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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY






    ENVIRONMENTAL HEALTH CRITERIA 5





    Nitrates, Nitrites and N-Nitroso Compounds









    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of either the World Health Organization or the United Nations
    Environment Programme

    Published under the joint sponsorship of the United Nations
    Environment Programme and the World Health Organization

    World Health Organization
    Geneva, 1978

    ISBN No. 92 4 154065 6

    (c) World Health Organization 1978

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    CONTENTS

    ENVIRONMENTAL HEALTH CRITERIA FOR NITRATES, NITRITES AND  N-NITROSO
    COMPOUNDS

    1. SUMMARY AND RECOMMENDATIONS FOR FURTHER STUDIES

         1.1. Summary
              1.1.1. Analytical methods
                      1.1.1.1   Nitrates and nitrites
                      1.1.1.2    N-nitroso compounds
              1.1.2. Sources and occurrence in the environment
                      1.1.2.1   Nitrates and nitrites
                      1.1.2.2    N-nitroso compounds
              1.1.3. Metabolism
                      1.1.3.1   Nitrates and nitrites
                      1.1.3.2    N-nitroso compounds
              1.1.4. Experimental studies in animals
                      1.1.4.1   Nitrates and nitrites
                      1.1.4.2    N-nitroso compounds
              1.1.5. Epidemiological studies
                      1.1.5.1   Nitrates and nitrites
                      1.1.5.2    N-nitroso compounds
              1.1.6. Evaluation of health risks
                      1.1.6.1   Nitrates and nitrites
                      1.1.6.2    N-nitroso compounds
         1.2. Recommendations for further studies
              1.2.1. Analytical problems
                      1.2.1.1   Nitrates and nitrites
                      1.2.1.2    N-nitroso compounds
              1.2.2. Sources and levels in the environment
                      1.2.2.1   Nitrates and nitrites
                      1.2.2.2    N-nitroso compounds
              1.2.3. Metabolism
                      1.2.3.1   Nitrates and nitrites
                      1.2.3.2    N-nitroso compounds
              1.2.4. Experimental studies
              1.2.5. Epidemiological and clinical studies
                      1.2.5.1   Nitrates and nitrites
                      1.2.5.2    N-nitroso compounds

    2. CHEMISTRY AND ANALYTICAL METHODS

         2.1. Chemical properties and reactions
              2.1.1. Nitrates and nitrites
              2.1.2.  N-nitroso compounds
              2.1.3. Formation of  N-nitroso compounds  in vitro
              2.1.4. The effects of other substances on the formation of
                       N-nitroso compounds
         2.2. Analytical methods
              2.2.1. Nitrates and nitrites
              2.2.2.  N-nitroso compounds

    3. SOURCES OF NITRATES, NITRITES AND  N-NITROSO COMPOUNDS IN AIR,
         WATER, SOIL, AND FOOD

         3.1. Natural occurrence
              3.1.1. Nitrates and nitrites
              3.1.2.  N-nitroso compounds
         3.2. Sources related to man's activities
              3.2.1. Nitrates and nitrites
                      3.2.1.1   Fertilizers
                      3.2.1.2   Animal wastes
                      3.2.1.3   Municipal, industrial, and transport
                                wastes
                      3.2.1.4   Deliberate addition of nitrates and
                                nitrites to food
              3.2.2.  N-nitroso compounds
                      3.2.2.1   Food
                      3.2.2.2   Tobacco
                      3.2.2.3   Industrial uses

    4. TRANSPORT AND TRANSFORMATION IN ENVIRONMENTAL AND BIOLOGICAL
         MEDIA

         4.1. Nitrogen Cycle
         4.2. Transformation in food
              4.2.1. Reduction of nitrates to nitrites
              4.2.2. Formation and degradation of  N-nitroso compounds
         4.3. Formation of  N-nitroso compounds from drugs and pesticides
         4.4. Formation of  N-nitroso compounds in animal organisms
              4.4.1. Formation of  N-nitroso compounds in simulated
                      gastric juice
              4.4.2. Formation of  N-nitroso compounds  in vivo
         4.5. Formation of  N-nitroso compounds by microorganisms
         4.6. The effects of other chemicals on the formation of
               N-nitroso compounds

    5. ENVIRONMENTAL LEVELS AND EXPOSURES

         5.1. Nitrates and nitrites
              5.1.1. Ambient air
              5.1.2. Water
              5.1.3. Selected foods
              5.1.4. Estimate of general population exposure
         5.2.  N-nitroso compounds
              5.2.1. Ambient air
              5.2.2. Water
              5.2.3. Selected foods
              5.2.4. Tobacco and tobacco smoke
              5.2.5. Estimate of general population exposure
              5.2.6. Occupational exposure to  N-nitroso compounds

    6. METABOLISM OF NITRATES, NITRITES, AND  N-NITROSO COMPOUNDS

         6.1. Gastrointestinal absorption
              6.1.1. Nitrates and nitrites
              6.1.2.  N-nitroso compounds
         6.2. Biotransformation and elimination
              6.2.1. Nitrates and nitrites
              6.2.2.  N-nitroso compounds

    7. EXPERIMENTAL ANIMAL STUDIES ON THE EFFECTS OF NITRATES, NITRITES,
         AND  N-NITROSO COMPOUNDS

         7.1. Nitrates and nitrites
              7.1.1. Acute and subacute toxicity studies
              7.1.2. Chronic toxicity and carcinogenicity studies
              7.1.3. Embryotoxicity
              7.1.4. Mutagenicity
              7.1.5. Interaction with nutritional factors
         7.2.  N-nitroso compounds
              7.2.1. Acute and subacute toxicity studies
              7.2.2. Carcinogenicity
                      7.2.2.1   Interspecies variation in response
                      7.2.2.2   Intraspecies variation in response
                      7.2.2.3   Dose-response relationships of  N-nitroso
                                compounds
                      7.2.2.4   Tumour induction by combined
                                administration of nitrites, and amines or
                                amides
                      7.2.2.5   Dose-response relationship for
                                combinations of nitrites and amines
                      7.2.2.6   Transplacental carcinogenesis
                      7.2.2.7   Morphological studies
                      7.2.2.8   Biochemical mechanisms
                      7.2.2.9   Interaction with various chemical factors
                      7.2.2.10  Miscellaneous modifying factors
              7.2.3. Embryotoxicity and teratogenicity
              7.2.4. Mutagenicity

    8. EFFECTS OF NITRATES, NITRITES, AND  N-NITROSO COMPOUNDS ON MAN

         8.1. Nitrates and nitrites
              8.1.1. Epidemiological studies
                      8.1.1.1   Exposure through water
                      8.1.1.2   Exposure through vegetables
                      8.1.1.3   High accidental exposures
                      8.1.1.4   Ambient air exposures
              8.1.2. Factors involved in susceptibility to nitrates
              8.1.3. Dose-response relationships for nitrates and
                      nitrites
         8.2.  N-nitroso compounds

    9. EVALUATION OF HEALTH RISKS TO MAN FROM EXPOSURE TO NITRATES,
         NITRITES, AND  N-NITROSO COMPOUNDS

         9.1. Nitrates and nitrites
              9.1.1. General considerations
              9.1.2. Assessment of health risks
         9.2.  N-nitroso compounds
              9.2.1. General considerations
              9.2.2. Assessment of health risks
         9.3. Reduction of exposure th

    REFERENCES
    

    NOTE TO READERS OF THE CRITERIA DOCUMENTS

        While every effort has been made to present information in the
    criteria documents as accurately as possible without unduly delaying
    their publication, mistakes might have occurred and are likely to
    occur in the future. In the interest of all users of the environmental
    health criteria documents, readers are kindly requested to communicate
    any errors found to the Division of Environmental Health, World Health
    Organization, Geneva, Switzerland, in order that they may be included
    in corrigenda which will appear in subsequent volumes.

        In addition, experts in any particular field dealt with in the
    criteria documents are kindly requested to make available to the WHO
    Secretariat any important published information that may have
    inadvertently been omitted and which may change the evaluation of
    health risks from exposure to the environmental agent under
    examination, so that the information may be considered in the event of
    updating and re-evaluating the conclusions contained in the criteria
    documents.

    WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR NITRATES,
    NITRITES, AND N-NITROSO COMPOUNDS

     Lyons, France, 16-21 February 1976

    Participants

     Members

    Dr. K. R. Bulusu, National Environmental Engineering Research
        Institute, Nagpur, India

    Mr T. J. Coomes, Food Chemistry, Composition and Safety, Ministry of
        Agriculture, Fisheries and Food, London, England  (Rapporteur)

    Dr R. Kroes, Department of Oncology, National Institute of Public
        Health, Bilthoven, Netherlands

    Dr W. Lijinsky, Frederick Cancer Research Center, Frederick, MD, USA

    Dr S.S. Mirvish, Eppley Institute for Research in Cancer, The
        University of Nebraska College of Medicine, Omaha, NE, USA

    Professor M. Nikonorow, Department of Food Research, State Institute
        of Hygiene, Warsaw, Poland

    Dr T. Petrova-Vergieva, Centre of Hygiene, Medical Academy, Sofia,
        Bulgaria

    Dr R. Preussmann, Institute for Toxicology and Chemotherapy, National
        Cancer Research Centre, Heidelberg, Federal Republic of Germany

    Dr P. Schmidt, Ministry of Health, Prague, Czechoslovakia

    Professor K. Symon, Centre of General and Community Hygiene, Institute
        of Hygiene and Epidemiology, Prague, Czechoslovakia  (Vice
         Chairman)

    Professor R. Truhaut, Toxicological Research Centre, Faculty of
        Pharmacy and Biology, Paris, France  (Chairman)

     Representatives of other organizations

    Mme M.-Th. van der Venne, Commission of the European Communities,
        Health Protection Directorate, Luxembourg

    Mr G. Dorin, Natural Resources and Pollution Control Division,
        Organization for Economic Cooperation and Development, Paris,
        France

     Secretariat

    Dr L. Griciute, Chief, Unit of Environmental Carcinogenesis, IARC,
        Lyons, France

    Dr A. Kolbye, Bureau of Foods, US Food and Drug Administration,
        Washington, DC, USA

    Dr F. C. Lu, Chief, Food Additives, World Health Organization, Geneva,
        Switzerland  (Secretary)

    Dr R. Montesano, Unit of Chemical Carcinogenesis, IARC, Lyons, France

    Dr I. C. Munro, Toxicology Research Division, Health Protection
        Branch, Department of National Health and Welfare, Ottawa, Canada

    Mr E. A. Walker, Unit of Chemical Carcinogenesis, IARC, Lyons, France

              

    a  Unable to attend:

        Dr E. Arrhenius, Director for Experimental Biology, Wallenberg
    Laboratory, Lilla Frescati, Stockholm, Sweden

        Dr V. Okulov, Petrov Research Institute of Oncology, Leningrad,
    USSR

     List of Abbreviations

    DMN     N-methyl- N-nitrosomethanamine ( N-nitrosodimethylamine,
           dimethylnitrosamine)

    DEN     N-ethyl- N-nitrosoethanamine   ( N-nitrosodiethylamine,
           diethylnitrosamine)

    DMA     N-methylmethanamine (dimethylamine)

    DEA     N-ethylethanamine (diethylamine)

    TMA     N,N-dimethylmethanamine (trimethylamine)

    TMAO   trimethyloxamine (trimethylamine oxide)

    ENVIRONMENTAL HEALTH CRITERIA FOR NITRATES, NITRITES, AND N-NITROSO
    COMPOUNDS

        A WHO Task Group on Environmental Health Criteria for Nitrates,
    Nitrites and  N-nitroso compounds met in Lyons from 16 to 20 February
    1976. Dr Higginson, Director of the International Agency for Research
    on Cancer opened the meeting on behalf of the Director-General. The
    Task Group reviewed and amended the second draft of the criteria
    document and made an evaluation of the health risks from exposure to
    these compounds.

