and 19 rats with fibroadenoma [control 5])

+

No effects on body weight or survival; solvent DMSO

King (1988); Imaida et al. (1991a)

n.g.

Rat; CD; 29 f; weanling

i.p., 0, 67 µmol (17 mg)/kg bw, thrice weekly (total dose 119 µmol/rat)

4 weeks;
61 weeks

No significant carcinogenic effects also with the metabolites (same dose) N-hydroxy-N-acetyl-1-aminopyrene and N-acetyl-1-aminopyrene

(–)

No significant effect on body or organ weight (liver, kidney, spleen); no altered liver foci; no data on survival; solvent DMSO

Imaida et al. (1991b)

n.g.

Rat; CD; 29–30 f; weanling

i.p., 0, 100 µmol (25 mg)/kg bw, once weekly

4 weeks;
87–90 weeks

Mammary adenocarcinomas:
f 2/30, 8/29*

+

No significant effect on body or organ weight (liver, kidney, spleen); no data on survival; solvent DMSO

Imaida et al. (1991b)

Mixture of 1-nitropyrene and small amounts of 1,3-, 1,6- and 1,8-dinitropyrene

     

0.11% 1,3-dinitropyrene, 0.27% 1,6-dinitropyrene, 0.23% 1,8-dinitropyrene

Rat; F-344/Jcl;
control 30 f, treated 40–46 f; 6 weeks

Gavage, 0, 5, 10, 20 mg/kg bw, twice weekly

55 weeks;
104 weeks

Mammary tumours:
f 4/28, 13/36*, 21/39**, 22/45** (mostly adenocarcinomas);
clitoral gland tumours:
f 1/28, 4/36, 11/39**, 12/45** (mostly squamous cell carcinomas);
leukaemia:
f 9/28, 23/36*, 22/39*, 27/45*

+

No significant effect on body weight, but dose-related decrease in survival from week 70 on associated with tumour formation; no effects on organ weight (brain, heart, liver, spleen, kidney, adrenal, uterus, ovary, thyroid); vehicle olive oil

Odagiri et al. (1986)

>99%; traces of dinitro-pyrenes

Rat; F344/DuCrj;
20 m; 8 weeks

s.c., 0, 2 mg/rat twice weekly

10 weeks;
lifetime

Tumours at injection site:
m 0/20, 8/17** (7 malignant fibrous histiocytoma, 1 extraskeletal osteosarcoma)

+

Decreased body weight gain (no data on survival) and reversible inflammation/ulcerations at the injection site in TS-treated rats;
solvent DMSO; authors’ comment: effect possibly due to dinitropyrene impurities

Ohgaki et al. (1982)

n.g.

Mouse; B6C3F1;
total of 266 mice (no further data)
prenatal, gestation day 15

i.p., 0, 20, 40, 50, 100 mg/kg bw

Single injection;
18 months

Significant (P < 0.05) induction of lung adenomas and adenocarcinomas (no further data)

(+)

Transplacental carcinogenesis studied; pregnant mice exposed; frequent abortions after injection of nitropyrene mixture; data from abstract, no further information available

Odagiri et al. (1993)

2-Nitropyrene

           

n.g.

Rat; CD; 28 f; weanling

Intramammary, 0, 2 µmol/injection, see remarks

Single application;
77 weeks

No effect on mammary tumour formation

(–)

No data on body or organ weight and survival; solvent DMSO; dose injected into tissue underlying each of the three thoracic nipples (left and right)

Imaida et al. (1991b)

n.g.

Rat; CD; 28–29 f; weanling

i.p., 0, 67 µmol (17 mg)/kg bw, thrice weekly (total dose 119 µmol/rat)

4 weeks;
61 weeks

Leukaemia/lymphoma:
f 0/29, 6/28***

+

No significant effect on body or organ weight (liver, kidney, spleen); no altered liver foci; solvent DMSO

Imaida et al. (1991b)

4-Nitropyrene

           

Purified by HPLC

Rat; CD; control 47 f, treated 27 f;
newborn

s.c., 0, 100 µmol (25 mg)/kg bw, once weekly

8 weeks;
86 weeks

Mammary tumours: f 17/47, 20/27**; malignant fibrous histiocytomas: f 0/47, 10/27***;
leukaemia: f 0/47, 5/27**; Zymbal gland carcinomas: f 0/47, 4/27*

+

No effects on body weight during treatment period, but reduced survival; solvent DMSO

King (1988); Imaida et al. (1995)

>99%

Mouse; CD-1;
28–29 m and 29–31 f; newborn

i.p., total dose 0, 2800 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 12 months

Liver carcinomas:
m 0/28, 20/29*; f 0/31, 1/29;
lung tumours:
m 1/28, 11/29*; f 0/31, 9/29*

+

54–85% survived to weaning (no further data on toxicity); vehicle DMSO; tumour incidence from weaning to month 12 recorded

Wislocki et al. (1986)

Purified by recrystallization

Rat; CD; 29 f; weanling

i.p., 0, 67 µmol (17 mg)/kg bw, thrice weekly (total dose 119 µmol/rat)

4 weeks;
61 weeks

Mammary adenocarcinomas:
f 1/29, 13/29**

+

No significant effect on body or organ weight (liver, kidney, spleen); altered liver foci in 1 treated rat; no data on survival; solvent DMSO

Imaida et al. (1991b)

Purified by recrystallization

Rat; CD; 28 f; weanling

Intramammary, 0, 2 µmol/injection, see remarks

Single application;
77 weeks

Mammary adenocarcinomas:
f 1/28, 19/28***

+

No data on body or organ weight and survival; solvent DMSO; dose injected into tissue underlying each of the three thoracic nipples (left and right)

Imaida et al. (1991b)

>99.8%

Rat; CD; 30 f; 30 days

Intramammary, 0, 2.04 µmol/injection, see remarks

Single application;
43 weeks

Mammary adenocarcinomas:
f 0/30, 7/30***;
mammary fibroadenomas:
f 5/30, 13/30*

+

No effects on body weight and survival; solvent DMSO; dose injected into tissue underlying each of the three thoracic nipples and three inguinal nipples (only left side, right side DMSO injection)

El-Bayoumy et al. (1993)

Mixture of 1,3-, 1,6- and 1,8-dinitropyrene

     

20.26% 1,3-dinitropyrene, 39.26% 1,6-dinitropyrene, 39.51% 1,8-dinitropyrene (>99%)

Mouse;
SENCAR;
control 22 m and 20 f, treated 37–40 m and 38–41 f;
7 weeks

Dermal, total dose 0, 0.05, 0.1, 0.5, 1, 2 mg/mouse

Single application (low dose) up to 2 applications per day for 5 days; 30 weeks

Skin papillomas:
m 0/22, 1/40, 1/38, 2/34, 6/38*, 10/39***;
f 0/20, 1/37, 0/37, 0/40, 6/39*, 11/38***

+

No data on toxicity; initiation/promotion study; vehicle acetone/DMSO; 1 week after initiation was complete, 2 µg tetradecanoylphorbol acetate applied twice weekly for 30 weeks (promotion)

Nesnow et al. (1984)

1,3-Dinitropyrene

           

>99%

Rat; CD; 35 f;
weanling

Gavage, 0, 10 µmol (2.9 mg)/kg bw, thrice weekly

4 weeks;
76–78 weeks

No significant carcinogenic effects

(–)

No effects on body weight and survival; solvent DMSO

King (1988); Imaida et al. (1991a)

>99.9%

Mouse; BALB/c; 13–18 m; 6 weeks

s.c., 0, 50 µg/mouse (1.7 mg/kg bw), once weekly

20 weeks;
60 weeks

No significant carcinogenic effects in contrast to the positive control BaP (same dose)

(–)

No effect on survival (no data on body weight); solvent DMSO

Tokiwa et al. (1986); Otofuji et al. (1987)

>99%

Rat; CD; 40–43 f; newborn

s.c., 0, 2.5–10 µmol (0.7–3 mg)/kg bw (see remarks), once weekly

8 weeks;
67 weeks

Malignant fibrous histiocytomas:
f 0/40, 5/43*;
mammary adenocarcinomas:
f 1/40, 6/43*

+

No effect on survival (no further data); solvent DMSO; 1st injection 2.5 µmol, 2nd and 3rd 5 µmol, 4th–8th 10 µmol/kg bw

King (1988); Imaida et al. (1995)

0.6% 1,6 dinitropyrene, 0.4% 1,8-dinitropyrene and 0.05% other nitropyrenes

Rat; F344/DuCrj;
control 20 m, treated 10 m; 6 weeks

s.c., 0, 0.2 mg/rat (~1 mg/kg bw), twice weekly

10 weeks;
347 days

Sarcomas at the site of injection #:
m 0/20, 10/10

+

No effect on body weight gain; in treated rats, reversible ulcer or scar formation at the site of injection (reversible); reduced survival due to local tumour development; solvent DMSO

Ohgaki et al. (1984)

>99%

Mouse; CD-1;
28–30 m and 31–39 f; newborn

i.p., total dose 0, 200 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 12 months

No significant carcinogenic effects

(–)

54–85% survived to weaning (no further data on toxicity); tumour incidence from weaning to month 12 recorded; vehicle DMSO

Wislocki et al. (1986)

>99%

Rat; CD; 31–36 f; weanling

i.p., 0, 10 µmol (2.9 mg)/kg bw, thrice weekly

4 weeks; 76–78 weeks

Mammary tumours: f 7/31, 19/36** (9 adeno-carcinomas, 12 fibroadenomas and 1 adenoma)

+

No effects on survival and body weight; solvent DMSO

King (1988); Imaida et al. (1991a)

1,6-Dinitropyrene

           

>99%

Rat; CD; 36 f; weanling

Gavage, 0, 10 µmol (3 mg)/kg bw, thrice weekly

4 weeks;
76–78 weeks

Mammary adenocarcinomas:
f 5/35, 11/36 (not significant)

(–)

No effects on body weight or survival; solvent DMSO

King (1988); Imaida et al. (1991a)

>99.9%

Syrian golden hamster; n.g.;
10 m and 10 f;
10 weeks

Intratracheal instillation, 0, 0.5 mg/hamster (~5 mg/kg bw), once weekly

26 weeks;
11 months

Lung adenocarcinomas #:
m 0/10, 10/10; f 0/10, 9/10;
leukaemia #:
m 0/10, 6/10; f 0/10, 6/10

+

Reduced survival due to tumour formation; no data on body weight; TS suspended in 0.2 ml saline

Takayama et al. (1985)

99.9%

Rat; F344/DuCrj; 28–31 m; 10–11 weeks

Intrapulmonary implant, 0, 0.15 mg/rat

Single implantation;
72 weeks

Lung tumours: m 0/31, 23/28** (21 squamous cell carcinomas, 2 undifferentiated carcinomas)

+

Reduced body and liver weight (no data on survival); TS in a mixture of beeswax–tricaprylin

Maeda et al. (1986)

99.8%

Rat; F344/NSlc;
26–40 m, high dose 9 m; 11 weeks

Intrapulmonary implant, 0, 0.003, 0.01, 0.03, 0.1, 0.15 mg/rat

Single implantation;
104 weeks

Lung tumours: m 0/40, 0/39, 4/30, 13/31, 22/26, 6/9 (mainly undifferentiated neoplasm), highly significant dose–response relationship (no further data)

+

No effect on body weight, but at >0.03 mg/rat, significantly reduced survival due to tumour formation; TS in a mixture of beeswax–tricaprylin

Iwagawa et al. (1989); Tokiwa et al. (1990b)

>99.9%

Mouse; BALB/c; 20 m; 6 weeks

s.c., 0, 0.1 mg/mouse, once weekly

20 weeks;
60 weeks

Malignant fibrous histiocytomas at the site of injection:
m 0/20, 10/20**

+

Reduced survival presumably due to tumour formation; no data on body weight; solvent DMSO

Tokiwa et al. (1984, 1986)

Purity checked by HPLC (no further data)

Rat; F344/DuCrj;
control 20 m, treated 10 m; 6 weeks

s.c., 0, 0.2 mg/rat twice weekly

10 weeks;
320 days (controls 650 days)

Sarcomas at the site of injection #:
m 0/20, 10/10;
no tumours in other organs

+

No effect on body weight; reduced survival due to tumour development; solvent DMSO

Ohgaki et al. (1985)

>99%

Rat; CD; 40–46 f; newborn

s.c., 0, 2.5–10 µmol (0.7–3 mg)/kg bw (see remarks), once weekly

8 weeks;
67 weeks

Malignant fibrous histiocytomas:
f 0/40, 46/46***;
leukaemia:
f 0/40, 9/46**

+

Survival reduced (149 versus 495 days) due to tumour development; enlarged liver and spleen (leukaemia); solvent DMSO; 1st injection 2.5 µmol, 2nd and 3rd 5 µmol, 4th–8th 10 µmol/kg bw

King (1988); Imaida et al. (1995)

>99%

Mouse; CD-1;
25–28 m and 29–31 f; newborn

i.p., total dose 0, 200 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected;
12 months

Liver carcinomas:
m 0/28, 5/25*; f 0/31, 0/29

+

54–85% survived to weaning (no further data on toxicity); vehicle DMSO; tumour incidence from weaning to month 12 recorded

Wislocki et al. (1986)

>99%

Rat; CD; control 31 f, treated 23 f; weanling

i.p., 0, 10 µmol (2.9 mg)/kg bw, thrice weekly

4 weeks;
76–78 weeks

Malignant fibrous histiocytomas:
f 0/31, 23/23 (P < 0.0001)

+

No effect on body weight, but average survival 135 days versus 535 days in controls due to tumour formation in the peritoneal cavity; solvent DMSO

King (1988); Imaida et al. (1991a)

1,8-Dinitropyrene

           

>99%

Rat; CD; 36 f; weanling

Gavage, 0, 10 µmol (2.9 mg)/kg bw, thrice weekly

4 weeks;
76–78 weeks

Mammary tumours:
f 12/35, 22/36* (12 adenocarcinomas and 12 fibroadenomas)

+

No effects on body weight and survival; solvent DMSO

King (1988); Imaida et al. (1991a)

0.6% 1,6-dinitropyrene, 0.4% 1,3-dinitropyrene and 0.05% other nitropyrenes

Rat; F344/DuCrj;
control 20 m, treated 10 m; 6 weeks

s.c., 0, 0.2 mg/rat, twice weekly

10 weeks;
169 days

Sarcomas at the site of injection #:
m 0/20, 10/10

+

No effect on body weight gain; in treated rats, reversible ulcer or scar formation at the site of injection; reduced survival due to tumour development; solvent DMSO