        The preparation of the first draft of the criteria document was
    based on national reviews of health effects research on nitrates,
    nitrites, and  N-nitroso compounds received from the national focal
    points for collaboration in the WHO Environmental Health Criteria
    Programme in Bulgaria, Canada, Czechoslovakia, the Federal Republic of
    Germany, Netherlands, New Zealand, Poland, the United Kingdom, the
    USA, and the USSR. Dr I. C. Munro, Toxicological Research Division,
    Health Protection Branch of the Department of National Health and
    Welfare, Ottawa, Ontario, Canada, prepared both the first draft and
    the second draft which took into account the comments received from
    the national focal points in Bulgaria, Canada, Czechoslovakia,
    Finland, Japan, New Zealand, Poland, Sweden, USA, and the USSR; from
    the United Nations Industrial Development Organization (UNIDO), the
    Food and Agriculture Organization of the United Nations (FAO), the
    United Nations Educational, Scientific and Cultural Organization
    (UNESCO), the International Atomic Energy Agency (IAEA), the Health
    Protection Directorate of the Commission of the European Communities
    (CEC), and from the International Federation of Pharmaceutical
    Manufacturers' Associations (IFPMA).

        At the request of the Secretariat, comments were also received
    from Dr S. Oden, Agricultural College, Department of Soil Science,
    Division of Ecochemistry, Uppsala, Sweden.

        The collaboration of these national institutions, international
    organizations and individual experts is gratefully acknowledged.
    Without their assistance this document could not have been completed.
    The collaboration of the International Agency for Research on Cancer
    in the preparation of the document and in acting as host to the Task
    Group is also greatly appreciated.

        The Secretariat wishes to thank Mr A. W. Kenny, Department of the
    Environment, London, England and Mr D. A. H. Price, Chorley Wood,
    Herts, England for their advice in the preparation of some sections of
    the document and Dr Munro for his help in the final phases of editing.

        This document is based primarily on national contributions and on
    original publications listed in the reference section. In addition,
    some recent publications reviewing the environmental health aspects of

    nitrates, nitrites and  N-nitroso compounds have been used. These
    include reviews and symposia by the US National Academy of Sciences,
    Washington, DC (Committee on Nitrate Accumulation, 1972), the US
    Department of Health, Education and Welfare (1970), the US
    Environmental Protection Agency,a the International Agency for
    Research on Cancer (Bogovski 8: Walker, 1974; Bogovski et al., 1972a;
    Walker et al., 1970), Druckrey et al. (1967), Lee (1970a), Magee &
    Barnes (1967), Magee et al. (1976), Montesano & Bartch (1976), and Sen
    (1974).

        Details concerning the WHO Environmental Health Criteria
    Programme including the definition of some terms frequently used in
    the documents may be found in the general introduction to the
    Environmental Health Criteria Programme published together with the
    environmental health criteria document on mercury (Environmental
    Health Criteria 1 -- Mercury, World Health Organization, Geneva,
    1976).

              

    a  "US Environmental Protection Agency (1976) Scientific and
       technical assessment report on nitrosamines. Preprint of document
       submitted for publication in the STAR series, Washington DC,
       Office of Reserch and Development, 210 pp.

    1.  SUMMARY AND RECOMMENDATIONS FOR FURTHER STUDIES

    1.1  Summary

    1.1.1  Analytical methods

    1.1.1.1  Nitrates and nitrites

        The Task Group recognized that interpretation of the results of
    analyses of nitrates and nitrites in environmental and biological
    media would vary according to the analytical methods employed (e.g.
    spectrophotometry, spectrofluorimetry, nitrate specific electrode) and
    that this would make meaningful comparisons of much data in the
    literature difficult.

        It was considered, however, that reported figures for water and
    for meat products could be compared as far as the assessment of health
    hazards was concerned. The Group also noted that, although the
    principle underlying a particular method of analysis may well be the
    same for a variety of substrates, difficulties usually arise during
    the sampling, extraction, and clean-up procedures which vary in
    complexity according to the nature of the substrate being analysed.

    1.1.1.2  N-nitroso compounds

        The detection and estimation of volatile  N-nitroso compounds is
    complicated by the following basic issues: they are likely to be
    present in environmental media in concentrations of only 1 part in
    109 parts; they occur in a complex matrix in food and biological
    samples many components of which will contain nitrogen and will react
    in a similar manner chemically; they must be isolated from this matrix
    in a form that permits their estimation and unequivocal
    identification. Whilst removal from the matrix is easy in the case of
     N-nitroso compounds of low molecular weight because they are steam-
    volatile, this approach cannot be used for non-volatile  N-nitroso
    compounds. Analysis of these compounds has received scant attention so
    far, although some work on clean-up procedures exists, and the use of
    the liquid chromatography technique is now under investigation
    following its successful application to the separation of model
    mixtures of  N-nitroso- N-alkyl ureas and the analogous urethanes.

        Irrespective of the isolation techniques used, the quantitative
    determination of  N-nitroso compounds requires a concomitant positive
    identification of the molecular species determined. For this reason,
    the preferred method of analysis, gas-liquid chromatography allied to
    a nitrogen-sensitive detector, must be linked to high-resolution mass
    spectrometry to confirm the presence of  N-nitroso compounds. Results
    should be considered positive only when or if mass-spectroscopic
    techniques have confirmed their unequivocal presence.

    1.1.2  Source and occurrence in the environment

    1.1.2.1  Nitrates and nitrites

        Nitrates are present naturally in soils, waters, all plant
    materials, and in meats. They are also found in small concentrations
    (1-40 µg/m3) in air as a result of air pollution. Levels in
    cultivated soils and thus, levels in water, (which normally do not
    exceed 10 mg/litre) may be increased by the use of commercial
    nitrogenous fertilizers and by the return of wastes, derived from
    animal husbandry or other sources, to the soil. Nitrate contents of
    crops are influenced by the plant species, by genetic and
    environmental factors, and by agricultural management practices. In
    certain crops the levels may be very high (1000 mg/kg or more).

        Nitrites are formed in nature by the action of nitrifying
    bacteria as an intermediate stage in the formation of nitrates, but
    concentrations in plants and water are usually very low. However,
    microbiological conversion of nitrate to nitrite may occur during the
    storage of fresh vegetables, particularly at room temperature, when
    nitrite concentrations may rise to exceptionally high levels (about
    3600 mg/kg dry weight). Both nitrates and nitrites are widely used in
    the production and preservation of cured meat products and of some
    fish. Such uses, which are controlled by law in many countries, are
    considered vital for the prevention of botulism caused by the growth
    of the toxin-producing strains of  Clostridium botulinum that are
    sometimes present in raw meat and that may persist in cooked meats.
    The weekly intake of nitrates by a member of the general population in
    England or in the USA has been roughly estimated to average about
    400-500 mg but these figures cannot be applied generally because of
    variations in feeding habits and in the nitrate concentrations in food
    and water.

    1.1.2.2  N-nitroso compounds

        Low concentrations of  N-nitroso compounds have been detected in
    air, water, and food, notably in nitrite-treated meat products and
    certain fish products. In most cases, the concentrations found in food
    have been in the µg/kg range. No effective estimate of general
    population exposure to  N-nitroso compounds can be made on the basis
    of these limited data.  N-methyl- N-nitrosomethanamine
    ( N-nitrosodimethylamine, DMN) has been detected in urban air
    samples and the presence of  N-nitroso compounds, tentatively
    identified as  N-nitroso derivatives of some pesticides, has been
    reported in samples from water treatment plants and river water in
    the USA.

        The  in vivo formation of  N-nitroso compounds from nitrates or
    nitrites and amines or amides has been demonstrated in experimental
    animals and in one case in man.

    1.1.3  Metabolism

    1.1.3.1  Nitrates and nitrites

        In normal healthy individuals, nitrates and nitrites are rapidly
    absorbed from the gastro-intestinal tract. Absorbed nitrite reacts
    with haemoglobin to form methaemoglobin which, in adults, is rapidly
    converted to oxyhaemoglobin by reducing systems such as NADH-
    methaemoglobin reductase. In infants up to three months old and in
    very young animals this enzyme system is not completely developed.
    Under these conditions, the methaemoglobin formed may increase in the
    body resulting in a characteristic clinical condition
    (methaemoglobinaemia). Microorganisms present in the food and
    gastrointestinal tract of very young infants may convert nitrates to
    nitrites and thus exacerbate the problem in this age group. In healthy
    individuals, absorbed nitrates are rapidly excreted by the kidneys.

    1.1.3.2  N-nitroso compounds

        Published information on the absorption, metabolism, and
    elimination of  N-nitroso compounds is limited. In cases where
    experimental animal data are available, they demonstrate that
     N-nitroso compounds are rapidly absorbed from the gastrointestinal
    tract and that their biological half-time appears to be less than
    24 h. A part of some compounds may be excreted unchanged via the
    kidney, or even exhaled, but the greater part is metabollically
    transformed (hydroxylation, chain shortening, ring-opening etc.) and
    several metabolites of  N-nitroso compounds have been identified
    in urine. Significant amounts of some compounds such as DMN may be
    degraded completely and the resulting carbon dioxide exhaled. The
    extent of such degradation varies depending on the structure of the
    compound and the animal species involved.

    1.1.4  Experimental studies in animals

    1.1.4.1  Nitrates and nitrites

        The major effect of nitrates and nitrites is the induction of
    methaemoglobinaemia, mostly readily observed in very young animals.
    Most experimental work has been connected with this problem although
    embryotoxic effects resulting in prenatal mortality, resorptions, and
    decreased birthweights have been noted in rat pups whose mothers
    received drinking water containing sodium nitrite. In adult rats given
    drinking water containing nitrite for 24 months, methaemoglobin levels
    were elevated but not to the point of producing overt toxic effects.
    Animal species studied appeared to be fairly resistant to the
    induction of methaemoglobinaemia by nitrites, since high doses were
    required to induce even minimal changes. However, very young animals
    have not been studied extensively or sufficiently. Nitrates and
    nitrites do not appear to be carcinogenic but nitrite mutagenicity has
    been demonstrated in several non-mammalian test systems.

    1.1.4.2  N-nitroso compounds

        In experimental animals, the most important biological actions of
     N-nitroso compounds are their carcinogenicity and teratogenicity.

        The carcinogenic action of  N-nitroso compounds in animals is
    known to occur in many different organs. In general, the routes of
    administration do not influence the localization of the tumours.
    However, both dose level and dose rate may affect the organ involved
    and the type of tumour produced. Specific structural changes in both
    dialkyl nitrosamines and cyclic nitrosamines affect their
    carcinogenicity.  N-nitroso compounds have been shown to be
    transplacentally carcinogenic, when given to animals in the second
    part of gestation, irrespective of the route of administration.
    Carcinogenicity following the combined administration of amines or
    amides and nitrites to animals has also been reported indicating the
     in vivo formation of  N-nitroso compounds.

        The mutagenic action of nitrosamides, noted in test systems,
    differs from that of nitrosamines in that the first group of compounds
    has been found to be mutagenic in almost all test systems, whereas
    nitrosamines seem only to be active in systems where metabolic
    activation occurs.

        Nitrosamines are known to have toxic and sometimes lethal effects
    on animal embryos, whereas nitrosamides cause malformations in several
    organs and systems.

    1.1.5  Epidemiological studies

    1.1.5.1  Nitrates and nitrites

        Adults do not appear to be harmed directly by exposure to the
    prevailing concentrations of nitrates and nitrites in the environment,
    although some recent studies have indicated that nitrate aerosols in
    the ambient air may act as respiratory irritants. However infants and
    very young children are particularly susceptible to the induction of
    methaemoglobinaemia by nitrates and nitrites, ingested in water and
    food, and several cases of illness and death have been reported. In
    most cases of methaemoglobinaemia, well-water containing high
    concentrations of nitrates had been used in the reconstitution of
    infant dried milk preparations. Most instances have been associated
    with water containing more than 90 mg per litre but a few cases of
    methaemoglobinaemia in infants have been associated with the
    consumption of water containing less than 50 mg per litre. Cases of
    methaemoglobinaemia in babies fed with spinach purée or carrot juice
    (both of which may contain very high levels of nitrates) have been
    reported, but there are too few data to establish dose-response
    relationships.

    1.1.5.2  N-nitroso compounds

        So far, correlations have not been established that link cancer
    in man with exposure to  N-nitroso compounds or their precursors, but
    the possible role of  N-nitroso compounds and in particular their
     in vivo formation in the development of nasopharyngeal, oesophageal,
    and stomach cancer has been suggested.

    1.1.6  Evaluation of health risks

    1.1.6.1  Nitrates and nitrites

        Epidemiological and clinical studies on man have shown that the
    main toxic manifestation resulting from the ingestion of nitrates and
    nitrites is methaemoglobinaemia. This has been confirmed by
    experimental animal studies. On the basis of available data, the Task
    Group concluded that the prevailing concentrations of nitrates and
    nitrites in food and water did not constitute a health risk for adult
    members of the general population and older children, but that the
    risk may be higher for infants under 6 months of age and particularly
    under 3 months. For this reason, the Group recommended that infant
    dried milk preparations should be reconstituted with low-nitrate water
    (at least below 45 mg/litre) and that low-nitrate vegetables should be
    used in baby foods.

        Also, the use of nitrates and nitrites as food additives should
    be reduced to the minimum, and avoided in fresh meat and fish. Nitrate
    levels in public water supplies should comply with the tentative limit
    of 45 mg/litre recommended by the 1971 International Standards for
    Drinking Water.

    1.1.6.2  N-nitroso compounds

        Although the precursors of  N-nitroso compounds (nitrites,
    amines, and amides) are known to be widely distributed in various
    environmental media, information concerning  N-nitroso compounds is
    limited. However, they are known to be present in certain foods and
    experimental animal studies have shown that they are formed in the
    body from a variety of precursors. This may also occur in man.

         N-nitroso compounds are carcinogenic in a wide range of animal
    species, most are mutagenic in test systems, and some have been shown
    to be teratogenic in animals.

        Although there is no epidemiological or clinical evidence at
    present, it is highly probable that these compounds may also be
    carcinogenic in man. A quantitative estimation of the carcinogenic
    risk to man associated with different levels of exposure is not
    possible, at this time, because of inadequate data. For these reasons,
    exposure to  N-nitroso compounds and their precursors, (nitrites,
    amines, and amides) should be kept as low as practically achievable.

    1.2  Recommendations for Further Studies

    1.2.1  Analytical problems

    1.2.1.1  Nitrates and nitrites

        The major need is for standardization of analytical methods. At
    present, it is difficult to compare the studies reported by one
    laboratory with those reported by another. While in many instances the
    principle underlying the determination is the same for many of the
    studies reported, the large variety of substrates containing nitrates
    and nitrites gives rise to difficulties with respect to sampling,
    extraction, and cleanup procedures. Further efforts are needed to
    standardize these analytical procedures on an international basis. To
    this end, the efforts of international and regional groups should be
    supported.

    1.2.1.2  N-nitroso compounds

        The principal problem associated with the determination of
     N-nitroso compounds in food and other environmental media results
    from interference by other components of the substrates. At present,
    positive identification of  N-nitroso compounds can be made only by
    mass spectroscopie techniques. Since such techniques are expensive and
    not generally available, alternative methods are required. In
    addition, methods for the detection and determination of nonvolatile
     N-nitroso compounds should be developed further.

    1.2.2  Sources and levels in the environment

    1.2.2.1  Nitrates and nitrites

        Research should be undertaken to find acceptable substitutes for
    nitrates and nitrites in the preservation of certain foods such as
    canned meats.

        National surveys of nitrate levels in soils, water, plant
    materials, foods, especially meat and milk products, and air are
    required together with quantitative data concerning other factors
    considered to have an effect on these levels. Similar information on
    nitrite levels is required with particular reference to foods and to
    areas where significant microbiological reduction of nitrates is
    likely.

        It is important that levels determined in survey work of this
    nature should be reported on the basis of standardized analytical
    methods to facilitate the eventual comparison of data from all
    sources. National authorities should be encouraged to publish survey
    data or to communicate them to the World Health Organization.

    1.2.2.2  N-nitroso compounds

        National surveys of food, air, and water for the presence of
    volatile and, where possible, nonvolatile  N-nitroso compounds are
    required and any results reported should be confirmed by mass
    spectroscopy. More studies are needed on the chemical conditions under
    which  N-nitroso compounds are formed (e.g. in mixtures of nitrites
    and amines or amides). The use of ascorbic acid for the prevention of
    nitrosamine formation and the inhibitory or catalytic effect of food
    constituents on the formation of  N-nitroso compounds also require
    studies. The role of oxides of nitrogen as possible nitrosating agents
    should be investigated in relation to the occurrence of  N-nitroso
    compounds in the environment (e.g. in the ambient and workroom air).

    1.2.3  Metabolism

    1.2.3.1  Nitrates and nitrites

        Further work on the influence of ascorbic acid and other
    ingredients of the stomach contents on the metabolism of nitrates and
    nitrites is required. The treatment of infant dried milk formulae with
    ascorbic acid or by the introduction of  Lactobacilli to prevent
    nitrate reduction should also be studied.

        Other areas requiring investigation include: the influence of
    gastro-enteric disease on the development of methaemoglobinaemia; the
    influence of the total gut flora on nitrate metabolism  in vivo; the
    relationship between ingested nitrate and salivary nitrate and nitrite
    levels.

    1.2.3.2  N-nitroso compounds

        More knowledge should be gained on the  in vivo formation of
     N-nitroso compounds in man and the factors involved. Studies
    comparing the metabolism of  N-nitroso compounds in experimental
    animals and in man are considered to be of the greatest importance.

    1.2.4  Experimental studies

        Further research on the biological action of  N-nitroso
    compounds should concentrate on dose-response relationships especially
    at low levels, and on their combined effects with other carcinogens,
    and environmental pollutants. The influence of nutritional factors on
    the carcinogenicity of  N-nitroso compounds should be studied in more
    detail.

        More inhalation studies are necessary to assess the importance of
    the recently reported occurrence of  N-nitroso compounds in air and
    further research is needed on the quantitative aspects of the
    mutagenic activity of  N-nitroso compounds and its possible
    significance for man.

    1.2.5  Epidemiological and clinical studies

    1.2.5.1  Nitrates and nitrites

        With respect to the adverse effects of nitrates and nitrites on
    infants, there is a need to investigate the relationship between
    methaemoglobinaemia and sudden infant death and to make further
    studies on the role of gastroenteritis in increasing infant
    susceptibility to nitrate poisoning. The role of acidified milk
    preparations and  Lactobacilli in protecting infants against
    methaemoglobinaemia, and the possible protective role of ascorbic acid
    fortification of infant milk preparations should also be elaborated.

    1.2.5.2  N-nitroso compounds

        Prospective and retrospective epidemiological studies in man,
    exposed to  N-nitroso compounds, are needed. Efforts should be made
    to determine whether cancers, that are peculiar to special areas of
    the world, might be due to exposure to  N-nitroso compounds. Chemical
    analyses of the environment for  N-nitroso compounds and their
    precursors should be carried out in conjunction with these
    epidemiological studies.

    2.  CHEMISTRY AND ANALYTICAL METHODS

    2.1  Chemical Properties and Reactions

    2.1.1  Nitrates and nitrites

        The nitrate ion (NO3-) is the conjugate base of nitric acid
    (HNO3). Nitric acid is a strong acid (pKa = -1.37) which
    dissociates in water yielding nitrate ions and hydroxonium ions
    (H3O+). Salts of nitric acid (nitrates) are readily soluble in
    water with the exception of the basic nitrates of mercury and bismuth.

        The nitrite ion is the conjugate base of nitrous acid (HNO2)
    which is a weak acid (pka = 3.37) and exists only in cold dilute
    aqueous solution because it decomposes readily to give water and
    dinitrogen trioxide (N2O3) or nitric acid, nitric oxide (NO), and
    water. Salts of nitrous acid (nitrites) are much more stable than the
    acid itself and are readily soluble in water with the exception of
    silver nitrite.

        In the environment (e.g. surface waters, soil) both nitrite and
    nitrate ions can be formed from the ammonium ion (NH4+) in a two
    step biological oxidation (nitrification) process:

    2 NH4+ + 2OH- + 3O2 <=> 2 NO2- + 2H+ + 4H2O               (1)

    2 NO2- + O2 <=> 2 NO3-                                      (2)

        These two reactions are mediated by different microorganisms:
    reaction (1) by an aerobic chemolithotroph  Nitrosomonas; reaction
    (2) by  Nitrobacter which obtains almost all its energy from the
    oxidation of nitrites.

        Higher plants assimilate nitrite from the soil by (a) reduction
    of nitrate to nitrite which is catalysed by nitrate reductase (NADPH)
    (1.6.6.3), and (b) reduction of nitrite to ammonia catalysed by
    nitrite reductase (1.7.99.3). Bacteria of many kinds can also reduce
    nitrate to nitrite. However, because nitrite is easily oxidised to
    nitrate the concentration of nitrites in environmental media such as
    surface waters is usually very low (about 1 mg/litre) even when the
    nitrate concentration is high (50-100 mg/litre).

        These biochemical reactions are a part of the nitrogen cycle
    which is further discussed in section 3.1.

    2.1.2  N-nitroso compounds

         N-nitroso compounds have a general structure

                            R1
                              \   N-N=O
                            R2/

        They can be divided into two classes with different chemical
    properties (Druckrey et al., 1967; Fridman et al., 1971):

         (1)  nitrosamines where R1 and R2 are alkyl or aryl groups;
              and

         (2)  nitrosamides where R1 is an alkyl or aryl group, and R2
              is an acyl group.

        Nitrosamines are generally stable compounds that only slowly
    decompose in the light or in aqueous acid solutions.

        In contrast, nitrosamides are much less stable in aqueous acids
    and unstable in basic solutions. Examples of nitrosamides are  N-
    alkyl- N-nitrosoureas (3) and  N-alkyl- N-nitrosourethanes (4).

                      R - N - C - NH2                              (3)
                          '   "
                         NO   O

                      R - N - C - OC2 H5                           (4)
                          '   "
                         NO   O

    The physical properties of  N-nitroso compounds vary widely depending
    on the substituent groups. Some like  N-methyl- N-nitrosomethanamine
    (dimethylnitrosamine, DMN) are oily liquids miscible with polar
    solvents. Some are solids e.g.  N-nitroso- N-phenylbenzenamine
    (diphenylnitrosamine) and are only slightly soluble in ethanol and
    practically insoluble in water. The lipid/water partition coefficients
    vary widely. Nitrosamines show ultraviolet absorption peaks in water
    at 230-240 nm and 330-350 nm. For nitrosamides, the long-wavelength
    absorption peak in water is at 390-420 nm. Some  N-nitroso compounds
    are volatile (Mirvish, 1975, 1976; Sen, 1974). Physical properties of
     N-nitroso compounds have been listed by Druckrey et al. (1967),
    Fieser & Fieser (1967), and Weast (1976).

        Nitrosamines may react by "transnitrosation" i.e. as nitrosating
    agents to nucleophilica species (Buglass et al., 1974). This
    reaction may have important biological implications.

              

    a  i.e. electron-rich.

    2.1.3  Formation of N-nitroso compounds  in vitro

        The formation of  N-nitroso compounds from amines and nitrites
    has been reviewed by Mirvish (1975), Sander (1971a, 1971b), and Sander
    & Schweinesberg (1972).