Ohgaki et al. (1984)

>99.9%

Mouse; BALB/c; 13–15 m; 6 weeks

s.c., 0, 50 µg/mouse, once weekly

20 weeks;
60 weeks

Local subcutaneous tumours:
m 0/13, 6/15*

+

No effect on survival (no data on body weight); solvent DMSO

Tokiwa et al. (1986); Otofuji et al. (1987)

0.4% 1,3-dinitropyrene (no further data)

Rat;
F344/DuCrj;
control 20 m, treated 10 m; 6 weeks

s.c., 0, 2, 20 µg/rat twice weekly

10 weeks;
320 days (controls 650 days)

Sarcomas at the site of injection #:
m 0/20, 9/10, 10/10

+

No effect on body weight; reduced survival due to local tumour development; solvent DMSO

Ohgaki et al. (1985)

>99%

Rat; CD; 37–40 f; newborn

s.c., 0, 2.5–10 µmol (0.7–3 mg)/kg bw (see remarks), once weekly

8 weeks;
67 weeks

Malignant fibrous histiocytomas:
0/40, 37/37***;
leukaemia: 0/40, 8/37**; mammary adenocarcinomas: f 1/40, 5/37*

+

Survival reduced (163 versus 495 days) due to tumour development; enlarged liver and spleen (leukaemia); solvent DMSO; 1st injection 2.5 µmol, 2nd and 3rd 5 µmol, 4th–8th 10 µmol/kg bw

King (1988); Imaida et al. (1995)

>99%

Mouse; CD-1;
28–31 m and 31–33 f; newborn

i.p., total dose 0, 200 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 12 months

No significant carcinogenic effects

(–)

54–85% survived to weaning (no further data on toxicity); vehicle DMSO; tumour incidence from weaning to month 12 recorded

Wislocki et al. (1986)

>99%

Rat; CD; 31–33 f; weanling

i.p., 0, 10 µmol (2.9 mg)/kg bw, thrice weekly

4 weeks;
76–78 weeks

Malignant fibrous histiocytomas:
0/31, 29/33
(P < 0.0001);
leukaemia: f 0/31, 7/33**;
mammary adenocarcinomas:
f 3/31, 14/33***

+

No effect on body weight, but average survival 236 days versus 535 days in controls due to local tumour formation; solvent DMSO

King (1988); Imaida et al. (1991a)

7-Nitrobenz[a]anthracene

           

>99%

Mouse; CD-1;
25–28 m and 31–33 f; newborn

i.p., total dose 0, 2800 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at day 1, 8 and 15 injected; 12 months

Liver tumours:
m 2/28, 7/25*; f 0/31, 0/33

+

54–85% survived to weaning (no further data on toxicity); vehicle DMSO; tumour incidence from weaning to month 12 recorded; parent PAH was more effective

Wislocki et al. (1986)

6-Nitrochrysene

           

>99%

Mouse; Crl/CD-1 (ICR)BR; 20 f; 50–55 days

Dermal, 0, 0.1 mg/mouse per day

10 days; 25 weeks

Skin tumours:
f 1/20, 12/20** (predominantly squamous cell papillomas)

+

No data on toxicity; initiation/promotion study; 10 days after initiation, 2.5 µg tetradecanoylphorbol acetate applied thrice weekly for 25 weeks (promotion); vehicle acetone

El-Bayoumy et al. (1982)

>99.8%

Rat, CD; 30 f;
30 days

Intramammary, 0, 2.04 µmol/injection, see remarks

Single application;
43 weeks

Mammary adenocarcinomas:
0/30, 15/30***;
mammary fibroadenomas: 5/30, 24/30***;
mammary spindle cell sarcomas:
0/30, 10/30***; significantly more mammary tumours on the left side compared with the right

+

No effects on body weight and survival; solvent DMSO; dose injected into tissue underlying each of the three thoracic nipples and three inguinal nipples (only left side, right side DMSO injection)

El-Bayoumy et al. (1993)

>99.9%

Mouse; BLU:Ha; control 91 m and 101 f, treated 26 m and 22 f; newborn

i.p., total dose 0, 7.7 µg/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 26 weeks

Total no. of mice with lung tumours 20/192, 32/48***; also significant increase in no. of lung tumours/mouse

+

No data on toxicity; solvent DMSO; lung tumours studied; no statistical evaluation for each sex; parent PAH revealed no carcinogenic effect

Busby et al. (1989)

>99%

Mouse; CD-1;
9–45 m and 11–34 f; newborn

i.p., total dose 0, 700, 2800 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 12 months

Liver adenomas and carcinomas:
m 2/28, 5/45 (2nd control), 25/33*, 3/9*; f 0/31, 0/34 (2nd control), 9/40*, 3/11*;
lung adenomas and carcinomas:
m 1/28, 4/45 (2nd control), 28/33*, 7/9*; f 0/31, 2/34 (2nd control), 36/40*, 9/11*;
malignant lymphomas:
m 1/28, 0/45 (2nd control), 6/33*, 3/9*; f 1/31, 0/34 (2nd control), 9/40*, 4/11*

+

54–85% survived to weaning, except high-dose group (25–30%); death occurred mainly between last injection and weaning (no further data); vehicle DMSO; two vehicle-treated control groups; tumour incidence from weaning to month 12 recorded; the parent PAH was less effective

Wislocki et al. (1986)

Purity checked by different methods (no further data)

Mouse; BLU:Ha; 22–23 m and 15–29 f; newborn

i.p., total dose 0, 38.5, 189 µg/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 26 weeks

Lung tumours #:
m 3/22, 22/22, 23/23;
f 1/15, 29/29, 21/21;
also increase in no. of lung tumours/mouse; in high-dose group, significant increase in lymphatic system tumours (no further data)

+

A few mice died 1st 3 months (no further data on toxicity); solvent DMSO; mainly lung tumours studied; no statistical evaluation for each sex

Busby et al. (1985)

Purity checked (no further data)

Mouse, Swiss-Webster BLU-Ha; 23–38 m and 24–46 f; newborn

i.p., total dose 0, 100, 700 nmol/mouse (0, 27.3, 191 µg/mouse)

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 32 weeks

Lung tumours:
m 7/38, 23/23**, 37/37**;
f 3/28, 24/24**, 21/21**;
liver tumours:
m 0/38, 15/23**, 31/37**;
f 0/28, 1/24, 1/46

+

No data on toxicity; solvent DMSO; liver and lung tumours studied; similar results with the metabolites trans-1,2-dihydro-1,2-dihydroxy-6-nitrochrysene and trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene; 6-nitroso- and 6-aminochysene not effective (equimolar doses)

El-Bayoumy et al. (1989a)

>99.5%

Mouse, Crj:CD-1(ICR);
control 22 m and 26 f, treated 9 m and 11 f; newborn

i.p., total dose 0, 1.4 µmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 24 weeks

Lung adenomas:
m 0/22, 9/9***;
f 0/26, 11/11***

+

No data on toxicity; solvent DMSO

Imaida et al. (1992)

n.g.

Mouse; CD-1;
control 51 m, treated 21 m; newborn

i.p., total dose 0, 0.25 µmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 12 months

Liver tumours #:
m 6/51, 21/21

+

No data on toxicity; solvent DMSO; only liver tumours evaluated; sequence analysis of mutations performed

Manjanatha et al. (1996)

>99%

Mouse; CD-1;
control 20 m, treated 16 m; newborn

i.p., total dose 0, 100 or 400 nmol/mouse

3 applications on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 1/20, 15/16***, 11/16***; liver carcinomas: 2/20, 14/16***, 15/16***; lung adenomas: 4/20, 16/16***, 16/16***; lung carcinomas: 0/20, 3/16, 10/16***

+

Mortality not compound related (no further data)

von Tungeln et al. (1999a)

n.g.

Mouse; B6C3F1;
control 18 m, treated 24 m; newborn

i.p., total dose 0, 0.3 µmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 12 months

Liver tumours #:
m 4/18, 24/24

+

No data on toxicity; solvent DMSO; only liver tumours evaluated; sequence analysis of mutations performed

Manjanatha et al. (1996)

Purified by recrystallization

Mouse; B6C;
control 4 m and 3 f, treated 9 m and 7 f; 9 weeks

i.p., 0, 2.2 µmol/mouse (20 mg/kg bw), every 2nd week

6 weeks;
25 weeks

Lung adenomas:
m 0/4, 6/9*, f 0/3, 3/7;
lung carcinomas: m 0/4, 0/9; f 0/3, 4/7*

+

No data on toxicity; solvent DMSO; transgenic mice used, DNA analysis performed

Ogawa et al. (1996)

>99%

Mouse; B6C3F1; 18–23 m; newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 12 months

Liver adenomas: 2/18, 19/19***
liver carcinomas: 0/18, 14/19***;
lung tumours: 1/18, 11/19***

+

No data on toxicity; solvent DMSO; caloric restriction inhibits tumour formation

Fu et al. (1994a)

>99%

Mouse; B6C3F1; 18–23 m; 8 days

i.p., total dose 0, 400 nmol/mouse

2 applications: 3/7 and 4/7 of the total dose at days 8 and 15 injected; 12 months

Liver adenomas: 2/18, 21/21***
liver carcinomas: m 0/18, 21/21***

+

No data on toxicity; solvent DMSO; caloric restriction inhibits tumour formation

Fu et al. (1994a)

>99%

Mouse, B6C3F1;
control 19 m, treated 24 m; newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 2/19, 19/19***; liver carcinomas: 0/19, 14/19***; lung tumours: 1/19, 11/19**

+

Mortality: 0%, 21%; no effects on body weight; solvent DMSO; only liver and lung tumours studied

von Tungeln et al. (1994b, 1999b)

>99%

Mouse, B6C3F1;
control 24 m, treated 22 m; newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 2/24, 5/22; liver carcinomas: 1/24, 20/22***; lung tumours: 0/24, 13/22***

+

Mortality: 0%, 8%; no effects on body weight; solvent DMSO; only liver and lung tumours studied

von Tungeln et al. (1999b)

n.g.

Mouse; C57BL/6; 15–19 m; 8 days

i.p., 0, 7.8, 15.7 mg/kg bw

2 applications at days 8 and 15; 12 months

Liver tumour incidence: 0/17, 15/18*, 16/18*

+

No data on toxicity; solvent DMSO; further test groups (sacrificed after 7 months after exposure) indicate that a deficiency in the p53 suppressor gene does not accelerate tumorigenesis

Dass et al. (1999)

>99.5%

Rat; Crj:CD (SD); control 40 m and 29 f, treated 31 m and 32 f; newborn

i.p., total dose 0 or 14.8 µmol/rat

5 applications: 1/37, 2/37, 4/37, 10/37, 20/37 of the total dose at days 1, 8, 15, 22, 29 injected;
32 weeks

Colon adenocarcinomas:
m 0/40, 5/31*, f 0/29, 2/32;
colon dysplasia/adenomas:
m 0/40, 7/31**; f 0/29, 14/32***

+

No data on toxicity; solvent DMSO

Imaida et al. (1992)

1-Nitrobenzo[a]pyrene

           

Purified (no further data)

Rat; F344/DuC1;
control 20 m, treated 10 m; 6 weeks

s.c., 0, 0.5, 1, 2 mg/rat (~0, 2.5, 5, 10 mg/kg bw)

Single injection;
100 weeks

Local subcutaneous tumours:
0/20, 0/10, 0/10, 1/10;
incidence of other tumours not significantly increased

(–)

No effect on body weight (no data on mortality); TS in beeswax–tricaprylin suspended; positive control

Horikawa et al. (1998)

>99%

Mouse; B6C3F1;
control 19 m, treated 23 m;
newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 2/19, 6/23; liver carcinomas: 0/19, 0/23; lung tumours: 1/19, 0/23

(–)

Mortality: 0%, 4%; no effects on body weight; solvent DMSO; only liver and lung tumours studied; carcinogenic effects not significant

von Tungeln et al. (1994b, 1999b)

>99%

Mouse; CD-1;
control 20 m, treated 23 m; newborn

i.p., total dose 0, 100 nmol/mouse

3 applications on days 1, 8 and 15;
12 months

No significant carcinogenic effects in liver and lung

(–)

Mortality not compound related (no further data); low dose tested; metabolite 1-nitrobenzo[a]pyrene-trans-7,8-dihydrodiol also negative

von Tungeln et al. (1999a)

2-Nitrobenzo[a]pyrene

           

>99%

Mouse, B6C3F1;
control 19 m, treated 21 m;
newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 2/19, 21/21***; liver carcinomas: 0/19, 15/21***; lung tumours: 1/19, 4/21

+

Mortality: 0%, 9%; no effects on body weight; solvent DMSO; only liver and lung tumours studied

von Tungeln et al. (1994b, 1999b)

3-Nitrobenzo[a]pyrene

           

Purified (no further data)

Rat; F344/DuC1;
control 20 m, treated 10 m; 6 weeks

s.c., 0, 0.5, 1, 2 mg/rat (~0, 2.5, 5, 10 mg/kg bw)

Single injection;
100 weeks

Local subcutaneous tumours:
0/20, 0/10, 0/10, 1/10;
incidence of other tumours not significantly increased

(–)

No effect on body weight (no data on mortality)
TS in beeswax–tricaprylin suspended; positive control

Horikawa et al. (1998)

>99%

Mouse; B6C3F1;
control 19 m, treated 24 m;
newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 2/19, 5/24; liver carcinomas: 0/19, 1/24; lung tumours: 1/19, 0/24

(–)

Mortality: 0%, 0%; no effects on body weight; solvent DMSO; only liver and lung tumours studied

von Tungeln et al. (1994b, 1999b)

>99%

Mousel CD-1;
control 20 m, treated 21 m;
newborn

i.p., total dose 0, 100 nmol/mouse

3 applications on days 1, 8 and 15; 12 months

No significant carcinogenic effects in liver and lung

(–)

Mortality not compound related (no further data); low dose tested; metabolite 3-nitrobenzo[a]pyrene-trans-7,8-dihydrodiol also negative

von Tungeln et al. (1999a)

6-Nitrobenzo[a]pyrene

           

>99%

Mouse; Crl/CD-1 (ICR)BR; 20 f; 50–55 days

Dermal, 0, 0.1 mg/mouse per day

10 days; 25 weeks

No significant effect on skin tumour formation

(–)

No data on toxicity; initiation/promotion study; 10 days after initiation was complete, 2.5 µg tetradecanoylphorbol acetate applied thrice weekly for 25 weeks (promotion); vehicle acetone

El-Bayoumy et al. (1982)

>99%

Mouse; CD-1;
28–29 m and 31–44 f; newborn

i.p., total dose 0, 560 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 12 months

Liver tumours:
m 2/28, 8/29*; f 0/31, 0/44

+

54–85% survived to weaning (no further data on toxicity); vehicle DMSO; tumour incidence from weaning to month 12 recorded; parent PAH was more effective

Wislocki et al. (1986)

>99.9%

Mouse; BLU:Ha; control 91 m and 101 f, treated 23–24 m and 26–30 f;
newborn

i.p., total dose 0, 14, 70 µg/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 26 weeks

No effects on lung tumour formation in contrast to parent PAH (equimolar dose)

(–)

No data on toxicity; solvent DMSO; only lung tumours studied

Busby et al. (1989)

1-Nitrobenzo[e]pyrene

           

>99%

Mouse; B6C3F1;
control 19 m, treated 24 m; newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 2/19, 4/24; liver carcinomas: 0/19, 1/24; lung tumours: 1/19, 0/24

(–)

Mortality: 0%, 0%; no effects on body weight; solvent DMSO; only liver and lung tumours studied

von Tungeln et al. (1994b, 1999b)

3-Nitrobenzo[e]pyrene

           

>99%

Mouse, B6C3F1;
control 19 m, treated 24 m;
newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 2/19, 4/24; liver carcinomas: 0/19, 0/24; lung tumours: 1/19, 1/24

(–)

Mortality: 0%, 0%; no effects on body weight; solvent DMSO; only liver and lung tumours studied

von Tungeln et al. (1994b, 1999b)

1,6-Dinitrobenzo[a]pyrene

           

n.g.