        For example, for  N-methylmethanamine (dimethylamine) (DMA) and
    sodium nitrite in dilute hydrochloric acid solutions, nitrositation is
    considered to proceed as follows (Mirvish, 1970):

    NaNO2 + HCl <=> HNO2 + NaCl                                  (5)

    2HNO2 <=> N2O3 + H2O                                         (6)

    (CH3)2NH + N2O3 <=> (CH3)2HN - NO + NO2-                     (7)

        The reaction rate depends on the concentration of nonionized
    amine and nitrous acid. At pH > 1, the main nitrosating agent is
    dinitrogen trioxide which is formed reversibly from 2 molecules of
    nitrous acid. The rate of reaction (7) is proportional to the
    concentration of dinitrogen trioxide, [N2O3], and hence to the
    square of nitrous acid concentration, [HNO2]2, i.e.

              rate (7) = k1[N2O3] [HNO2]2                       (8)

    The concentrations of nonionized amine and of free nitrous acid vary
    with pH but k1, is independent of pH. For practical purposes it is
    more convenient to rewrite equation (8) in terms of the total
    concentrations of nitrite and DMA i.e.

              rate (3) = k2 [total amine] [total nitrite]2      (9)

    where k2 depends on pH; k2 and the reaction rate show maximum
    values at pH = 3.4 corresponding to the strength of nitrous acid
    (pka = 3.37). The reaction rate decreases tenfold for each 1-unit
    increase in pH above pH = 3.4. Below this pH level, the nitrite is
    almost completely converted to nitrous acid. The main effect of a
    further reduction in pH is a continuous drop in nonionized amine
    concentration, causing a decrease in the reaction rate. There is no
    sharp pH limit for nitrosation. It can occur slowly at a pH of 5 or
    even 6, as observed for DMA (Mirvish, 1970).

        The nitrosation of amides, such as  N-alkylureas and
     N-alkylurethanes proceeds rapidly (Challis & Challis, 1970;
    Mirvish, 1971; Sander & Burkle, 1969). In this case the nitrosating
    agent is probably the nitracidium ion (H2NO2)+:

              2HNO2 <=> (H2NO2)+ + NO2-                      (10)

              or

              HNO2 + H+ <=> (H2NO2)+                         (10a)

    and nitrosation is accomplished by the following reaction:

    RNH.COR' + (H2NO2)+ -> RN(NO).COR' + H2O + H+            (12)

        The reaction rate is again proportional to the concentrations of
    nonionized alkylurea and nitracidium ions the formation of which can
    be considered to proceed by equation (10a). Hence

              rate (12) = k3 [RNH.COR'] [HNO2] [H+]                 (13)

              or

              rate (12) = k4 [total amide] [total nitrite] [H+]     (14)

    The reaction rate, which increases about tenfold for each 1-unit drop
    in pH from 3 to 1, does not show a pH maximum; k4 depends on the
    ionization of nitrite and, hence, on pH but it does not depend on the
    ionization of amides, which are only slightly ionized above pH = 2.

        Tables giving the rate constants for 15 amines and 21 amides
    according to the above equations (Mirvish, 1975), indicate that the
    most rapidly nitrosated classes of compounds are the  N-alkylureas,
     N-arylureas,  N-alkylcarbamates, secondary aromatic amines,
    secondary amine piperazine, morpholine derivatives, and tertiary
    enamines.

        It has been suggested that under mildly acidic conditions
    tertiary amines also react with nitrous acid to produce nitrosamines
    (Hein, 1963; Lijinsky, 1974; Lijinsky & Greenblatt, 1972; Lijinsky &
    Singer, 1974; Lijinsky et al., 1972b; Roberts & Caserio, 1964; Smith &
    Loeppky, 1967). Ender et al. (1967) studied the reaction between
    nitrites and various methylamines including: methanamine
    (monomethylamine); DMA;  N,N-dimethylmethanamine (trimethylamine,
    TMA); and trimethyloxamine (trimethylamine oxide, TMAO); they found
    that DMN was produced in all cases. However, the rate of production
    was proportional to the amount of nitrite present and increased with
    decreasing pH values and increasing temperature. DMA was the most
    reactive followed by TMA. Small amounts of DMN were formed from DMA
    and sodium nitrite under very mild conditions (e.g. at 4°C). At 

    pH = 6.0, 2 to 2.5 times more DMN was formed than at pH = 6.5.
    However, Malins et al. (1970), who failed to detect DMN formation at
    pH levels of 5.8-6.4 after heating an aqueous mixture of sodium
    nitrite and TMAO or DMA, found that trace amounts of DMN were
    detectable in reaction mixtures consisting of TMA at concentrations of
    400-2000 mg/litre and sodium nitrite at 400 mg/litre.

        Fiddler et al. (1972) demonstrated the formation of DMN from
    quaternary ammonium compounds and nitrite. The compounds studied
    included  N,N,N-trimethylethaminium chloride (neurine chloride),
    2-(acetyloxy)- N,N,N-trimethylethanaminium chloride (acetylcholine
    chloride), choline chloride, 1-carboxy- N,N,N-trimethylmethanaminium
    hydroxide (betaine), and 3-carboxy-2-hydroxy- N,N,N-trimethyl-1-
    propan-aminium chloride (carnitine chloride).

        Nitrites are present in various foods and in saliva (Tannenbaum
    et al., 1974) and can be formed in the infected bladder by bacterial
    reduction. They may also be present in the stomach of infants and of
    achlorhydric subjects where they are formed from nitrates, lower
    acidity allowing the growth of nitrate-reducing bacteria.

        Secondary amines are widely distributed in foods and have been
    found in fish, eggs, rolls, biscuits, chocolate, soup cubes, meats,
    and potatoes (Heyns, 1973, Lijinsky & Epstein, 1970). Tobacco and
    tobacco smoke contain several secondary amines including pyrrolidine,
    DMA, and piperidine (Neurath, 1972). Some aliphatic and heterocyclic
    amines were identified in human blood and urine (Asatoor & Simenhoff,
    1965; Perry et al., 1962). Other sources of secondary amines have been
    given by Sander et al. (1971).

        Methylguanidine, a natural constituent of beef (Kapeller-Adler &
    Krael, 1930a) and shark, rayfish, and cod (Kapeller-Adler & Krael,
    1930b), reacted with nitrite to produce  N-methyl- N-nitrosourea and
     N-methyl- N-nitrosocyanamide (Mirvish, 1971). The amino acids
    1-proline, 1-hydroxyproline, and  N-methylglycine (sarcosine) were
    nitrosated 140-230 times more quickly than DMA at pH = 2.2-2.5
    (Mirvish et al., 1973a).

         N-nitroso compounds formed from 22 natural compounds were
    listed by Mirvish (1975). In addition, nitrosation of  N,N-bis (3
    aminopropyl)-1, 4-butanediamine (spermine) and spermidine, two
    polyamines, was reported by Ferguson et al. (1974) and Hildrum et al.
    (1975).

    2.1.4  The effects of other substances on the formation of N-nitroso
           compounds

        Several substances have been shown to catalyse the formation of
    nitroso compounds from secondary amines and nitrite. Boyland & Walker
    (1974) and Fan & Tannenbaum (1973) noted that chloride, bromide,
    iodide, and thiocyanate catalysed the reaction while sulfate and
    perchlorate ions did not have any effect. The effects of thiocyanate 

    have been studied more extensively; in its absence, the nitrosation of
     N-methylbenzenamine ( N-methylaniline) and other secondary amines
    is at a maximum at pH = 3, but in its presence, the reactions proceed
    much more rapidly between pH levels of 1 and 2. Thiocyanate is present
    in amounts of 110-330 mg/litre in human saliva. It has been estimated
    that the thiocyanate concentration in the stomach is 3 times higher in
    smokers than in nonsmokers.

        Roller & Keefer (1974) reported a pronounced increase in the rate
    of formation of DMN from DMA and nitrite in the presence of certain
    carbonyl compounds and at a pH level higher than 3. Formaldehyde was
    the most effective catalyst and the effect was appreciable even at 
    pH = 9. Challis & Bartlett (1975) reported that 3-[[3-(3,4-
    dihydroxyphenyl)-1-oxo-2-propenyl]oxy]-1,4,5-trihydroxycyclohexane-
    carboxilic acid (chlorogenic acid), a constituent of coffee was a
    potent catalyst and in studies by Walker et al. (1975) 3,4,5-
    trihydroxybenzoic acid (gallic acid) catalysed the nitrosation of
    amines but only within a restricted pH range (around pH = 4).

        On the other hand, Bogovski et al. (1972b) noted that tannins,
    which are present in many foods, competed with secondary amines for
    nitrite and thus led to a reduction in the amount of nitrosamine
    formed. Similarly Challis (1973) demonstrated the preferential
    nitrosation of phenols in the presence of amine to form
     p-nitrosophenols suggesting a scavenging effect of phenols at
    low pH.

        Ascorbic acid inhibited the formation of DMN from oxytetracycline
    and nitrite and also from aminophenazone (aminopyrine) and nitrite
    (Mirvish et al., 1972, 1974). The same authors reported that gallic
    acid, the active ingredient in tannins, completely inhibited
    nitrosomorpholine formation from the parent amine and nitrite and that
    sodium sulfite had a similar blocking activity.

        The inhibitory effects of ascorbic acid and other inhibitory
    agents on chemical nitrosation have recently been compared by Mirvish
    et al., 1975 and it would seem, at present, that ascorbic acid is the
    most effective and useful inhibitor of amine nitrosation.

    2.2.  Analytical Methods

    2.2.1  Nitrates and nitrites

        Methods for the determination of nitrates and nitrites in surface
    and waste waters have been reviewed by Marculescu (1971). The most
    suitable methods are colorimetric procedures using sodium salicylate
    for nitrates and 4-aminobenzenesulfonic acid (sulfanilic acid) and
    1-naphthalenamine (l-naphthylamine) for nitrites.

        A standard procedure for determining nitrates in plants (HMSO,
    1973) is based upon the reduction of nitrates to ammonia which is


    removed by steam distillation and determined titrimetrically. The
    nitrate electrode has been used in the determination of nitrates in
    extracts of soils and herbage, and drainage water in the United
    Kingdom (HMSO, 1974) and in the Federal Republic of Germany (Weil &
    Quentin, 1973). Results indicated that several extraction procedures
    applied to herbage gave higher values with the nitrate electrode than
    with the standard distillation procedure. For drainage waters, better
    agreement was obtained between the electrode and a spectrophotometric
    procedure involving, 3,4-xylenol.

        A variety of methods is available for the determination of
    nitrates and nitrites in foods. A nitrate specific electrode for the
    electrochemical determination of nitrate in spinach suspensions was
    tested by Voogt (1969). Other anions Present in the spinach did not
    have any direct influence on the precision of the results. Variations
    in nitrate activity due to variations in the ionic strength of the
    spinach extracts could be minimized by measuring the potential of the
    extract in a 1% sodium sulfate solution. The precision of the method
    was ± 2%. Kamm et al. (1965) developed a new method for the
    determination of nitrates and nitrites in foods that would accurately
    determine concentrations as low as 1 mg/kg. 1-Naphthylamine was
    diazotized by nitrite and coupled with excess amine to give 4-(1-
    naphthylazo)-1-naphthylamine which was measured spectrophoto-
    metrically. Nitrate was quantitatively reduced by passage through a
    cadmium column and determined as nitrite. Nitrite passed through the
    column unaltered; thus nitrate was determined by difference.
    Spectrophotometric and spectrofluorimetric methods for the
    determination of low levels of nitrite in cheese were developed by
    Rammel & Joerin (1972). The limits of detection for the two methods
    were 50 µg and 3.0 µg of nitrite-N respectively, per kg of cheese or
    milk products. A method to determine free and bound nitrite in meats
    was published by Mirna (1974). Free nitrite was determined with the
    Griess reagent whereas bound nitrite was liberated with Hg2+ in
    aqueous acetone solution prior to diazotization. Methods of analysis
    for nitrates and nitrites in several food products including meats,
    cured meats, dry cure mix of curing pickle, flours, and baby foods
    have been described (Horwitz, 1975) and adequate methods for the
    determination of nitrates and nitrites in urine and blood are also
    available (Shechter et al., 1972; Schneider & Yeary, 1973; Wegner,
    1972).

    2.2.2  N-nitroso compounds

        The problems of estimating  N-nitroso compounds in food and
    other environmental media were recently reviewed by Bogovski & Walker
    (1974), Bogovski et al. (1971a), Eisenbrand (1973), Fiddler (1975),
    Scanlan (1975), Sen (1974), and Walker et al., (1976). The analytical
    process can be divided into three major steps: extraction and
    distillation from the specimen; purification; and qualitative and
    quantitative determination.

        The main difficulties in such analyses arise from the fact that
    nitrosamines occur at very low concentrations and that they lack
    suitable characteristics for trace analysis. They also suffer from
    interference from other chemicals in the substrate which gives rise to
    a considerable number of false-positive reports of the presence of
    nitrosamines.

        Most of the nitrosamines so far detected in foods are steam
    volatile. Many analytical methods take advantage of this fact and, in
    most of them, nitrosamines are isolated by distillation from an
    aqueous, acidic, or basic solution. Distillation from an acidic
    solution has the additional advantage of removing interfering amines.
    Howard et al. (1970) digested fish samples with methanolic potassium
    hydroxide before subjecting them to distillation. Telling et al.
    (1971) reported improved recoveries of nitrosamines by vacuum
    distillation. Other workers (Kröller, 1967; Sen et al., 1969a, 1972)
    preferred initial extraction of the nitrosamines with ether or
    methylene chloride prior to aqueous distillation. However, these
    techniques cannot be used for the analysis of nonvolatile nitrosamines
    such as nitrosoproline and nitrososarcosine.

        Various column chromatographic procedures have been reported for
    the clean-up of nitrosamines isolated from foods and biological
    materials. These include ion-exchange (Alam et al., 1971a, 1971b; Sen
    et al., 1969a, 1969b) or basic alumina columns (Sen, 1970; Sen et al.,
    1970; Telling et al., 1972). These preliminary clean-ups have proved
    to be extremely useful in cases where nitrosamines were estimated by
    the conventional thin layer chromatography (TLC) and gas-liquid
    chromatography (GLC) methods but such clean-ups were thought to be
    unnecessary when a highly specific method such as high resolution gas-
    liquid chromatography-mass spectroscopy (GLC-MS), was used for the
    analysis.

        Detection techniques can be divided into screening methods
    suitable for routine surveys, and confirmation techniques to be used
    if the results of the preliminary screening technique are positive.
    Combined high resolution GLC-MS is believed to be the only reliable
    confirmation technique available at the moment.

        Eisenbrand & Preussman (1970) have described a colorimetric
    technique in which nitrosamines are cleaved to nitrosyl bromide and
    secondary amines, and the liberated NO+ ion is measured
    colorimetrically after reacting with  N-1-naphthalenyl-1,2-
    ethanediamine (N(naphthyl-(1)-) ethylenediamine). The technique
    appears to be reliable and applicable to a wide variety of
    nitrosamines. The aminies formed after splitting may also be used to
    estimate nitrosamines through the formation of fluorescent
    5-(dimethylamino)-1-naphthalenesulphonyl (dansyl) derivatives.

        Various TCL methods have been used for the detection and semi-
    quantitative estimation of nitrosamines (Eisenbrand & Preussman, 1970;
    Kröller, 1967; Möhler & Mayrhofer, 1968; Sen & Dalpé, 1972; Sen et
    al., 1969a, 1973a; Yang & Brown, 1972). Most of these methods are
    based on the principle of splitting the nitrosamines by UV radiation
    into the parent secondary amines and nitrous acid, and subsequently
    detecting these breakdown products with 2,2-dihydroxy-IH-indene-
    1,3(2H)-dione (ninhydrin)  N-phenylbenzeneamine (diphenylamine), and
    Griess reagent, respectively. In some methods, nitrosamines are
    reduced to hydrazines which are detected on TLC plates after the
    formation of suitable derivatives.

        GLC offers a rapid and sensitive technique for the analysis of
    nitrosamines. In earlier work, the flame ionization detector was used
    but it was later abandoned because of the lack of sensitivity, and
    specifity. In more recent studies, various nitrogen-specific detectors
    have been used such as the alkaline-flame ionization detector (Fiddler
    et al., 1971; Howard et al., 1970; Kawabata, 1974), the Coulson
    electrolytic conductivity detector (Crosby et al., 1972; Issenberg &
    Tannenbaum, 1972; Panalaks et al., 1972; Rhoades & Johnson, 1970; Sen
    et al., 1972, 1973a), and the microcoulometric detector (Newall &
    Sisken, 1972). Each has some advantages and disadvantages, and the
    reader is advised to consult the original papers for further details.
    In one technique, nitrosamines were oxidized to the corresponding
    nitramines which were then detected by an electron capture detector
    (Althorpe et al., 1970; Castegnaro et al., 1972; Sen, 1970; Telling,
    1972). Alliston et al. (1972) and Eisenbrand (1972) converted the
    nitrosamines to the parent amines from which the heptafluoro-butyryl
    derivatives were prepared and determined by electron capture detector.

        Recently, Fine & Rufeh (1974) and Fine et al. (1974) have
    reported a new instrument which is specific to the  N-nitroso
    functional group and is capable of detecting  N-nitroso compounds in
    foodstuffs at the µg/kg level with little or no concentration or
    purification. In this technique, the  N-nitroso compounds are cleaved
    at the  N-NO bond in the presence of a specific catalyst, and the
    liberated NO is converted to excited nitrogen dioxide (NO2*) by
    reaction with ozone. As the excited nitrogen dioxide rapidly decays,
    it emits light in the near infrared region of the spectrum which can
    be detected and measured. The instrument can be coupled either to a
    GLC or a high pressure liquid chromatograph thus making it suitable
    for the analysis of both volatile and nonvolatile nitrosamines.

        The nonvolatile nitrosamines constitute a more varied group of
    compounds than the volatile nitrosamines and, as such, cause
    additional analytical problems. Nitrosoamino acids, such as
    nitrososarcosine, nitrosoproline, and nitroso-2-hydroxyproline may be
    analysed by conversion to volatile derivatives such as silyl ethers
    (Eisenbrand et al., 1975).

        Methods have been proposed for the determination of total
     N-nitroso compounds, the general approach being to cleave the nitroso
    group and measure the nitric oxide formed. Fan & Tannenbaum (1971)
    eliminated the problem of nitrate interference by using long-wave
    ultraviolet irradiation (360 nm) to split the nitroso group. The
    released nitrite was diazotized and coupled to form a dyestuff before
    colorimetric estimation. The method was designed for automation.
    Eisenbrand & Preussmann (1970) used hydrobromic acid in a nonaqueous
    medium to split the nitroso group. A method for splitting the nitroso
    group which does not require any anhydrous medium has been proposed by
    Fine et al. (1976a).

    3.  SOURCES OF NITRATES, NITRITES AND N-NITROSO COMPOUNDS IN AIR,
        WATER, SOIL AND FOOD

    3.1  Natural Occurrence

    3.1.1  Nitrates and nitrites

        Nitrates in soil and in surface and groundwater result from the
    natural decomposition by microorganisms of organic nitrogenous
    material such as the protein in plants, animals, and animal excreta.
    The ammonium ion formed is oxidized to nitrites and nitrates (section
    2.1.1). Natural occurrence of nitrates and nitrites in the environment
    is a consequence of the nitrogen cycle (section 4.1) but normally
    nitrites are only found in very low concentrations.

    3.1.2  N-nitroso compounds

        Systematic studies on the natural occurrence of  N-nitroso
    compounds have not been reported but a few studies show that these
    compounds may occur in certain microorganisms (Murthy et al., 1966;
    Vavra et al., 1960) and in one variety of mushroom (Hermann, 1960). At
    least one of these compounds, strephozotrin, is a potent carcinogen
    (Arison & Feudale, 1967; Sibay & Hayes, 1969). Other reports on the
    natural occurrence of diethylnitrosamine (DEN) in certain plants have
    still to be confirmed by modern analytical methods.

    3.2  Sources Related to Man's Activities

    3.2.1  Nitrates and nitrites

    3.2.1.1  Fertilizers

        Artificial fertilizers, a major source of environmental nitrates,
    may be composed of a variety of chemicals including ammonium, calcium,
    potassium, and sodium nitrates, and urea. The production of
    nitrogenous fertilizers in the world has increased in terms of N from
    15.8 million tonnes in 1961/62-1965/66 to 42.3 million tonnes in
    1974/75 (United Nations, 1976).

        The fact that plants cannot use soil nitrogen completely is of
    great significance; nitrogen utilization may vary from 25 to 85%
    depending on the crop and on agricultural techniques. Thus, to obtain
    maximum production, a great excess of nitrogen fertilizer must be
    applied to the soil and the resulting nitrogen runoff will be
    substantially increased. For example, Kohl et al. (1971) showed that
    as much as 55-60% of the nitrogen input in the Sangamon River feeding
    Lake Decatur, IL, USA, was of fertilizer origin. Lee (1970b), Sawyer
    (1947), and Sylvester (1961), have all published data showing that
    nitrogen runoff is 3-10 times higher from fertilized areas than from
    unfertilized areas in the same region. However, analysis of stream 

    waters did not show a clear relationship between the nitrate
    concentrations in British rivers and the amounts of fertilizers used
    on adjacent land (Tomlinson, 1970).

        Brown & Smith (1967) observed that nitrogen fertilization tended
    to increase the nitrate content of vegetables and attempts have been
    made to correlate nitrogen application rates with the nitrate contents
    of lettuce, radish, and spinach. In studies in Bulgaria, Biocev &
    Pocinkova (1972) noted that the nitrate levels in spinach increased
    when as little as 20 kg of nitrogen per ha was added to the soil.
    Schuphan (1969) observed that application of four times the normal
    amount of fertilizer resulted in considerably higher nitrate levels in
    spinach but that the nitrite levels remained low.

    3.2.1.2  Animal wastes

        Another major source of nitrates is farm animal wastes which
    contain large amounts of nitrogenous materials that may be converted
    into nitrates. The problem is more acute where farming is carried out
    intensively, a common practice in North America for both livestock and
    poultry. Since a 450 kg steer excretes about 43 kg of nitrogen per
    year, a 3200 head feedlot would produce 1400 tonnes annually on a
    relatively small area--an amount equivalent to about 260 000 people.
    Thus, such feedlots become "small area" sources of nitrogen runoff.
    Only 10% of these wastes is returned to cultivated land (Standford et
    al., 1969) and runoff studies demonstrate a considerable problem of
    environmental pollution. Nye (1973) reported Gilbertson et al. (1970),
    who found that the total nitrogen concentration in runoffs ranged from
    about 50 to over 5500 mg/litre. Animal husbandry, even when carried
    out on pastures or with the return of the animal wastes to cultivated
    land, may still impose problems. Adriano et al. (1971) concluded that
    wastes from a maximum of 7-8 cows could efficiently be used per
    hectare of farmland or pasture and that higher application rates might
    raise nitrate levels above 10 mg/litre in the subsoil waters.

    3.2.1.3  Municipal, industrial, and transport wastes

        Discharges of municipal and industrial wastes are concentrated
    sources of nitrogen compounds that are, to a large extent, released
    directly into surface waters. The amount of nitrogen in human wastes
    is estimated to be about 5 kg per person per year (Committee on
    Nitrate Accumulation, 1972). Even if treated, this waste will
    represent a heavy water pollution load since secondary treatment
    removes less than half of the nitrogen. Ammonium ions in the effluent
    of septic tanks may be rapidly converted to nitrate which may
    penetrate some distance from the tank. Sludge from treatment plants
    and septic tanks has also to be disposed of and represents another
    significant source of nitrogen pollution. Solid waste disposal
    practices, particularly sanitary landfills and dumps, may represent a
    source of water pollution by nitrogen compounds.