Rat; F344/DuCrj;
control 20 m, treated 11 m; 6 weeks

s.c., 0, 8, 40, 200, 1000 µg/rat

Single injection;
100 weeks

No subcutaneous tumours at the site of injection in contrast to the parent PAH

(–)

No data on toxicity; TS dissolved in a mixture of equal volumes beeswax and tricaprylin

Horikawa et al. (1993); Tokiwa et al. (1994)

99.98%

Rat; F344/DuC1;
control 20 m, treated 11 m; 6 weeks

s.c., 0, 8, 40, 200, 1000 µg/rat (~0, 0.04, 0.2, 1, 5 mg/kg bw)

Single injection;
100 weeks

Subcutaneous malignant fibrous histiocytoma:
0/20, 0/11 0/11, 0/11;
incidence of other tumours not significantly increased

(–)

No effect on body weight (no data on mortality); TS suspended in beeswax–tricaprylin; positive control

Horikawa et al. (1998)

3,6-Dinitrobenzo[a]pyrene

           

n.g.

Rat; F344/DuCrj; 20–21 m; 6 weeks

s.c., 0, 8, 40, 200, 1000 µg/rat

Single injection;
100 weeks

Subcutaneous malignant fibrous histiocytomas at the site of injection #: 0/20, 1/21, 5/21, 8/21, 14/20

+

No data on toxicity; TS dissolved in a mixture of equal volumes beeswax and tricaprylin

Horikawa et al. (1993); Tokiwa et al. (1994)

99.99%

Rat; F344/DuC1;
control 20 m, treated 20–21 m; 6 weeks

s.c., 0, 8, 40, 200, 1000 µg/rat (~0, 0.04, 0.2, 1, 5 mg/kg bw)

Single injection;
100 weeks

Subcutaneous malignant fibrous histiocytoma:
0/20, 1/21 6/21*, 9/20**, 14/20**; incidence of other tumours not significantly increased

+

Dose-dependent reduction in body weight in all treatment groups, presumably due to tumour formation (no data on mortality);
TS suspended in beeswax–tricaprylin

Horikawa et al. (1998)

7-Nitrodibenz[a,h]anthracene

         

n.g.

Mouse, B6C3F1;
n.g. (only m used); newborn

i.p., total dose 0, 400 nmol/mouse

3 applications on days 1, 8 and 15; n.g.

Increased incidence of liver tumours (no further data); parent PAH more active (100% liver tumour incidence)

(+)

Solvent DMSO; only liver tumours studied; data from abstract, no further information available

von Tungeln et al. (1994b)

n.g.

Mouse, B6C3F1;
24 m; newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose at days 1, 8 and 15 injected; 12 months

Liver adenomas: 2/24, 12/24**;
liver carcinomas: 1/24, 2/24; lung tumour incidence not increased

+

No treatment-related effect on mortality or body weight; solvent DMSO; parent PAH more effective

Fu et al. (1998b)

9-Nitrodibenz[a,c]anthracene

         

>99%

Mouse, B6C3F1;
24 m; newborn

i.p., total dose 0, 400 nmol/mouse

3 applications: 1/7, 2/7 and 4/7 of the total dose on days 1, 8 and 15, respectively;
12 months

Liver adenomas: 2/24, 2/24; liver carcinomas: 1/24, 0/24; lung tumours: 0/24, 0/24

(–)

Mortality: 0%, 0%; no effects on body weight; solvent DMSO; only liver and lung tumours studied

von Tungeln et al. (1994b, 1999b)

3-Nitroperylene

           

>99%

Mouse; Crl/CD-1 (ICR)BR;
20 f; 50–55 days

Dermal, 0, 0.1 mg/mouse per day

10 days; 25 weeks

Local skin tumours:
f 1/20, 8/19** (predominantly squamous cell papillomas)

+

No data on toxicity; initiation/promotion study; 10 days after initiation was complete, 2.5 µg tetradecanoylphorbol acetate applied thrice weekly for 25 weeks (promotion); vehicle acetone

El-Bayoumy et al. (1982)

a

All studies used vehicle-treated controls unless otherwise stated; m = male; f = female; DMSO = dimethyl sulfoxide; # = no data on statistical evaluation; MTD: maximum tolerated dose; n.g. = not given; TS: test substance; significant compared with control, * (P < 0.05), ** (P < 0.01), *** (P < 0.001); n.g.= data not given.

b

Age of animals at start of the experiment.

c

The Task Group evaluated the strength of each of the in vivo studies based on the following criteria: a) numbers of animals in each group, b) duration of study, c) adequate dosing (e.g., MTD in negative studies) and d) appropriateness of controls. Specific comments on the strengths and weaknesses of the studies are included in the table. The Task Group recognized that some of the studies did not report statistical evaluations, and this is noted in the table:

 

+, study positive and acceptable to Task Group (data are not from an abstract, more than 10 animals per group per sex, and included an appropriate control) for assessment of carcinogenicity

 

(+), study claimed positive results; however, the Task Group considered the data "less than acceptable" for assessment (data from abstract, less than 10 animals per group per sex and/or no control)

 

–, although none of the studies met this criterion, study negative and acceptable to Task Group (data are from manuscript, >25 surviving (2 years) animals per group per sex, and included a control) for assessment of carcinogenicity

 

(–), study claimed negative results; however, the Task Group considered the data "less than acceptable" for assessment (data from abstract, insufficient number of animals per group per sex and/or no control).

No effect on survival or body weight was observed in rats after repeated s.c. application (twice weekly for 10 weeks) of 20 mg/kg bw (Ohgaki et al., 1985).

7.2.5 1,3-Dinitropyrene

Rats treated via gavage with about 3 mg/kg bw 3 times weekly for 4 weeks showed no effects on body weight or survival (King, 1988).

Local effects preceding tumour formation were noted in rats after repeated s.c. injection of 0.2 mg per animal. Rats showed ulcer and scar formation at the site of injection (Ohgaki et al., 1984).

7.2.6 1,6-Dinitropyrene

Imaida et al. (1991a) reported no effects on body weight or survival in a gavage study on rats. Rats were treated 3 times per week for 4 weeks at a dose level of 3 mg/kg bw.

7.2.7 1,8-Dinitropyrene

Ulcer and scar formation at the site of injection were noted in rats after repeated s.c. injection of 0.2 mg per animal (Ohgaki et al., 1984).

7.2.8 6-Nitrochrysene

In the mouse newborn assay (Wislocki et al., 1986), the total dose of 2.8 µmol per mouse resulted in increased mortality shortly after the exposure period.

7.2.9 1- and 3-nitrobenzo[a]pyrene

Subcutaneous bolus injection of 2.5–10 mg/kg bw did not affect body weight gain in rats (Horikawa et al., 1998).

7.2.10 1,6-Dinitrobenzo[a]pyrene

In the same study (Horikawa et al., 1998), s.c. bolus injection of 0.04–5 mg/kg bw also resulted in no changes in body weight gain in rats.

7.3 Skin and eye irritation and sensitization

No studies are available.

7.4 Reproductive toxicity, embryotoxicity and teratogenicity

No studies are available.

7.5 Mutagenicity and related end-points

The Task Group was aware that the calculations for mutagenic potency could only be estimated and were not exact, due to the following shortcomings: (1) uncontrolled interlaboratory variation among protocols; (2) absence of information about the mutability of the bacterial strains used in several laboratories (lack of use of different positive controls); and (3) differences in the purity of the nitroPAH (e.g., contamination by dinitroPAHs). The Task Group recognized that, historically, contamination of a mononitroPAH with small amounts of dinitroPAHs has dramatically affected the reported mutagenicity of the particular mononitroPAH (for discussion, see Rosenkranz & Mermelstein, 1983; Tokiwa & Ohnishi, 1986). For instance, the average mutagenicity of 1-nitropyrene (690 revertants/nmol in TA98; Table 40) is approximately 400-fold less than the mutagenicity of 1,6- or 1,8-dinitropyrene (average approximately 235 000 revertants/nmol; Table 40). Therefore, considering the mutagenicity of highly purified 1-nitropyrene was reported as 490 revertants/nmol in Tokiwa & Ohnishi (1986) and that for the 1,6- and 1,8-dinitropyrene averaged 216 000 revertants/nmol in TA98, the mutagenicity of 1400 revertants/nmol for 1-nitropyrene in TA98 reported by Ho et al. (1981) could be due to as little as 0.4% contamination with dinitropyrenes. Historically, the use of strains TA98NR– (nitroreductase deficient) and TA98AT– (O-acetyltransferase deficient) has allowed an assessment of the relative level of contamination.

Table 40. Summary of mutagenic potency of nitroPAHs in Salmonella microsome assay

Substancea (referencesb)

Revertants per nmol nitroPAH in Salmonella typhimurium strains with and without metabolic activation (MA)c

Predominant type of histidine mutatione

TA98d

TA100d

TA1535d

TA1537d

TA1538d

–MA

+MA

–MA

+MA

–MA

+MA

–MA

+MA

–MA

+MA

1-Nitronaphthalene (a–l)

0–1.0f
0.27
(n = 10)

0.1–2.7
0.99
(n = 5)

0.8–9.9
3.3
(n = 9)

0.55–6.3
2.2
(n = 5)

0–0.05
0.016
(n = 3)

0.04–0.07
0.055
(n = 2)

0–<0.01
0
(n = 2)

0
0
(n = 2)

0.05–0.24
0.12
(n = 4)

0.13
0.13
(n = 1)

base pair substitution

2-Nitronaphthalene (b–f, h, j, m–z)

0.12–1.0g
0.46
(n = 8)

0.09–0.69
0.39
(n = 2)

1.1–8.7
3.6
(n = 9)

1.1–4.4
3.2
(n = 3)

0.3–3.7
1.6
(n = 4)


1.6
(n = 1)

0.03–0.15
0.09
(n = 2)

 

0.02–0.4
0.12
(n = 4)


0.045
(n = 1)

base pair substitution

1,3-Dinitronaphthalene (h, s)

0.89–0.9
0.89
(n = 2)

 

6.7–7.3
7.0
(n = 2)

 

0.15–3.1
1.6
(n = 2)

 

0.7–0.76
0.73
(n = 2)

     

base pair substitution

1,5-Dinitronaphthalene (h, k, s, aa)

2.2–4.0
3.2
(n = 4)


2.2
(n = 1)

4.7–14
8.7
(n = 3)


8.3
(n = 1)

0h

 


0.22
(n = 2)

     

base pair substitution

1,8-Dinitronaphthalene (h, k, aa)

4.4–7.9
6.1
(n = 2)


7.5
(n = 1)


13
(n = 1)


6.8
(n = 1)

0h

 

0h

     

base pair substitution

2,3,5-Trinitronaphthalene (bb, cc)

32–65
48
(n = 2)

 


31
(n = 1)

 


0.6
(n = 1)

 


17
(n = 1)

 


94
(n = 1)


99
(n = 1)

frameshift

1,3,6,8-Tetranitronaphthalene (h, cc)

0.24–30
15
(n = 2)

 


24
(n = 1)

 


0.6
(n = 1)

 


6
(n = 1)

 


33
(n = 1)


8
(n = 1)

_* i

3-Nitroacenaphthene (l)


10
(n = 1)

 


26
(n = 1)

 

<0.01

 


0.8
(n = 1)

 


3.8
(n = 1)

 

base pair substitution

5-Nitroacenaphthene (a, g, k, l, r, dd–hh)

1.6–30
7.2
(n = 8)

4.3–25
16
(n = 8)

2.0–40
17
(n = 8)

14–71
29
(n = 9)

<0.01–0.6
0.26
(n = 3)

0.27–0.32
0.29
(n = 2)

0.1–1.54
0.63
(n = 4)

1.1–1.2
1.2
(n = 2)

0.78–8.3
3.1
(n = 4)

6.1–27
17
(n = 2)

_* i

2-Nitrofluorene (e–g, j–l, o, r, t, u–x, z, aa, ii, jj, ll–tt, vv–zz)

2.8–430
57
(n = 27)

6–72
25
(n = 8)

4.6–200
36
(n = 11)

7.5–35
22
(n = 5)

0–0.01
0.005
(n = 2)

 

0.04–1
0.61
(n = 3)

 

6.5–270
65
(n = 10)

15–35
28
(n = 3)

_* i

3-Nitrofluorene (yy)

7.4
(n = 1)

                   

2,5-Dinitrofluorene (g, xx)

1600–2500
2000
(n = 2)


770
(n = 1)


200
(n = 1)


60
(n = 1)

       


1700
(n = 1)

 

frameshift

2,7-Dinitrofluorene (g, k, ll, pp, qq, xx, A)

470–5200
2000
(n = 7)

38–2600
1100
(n = 3)

6–174
100
(n = 4)

14–90
52
(n = 2)

0h

 

290–310
300
(n = 2)

 

350–3000
2100
(n = 3)