        The nitrogen content of industrial wastes is highly variable;
    fuel and food processing industries and petroleum refineries may
    constitute important sources of nitrogen pollution. The
    nitrogen/BODa ratio of food processing plant wastes is about 0.05
    while for animal processing wastes this ratio amounts to 0.5.
    (Committee on Nitrate Accumulation 1972). Oxides of nitrogen released
    into the atmosphere from man-made sources such as motor vehicles,
    fossil fuel combustion, and industrial processes amount to about 50
    million tonnes per year on a global scale (Robinson & Robbins, 1972).
    A considerable proportion of this fixed nitrogen is eventually
    returned to the earth's surface as nitrate.

    3.2.1.4  Deliberate addition of nitrates and nitrites to food

        Nitrates and nitrites are widely used in the production of
    certain meat products and in the preservation of fish in some
    countries. Reasons for using these salts in food production have been
    reviewed by Ingram (1974). Nitrite is used in meat curing to obtain
    the characteristic pink colour and flavour of cured meat. While a
    nitrite content of less than 5 mg/kg of meat is sufficient to give a
    satisfactory colour for a limited period of time, up to 20 mg/kg may
    be necessary to give commercially adequate colour stability and about
    50 mg/kg to produce the characteristic flavour. However, detailed
    experimental confirmation of these figures is lacking.

        Curing meat gives an important degree of protection against
    botulism and may provide similar protection against other bacteria
    such as  Clostridium welchii and staphylococci, although the
    importance of this has not yet been assessed. The question of how much
    nitrite is necessary to protect against botulism is very complex
    because of several associated factors.

        The addition of nitrates and nitrites to meats, meat products,
    and cheese is governed by legislation in most countries, some of which
    also allow the addition of these salts to fish products.

    3.2.2  N-nitroso compounds

    3.2.2.1  Food

        The formation of  N-nitroso compounds from nitrites and amines
    during the storage and processing of food is discussed in section
    4.2.2.

              

    a  BOD = biological oxygen demand.

    3.2.2.2  Tobacco

        Nitrosonornicotine has been found in unburned smoking tobacco,
    chewing tobacco, and in snuff. The same compound has been identified
    in the mainstream smoke of a popular nonfilter cigarette in the USA
    (Hoffman et al., 1974).

    3.2.2.3  Industrial uses

        Although  N-nitroso compounds do not appear to be extensively
    used at present, Magee (1972) reports that patent applications have
    been made in the UK for their use in the manufacture of dyestuffs,
    lubricating oils, explosives, insecticides, and fungicides. Some
    nitrosamines (nitrosodiphenylamine,  N-N-dinitrosopentamethylenete-
    tramine, polymerized  N-nitroso 2,2,4-trimethyl-1,2-dihydroquinoline
    and  N-methyl- N, 4-dinitrosoaniline) are used as organic
    accelerators and antioxidants in the production of rubber (Boyland et
    al., 1968). DMN has been used as an industrial solvent, as a
    nematocide, and in the synthesis of the rocket fuel 1,1-
    dimenthylhydrazine. There is some evidence that DMN might also be
    formed during the combustion of this rocket fuel (Simoneit &
    Burlingame, 1971). There are patents for the use of DMN as a solvent
    in the plastics and fibre industry, as an additive for lubricants, and
    to increase the dielectric constant in condensers (Daiber, 1966).

        Industrial uses may result in the occurrence of  N-nitroso
    compounds in the work environment and in industrial effluents. Fine et
    al. (1976b) reported point sources of air pollution by DMN in
    Baltimore, MD, and Belle, WV, in the USA. A factory using DMN as an
    intermediate was shown to be the source in Baltimore and was shut
    down; in Belle the source of DMN was an amine-manufacturing facility.

    4.  TRANSPORT AND TRANSFORMATION IN ENVIRONMENTAL
        AND BIOLOGICAL MEDIA

    4.1.  Nitrogen Cycle

        The continuous interchange between atmospheric and terrestrial
    nitrogen takes place along a number of different pathways including
    air, water, soil, microorganisms, plants, animals, and man. This
    transfer and transformation of nitrogen is referred to as the nitrogen
    cycle (Fig. 1).

        The main factors affecting it are the climatic conditions, the
    type and density of animal and plant populations, agricultural
    practices, and animal husbandry. The nitrogen cycle has undergone
    profound modifications through the agricultural and industrial
    activities of man (Bolin & Arrhenius, 1977; Committee on Nitrate
    Accumulation, 1972; Commoner, 1970; FAO/IAEA Panel of Experts, 1974).

        Atmospheric nitrogen is in the form of dinitrogen (N2); the
    great strength of the N = N bond is mainly responsible for its
    chemical inertness. A part of atmospheric nitrogen is transformed by
    microbial action and incorporated into living organisms. This process
    is called nitrogen "fixation" and is estimated to amount globally to
    150 million tonnes of fixed nitrogen per year. In industrial nitrogen
    fixation, atmospheric nitrogen is combined with hydrogen at high
    temperatures and pressures in the presence of suitable metal catalysts
    (Haber-Bosch process) to produce ammonia. Industrial nitrogen fixation
    accounts for about one quarter of the total world production of fixed
    nitrogen (Bolin & Arrhenius, 1977). Various atmospheric processes
    which have been discussed elsewhere (WHO, 1977) are minor sources of
    fixed nitrogen.

        Biological nitrogen fixation, i.e. its reduction to ammonia, can
    be accomplished by only a limited number of organisms. Symbiotic
    nitrogen fixation takes place in the root nodules of legumes such as
    soya bean, clover, and alfalfa, which contain bacteria of the
     Rhizobium species. There are also symbiotic processes with plants
    other than legumes involving, for example, some cyanobacteria. A
    number of free living bacteria and algae can also fix nitrogen (Burns
    & Hardy, 1975; Quispel, 1974). The fixation is catalysed by a complex
    enzyme nitrogenase (1.7.99.2). Ammonia produced by biological fixation
    is then converted to nitrite and nitrate by the process of
    nitrification (see section 2.1.1). Plants can assimilate only a part
    of the nitrates present in soils; some leaches into ground water and
    rivers and may reach estuaries and oceans, the rest is subjected to
    denitrification, another natural biochemical process that degrades
    nitrates to nitrogen or nitrous oxide (dinitrogen oxide) which are
    released into the atmosphere. Denitrification takes place in the soil
    and also at the interface between water and sediment in oceans,
    rivers, lagoons, and lakes. Nitrates from natural fixation and 

    FIGURE 1

    artificial fertilizers are ultimately used for the synthesis of
    biological molecules, particularly proteins. Plants and animal waste
    and dead tissues return fixed nitrogen to the soils, where part of it
    is recycled and part returned to the atmosphere, thus completing the
    nitrogen cycle. According to Delwiche (1970), nitrogen fixation on a
    world basis may exceed denitrification by about 10%. The increased use
    of industrial fertilizers has resulted in some areas in increased
    concentrations of nitrates in bodies of water, resulting, in some
    cases, in eutrophication.

    4.2  Transformation in Foods

    4.2.1  Reduction of nitrates to nitrites

        Because of the ability of spinach to accumulate large quantities
    of nitrates and reported cases of intoxication associated with the
    consumption of this vegetable, several studies have been undertaken on
    the conversion of nitrates to nitrites in spinach.

        Data presented by Phillips (1968a) indicated that initial
    nitrite-N contents of fresh, frozen, canned, and baby-food spinach
    were generally less than 1 mg/kg fresh weight. However, several
    authors have reported a rapid fall in nitrate levels and increase in
    nitrite levels in fresh spinach during the first 4 days of storage at
    room temperature (Achtzehn & Hawat, 1970; Phillips, 1968a; Schuphan,
    1965). Higher nitrite levels occurred in spinach from fertilized
    ground (Brown & Smith, 1967) and these could reach exceptionally high
    values (3600 mg/kg dry weight) with excessive fertilization (Schuphan,
    1965).

        Under refrigeration, the nitrite-N contents of fresh spinach
    increased very gradually throughout a storage period of 28 days
    (Phillips, 1968a). Significant increases in nitrite-N levels did not
    occur during the storage of frozen, canned, or baby-food spinach, but
    increased concentrations were found in frozen spinach, that had been
    left to thaw at room temperature for an excessively long period (39 h)
    (Phillips, 1968a).

        There was also a slight rise in nitrite levels when partially
    consumed jars of commercial baby foods containing nitrates were stored
    for 7 days at room temperature instead of under refrigeration
    (Phillips, 1969). Selenka (1970) noted that nitrite formation in baby
    foods was rapid in the presence of  Escherichia coli and  Pseudomonas
     fluorescens, less rapid with  Bacillus subtilis, and very slow with
     Staphylococcus albus.

        When foods consisted of a solid immersed in a liquid (e.g. canned
    foods or frozen foods after thawing) nitrates were partially
    transferred to the liquid portion or into the water in which the food
    was cooked (Bodiphala & Ormrod, 1971). When large volumes of water and

    long cooking times were employed (Kilgore et al., 1963), and when
    canned vegetables were blanched in hot water instead of steam
    (Johnson, 1966), significant amounts of nitrates were leached out of
    the foods. Nitrate reductase (NADPH) (1.6.6.3) activity was rapidly
    destroyed during cooking, thereby greatly diminishing further
    conversion of nitrates to nitrites (Bodiphala & Ormrod, 1971). It is
    also well known that the sterilization treatments necessary for
    canning destroy microorganisms that could convert nitrates to
    nitrites.

        Conversion of nitrates to nitrites occurred more slowly in
    vacuum-packed bacon than in unpackaged bacon, presumably due to the
    low reducing ability of anaerobes (Cavett, 1962). Spencer (1967) found
    that the nitrite content of vacuum-packed bacon decreased slowly on
    storage. It has also been reported by Sebranek et al. (1974) that
    nitrite levels in meat, determined 2 days after processing, were less
    than half those originally added to frozen samples and samples
    processed at 71°C, and that they decreased further during storage.
    Frying, grilling, or boiling bacon or ham reduced the nitrites content
    by 20-90% (Food Standards Committee, 1959).

        When direct gas firing of spray dryers was employed, the nitrate-
    N contents of dried milk products increased by 1-3 mg/kg compared with
    those obtained using indirectly heated sprayers but nitrite-N levels
    were unaffected (Manning et al., 1968). Air drying of potato and corn
    starch led to the formation of only trace amounts of nitrite
    (Gerritsen & De Willingen, 1969).

        Nitrates may be reduced to nitrites when cooking is carried out
    in aluminium utensils (Osteryoung, unpublished data)a. This
    observation appears to be significant since some countries use
    aluminium utensils for boiling milk and water, a practice which could
    lead to the formation of sizeable quantities of nitrites. This effect
    of aluminium should be investigated further.

    4.2.2  Formation and degradation of N-nitroso compounds

        The conditions under which various amines, amino acids, and
    proteins in food could react with nitrite to form nitrosamines were
    studied by Ender & Ceh (1971) and by Sen et al. (1970) who showed that
    when cod, herring, hake, halibut, mackerel, or salmon were treated
    with sodium nitrite at 200 mg/kg and cooked at 110°C for 60-70 min
    there were only trace amounts (2.5-25 µg/kg) of DMN in the cooked
    product. The highest levels were found in mackerel and hake, both of
    which contain large amounts of DMA and TMA. Samples without added
    nitrite did not contain any detectable nitrosamines.

              

    a  Nitrates as human and animal health hazards, Paper presented at
       the Second Conference on Environmental Chemicals, Colorado State
       University, 1973.

        The formation of DMN was studied in aqueous model systems
    containing methyl amines and sodium nitrite under conditions which
    were more severe than those employed in the commercial processing of
    nitrite-treated smoked chub (a fresh water fish containing small
    amounts of TMA, TMAO, and DMA). The results of the studies showed that
    not more than 10 µg of DMN per kg of the final product would be formed
    during the smoking process (Malins et al., 1970).

        Recently, Sen et al. (1973b) suggested that a major source of
    nitrosamines in cured meat might arise from an interaction between the
    nitrites and spices, such as black pepper and paprika, that are
    present in curing mixtures. Nitrosopyrrolidine, nitrosopiperidine, and
    DMN were found in a curing mixture used by a meat manufacturer in
    Canada. The same authors (Sen et al. 1974a) have also studied the
    effect of sodium nitrite concentration on the formation of
    nitrosopyrrolidine and DMN in fried bacon. Bacon samples prepared with
    sodium nitrite at 0, 50, 100, 150, or 200 mg/kg were analyzed for
    nitrosopyrrolidine and DMN. No nitrosamine was detected in samples
    prepared without nitrite but all treated samples contained 2-20 µg/kg
    of nitrosamines. The level of nitrosopyrrolidine was related to the
    initial concentration of nitrite in the bacon. It has also been shown
    that nitrosamine formation in bacon increases with increasing
    temperature and time of frying and that whereas baking, broiling, or
    frying produce variable amounts of nitrosamines none is produced with
    cooking in a microwave oven (Pensabene et al., 1974).

        After deliberate nitrosation of eggs and meat with unusually
    large amounts (1%) of sodium nitrite, some  N-nitroso compounds
    appeared to have been formed but the chemical nature of the compounds
    detected was not clear (Walters, 1971).

        No systematic studies on the formation of  N-nitroso compounds
    in cheese have been performed, although some types of cheese are known
    to be processed with nitrate and nitrite.

        There are few data on the fate of  N-nitroso compounds during
    the cooking, processing, or storage of food but some studies have
    demonstrated that the volatile nitrosamine DMN and nitrosopyrrolidine
    may be lost during the frying of bacon (Sen et al., 1973a).

    4.3  Formation of N-nitroso Compounds from Drugs and Pesticides

        Reaction with nitrite to form nitrosamines is not restricted to
    food components. Lijinsky & Greenblatt (1972) and Lijinsky (1974)
    reported that some antibiotics and other drugs that are widely used
    can react with nitrites to form nitrosamines in alarmingly high
    quantities. The drugs examined included oxytetracycline,
    aminophenazone (aminopyrine), disulfiram,  N,N-diethyl-3-
    pyridinecarboxamide (nikethamide), tolazamide, and  (E,E)-1-[5-(1,3-
    benzodioxol-5-yl)-1-oxo-2,4-pentadienyl] piperidine (piperine).

    Optimum conditions of temperature, pH, and concentration for these
    reactions have been reported by Lijinsky et al. (1972a, 1972b, 1972c)
    who more recently (Lijinsky, 1974) studied the reactions of
    aminopyrine and other commonly used drugs with nitrous acid at rather
    low concentrations to assess the magnitude of the hazard to man from
    such interactions. The topic has been reviewed by Mirvish (1975) who
    has listed 41 drugs and pesticides that have been nitrosated.
    Pesticides listed include atrazine, simazine, ziram, and thiram.

    4.4  Formation of N-nitroso Compounds in Animal Organisms

    4.4.1  Formation of N-nitroso compounds in simulated gastric juice

        Formation of DEN was demonstrated when DEA and nitrite were
    incubated in the gastric juice of the rat, rabbit, cat, dog, and man.
    More DEN was formed in human and rabbit gastric juices (pH 1-2 in both
    cases) than in rat gastric juice (pH 4-5). (Sen et al., 1969a, 1969b).

        The formation of nitrosamines by the interaction of some drugs
    with nitrite in the presence of human gastric juice have been studied
    by Scheunig & Ziebarth (1976). At 37°C and a pH = 2, for 1 h,
    aminopyrine, sodium [(2,3 dihydro-1,5-dimethyl-3-oxo-2-phenyl-IH-
    pyrazol-4-yl)methylamino] methanesulfonate (analgin), and piperazine
    gave nitrosamine yields (calculated on the basis of nitrite used) of
    69%, 11%, and 74.8% respectively.

         In vitro studies have been carried out (Wells et al., 1974) in
    which several foods (pork, egg, bread, milk, and cheese) were
    incubated under simulated gastric conditions with concentrations of
    nitrite similar to those used as food preservatives. The effect of the
    thiocyanate ion as a catalyst for nitrosation was also studied since
    it is secreted in saliva. Of the foods studied, only cheese produced
    detectable amounts of volatile nitrosamines. The identity of the
    nitrosamines was not indicated.

    4.4.2  Formation of N-nitroso compounds  in vivo

        When DEA and nitrite were fed to cats and rabbits, considerable
    amounts of DEN were detected in the stomach of the experimental
    animals (Sen et al, 1969a, 1969b). Similar results were reported by
    Sander & Sief (1969). Epstein (1972) reported the formation of
    nitrosopiperidine in the gastrointestinal tract of rats treated with
    nitrite and piperidine hydrochloride. When the nitrite concentration
    was constant, nitrosopiperidine formation in the small intestine
    increased with increasing concentrations of piperidine.
    Nitrosopiperidine was also found in the stomach. Recently, Sander et
    al. (1974a) demonstrated the formation of  N-N'-dinitrosopiperazine,
    DMN, and  N-nitroso- N-methylbenzylamine in the stomach contents of
    rats given the parent amines combined with nitrite. Considerable
    individual variation in the degree of synthesis of  N-N'-
    dinitrosopiperazine was noted in the animals. In another recent 

    report,  N-nitrosopyrrolidine was formed very rapidly in the stomach
    of dogs from sodium nitrite and pyrrolidine (within 2-6 min) but after
    30 min nearly all of it had disappeared, presumably due to its rapid
    absorption (Mysliwy et al., 1974).

        Indirect evidence of  in vivo formation of  N-nitroso compounds
    has also been provided by some toxicity studies. Thus, hepatic
    lesions, formed following administration of nitrite and some amines,
    were similar to those produced by DMN or  N-nitroso- N-
    methylbenzylamine (Asahina et al., 1971). Similar effects were noted
    where nitrite was administered up to 3 h after DMA, but the effect was
    markedly reduced if the nitrite was given prior to the amine.

    4.5  Formation of N-nitroso Compounds by Microorganisms

        Studies conducted by Hawksworth & Hill (1971a), Klubes & Jondorf
    (1971), and Sander & Sief (1969) suggested that nitrosamines could be
    synthesized from secondary amines and nitrates or nitrites by
     Escherichia coli and some species of streptococci. Fong & Chan
    (1973b) demonstrated that homogenized Chinese salt fish inoculated
    with  Staphylococcus aureus (a nitrate-reducing bacterium) produced
    considerable amounts of DMN.

        Formation of nitrosamines in the presence of bacteria is unlikely
    to occur in the large intestine, but the infected bladder and
    achlorhydric stomach are likely sites (Hawksworth et al., 1974).

        The ultimate mechanism of bacterial production of nitrosamines
    remains to be ascertained. According to Hawksworth et al. (1974),
    certain bacteria do reduce nitrate to nitrite but the formation of the
    nitrosamine may be nonenzymatic and involve some heat-resistant
    metabolite.

    4.6  The Effects of Other Chemicals on the Formation of N-nitroso
         Compounds

        Fiddler et al. (1973) and Greenberg (1974) showed that high
    levels of ascorbic acid reduced nitrosamine formation in frankfurter
    sausages and in fried bacon. On the other hand, Nagata & Mirna (1974)
    reported an increase in nitrosamine formation in meat products in the
    presence of ascorbic acid. Other studies conducted on the inhibition
    of nitrosamine formation by various compounds include a report by Sen
    & Donaldson (1974) in which nitrosamine formation in human saliva was
    inhibited by ascorbic acid. Ziebarth & Scheunig (1976) tested a number
    of substances and beverages for the inhibition of the nitrosation of
    several drugs under simulated gastric conditions. Of all the
    substances investigated, ascorbic acid was regarded as the best
    inhibitor because of its pronounced activity at pH values occurring in
    the stomach and because it was not toxic in the amounts used.

    5.  ENVIRONMENTAL LEVELS AND EXPOSURES

    5.1  Nitrates and Nitrites

    5.1.1  Ambient air

        Nitrate aerosols are the final stage in the atmospheric oxidation
    of gaseous oxides of nitrogen, and substantial amounts of particulate
    nitrates may be formed in urban areas affected by photochemical
    pollution (Pitts & Lloyd, 1973). The concentration of nitrates in air
    may range from about 1 to 40 µg/m3, depending on the sampling and
    averaging periods. For example, the estimated annual mean values
    (1968-1972) in Chattanooga, TN, USA, were between 1 and 6 µg/m3
    (French et al., unpublished)a. The daily mean concentrations of
    airborne nitrates in the central part of Tokyo ranged, in 1973, from
    0.9 to 41.8 µg/m3 with an annual mean of 8.2 µg/m3. On the other
    hand, in a small city with few industries (Matsue City) the daily
    means were in the range of 1.1 -- 9.2 µg/m3, with an annual mean of
    2.6 µg/m3 (Japan Environmental Sanitation Center, 1974).

    5.1.2  Water

        The concentrations of nitrates and nitrites in surface and ground
    waters vary within wide limits, depending on geochemical conditions,
    human and animal waste management practices, the extent to which
    nitrogen-containing agriculture fertilizers are used locally, and on
    industrial discharges of nitrogen compounds (section 3.2.1.).

        In general, surface waters do not usually contain nitrate in
    concentrations higher than 10 mg/litre, and nitrite concentrations
    rarely exceed 1 mg/litre. However, a steady upward trend of nitrate
    levels has been reported in recent years in some countries, both in
    surface and ground waters. Thus, for example, in the River Thames,
    England, nitrate concentrations increased from an average of
    4 mg/litre in 1968 to an average of 9 mg/litre for the last quarter of
    1973 (Water Research Centre, 1974). Similar increases have been
    observed in several other English rivers (Casey, 1975; Owens, 1970;
    Tomlinson, 1970). The nitrate concentrations are increasing in some
    rivers that drain the great agricultural section of the centre of the
    USA, and in selected small rivers the 45 mg/litre limit is sometimes
    exceeded (Viets & Aldrich, 1973). A small increase in the nitrate
    concentration of the Tamagawa River, Tokyo, Japan has also been
    reported. From 1951-1965, the nitrate ion concentration rose from
    7.9 mg/litre to 9.1 mg/litre. During the same period, the nitrite
    concentration increased from 0.049 mg/litre to 0.53 mg/litre, i.e by a
    factor of about 10 (Goto, 1973).

              

    a  French, J. G., Hasselblad, V., & Johnson, R. Aggravation of
       asthma by air pollutants. 1971 -72 Southeastern CHESS studies.

        Studies of 991 settlements in Bulgaria indicated that only 64
    towns and villages had drinking water levels of nitrates between
    30 mg/litre (Bulgarian standard) and 50 mg/litre. In 20 settlements,
    situated in areas with intensive agriculture and stock breeding, the
    nitrate concentrations exceeded 50 mg/litre. The reportb points out
    that such problems did not exist some 10 years ago when smaller
    quantities of nitrogen fertilizers were used in agriculture.