 

frameshift

2-Nitroanthracene (j, B)

890–1600
1200
(n = 2)


64
(n = 1)


1100
(n = 1)

 


790
(n = 1)

     


840
(n = 1)

 

_* i

9-Nitroanthracene (g, k, p, qq, ss, tt, C, D, F)

0.01–2.7
0.66
(n = 7)

0.07–2.0
0.59
(n = 6)

0.89–3.6
1.9
(n = 3)

0.89–3.0
2.0
(n = 3)

           

base pair substitution

9,10-Dinitroanthracene (ff)

0
(n = 1)

<1

               

_* i

1-Nitrophenanthrene (G)

110
(n = 1)

 

330
(n = 1)

             

base pair substitution

2-Nitrophenanthrene (j, ll)

130–330
230
(n = 2)

 


62
(n = 1)

 


2.7
(n = 1)

     


140
(n = 1)

 

frameshift

3-Nitrophenanthrene (G)

330
(n = 1)

 

620
(n = 1)

             

_* i

9-Nitrophenanthrene (ll, vv, D, G)

<0.45–290
180
(n = 3)


44
(n = 1)

<0.89–981
710
(n = 2)

1.6–22
12
(n = 1)

           

base pair substitution

1,5-Dinitrophenanthrene (G)

4
(n = 1)

 

5
(n = 1)

             

_* i

1,6-Dinitrophenanthrene (G)

120
(n = 1)

 

1200
(n = 1)

             

base pair substitution

1,10-Dinitrophenanthrene (G)

0.8
(n = 1)

 

2
(n = 1)

             

base pair substitution

2,6-Dinitrophenanthrene (G, H)

730
(n = 2)

 

1800
(n = 1)

             

base pair substitution

2,7-Dinitrophenanthrene (ll)

3900
(n = 1)

                 

_* i

2,9-Dinitrophenanthrene (G)

3
(n = 1)

 

590
(n = 1)

             

base pair substitution

2,10-Dinitrophenanthrene (G)

120
(n = 1)

 

240
(n = 1)

             

base pair substitution

3,5-Dinitrophenanthrene (G)

70
(n = 1)

 

240
(n = 1)

             

base pair substitution

3,6-Dinitrophenanthrene (G)

90
(n = 1)

 

2100
(n = 1)

             

base pair substitution

3,10-Dinitrophenanthrene (G)

110
(n = 1)

 

1400
(n = 1)

             

base pair substitution

4,9-Dinitrophenanthrene (G)

1
(n = 1)

 

2
(n = 1)

             

base pair substitution

4,10-Dinitrophenanthrene (G)

5.3
(n = 1)

 

160
(n = 1)

             

base pair substitution

1,5,9-Trinitrophenanthrene (G)

170
(n = 1)

 

110
(n = 1)

             

_* i

1,5,10-Trinitrophenanthrene (G)

14
(n = 1)

 

4
(n = 1)

             

frameshift

1,6,9-Trinitrophenanthrene (G)

360
(n = 1)

 

720
(n = 1)

             

base pair substitution

1,7,9-Trinitrophenanthrene (G)

1000
(n = 1)

 

1300
(n = 1)

             

_* i

2,5,10-Trinitrophenanthrene (G)

63
(n = 1)

 

370
(n = 1)

             

base pair substitution

2,6,9-Trinitrophenanthrene (G)

180
(n = 1)

 

1200
(n = 1)

             

base pair substitution

3,5,10-Trinitrophenanthrene (G)

50
(n = 1)

 

44
(n = 1)

             

_* i

3,6,9-Trinitrophenanthrene (G)

530
(n = 1)

 

1300
(n = 1)

             

base pair substitution

1-Nitrofluoranthene (l, gg, D, I)

74–540
370
(n = 4)


170
(n = 1)

124–988
680
(n = 3)

 

<0.1

 


84
(n = 1)

 


180
(n = 1)

 

_* i

2-Nitrofluoranthene (zz, I–L)

61–1000
400
(n = 4)

89–760
270
(n = 4)

               

_* i

3-Nitrofluoranthene (f, k, l, aa, qq, vv, ww, D, H–P)

1400–14 000
5900
(n = 17)

31–590

250
(n = 7)

747–5800

2400
(n = 8)

31–640

200
(n = 5)

<1–25

25
(n = 1)



1.2
(n = 1)

46–980

350
(n = 4)



3.6
(n = 1)

810–4500

2400
(n = 6)

12–338

130
(n = 3)

frameshift

7-Nitrofluoranthene (l, gg, D, I)

4–544
170
(n = 4)


24
(n = 1)

123–990
420
(n = 3)

 

<0.1

 


11
(n = 1)

 


14
(n = 1)

 

base pair substitution

8-Nitrofluoranthene (l, D, I, L, M)

2200–18 000
10 000
(n = 4)



460
(n = 1)

400–750

570
(n = 2)



350
(n = 1)

<25


(n = 2)

 

<123–210

210
(n = 1)

 

9900–14 000
12 000
(n = 2)

 

frameshift

1,2-Dinitrofluoranthene (I, L)

1000–1300
1100
(n = 2)

180–190
180
(n = 2)

               

_* i

1,3-Dinitrofluoranthene (I)

2600
(n = 1)

470
(n = 1)

               

_* i

2,3-Dinitrofluoranthene (L)

420
(n = 1)

80
(n = 1)

               

_* i

2,4-Dinitrofluoranthene (L)

6000
(n = 1)

550
(n = 1)

               

_* i

2,5-Dinitrofluoranthene (L)

210
(n = 1)

540
(n = 1)

               

_* i

3,4-Dinitrofluoranthene (H, O–Q)

4100–4500
4300
(n = 3)


28
(n = 1)

3200–3300
3200
(n = 2)


24
(n = 1)

0h

0h


26
(n = 2)


3.6
(n = 1)

440–610
520
(n = 3)


15
(n = 1)

_* i

3,7-Dinitrofluoranthene (H, O, P)



123 000
(n = 2)



100
(n = 1)

25 000–31 000
28 000
(n = 2)



76
(n = 1)

0h

0h

4900–6800

5800
(n = 2)



1.2
(n = 1)

13 000–19 000
16 000
(n = 2)



20
(n = 1)

frameshift

3,9-Dinitrofluoranthene (H, O, P)


100 000
(n = 2)


130
(n = 1)


21 000
(n = 2)


100
(n = 1)

0h

0h

2800–3200
3000
(n = 2)


12
(n = 1)


31 000
(n = 2)


68
(n = 1)

frameshift

1,2,4-Trinitrofluoranthene (L)

3600
(n = 1)

140
(n = 1)

               

_* i

1,2,5-Trinitrofluoranthene (L)

1400
(n = 1)

69
(n = 1)

               

_* i

2,3,5-Trinitrofluoranthene (L)

2700
(n = 1)

720
(n = 1)

               

_* i

1-Nitropyrene (d–f, k, o, p, q, s, v–z, aa, ll, qq, ss, vv, ww, B, D, E, H, J, M, N, P, R–Z, AA–OO)

50–4400
690
(n = 41)

17–1700
370
(n = 14)

41–910
210
(n = 15)

29–98
55
(n = 5)

0h

0h

1–500
160
(n = 4)

 

70–1200
250
(n = 10)

5–87
46
(n = 2)

frameshift

2-Nitropyrene (ll, oo, pp, D)

1800–2800
2400
(n = 4)


170
(n = 1)

390–740
510
(n = 3)


260
(n = 1)

0h

     


3800
(n = 1)

 

frameshift

4-Nitropyrene (d, H, QQ)

2500–2700
2600
(n = 2)

 


580
(n = 1)

             

frameshift

1,3-Dinitropyrene (e, f, q, s, aa, ll, qq, vv, H, M, P, S, FF, HH–KK, RR, SS)

29 000–16 0000
93 000
(n = 12)



4500
(n = 1)

8300–37 000
22 000
(n = 6)



5400
(n = 1)

0h

 

3000–13 000
8000
(n = 2)

 

11 000–79 000
35 000
(n = 3)

 

frameshift

1,6-Dinitropyrene (e, f, k, q, s, vv, aa, B, H, M, P, S, U, W, Z, EE, FF, HH–KK, MM, RR–TT)

25 000–250 000
120 000
(n = 15)

380–37 000
14 000
(n = 3)

4300–33 000
15 000
(n = 8)

61–1300

760
(n = 3)

0h

 

9200–33 000
18 000
(n = 3)

 

12 000–61 000
24 000
(n = 4)

 

frameshift

1,8-Dinitropyrene (e, f, k, q, s, w–z, aa, nn, qq, rr, vv, zz, H, M, P, S, W, Z, BB, FF, HH–KK, UU, RR, SS)

55 000–2 000 000
350 000
(n = 18)

120–77 000
39 000
(n = 2)

5500–
55 000
25 000
(n = 7)

42–4400

2200
(n = 2)

0h

 

12 000–21 000
16 000
(n = 2)

 

9900–35 000
25 000
(n = 3)

 

frameshift

2,7-Dinitropyrene (ll)

38 000
(n = 1)

                 

_* i

1,3,6-Trinitropyrene (e, f, s, vv, FF, HH–KK)

17 000–240 000
61 000
(n = 6)

 

1100–40 000
12 000
(n = 4)

 

0h

 



20 000
(n = 1)

 

16 000–20 000
18 000
(n = 2)

 

frameshift

1,3,6,8-Tetranitropyrene (f, q, s, vv, FF, HH–KK)

3200–84 000
23 000
(n = 6)

 

100–13 000
3500
(n = 4)

 

0h

 



3300
(n = 1)

 



1350
(n = 1)

 

frameshift

7-Nitrobenz[a]anthracene (D, QQ, VV)

<1


1.4
(n = 1)

0.55–<1
0.55
(n = 1)


11
(n = 1)

           

base pair substitution

2-Nitrochrysene (gg, PP)


2100
(n = 1)

27–450
240
(n = 2)


440
(n = 1)

100–350
220
(n = 2)

           

frameshift

5-Nitrochrysenej (D)

<0.55

2.7
(n = 1)

2.7
(n = 1)

5.5
(n = 1)

           

base pair substitution

6-Nitrochrysene (d, k, qq, vv, ww, D, VV–XX)

5–270
66
(n = 7)

12–110
42
(n = 5)

38–270
130
(n = 5)

40–410
160
(n = 5)

       


140
(n = 1)


12
(n = 1)

base pair substitution

3-Nitrobenzo(k)-fluoranthene (l)

580
(n = 1)

 

990
(n = 1)

 

<1

 

35
(n = 1)

 

150
(n = 1)

 

_* i

7-Nitrobenzo(k)-fluoranthene (l)

<0.01

 

<0.01

 

<0.01

 

<0.01

 

<0.01

 

_* i

1-Nitrobenzo[a]pyrene (d, rr, H, QQ, YY, ZZ, aaa, bbb)

73–840
590
(n = 7)

150–920
470
(n = 4)

34–2380
980
(n = 3)


14
(n = 1)

           

_* i

2-Nitrobenzo[a]pyrene (PP)

2500
(n = 1)

610
(n = 1)

190
(n = 1)

720
(n = 1)

           

frameshift

3-Nitrobenzo[a]pyrene (d, rr, H, QQ, YY, aaa–fff)

93–1900
890
(n = 9)

250–861
550
(n = 5)

28–3100
960
(n = 4)

26–41
33
(n = 2)

           

_* i

6-Nitrobenzo[a]pyrene (k, rr–tt, D–F, VV, YY, bbb, ggg, hhh)

<1–68
33k
(n = 3)

30–440
170
(n = 7)

<1–59
22k
(n = 3)

19–440
160
(n = 6)

   


7
(n = 1)


7
(n = 1)

   

_* i

1-Nitrobenzo[e]pyrene (D, Y, iii)

1.3–39
19
(n = 3)

11–45
28
(n = 2)


44
(n = 1)


50
(n = 1)

<1.5

 


50
(n = 1)

 


6
(n = 1)

 

_* i

3-Nitrobenzo[e]pyrene (D, Y, QQ)

890–3100
1800
(n = 3)


1000
(n = 1)


30
(n = 1)


210
(n = 1)

<1.5

 

<30

 

<30

 

frameshift

4-Nitrobenzo[e]pyrene (d)

980
(n = 1)

 

300
(n = 1)

             

frameshift

1,3-Dinitrobenzo[e]pyrene (d)

8500
(n = 1)

                 

_* i

1,6-Dinitrobenzo[e]pyrene (Y)

98
(n = 1)

92
(n = 1)

               

_* i

1,8-Dinitrobenzo[e]pyrene (Y)

0h

0h

               

_* i

3,6-Dinitrobenzo[a]-pyrene (H)

140 000
(n = 1)

                 

_* i

3-Nitrodibenz[a,h]-anthracene (PP)

320
(n = 1)

960
(n = 1)

<1

560
(n = 1)

           

_* i

7-Nitrodibenz[a,h]-anthracene (d)

<1

 

<1

             

_* i

9-Nitrodibenz[a,c]-anthracene (d)

<1

 

<1

             

_* i

3-Nitroperylene (p, tt, D, E, bbb, ggg)

4.4–30
51
(n = 3)

260–3600
1700
(n = 4)


59
(n = 1)

890–1000
940
(n = 2)

 

0h

 


100
(n = 1)


30
(n = 1)

110–500
300
(n = 2)

_* i

3,6-Dinitroperylene (bbb)

1500
(n = 1)

1600
(n = 1)

270
(n = 1)

890
(n = 1)

           

_* i

3,7-Dinitroperylene (bbb)

1600
(n = 1)

<68

240
(n = 1)

<34

           

frameshift

1-Nitrocoronene (D)

2.8
(n = 1)

6.9
(n = 1)

1.4
(n = 1)

3.5
(n = 1)

           

_* i

4-Nitrobenzo[ghi]-perylene (gg)

 

1900
(n = 1)

 

49
(n = 1)

           

frameshift

7-Nitrobenzo[ghi]-perylene (gg)

 

<0.6

 

<0.6

           

_* i

a

Substances with adequate database (>5 data with at least one strain, at least 3 data with the strain showing the maximal mutagenic activity) marked by bold-typed substance name.

b

Studies used for calculation of means are as follows:

 

a) Dunkel et al. (1985); b) El-Bayoumy et al. (1981); c) Gupta et al. (1996); d) Jung et al. (1991); e) Klopman et al. (1984); f) Massaro et al. (1983); g) Matsuda (1981); h) McCoy et al. (1981a) ; i) Mortelmans et al. (1987); j) Scribner et al. (1979); k) Tokiwa et al. (1981b); l) Vance & Levin (1984); m) De Flora (1979); n) De Flora et al. (1984); o) Hagiwara et al. (1993); p) Ho et al. (1981); q) Löfroth (1981); r) McCann et al. (1975); s) Mermelstein et al. (1982); t) Rosenkranz & Poirier (1979); u) Simmon (1979a); v) Wang et al. (1980); w) Watanabe et al. (1989); x) Watanabe et al. (1990) ; y) Watanabe et al. (1993) ; z) Yamada et al. (1997); aa) Tokiwa et al. (1985); bb) Sorenson et al. (1983); cc) Whong & Edwards (1984); dd) McCoy et al. (1983a); ee) McCoy et al. (1983b); ff) Möller et al. (1985); gg) Rosenkranz & Mermelstein (1983); hh) Yahagi et al. (1975); ii) Castellino et al. (1978); jj) Cui et al. (1996); kk) Edenharder & Tang (1997); ll) Hirayama et al. (1988); mm) Hughes et al. (1997); nn) Jurado et al. (1994); oo) LaVoie et al. (1981); pp) McCoy et al. (1981b); qq) Pederson & Siak (1981); rr) Pitts et al. (1984); ss) Pitts et al. (1982); tt) Pitts (1983); uu) Schleibinger et al. (1989) ; vv) Sugimura & Takayama (1983); ww) Tokiwa et al. (1981a); xx) Vance et al. (1987); yy) Watanabe et al. (1997a); zz) Zielinska et al. (1987); A) Levin et al. (1979); B) Fu et al. (1986); C) Fu et al. (1985a); D) Greibrokk et al. (1984); E) Pitts et al. (1978); F) Wang et al. (1978); G) Sera et al. (1996); H) Tokiwa et al. (1994); I) Shane et al.. (1991); J) Ball et al. (1994); K) Ball et al. (1995); L) Zielinska et al. (1988) ; M) Ball & Young (1992); N) Sangaiah et al. (1996) ; O) Nakagawa et al. (1987); P) Tokiwa et al. (1986); Q) Horikawa et al. (1987); R) Ball et al. (1984b); S) Crebelli et al. (1995); T) DeMarini et al. (1996); U) El-Bayoumy & Hecht (1986); V) El-Bayoumy & Hecht (1983); W) Fifer et al. (1986a); X) Fifer et al. (1986b); Y) Fu et al. (1989); Z) González de Mejía et al. (1998); AA) Harris et al. (1984); BB) Heflich et al. (1985a); CC) King et al. (1984); DD) Lee et al. (1994); EE) Manabe et al. (1985); FF) McCoy et al. (1985a); GG) McCoy et al. (1984); HH) Mermelstein et al. (1981); JJ) Nakayasu et al. (1982); KK) Rosenkranz et al. (1980); LL) Taylor et al. (1995); MM) Tokiwa et al. (1984); NN) Urios et al. (1994); OO) Yu et al. (1991); PP) Yu et al. (1992); QQ) Fu et al. (1985b); RR) Shah et al. (1991); SS) Shane & Winston (1997); TT) Ashby et al. (1983); UU) Rosenkranz et al. (1982); VV) Fu et al. (1988b); WW) El-Bayoumy & Hecht (1984b); XX) El-Bayoumy et al. (1989b); YY) Chou et al. (1984); ZZ) Chou et al. (1986); aaa) Hass et al. (1986a); bbb) Löfroth et al. (1984); ccc) Chou et al. (1985); ddd) Fu et al. (1997); eee) Hass et al. (1984); fff) Heflich et al. (1989); ggg) Anderson et al. (1987); hhh) Fu et al. (1982); iii) Fu et al. (1988c).

c

Study results revealing no mutagenicity were not included (exception see below) if the strain was presumably not tested up to cytotoxicity; study results with no explicitness (e.g., < or > 1) not used for calculation of the mean; MA = metabolic activation (rodent liver microsomes).

d

In each of these columns, the following values are presented (separated in data with or without MA): a) range of mutagenic potency in revertants per nmol (2 or more data available), b) mean number of revertants per nmol and c) in parentheses the number n of data used for calculation of the mean.

e

Bold-typed letters indicate clearly more revertants (difference at least 10-fold) in strains detecting base pair substitutions (TA100, TA1535) relative to strains detecting frameshift mutations (TA98, TA1537, TA1538) or the reversal (comparison in dependence on metabolic activation); a difference of at least 2-fold is marked by normal-typed letters.

f

30 revertants/nmol after 90-min preincubation.

g

680 revertants/nmol after 90-min preincubation.

h

Presumably not tested up to cytotoxicity (included in the table if no other data were available).

i

_* = less than 2-fold increase.

j

Structural assignment not given.

k

In further studies, no mutagenicity was observed without MA, but the substance was not tested up to cytotoxicity.

7.5.1 In vitro genotoxicity studies

Details on the genotoxicity of nitroPAHs in vitro are presented in four tables. The results of the numerous studies on the Salmonella microsome assay (Maron & Ames, 1983) with nitroPAHs are summarized in Table 40. Data on the genotoxicity of nitroPAHs in other bacterial systems are documented in Table 41. Data on eukaryotic test systems, including human cells, are presented in Tables 42 and 43 (genotoxicity) and 44 (cell transformation). A summary of all data on genotoxicity in vitro is given in Table 45. The final evaluation of genotoxicity and a scaling of the mutagenic potency in the Salmonella microsome assay are presented in Table 46.

Table 41. Genotoxicity of nitroPAHs in bacteria, other than the Salmonella microsome test

Substance

Test type

Bacteriaa

Resultsb

Reference

–MAc

+MAc

1-Nitronaphthalene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

±

Dunkel et al. (1985)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

(±)

n.g.

Nakamura et al. (1987)

SOS DNA repair (SOS chromotest)

E. coli PQ37

(±)

Mersch-Sundermann et al. (1991, 1992)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+d

n.g.

Oda et al. (1992, 1993)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+

n.g.

Schmid et al. (1997)

2-Nitronaphthalene

DNA damage/repair (rec assay)

E. coli polA– (DNA repair deficient) or polA+ (proficient)

+

n.g.

Rosenkranz & Poirier (1979)

DNA damage/repair (rec assay)

E. coli WP67 (DNA repair deficient) or WP2 (proficient)

+

+

De Flora et al. (1984)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+

n.g.

Nakamura et al. (1987)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

(±)

Mersch-Sundermann et al. (1991, 1992)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+d

n.g.

Oda et al. (1992, 1993)

1,3-Dinitronaphthalene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

1,5-Dinitronaphthalene

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

Mersch-Sundermann et al. (1991, 1992)

DNA damage (prophage induction)

E. coli (induction of prophage in lysogenic strain B/r.WP2(lambda), indicator strain SR714)

+

n.g.

Rossmann et al. (1991)

2,7-Dinitronaphthalene

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

(±)

Mersch-Sundermann et al. (1991, 1992)

2,3,5-Trinitronaphthalene

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

+

n.g.

Sorenson et al. (1983)

5-Nitroacenaphthene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

(±)

+

McCoy et al. (1983b)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

+

Dunkel et al. (1985)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+d

n.g.

Oda et al. (1992, 1993)

2-Nitrofluorene

DNA cell binding

E. coli Q13

+

+e

Kubinski et al. (1981)

DNA damage (K12 inductest)

E. coli (induction of prophage in lysogenic strain GY5027, indicator strain GY4015)

Mamber et al. (1984)

DNA damage (prophage induction)

E. coli K12 (lysogen; induction of prophage lambdaclts857)

+

+e

Ho & Ho (1981)

DNA damage (prophage induction)

E. coli (induction of prophage in lysogenic strain B/r.WP2(lambda), indicator strain SR714)

+

n.g.

Rossmann et al. (1991)

DNA damage/repair (rec assay)

E. coli polA– (DNA repair deficient) or polA+ (proficient)

+

n.g.

Rosenkranz & Poirier (1979)

DNA damage/repair (rec assay)

E. coli WP100 (DNA repair deficient) or WP2 (proficient)

+

McCarroll et al. (1981a)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

McCarroll et al. (1981b)

 

DNA damage/repair (rec assay)

E. coli WP100 (DNA repair deficient) or WP2 (proficient)

+

n.g.

Doudney et al. (1981)

DNA damage/repair (rec assay)

E. coli JC (DNA repair deficient) or AB1157 (proficient)

n.g.

±

Suter & Jaeger (1982)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

n.g.

+

Suter & Jaeger (1982)

DNA damage/repair (rec assay)

E. coli WP100 (DNA repair deficient) or WP2 (proficient)

+

n.g.

Mamber et al. (1983, 1984)

Forward mutation assay

S. typhimurium TA1537, 100, 98 (resistance to 8-azaguanine)

n.g.

Castellino et al. (1978)

Forward mutation assay

S. typhimurium TA1538, 1535 (resistance to 8-azaguanine)

+

n.g.

Castellino et al. (1978)

Forward mutation assay

E. coli WP2 (resistance to L-azetidine-2-carboxylic acid)

+

n.g.

Mitchell (1980)

Forward mutation assay

S. typhimurium BA8 (resistance to L-arabinose)

+

n.g.

Ruiz-Rubio et al. (1984)

Forward mutation assay

S. typhimurium SV50 (resistance to arabinose)

n.g.

Xu et al. (1984)

Forward mutation assay

E. coli WP2 or CM891 (resistance to L-azetidine-2-carboxylic acid)

(±)

n.g.

Mitchell & Gilbert (1985)

Forward mutation assay

S. typhimurium BA3 or BA9 (resistance to L-arabinose)

+

n.g.

Hera & Pueyo (1986)

Reverse mutation assay

E. coli WP2uvrA (trp reversion)

+

n.g.

McCoy et al. (1981b)

Reverse mutation assay

E. coli CM891 (trp reversion)

+

n.g.

Mitchell & Gilbert (1984)

Reverse mutation assay

S. typhimurium BA3, 8 (reversion to histidine auxotrophy)

+

n.g.

Ruiz-Rubio et al. (1984)

Reverse mutation assay

E. coli WP2 or CM891 (trp reversion, positive only in CM891)

+

n.g.

Mitchell & Gilbert (1985)

Reverse mutation assay

S. typhimurium TA1538 with plasmid pYG8031, pYG8011, pSE117, pKM101, pBR322

+

n.g.

Nohmi et al. (1995)

Reverse mutation assay

E. coli MX100 (reversion of arginine auxotrophy)

+

n.g.

Kranendonk et al. (1996)

Reverse mutation assay

E. coli strains (reversion to Lac+; induction of frameshifts enhanced in strains with higher acetyltransferase activity)

+

n.g.

Hoffmann et al. (2001)

SOS DNA repair (SOS chromotest)

E. coli PQ37

n.g.

+

Ohta et al. (1984)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

n.g.

Quillardet et al. (1985)

SOS DNA repair (SOS chromotest)

E. coli K12 PQ37

+

n.g.

Mamber et al. (1986)

SOS DNA repair (SOS chromotest)

E. coli K12 PQ37

+

+

Marzin et al. (1986)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+

n.g.

Nakamura et al. (1987)

SOS DNA repair (SOS chromotest)

E. coli PQ37

Schleibinger et al. (1989)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+

+

Schleibinger et al. (1989)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

(±)

Mersch-Sundermann et al. (1991, 1992)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+d

n.g.

Oda et al. (1992, 1993, 1996)

2,7-Dinitrofluorene

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion, no data on cytotoxicity)

n.g.

McCoy et al. (1981b)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

+e

Mersch-Sundermann et al. (1991, 1992)

9-Nitroanthracene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

SOS DNA repair (SOS chromotest)

E. coli PQ37

Mersch-Sundermann et al. (1991, 1992)

2-Nitrofluoranthene

DNA damage

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Nakagawa et al. (1987)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

+

Schleibinger et al. (1989)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+

+f

Schleibinger et al. (1989)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

+e

Mersch-Sundermann et al. (1991, 1992)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+d

n.g.

Oda et al. (1992, 1993)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by human CYP1A1, CYP1A2 or CYP1B1 co-expressed with NPR)

+

+f

Yamazaki et al. (2000)

3-Nitrofluoranthene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1986)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002; NM2009 & 3009 (acetyltransferase activity Up Arrow)

+

n.g.

Shimada et al. (1994)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by human CYP1A1, CYP1A2 or CYP1B1 co-expressed with NPR)

+

+e

Yamazaki et al. (2000)

8-Nitrofluoranthene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

3,4-Dinitrofluoranthene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Nakagawa et al. (1987)

3,7-Dinitrofluoranthene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Nakagawa et al. (1987)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+d

n.g.

Oda et al. (1992, 1993)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002; NM2009 and 3009 (acetyltransferase activity Up Arrow)

+

n.g.

Shimada et al. (1994)

3,9-Dinitrofluoranthene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Nakagawa et al. (1987)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+d

n.g.

Oda et al. (1992, 1993)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002; NM2009 and 3009 (acetyltransferase activity Up Arrow)

+

n.g.

Shimada et al. (1994)

1-Nitropyrene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Horikawa et al. (1986)

Forward mutation assay

S. typhimurium TM677 (resistance to 8-azaguanine)

+

+e

Busby et al. (1994a)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

n.g.

Mermelstein et al. (1981)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

(±)

n.g.

Tokiwa et al. (1984)

Reverse mutation assay

E. coli WP2uvrA, 3001-04 (trp+ reversion; positive only in 3003 and 3004g)

(±)

n.g.

McCoy et al. (1985b)

Reverse mutation assay

S. typhimurium TA1538 with plasmid pYG8031, pYG8011, pSE117, pKM101, pBR322

+

n.g.

Nohmi et al. (1995)

SOS DNA repair (SOS umu test)

E. coli PQ37

+

n.g.

Ohta et al. (1984)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+

n.g.

Nakamura et al. (1987)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

(±)

(±)

Schleibinger et al. (1989)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

+f

Schleibinger et al. (1989)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

+e

Mersch-Sundermann et al. (1991, 1992)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+

n.g.

Fretwurst & Ahlf (1996)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by human CYP1A1, CYP1A2 or CYP1B1 co-expressed with NPR)

+

+e

Yamazaki et al. (2000)

2-Nitropyrene

Forward mutation assay

S. typhimurium TM677 (resistance to 8-azaguanine)

+

Busby et al. (1994a)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by CYP1B1 or CYP1A1, not CYP1A2)

n.g.

+

Shimada et al. (1996)

4-Nitropyrene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Horikawa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

Forward mutation assay

S. typhimurium TM677 (resistance to 8-azaguanine)

+

+e

Busby et al. (1994a)

1,3-Dinitropyrene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Horikawa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

Forward mutation assay

S. typhimurium TM677 (resistance to 8-azaguanine)

+

+e

Busby et al. (1994a)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

n.g.

Mermelstein et al. (1981)

Reverse mutation assay

E. coli WP2uvrA, 3001-04 (trp+ reversion; positive only in 3003 and 3004g)

+

n.g.

McCoy et al. (1985b)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+

n.g.

Nakamura et al. (1987)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

+e

Mersch-Sundermann et al. (1991, 1992)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+d

n.g.

Oda et al. (1992, 1993)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002; NM2009 and 3009 (acetyltransferase activity ALT="Up Arrow">)

+

n.g.

Shimada et al. (1994)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+

+e

Shane & Winston (1997)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by human CYP1A1, CYP1A2 or CYP1B1 co-expressed with NPR)

+

+e

Yamazaki et al. (2000)

1,6-Dinitropyrene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Horikawa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

Forward mutation assay

S. typhimurium TM677 (resistance to 8-azaguanine)

+

+e

Busby et al. (1994a)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

n.g.

Mermelstein et al. (1981)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

+

n.g.

Tokiwa et al. (1984)

Reverse mutation assay

E. coli WP2uvrA, 3001-04 (trp+ reversion; positive only in 3003 and 3004g)

+

n.g.

McCoy et al. (1985b)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+

n.g.

Nakamura et al. (1987)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

+e

Mersch-Sundermann et al. (1991, 1992)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+d

n.g.

Oda et al. (1992, 1993)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002; NM2009 and 3009 (acetyltransferase activity Up Arrow)

+

n.g.

Shimada et al. (1994)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+

+e

Shane & Winston (1997)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by human CYP1A1, CYP1A2 or CYP1B1 co-expressed with NPR)

+

+e

Yamazaki et al. (2000)

1,8-Dinitropyrene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Horikawa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

Forward mutation assay

S. typhimurium TM677 (resistance to 8-azaguanine; mutagenicity proportional to DNA binding)

+

n.g.

Sanders et al. (1983)

Forward mutation assay

S. typhimurium BA15 (resistance to L-arabinose)

+

n.g.

Jurado et al. (1993)

Forward mutation assay

S. typhimurium TM677 (resistance to 8-azaguanine)

+

+e

Busby et al. (1994a)

Forward mutation assay

S. typhimurium BA14 (resistance to L-arabinose)

+

n.g.

Jurado et al. (1994)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

n.g.

Mermelstein et al. (1981)

Reverse mutation assay

E. coli WP2uvrA, 3001-04 (trp+ reversion; positive only in 3003 and 3004g)

+

n.g.

McCoy et al. (1985b)

Reverse mutation assay

S. typhimurium TA1538 with plasmid pYG8031, pYG8011, pSE117, pKM101, pBR322

+

n.g.

Nohmi et al. (1995)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+

n.g.

Nakamura et al. (1987)

SOS DNA repair (SOS chromotest)

E. coli PQ37

+

+e

Mersch-Sundermann et al. (1991, 1992)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002

+d

n.g.

Oda et al. (1992, 1993)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (umu gene expression)

+

+e

Shane & Winston (1997)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by human CYP1A1, CYP1A2 or CYP1B1 co-expressed with NPR)

+

+e

Yamazaki et al. (2000)

1,3,6-Trinitropyrene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Horikawa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

+

n.g.

Mermelstein et al. (1981)

1,3,6,8-Tetranitropyrene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Horikawa et al. (1986)

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

Reverse mutation assay

E. coli WP2uvrA (trp+ reversion)

n.g.

Mermelstein et al. (1981)

7-Nitrobenz[a]anthracene

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by human CYP1A1, CYP1A2 or CYP1B1 co-expressed with NPR)

+

Yamazaki et al. (2000)

6-Nitrochrysene

DNA damage/repair (rec assay)

B. subtilis M45 (DNA repair deficient) or H17 (proficient)

+

n.g.

Tokiwa et al. (1987b)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by CYP1B1, not CYP1A1 or CYP1A2)

n.g.

+

Shimada et al. (1996)

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by human CYP1A1, CYP1A2 or CYP1B1 co-expressed with NPR)

+

+e

Yamazaki et al. (2000)

6-Nitrobenzo[a]pyrene

SOS DNA repair (SOS umu test)

S. typhimurium TA1535 pSK1002 (MA by human CYP1A1, CYP1A2 or CYP1B1 co-expressed with NPR)

+

Yamazaki et al. (2000)

a

trp = tryptophan; NPR = NADPH-cytochrome P450 reductase; Up Arrow = increased.

b

The results in Table 41 are derived as follows:

 

+

is a clear positive result, also given as + or positive by the authors of the publication.

 

(±)

is a weak positive result, also classified by the authors of the publication as ± or ?.

 

(+)

given where there were weak positive results with dose–response.

 

±

given when the result was inconclusive, which means that the result was negative, but testing was not performed up to cytotoxic concentrations. Further ± was given for weak positive effects without dose–response or without sufficient documentation for further assessment.

 

is a negative result.

c

MA = metabolic activation (preparation of liver microsomes [S9-mix] from rodent species); n.g. = not given.

d

Highly sensitive in strains having high O-acetyltransferase and nitroreductase activities.

e

Reduced by metabolic activation.

f

Increased by metabolic activation.

g

Positive results only in E. coli strains that have increased permeability to large molecules and DNA repair deficiency.

The results in Tables 41–44 are derived as follows:

+

is a clear positive result, also given as + or positive by the authors of the publication;

(±)

is a weak positive result, also classified by the authors of the publication as ± or ?;

(+)

is a weak positive result with dose–response;

±

is an inconclusive result, which means that the result was negative, but testing was not performed up to cytotoxic concentrations; further ± was given for weak positive effects without dose–response or without sufficient documentation for further assessment; and

represents a negative result.

7.5.1.1 Salmonella typhimurium microsome assay

The majority of the studies on the genotoxicity of nitroPAHs (more than 100 studies on a total of 91 nitroPAHs) have used the Salmonella typhimurium microsome assay with a standardized test protocol using the strains TA98, TA100, TA1535, TA1537 or TA1538. For conciseness, the details of each individual study are not given here but are compiled in Table 40. A summary of the data on the Salmonella microsome assay is presented in Table 45.

7.5.1.2 Comparison of the mutagenic potency of nitroPAHs in the Salmonella microsome assay

From the extensive data, it is possible to compare the mutagenic potency of nitroPAHs in Tables 40 and 46. In analogy to other publications (Tokiwa et al., 1981b, 1993b; Rosenkranz & Mermelstein, 1983; Purohit & Basu, 2000), the mutagenic potency of the examined nitroPAHs in the Salmonella microsome assay is determined in this documentation by the number of revertants per nanomole for each S. typhimurium strain (see Table 40). For different doses, the number of revertants per nanomole has been calculated and the number of spontaneous revertants subtracted. The dose resulting in the maximum number of revertants per nanomole is used for further calculation and assessment. Only those studies that fulfilled the following minimum requirements were documented (and mutagenic potencies determined): at least three doses tested with the corresponding strain, negative control, spontaneous reversion rate at least doubled, and graphical or tabulated presentation of results. Less valid studies were also documented if the database of the nitroPAH was small (<4 studies). Data from studies in which the authors calculated the mutagenic potency by more sophisticated methods (e.g., calculated from the linear regression analysis of the data) were also added to the data pool without change, except for conversion to the unit revertants per nanomole where necessary.

For most nitroPAHs, only a few data are available for one or more of the S. typhimurium strains (74 out of 91 nitroPAHs; see Table 40). Only 17 nitroPAHs revealed a large database on mutagenicity in the Salmonella microsome assay (>5 tests with at least one strain; bold type substance name in Table 40). There are five nitroPAHs that showed exceptionally high mutagenic potency (>100 000 revertants/nmol, highest scale in Table 40) in this test system: 3,7-, and 3,9-dinitrofluoranthene, 1,6- and 1,8-dinitropyrene, and 3,6-dinitrobenzo[a]pyrene (shown by ++++++ in Table 46). A somewhat lower potency was seen with 8-nitrofluoranthene, 1,3-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene, where the limit 100 000 revertants/nmol was not reached but >10 000 was (second highest scale in Table 40; shown by +++++ in Table 46).

Most nitroPAHs showed medium mutagenic potency (24 nitroPAHs marked in Table 46 with +++ [>100, <1000 revertants/nmol] or 29 nitroPAHs marked by ++++ [>1000, <10 000 revertants/nmol]). Eleven nitroPAHs reached the second lowest scale (marked by ++ [>10, <100 revertants/nmol]).

Eleven out of 91 nitroPAHs showed only a weak mutagenic potency relative to other nitroPAHs (lowest scale in Table 46, <10 revertants/nmol, marked by +): 1- and 2-nitronaphthalene, 1,3- and 1,5-dinitronaphthalene, 3-nitrofluorene, 9-nitroanthracene, 1,5-, 4,9- and 1,10-dinitrophenanthrene, 5-nitrochrysene and 1-nitrocoronene. In seven nitroPAHs, the mutagenic potency could not be calculated. At least with 1- and 2-nitronaphthalene, the weak mutagenic effect is influenced by the volatility (see also section 2), leading consequently to a short period of exposure of the bacteria to the substance. Gupta et al. (1996) reported an increase in the mutagenic activity of about 1 (1-nitronaphthalene) or 2 (2-nitronaphthalene) orders of magnitude by prolongation of the preincubation time to 90 min. With none of the nitroPAHs listed in Table 40 were clearly negative results obtained in the Salmonella microsome assay. Further, the validity of negative results is limited, for example, if the substance was not tested up to cytotoxicity (see 1,8-dinitrobenzo[e]pyrene in Table 40).

For comparison, it should be noted that the mutagenicity of BaP with exogenous metabolic activation is ~2.3 revertants/nmol, and no mutagenic activity is shown in the absence of metabolic activation.

7.5.1.3 Studies into the pathways of microbial metabolism

Various strategies have been used to investigate the metabolism and "mutagenic activation pathways" leading to histidine reversion in bacteria. As well as through comparison of the mutagenic effect of individual nitroPAHs using the Ames Salmonella typhimurium reversion assay, other attempts include:

These and other studies have shown that all nitroPAHs do not follow the same metabolic and activation pathways.

1) The effect of metabolic activation on mutagenicity

The effect of metabolic activation in the Salmonella microsome assay can be assessed by comparing the maximum mutagenic potency with and without exogenous metabolic activation in the same strain (see Table 40 and summary in Table 46). For this, comparative data are available on 48 nitroPAHs (see column "mutagenic potency" in Table 46). No data are available on 43 nitroPAHs; 41 were tested only without and 2 only with metabolic activation in the same strain. It should be noted that parent PAHs need metabolic activation for mutagenic effects in S. typhimurium (IPCS, 1998).

Most nitroPAHs (35 out of 48), including those with exceptionally high mutagenic potency (e.g., dinitropyrenes), were clearly more effective in the Salmonella microsome assay without metabolic activation (marked in Table 46 by –S9) or exhibited a moderate difference (10 nitroPAHs, marked by –S9). This indicates that mutagenicity shown in the Salmonella microsome assay was induced mainly by the enzymes in the bacteria itself and not by the added mammalian activation system. Furthermore, there is evidence that with some nitroPAHs, the metabolic activation system inhibited mutagenicity — for example, see 3,7- and 3,9-dinitrofluoranthene in Table 40.

Only 3 out of these 48 nitroPAHs induced clearly more reverse mutations with metabolic activation (marked by +S9 in Table 46): 7-nitrobenz[a]anthracene, 6-nitrobenzo[a]pyrene and 3-nitroperylene. Metabolic activation of 6-nitrobenzo[a]pyrene (Fu et al., 1982) and 3-nitroperylene (Anderson et al., 1987) was investigated in more detail. In contrast to most other nitroPAHs, where bacterial nitroreduction is the first step in metabolic activation, for these nitroPAHs it is suggested that predominantly a ring oxidation by mixed-function oxidase enzymes is involved, followed by further bacterial metabolism to form the ultimate mutagen (Anderson et al., 1987). For comparison, most parent PAHs are also metabolized by a ring oxidation (IPCS, 1998).

With 10 nitroPAHs, the mutagenic potency was higher with metabolic activation but in the same order of magnitude as without (marked by normal type +S9 in Table 46), indicating a) activation by both bacterial enzymes and mammalian microsomal enzymes or b) that S9-mix has no inhibitory effect on mutagenicity.

It should be pointed out that the higher mutagenic activity (marked by +S9 in Table 46) of exogenous metabolic activation with mammalian microsomes is predominantly observed in nitroPAHs with five aromatic rings.

2) Activation of nitroPAHs in strains with altered enzyme activity

For several nitroPAHs (n = 17), a large database on the standard tester strains of S. typhimurium is available (bold type substance name in Table 40); for some of these nitroPAHs, additional studies have been carried out using nitroreductase- or O-acetyltransferase-deficient strains as well as strains overproducing both enzymes. Examples of these results are shown in Figure 17. The direct mutagenic activity of most nitroPAHs in the Salmonella reversion test is a consequence of reduction of the nitro group to a hydroxylamine, which is catalysed by bacterial nitroreductase (Rosenkranz & Mermelstein, 1983). This nitroreduction and the binding of the corresponding electrophilic arylnitrenium ion (R-NH+) to DNA are discussed as one of several possible metabolic activation pathways in S. typhimurium (Fu, 1990; Ball et al., 1995).

The use of a nitroreductase-deficient strain (marked with NR– in Figure 17) revealed a clear decrease (difference at least 5-fold) in mutagenic activity compared with the standard tester strain showing the maximal mutagenic potency for three nitroPAHs: 2-nitronaphthalene (McCoy et al., 1981a), 2,7-dinitrofluorene (McCoy et al., 1981b) and 1,3-dinitropyrene (Crebelli et al., 1995). A less clear decrease (only 2- to 5-fold) was seen with 5-nitroacenaphthene (McCoy et al., 1983a), 3-nitrofluoranthene (Tokiwa et al., 1986) and 1-nitropyrene (Figure 17). No data for direct comparison between the standard tester strain with maximal response and the corresponding nitroreductase-deficient strain are available with 2-nitrofluorene, but the decreasing effect was obvious when comparing TA98 versus TA98NR– (see Figure 17).

Figure 17

Figure 17

Fig. 17. Examples of mutagenicity studies on nitroPAHs using nitroreductase- or O-acetyltransferase-deficient strains as well as strains overproducing both enzymes. All experiments without metabolic activation system.
NR– = nitroreductase-deficient strain; NR+ = nitroreductase-overproducing strain; AT– = O-acetyltransferase-deficient strain; AT+ = O-acetyltransferase-overproducing strain.

Furthermore, using TA98 with high nitroreductase activity (e.g., TA98NR+ in Figure 17), these nitroPAHs showed an elevated mutagenic effect compared with the corresponding standard tester strain — for example, 2-nitronaphthalene (4-fold increase; Hagiwara et al., 1993), 2-nitrofluorene (12-fold; Figure 17) and 1-nitropyrene (30-fold; see Figure 17) (no further data available for comparison between the standard tester strain with maximal response and the corresponding strain with high nitroreductase activity).

Overall, these examples show that nitroreduction is an important mutagenic activation pathway in S. typhimurium.

Nitroreductases and O-acetyltransferases have been shown to be important enzymes in the mutagenic activation of nitroPAHs in bacteria. It is postulated that after reduction of the nitro group to hydroxylamine, this proximate mutagen requires further activation to form the ultimate mutagen (an arylnitrenium ion). For some nitroPAHs, this activation step occurs through O-acetylation of the hydroxylamine (McCoy et al., 1985a; Fu, 1990; Ball et al., 1994, 1995).

There are a limited number of nitroPAHs with available data on O-acetyltransferase-overproducing and -deficient strains. Several nitroPAHs showed a decrease in mutagenicity with O-acetyltransferase-deficient strains (e.g., TA98AT–) as well as with nitroreductase-deficient strains. For example, pronounced decreases were seen with 5-nitroacenaphthene (McCoy et al., 1983a), 3-nitrofluoranthene (Shane et al., 1991) and 1,3-dinitropyrene (Crebelli et al., 1995), whereas the decreases were not as pronounced with 2-nitrofluorene (see Figure 17), 2,7-dinitrofluorene (Hirayama et al., 1988) and 1-nitropyrene (see Figure 17).

Some nitroPAHs showed a clear increase in mutagenic potency (about 1 order of magnitude) relative to the standard tester strain in strains overproducing O-acetyltransferase (marked by AT+ in Figure 17) — for example, 2-nitrofluorene (20-fold increase; Figure 17), 3-nitrofluoranthene (20-fold; Ball et al., 1994; Sangaiah et al., 1996), 1,6-dinitropyrene (30-fold; Tokiwa et al., 1985, 1986, 1994) and 1,8-dinitropyrene (15-fold; see Figure 17). For these nitroPAHs, O-acetylation seems to be a mutagenic activation pathway in S. typhimurium.

For 1,6- and 1,8-dinitropyrene, no significant difference between nitroreductase-deficient strains, standard tester strains and nitroreductase-overproducing strains were seen (similar results with both nitroPAHs; data on 1,8-dinitropyrene, see Figure 17); with these two nitroPAHs, however, a clear decrease in mutagenic activity compared with the standard tester strain was obtained with O-acetyltransferase-deficient (AT–) strains (see Figure 17). Together with data on strains overproducing O-acetyltransferase (see above), this suggests that 1,6- and 1,8-dinitropyrene are examples of nitroPAHs that do not depend on nitroreductase for expressing maximal direct mutagenicity in S. typhimurium (Rosenkranz & Mermelstein, 1983). In accordance with this activation mechanism, 1,8-dinitropyrene was highly mutagenic in the O-acetyltransferase-overproducing strain (highest measured mutagenic potency, 15-fold increase relative to TA98), but no increasing effect was detected in the nitroreductase-overproducing strain (see Figure 17).

6-Nitrobenzo[a]pyrene (see Figure 17) is an example of a nitroPAH that needed an exogenous mammalian metabolic activation system for mutagenic effects in S. typhimurium. The mutagenic potency was only moderately increased in nitroreductase- and O-acetyltransferase-deficient strains compared with the standard tester strain.

Additional data on mechanisms of mutagenicity are presented in chapter 6.

3) Use of tester strains to determine frameshift versus base pair substitution

Data on standard tester strains recommended for general mutagenesis screening by Ames et al. (1975) are documented in Table 40. Strains TA1535 and TA100 detect mutagens that cause base pair substitutions, primarily at the G:C base pair in the hisG gene, and the other three strains detect various frameshift mutagens (Maron & Ames, 1983). Base pair substitutions induced by exposure to nitroPAHs were in general not detected by TA1535 but by its R-factor derivative TA100 (see Table 40) containing plasmid pKM101, which enhances an error-prone DNA repair and increases spontaneous and chemically induced mutations.

NitroPAHs that clearly induced more base pair substitutions than frameshift mutations (difference 10-fold or more) as well as nitroPAHs inducing more frameshift mutations (mainly detected by TA98, a strain also including plasmid pKM101) are tabulated in Table 40 (right column; marked by bold type letters). Six nitroPAHs clearly induced more base pair substitutions than frameshift mutations, and seven nitroPAHs were clearly more effective in strains detecting frameshift mutations. A difference of at least 2-fold is marked (more base pair substitutions, 21 nitroPAHs; more frameshift mutations, 16 nitroPAHs) also in Table 40 (normal type letters in the right column). No clear differences were observed with the remaining nitroPAHs, or a comparison was not possible (only one strain tested, 14 nitroPAHs).

There is a tendency in nitroPAHs with two or three aromatic rings towards more base pair substitutions and in nitroPAHs with three or four rings towards more frameshift mutations. For example, nitrated naphthalenes and phenanthrenes induced more base pair substitutions, and nitrated pyrenes induced more frameshift mutations.

Watanabe et al. (1997b) examined the mutational specificity of 12 nitroPAHs in six S. typhimurium strains (TA7001–7006) that reverted only by one specific base substitution. All tested nitroPAHs induced TA Right Arrow AT, CG Right Arrow AT and CG Right Arrow GC transversions and GC Right Arrow AT transition in his genes. AT Right Arrow GC transition and TA Right Arrow GC transversion were weakly or not induced by the tested nitroPAHs.

It is suggested that frameshift mutations induced by nitroPAHs are not due to simple intercalation but rather to DNA adduct formation (Rosenkranz & Mermelstein, 1983; see also chapter 6), mainly at the C8 position of guanine (Rosenkranz et al., 1985). The most frequent frameshift mutation in S. typhimurium TA98 induced, for example, by 1-nitropyrene is a –2 deletion of a G:C or C:G base pair within a CGCGCGCG hot-spot sequence of the hisD3052 mutation (Bell et al., 1991).

7.5.1.4 Relationship between mutagenic potency in S. typhimurium and the chemical structure of nitroPAHs

1) Number of aromatic hydrocarbon rings in nitroPAHs

There seems to be some relationship between mutagenic potency and the number of aromatic rings within the nitroPAH molecule.

In Table 46, nitrated (mono-, di-, tri-, tetra-) naphthalenes (two rings) showed only low mutagenic potency (two lowest scales in column "mutagenic potency"; <100 revertants/nmol; marked by + and ++).

NitroPAHs with three rings (acenaphthene, fluorene, anthracene, phenanthrene) revealed a low mutagenic potency (10 out of 32 examined nitroPAHs) or, in most cases (22 out of 32 investigated nitroPAHs), a medium mutagenic potency (two medium scales; >100, but <10 000 revertants/nmol; marked by +++ and ++++ in Table 46). The two highest scales (>10 000 revertants/nmol) were not reached in this group of nitroPAHs.

In contrast, 9 out of 30 examined nitroPAHs with four rings (fluoranthene, pyrene, benz[a]anthracene, chrysene) showed a high mutagenic potency (two highest scales in Table 46, marked by +++++ and ++++++), 19 a medium mutagenic potency and only 2 a low mutagenic potency. Vance & Levin (1984) also examined the effect of the number and arrangement of aromatic nuclei in nitroPAHs on mutagenicity in the Salmonella microsome assay. They presented evidence for highest activity in nitroPAHs with four aromatic rings and an overall length of three rings.

This tendency towards a higher mutagenic potency with increasing number of rings is not followed for nitroPAHs with five rings (e.g., benzo[x]fluoranthenes, benzo[x]pyrenes, perylene). However, 12 out of 15 nitroPAHs in Table 46 with available data revealed medium mutagenic potency, 1 nitroPAH high mutagenic potency and only 2 nitroPAHs low mutagenic potency.

Data on nitroPAHs with six or more rings (e.g., coronene) are not sufficient for assessment of a correlation (Table 46).

2) Number of nitro groups

There are several examples in Table 46 revealing an increased mutagenic potency for dinitration compared with mononitration of PAHs. Although influenced by the orientation of the nitro groups (see below), a second nitro group elevated mutagenic potency in fluorenes, phenanthrenes, fluoranthenes, pyrenes and benzo[a]pyrenes. The highest scale for mutagenic potency was observed in the dinitrated PAHs 3,7- and 3,9-dinitrofluoranthene, 1,6- and 1,8-dinitropyrene and 3,6-dinitrobenzo[a]pyrene.

There is no case for a further increase in mutagenic potency with a third or a fourth nitro group. Comparing, for example, the mononitrated pyrenes with 1,3,6-trinitro- and 1,3,6,8-tetranitropyrene, the mutagenic potency increased with further nitro groups, although the potency of the dinitropyrenes is not reached (Table 46).

3) Orientation of nitro group

The orientation of the nitro group is an important structural factor in determining the mutagenic activity of nitroPAHs. Comparing experimental results on direct mutagenic activity of nitroPAHs in the Salmonella microsome assay, it is suggested that nitroPAHs with their nitro group oriented perpendicular or nearly perpendicular to the plane of the aromatic rings generally exhibit weak or no mutagenicity (Fu et al., 1985b, 1988b, 1989, 1997; Jung et al., 1991). The decreased mutagenicity of nitroPAHs with nitro groups perpendicular to the aromatic ring system is thought to be due to their inability to fit into the active site of the bacterial nitroreductases because of steric interactions (Fu et al., 1985b).

In a recent study on 22 different nitrated phenanthrenes (Sera et al., 1996), three dinitrophenanthrenes showed weak mutagenic potency: 1,5-, 1,10- and 4,9-dinitrophenanthrene (Tables 40 and 46). In these phenanthrene derivatives, substituents were oriented perpendicular or nearly perpendicular to the aromatic system. In contrast, dinitrophenanthrenes with substituents showing a dihedral angle (calculated by the authors) below 10 degrees exhibited high mutagenic activity — for example, 3,6-dinitrophenanthrene. Similar results were presented with mononitrated phenanthrenes (highest mutagenicity with 3-nitrophenanthrene, revealing the lowest dihydral angle) and trinitrated phenanthrenes (lowest mutagenic potency in 1,5,10-trinitrophenanthrene, in which all three substituents reached a dihydral angle above 50 degrees) (Sera et al., 1996).

Further studies suggest that the nitroPAHs with the nitro group situated at the longest axis of the molecule exhibited the highest mutagenicity in S. typhimurium (Vance & Levin, 1984; Hirayama et al., 1988; Yu et al., 1992). For example, 2-nitropyrene has a higher mutagenic potency than 1- or 4-nitropyrene, and 2-nitrochrysene revealed a higher mutagenicity than 6-nitrochrysene. This conclusion is in accord with the hypothesis above, since nitroPAHs with nitro substituents oriented perpendicular to the plane of aromatic rings in general have their nitro group situated either at the shortest axis of the molecule or near the shortest axis with weak mutagenic activity (Yu et al., 1992).

Klopman et al. (1984) presented evidence for an inverse linear relationship between the calculated energies of the lowest unoccupied molecular orbital (LUMO) and the logarithm of the direct-acting mutagenicity of nitroPAHs in Salmonella strains. In a recent study on 11 dinitrophenanthrene derivatives (Sera et al., 1996), it could be shown that nitroPAHs with no nitro substituent perpendicular to the aromatic ring have a lower LUMO energy level.

Furthermore, structurally similar nitroPAHs showed a positive correlation between reduction potential measured by polarographic methods and direct mutagenicity (Jung et al., 1991).

7.5.1.5 Bacterial test systems other than the Salmonella microsome assay

The available data on the genotoxicity of nitroPAHs in bacterial test systems other than the Salmonella microsome assay are presented in Table 41. The literature is focused on two end-points: gene mutation and DNA damage/repair. Although the database is smaller than that on the Salmonella microsome assay (91 nitroPAHs), data on 27 nitroPAHs were given (see Table 41 and summary in Table 45).

With three exceptions, gene mutation assays (data on 13 nitroPAHs) in Table 41 confirmed the positive results in the Salmonella microsome assay (see Table 45). Exclusively positive results were obtained with DNA damage/repair assays on 26 nitroPAHs (Tables 41 and 45).

The effect of metabolic activation in the Salmonella microsome assay is discussed in detail in section 7.5.1.3. Bacterial systems other than the Salmonella microsome assay did not need metabolic activation systems (see Tables 41 and 45). In those studies where it was possible to compare genotoxic activities with and without exogenous metabolic activation, it was found that most nitroPAHs showed genotoxic effects irrespective of the presence of metabolic activation (11 nitroPAHs) and seven nitroPAHs were more active without, indicating the important role of bacterial enzymes in the metabolic activation of nitroPAHs in vitro.

7.5.1.6 Eukaryotic test systems

Genotoxicity of nitroPAHs in eukaryotic cells, including fungi, plants and mammalian cells, is presented in Table 42, and genotoxicity of nitroPAHs in human cells is shown in Table 43 (summary of data in Table 45). These data are available for 28 nitroPAHs. Most of these nitroPAHs have been studied in more detail because they were detected in diesel particulate extracts (IARC, 1989) and were discussed as possible contributors to the carcinogenic risk of diesel exhaust. Twenty-five of the 28 nitroPAHs showing positive results in bacterial systems gave predominantly positive results in eukaryotic test systems, with the exception of 1,3,6,8-tetranitropyrene (two negative end-points), 9-nitroanthracene (one positive and one negative end-point) and 7-nitrobenz[a]anthracene (one inconclusive result; see Table 45).

Table 42. In vitro genotoxicity of nitroPAHs in eukaryotic cells, excluding human cells (see Table 43)

Substance

End-pointa

Tested cell type (remarks)

Resultsb

Reference

–MAc

+MAc

1-Nitronaphthalene

GM

Mouse lymphoma cells L5178Y (TK test)

d

n.g.

Shelby & Stasiewicz (1984)

GM

Chinese hamster lung cells V79 (MA by co-culture of rat hepatocytes, HPRT test)

±

±

Boyes et al. (1991)

GM

Chinese hamster lung cells V79 (MA by co-culture of hamster hepatocytes, HPRT test)

±

+

Boyes et al. (1991)

SCE

Cultured mammalian cells (no details available)

d

n.g.

Shelby & Stasiewicz (1984)

SCE

Chinese hamster lung cells V79 (MA by co-culture with rat or hamster hepatocytes)

+

Boyes et al. (1991)

CA

Cultured mammalian cells (no details available)

+d

n.g.

Shelby & Stasiewicz (1984)

2-Nitronaphthalene

MR

S. cerevisiae D3

+

+

Simmon (1979b)

UDS

Primary rat hepatocytes

 

Mori et al. (1987)

UDS

Primary mouse hepatocytes

 

Mori et al. (1987)

1,8-Dinitronaphthalene

UDS

Primary mouse hepatocytes

 

Mori et al. (1987)

 

UDS

Primary rat hepatocytes

 

+

Mori et al. (1987)

5-Nitroacenaphthene

UDS

Primary mouse hepatocytes

 

+

Mori et al. (1987)

CA

Chinese hamster lung cells CHL

+

+e

Matsuoka et al. (1991)

1-Nitrofluorene

GM

Mouse lymphoma cells L5178Y (TK test)

+

n.g.

Amacher et al. (1979)

2-Nitrofluorene

GM

A. nidulans haploid strain35 (forward mutation; resistance to 8-azaguanine)

n.g.

Bignami et al. (1982)

 

GM

A. nidulans haploid strain35 (forward mutation; induction of methA1 suppressors)

n.g.

Bignami et al. (1982)

 

MR

S. cerevisiae D3

+

+f

Simmon (1979b)

 

GC

S. cerevisiae D4

n.g.

Mitchell (1980)

 

GM

Tradescantia clone 4430 (plant cuttings exposed by inflorescence immersion)

+

n.g.

Schairer & Sautkulis (1982)

 

GM

Mouse lymphoma cells L5178Y (TK test)

+

n.g.

Oberly et al. (1984)

 

GM

Chinese hamster ovary cells (CHO, HPRT test)

+

Oberly et al. (1990)

 

GM

Mouse lymphoma cells L5178Y (TK test)

+

n.g.

Oberly et al. (1996)

 

GM

Chinese hamster V79 cells (cells with different enzyme activities tested: CYP1A2, CYP2C9 or CYP3A4 with and without endogenous acetyltransferase; HPRT test)

 

Kappers et al. (2000)

 

UDS

Primary rat hepatocytes

 

Probst et al. (1981)

 

UDS

Primary rat hepatocytes

 

+

Mori et al. (1987)

 

UDS

Primary mouse hepatocytes

 

+

Mori et al. (1987)

2-Nitrofluorene

SSB

Alveolar type II cells, Clara cells or macrophages isolated from rabbit lung (alkaline elution assay, tested up to cytotoxicity)

n.g.

Becher et al. (1993)

VE

C3H2K cells (no further data) infected with mouse leukaemia virus

(±)

n.g.

Yoshikura et al. (1979)

 

SCE

Chinese hamster ovary (CHO) cells

+

+e

Nachtman & Wolff (1982)

SCE

Chinese hamster lung cells V79

+

Schleibinger et al. (1989)

 

CA

Chinese hamster lung cells CHL

+

+e

Matsuoka et al. (1991)

MN

Chinese hamster lung cells V79

+

n.g.

Glatt et al. (1990)

 

MN

Rat intestinal cells IEC-17/-18 (metabolically competent)

 

+

Glatt et al. (1990)

 

MN

Embryonal human liver cells

 

+

Glatt et al. (1990)

 

MN

Mouse BALB/c-3T3 cells

+

n.g.

Gu et al. (1992)

 

MN

Chinese hamster CHL cells (sublines expressing bacterial or human N-acetyltransferases more effective than parent CHL cells)

+

n.g.

Watanabe et al. (1994)

3-Nitrofluorene

CA

Chinese hamster lung cells CHL

+

+e

Matsuoka et al. (1991)

4-Nitrofluorene

CA

Chinese hamster lung cells CHL

+

+e

Matsuoka et al. (1991)

2,7-Dinitrofluorene

UDS

Primary rat hepatocytes

 

+

Probst et al. (1981)

UDS

Primary rat hepatocytes

 

+

Mori et al. (1987)

UDS

Primary mouse hepatocytes

 

+

Mori et al. (1987)

9-Nitroanthracene

UDS

Primary rat hepatocytes

 

Mori et al. (1987)

 

UDS

Primary mouse hepatocytes

 

Mori et al. (1987)

3-Nitrofluoranthene

GM

Chinese hamster lung cells V79 (HPRT test)

+g

Berry et al. (1985)

UDS

Primary rat hepatocytes

 

+

Mori et al. (1987)

UDS

Primary mouse hepatocytes

 

+

Mori et al. (1987)

SCE

Chinese hamster lung cells V79

+

Schleibinger et al. (1989)

8-Nitrofluoranthene

GM

Chinese hamster lung cells V79 (HPRT test)

+g

Berry et al. (1985)

3,7-Dinitrofluoranthene

GM

Chinese hamster lung cells V79 (HPRT test)

n.g.

+

Tokiwa et al. (1988)

CA

Chinese hamster lung cells CHL (no induction of polyploid cells)

+

(±)

Matsuoka et al. (1993)

3,9-Dinitrofluoranthene

GM

Chinese hamster lung cells V79 (HPRT test)

n.g.

+

Tokiwa et al. (1988)

 

CA

Chinese hamster lung cells CHL (no induction of polyploid cells)

+

(±)

Matsuoka et al. (1993)

1-Nitropyrene

GC

S. cerevisiae D4 (trp locus; cells exposed in suspension or in plates)

n.g.

McCoy et al. (1983c)

 

GC

S. cerevisiae D4 (trp locus)

h

n.g.

McCoy et al. (1984)

 

GM

Heterozygous soybean strain T219 (seeds exposed, mutated spots evaluated, MA by rat S9-mix; no effect with pyrene)

+

Katoh et al. (1994)

 

GM

Chinese hamster ovary cells (CHO, HPRT test)

+

+e

Marshall et al. (1982)

 

GM

Chinese hamster lung cells (maximum concentration of 20 µg/ml not cytotoxic, DT tested)

Nakayasu et al. (1982)

 

GM

Chinese hamster ovary cells (CHO, HPRT test)

+

+e

Li & Dutcher (1983)

 

GM

Chinese hamster lung cells (not tested up to cytotoxicity, DT tested)

n.g.

Sugimura & Takayama (1983)

 

GM

Chinese hamster lung cells V79 (MA by Syrian hamster embryo cells, NA test)

Takayama et al. (1983)

 

GM

Chinese hamster lung cells V79 (MA by co-cultivated rat hepatocytes, HPRT test)

+

Ball et al. (1985)

 

GM

Chinese hamster lung cells V79 (HPRT test)

(±)

+

Berry et al. (1985)

 

GM

Chinese hamster ovary cells (not tested up to cytotoxicity; high activity of the metabolite 1-nitrosopyrene, HPRT test)

n.g.

Fifer et al. (1986a)

 

GM

Chinese hamster ovary cells (CHO; not tested up to cytotoxicity, metabolites mutagenic without MA, HPRT test)

n.g.

Heflich et al. (1985a, 1986a)

 

GM

Chinese hamster ovary cells (CHO; no data on cytotoxicity, metabolite 1-nitrosopyrene mutagenic without MA, HPRT test)

n.g.

Heflich et al. (1986c)

 

GM

Chinese hamster ovary cells (CHO, HPRT test)

+e

Heflich et al. (1990)

 

GM

Chinese hamster CHO-K1 (DNA repair proficient, HPRT test)

n.g.

Thornton-Manning et al. (1991a)

 

GM

Chinese hamster CHO-UV5 (DNA repair deficient, HPRT test)

+

n.g.

Thornton-Manning et al. (1991a)

 

GM

Chinese hamster CHO-K1 (DNA repair proficient, HPRT test)

+

Thornton-Manning et al. (1991b)

 

GM

Chinese hamster CHO-UV5 (DNA repair deficient; more effective at anaerobic conditions, HPRT test)

+

Thornton-Manning et al. (1991b)

 

GM

Chinese hamster V79 cells (cells with different enzyme activities tested: CYP1A2 or CYP3A4 with and without endogenous acetyltransferase; HPRT test; positive with CYP1A2 plus acetyltransferase activity)

 

+

Kappers et al. (2000)

 

GM

Mouse NIH/3T3 cells (cells with two different enzyme activities tested: a) only acetyltransferase or b) CYP1A2; HPRT test; positive only with CYP1A2)

 

+

Kappers et al. (2000)

 

UDS

Primary rat hepatocytes (higher potency than in hamster hepatocytes)

 

+

Kornbrust & Barfknecht (1984)

 

UDS

Primary Syrian golden hamster hepatocytes (see above)

 

+

Kornbrust & Barfknecht (1984)

 

UDS

Primary rat tracheal epithelial cells

+

n.g.

Doolittle & Butterworth (1984)

 

UDS

Clara cells isolated from rabbit lung (primary cell culture)

+

n.g.

Haugen et al. (1986)

 

UDS

Alveolar type-II cells isolated from rabbit lung (primary cell culture)

n.g.

Haugen et al. (1986)

 

UDS

Primary rat hepatocytes

 

+

Mori et al. (1987)

 

UDS

Primary mouse hepatocytes

 

+

Mori et al. (1987)

 

SSB

Chinese hamster lung cells V79 (alkaline elution assay)

+

n.g.

Saito et al. (1984b)

 

SSB

Rat hepatoma cell line H4-II-E (alkaline elution assay)

 

+

Möller & Thorgeirsson (1985)

 

SSB

Primary mouse hepatocytes (alkaline elution assay)

 

+

Möller & Thorgeirsson (1985)

 

SSB

Chinese hamster lung fibroblasts Don

+

+

Edwards et al. (1986b)

 

SSB

Alveolar type II cells, Clara cells or macrophages isolated from rabbit lung (alkaline elution assay, tested up to cytotoxicity)

n.g.

Becher et al. (1993)

 

VE

Rat fibroblasts H3 transformed by ts-a mutant of polyoma virus

+

+e

Lambert & Weinstein (1987)

 

SCE

Chinese hamster lung cells V79 (abstract)

+

n.g.

Heidemann & Miltenburger (1983)

 

SCE

Chinese hamster lung cells V79

+

Schleibinger et al. (1989)

 

CA

Chinese hamster pulmonary cell line Don:Wg3h

+

n.g.

Lafi & Parry (1987)

 

CA

Chinese hamster lung cells CHL

i

+

Matsuoka et al. (1991)

2-Nitropyrene

MN

Isolated rodent hepatocytes (abstract)

 

+

Müller et al. (1995)

1,3-Dinitropyrene

GC

S. cerevisiae D4 (trp locus; cells exposed in suspension or in plates)

n.g.

McCoy et al. (1983c)

 

GM

Heterozygous soybean strain T219 (seeds exposed, mutated spots evaluated, MA by rat S9-mix)

+

+

Katoh et al. (1994)

 

GM

Chinese hamster lung cells (DT tested)

+

n.g.

Nakayasu et al. (1982)

 

GM

Chinese hamster ovary cells (CHO; mutagenicity increased with low amounts of MA system, HPRT test)

+

+f

Li & Dutcher (1983)

 

GM

Chinese hamster lung cells (DT tested)

+

n.g.

Sugimura & Takayama (1983)

 

GM

Chinese hamster lung cells V79 (MA by Syrian hamster embryo cells, NA test)

(±)

+

Takayama et al. (1983)

 

GM

Chinese hamster lung cells V79 (inhibition by haemin, NA test)

+

n.g.

Katoh et al. (1984)

 

UDS

Primary rat hepatocytes

 

+

Mori et al. (1987)

 

UDS

Primary mouse hepatocytes

 

+

Mori et al. (1987)

 

SSB

Primary mouse hepatocytes (alkaline elution assay)

 

(±)

Möller & Thorgeirsson (1985)

 

VE

Rat fibroblasts H3 transformed by ts-a mutant of polyoma virus

+

n.g.

Lambert & Weinstein (1987)

 

CA

Chinese hamster lung cells CHL

+

+f

Matsuoka et al. (1991)

 

MN

mouse hepatoma cells BWI-J (not tested up to cytotoxicity)

 

Roscher & Wiebel (1992)

 

MN

Rat hepatoma cells 5L (not tested up to cytotoxicity)

 

Roscher & Wiebel (1992)

 

MN

Rat hepatoma cells H4IIEC3/G-

 

+

Roscher & Wiebel (1992)

 

MN

Chinese hamster lung cells V79

+

n.g.

Roscher & Wiebel (1992)

 

MN

Rat fibroblastoid cells 208F (not tested up to cytotoxicity)

n.g.

Roscher & Wiebel (1992)

1,6-Dinitropyrene

GC

S. cerevisiae JD1 (trp and his loci)

+j

n.g.

Wilcox & Parry (1981)

 

GC

S. cerevisiae JD1 (trp and his loci)

+k

+e

Wilcox et al. (1982)

 

GC

S. cerevisiae D4 (trp locus; cells exposed in suspension or in plates)

n.g.

McCoy et al. (1983c)

 

GM

Heterozygous soybean strain T219 (seeds exposed, mutated spots evaluated, MA by rat S9-mix)

+

+

Katoh et al. (1994)

 

GM

Chinese hamster lung cells (DT tested)

+

+f

Nakayasu et al. (1982)

 

GM

Chinese hamster ovary cells (CHO, HPRT test)

+

+f

Li & Dutcher (1983)

 

GM

Chinese hamster lung cells (DT tested)

+

n.g.

Sugimura & Takayama (1983)

 

GM

Chinese hamster lung cells V79 (inhibition by haemin, NA test)

+

n.g.

Katoh et al. (1984)

 

GM

Chinese hamster ovary cells (CHO, HPRT test)

+

n.g.

Edgar & Brooker (1985)

 

GM

Chinese hamster ovary cells (CHO; higher activity of the metabolite 1-nitro-6-nitrosopyrene, HPRT test)

(±)

n.g.

Fifer et al. (1986a)

 

UDS

Primary rat hepatocytes (higher potency than the positive control acetylaminofluorene)

 

+

Butterworth et al. (1983)

 

UDS

Primary rat tracheal epithelial cells

+

n.g.

Doolittle & Butterworth (1984)

 

UDS

Prima