        Much higher concentrations of nitrates are sometimes found in
    ground water, particularly in water derived from dug wells. A survey
    of over 2000 rural wells in Saskatchewan, Canada, revealed that 18.8%
    contained nitrate concentrations of more than 50 mg/litre and 5.3% had
    nitrate levels exceeding 300 mg/litre (Robertson & Draycott, 1948).
    Hedlin (1971) also reported levels above 45 mg/litre in some wells in
    a rural area of Manitoba, Canada. In many farm wells in central USA,
    nitrate concentrations may range from 45-450 mg/litre. This problem is
    neither new nor local, since such conditions have been recorded from
    1895 to 1970 in Illinois, in 1939 in Iowa, and in 1970 in Minnesota
    (Viets & Aldrich, 1973). The mean nitrate concentration in ground
    water consumed by children affected by methaemoglobinaemia in
    Czechoslovakia ranged from 18-257 mg/litre (Schmidt & Knotek, 1970).
    According to Gruenar & Shuval (1970), about 180 wells for community
    water supplies in the densely populated central and southern coastal
    plain in Israel had nitrate concentrations exceeding 45 mg/litre. In
    England, nitrate concentrations in some ground waters have been
    reported to range from 12 mg/litre (Foster & Crease, 1974) to over
    22 mg/litre (Reeves et al., 1974). Nitrate concentrations exceeding
    45 mg/litre have not been reported in centralized water supplies in
    the USSRc. However, high concentrations have been found from time
    to time in dug wells, for example, 310-400 mg/litre in Leningrad
    Oblast (Motylev, 1969), 110-200 mg/litre in the Tatar SSR (Petukhov
    et al., 1972) and up to 430 mg/litre in the Moldvian SSR (Diskalenko,
    1969).

    5.1.3  Selected foods

        According to the data compiled by the National Institute of
    Environmental Health Sciences (NIEHS, 1970), the levels of nitrates in
    vegetables vary considerably. The highest levels were found in beets,
    egg plant, kale, and spinach and the lowest in tomatoes and peas;
    similar findings were obtained in the German Democratic Republic by
    Achtzehn & Hawat (1969). It is of interest to note that the nitrate
    levels in vegetables reported by Jackson in 1967 were similar to those
    reported by Richardson in 1907, when manure was used instead of
    chemical fertilizers.

              

    b  Contribution to the WHO environmental health criteria document on
       nitrates, nitrites and nitrosamines, Sofia, 1974.

    c  Contribution to the WHO environmental health criteria document on
       nitrates, nitrites and nitrosamines, Moscow, 1974.
        Nitrate contents vary not only between vegetable species but also
    widely within a given species. This variation within a species may be
    accounted for by such factors as temperature, sunlight, soil moisture,
    and the level of available nitrogen in the soil (US Department of
    Agriculture, 1965). A relationship between nitrate accumulation in
    spinach and levels of fertilizer applied to the soil has been
    demonstrated by a number of authors (Brown & Smith, 1967; Phillips,
    1971). Furthermore, Schuphan (1965) reported exceptionally high levels
    of nitrites (3600 mg/kg dry weight) in excessively fertilized fresh
    spinach stored at room temperature.

        A survey of the nitrate contents of fruits in the German
    Democratic Republic, revealed that they were high in bananas and
    strawberries but could not be detected in the other fruits examined
    (Achtzehn & Hawat, 1969).

        Cow's milk contained nitrate levels of 0-0.5 mg/litre (Simon et
    al., 1964).

        The levels of nitrates and nitrites in baby foods are of special
    concern since infants are considerably more sensitive to the toxic
    effects of nitrates than adults. Kamm et al. (1965) studied 194
    prepared infant foods and found that, on average, fruits, dairy
    products, puddings, egg products, meats, dry and concentrated food
    supplements, and precooked cereal products contained nitrate levels of
    less than 90 mg/kg. Vegetables, however, had a wide range of nitrate
    contents varying from 0.9 to 2165 mg/kg, but nitrite levels never
    exceeded 7 mg/kg. In studies on a number of canned foods, baby foods,
    frozen foods, and vegetables, several varieties of fruit, spinach, and
    beets generally had the highest nitrate contents (Bodiphala & Ormrod,
    1971). Additional information may be found in articles by NIEHS (1970)
    and Ashton (1970).

        In a survey of various cured meats (Table 1, Ashton, 1970), the
    highest nitrate content of 370-511 mg/kg was found in ham. In
    analysing 197 samples of cured meat products, Panalaks et al. (1972)
    found that the levels of nitrates and nitrites ranged from
    0-3467 mg/kg and 0-252 mg/kg, respectively.

        Dubrow & Kakisch (1960) analysed 338 samples of cheese and
    reported that all were free of nitrites (less than 1 mg/kg). However,
    40% of the samples contained nitrate levels of more than 1 mg/kg.
    Rammell & Joerin (1972) also found low nitrite levels in cheese.

    Table 1.  Nitrate and nitrite contents of cured meats
                                                                    

    Meat type                     Nitrate (mg/kg)     Nitrite (mg/kg)
                                                                    

    silverside                       133-303                9-26

    ham                              370-511               7-150

    luncheon meat                     59-214              3.1-47

    chopped ham & pork                53-101               22-62

    corned beef                      118-135              18-208

    frankfurter sausage              119-141            8.5-10.3

                                                                    

    From: Ashton (1970)


    5.1.4  Estimate of general population exposure

        One of the important sources of exposure to nitrates for man is
    water. The level of nitrates in water may vary from practically nil to
    over 200 mg/litre. In water from municipal supplies, however, it is
    likely to be under 10 mg/litre. Thus, assuming an intake of 2 litres
    of water per day, the daily intake of nitrates from this source would
    normally be less than 20 mg, but with extremes of 0 and over 400 mg.

        The other main sources of nitrates and nitrites are certain
    vegetables and meat products. The intake from these sources is even
    more variable because of marked differences not only in levels in
    these foods but also in dietary patterns. However, Ashton (1970)
    estimated the weekly intake of nitrates for a member of the general
    population in the USA to be about 400 mg including 210 mg from
    vegetables, 110 mg from meat products, and 85 mg from water (7 litres
    per week). The estimate of Hill et al. (1973) for a member of the
    general population in England included 225 mg from vegetables, 110 mg
    from meat, and 105 mg from water in "control towns" and 645 mg from
    water in Worksop, England. However these figures cannot be applied
    generally because of variations in feeding habits and nitrate levels
    in environmental media. Since the intake of nitrites is even more
    variable, no estimates have been reported.

    5.2  N-nitroso Compounds

    5.2.1  Ambient air

        The occurrence of  N-nitroso compounds in urban air was reported
    first by Bretschneider & Matz (1973) and confirmed by Fine et al.
    (1976b) who found DMN at concentrations of about 1.2-3.5 µg/m3
    (0.33-0.96 ppb) in an industrial district in Baltimore, MD, USA, and
    about 0.06-0.17 µg/m3 (0.014 ppb - 0.051 ppb) in Belle, WV, USA.
     N-nitroso compounds may be present in air, either due to their
    formation in the air from secondary amines and oxides of nitrogen
    (Neurath, 1972) or due to industrial omissions as in the instances
    referred to.

    5.2.2  Water

        There are few reports on the occurrence of  N-nitroso compounds
    in water. Fine et al. (1976b) analysed samples from the Mississippi
    river (New Orleans, LA) and from 3 water treatment plants in
    Louisiana. Using the new  N-nitroso compound-specific thermal energy
    analyser (TEA) interfaced to both a gas chromatograph (GC) and a high
    performance liquid chromatograph (LC), several peaks were tentatively
    identified as belonging to  N-nitroso derivatives of some pesticides.
    The estimated concentrations were of the order of 0.1 µg/kg.

    5.2.3  Selected foods

        A summary of the reported occurrences of nitrosamines in meat and
    fish products, adapted from Sen (1974) and updated, is presented in
    Table 2. Only the results that were confirmed by mass spectroscopy are
    quoted in this table. It was noted that the majority of some 50
    publications dealt with the determination of nitrosamines,
    particularly DMN, in processed pork meat. The methods employed for
    analysis mainly involved gas chromatography. A few results were
    confirmed by mass spectroscopic techniques. However, mass spectroscopy
    confirmation is currently being employed more frequently than in the
    past.


        Table 2.  Levels of nitrosamines in various meat and fish productsa
                                                                                                                       

                                                            N-nitroso
    Meat                          Country                  compounds      Levels (c)          Reference
                                  or area                  foundb
                                                                                                                       

    dry sausage                   Canada                   DMN            10-20 µg/kg         Sen (1972)
    uncooked salami sausage       Canada                   DMN            20-80 µg/kg         Sen (1972)
    salami sausage                Netherlands              DMN            0.3 µg/kg(e)        Stephany et al. (1976)
                                                           DMN            0.1(e)              Stephany et al. (1976)
                                                           NDBA           1.1(e)              Stephany et al. (1976)
                                  Netherlands              NPY            0.4(e)              Stephany et al. (1976)
                                                           NPIP           0.3(e)              Stephany et al. (1976)

    bacon                         Canada                   NPY            4-25 µg/kg          Sen et al. (1973a)
                                  Canada                   NPY            25-40 µg/kg         Sen et al. (1974a)
                                  Netherlands              DMN            0.8 µg/kg(e)        Stephany et al. (1976)

    bacon                                                  DEN            0.2(g)
                                  Netherlands              NDBA           0.6(g)              Stephany et al. (1976)
                                                           NPY            0.4(g)
                                  Netherlands              NPIP           0.6(g)              Stephany et al. (1976)
                                  USA                      NPY            7-35 µg/kg          Pensabene et al. (1974)

    bacon                         USA                      NPY            2, 28, 13           Fiddler et al. 1974

    uncooked bacon                Canada                   DMN            30 µg/kg            Sen et al. (1973a)

    fried bacon                   Netherlands              DMN            2.4 µg/kg           Groenen et al. (1976)
                                                           DEN            4.43 µg/kg
                                  Netherlands              DMN            1.1 µg/kg(e)        Stephany et al. (1976)
                                                           DEN            0.2(g)
                                  Netherlands              NDBA           0.7(g)              Stephany et al. (1976)
                                                           NPY            16.4(g)

                                                                                                                       

    Table 2 (Cont'd)
                                                                                                                       

                                                            N-nitroso
    Meat                          Country                  compounds      Levels (c)          Reference
                                  or area                  foundb
                                                                                                                       

                                  Netherlands              NPIP           3.9(g)              Stephany et al. (1976)
                                  UK                       NPY            1-40 µg/kg          Crosby et al. (1972)
                                  USA                      NPY            20-207 µg/kg        Fazio et al. (1973)

    smoked meat                   Netherlands              DEN            7.91 µg/kg          Groenen et al. (1976)
                                  Netherlands              DMN            3                   Groenen et al. (1976)

    smoked horse and              Netherlands              DMN            7.3 µg/kg(d)        Stephany et al. (1976)
    beef meat                                              DEN            0.6(d)              Stephany et al. (1976)
                                  Netherlands              NDBA           0.4(d)              Stephany et al. (1976)
                                                           NPY            0.1(d)              Stephany et al. (1976)
                                  Netherlands              NPIP           0.1(d)              Stephany et al. (1976)

    ham with layer                Germany                  DMN            3 µg/kg             Eisenbrand et al. (1975)
    of pepper grains              (Federal Republic of)
    on the outside.
    Only fat portion.

    ham with layer                Germany                  NPIP           6 µg/kg             Eisenbrand et al. (1975)
    of pepper grains              (Federal Republic of)
    on the outside.

    Whole product                 Germany                  NPY            6
    homogenized.                  (Federal Republic of)

    ham, fried                    Germany                  DMN            1 µg/kg             Eisenbrand et al. (1975)
                                  (Federal Republic of)    NPIP           8
                                                           NPY            19

                                                                                                                       

    Table 2 (Cont'd)
                                                                                                                       

                                                            N-nitroso
    Meat                          Country                  compounds      Levels (c)          Reference
                                  or area                  foundb
                                                                                                                       

    ham with layer of             Germany                  NPIP           4 µg/kg             Eisenbrand et al. (1975)
    pepper grains                 (Federal Republic of)
    on the outside.               Germany                  NPY            9
    Whole product                 (Federal Republic of)
    homogenized.

    German type of                Germany                  DMN            1 µg/kg             Eisenbrand et al. (1975)
    bacon, raw                    (Federal Republic of)

    German type of                Germany                  DMN            1 µg/kg             Eisenbrand et al. (1975)
    bacon, fried                  (Federal Republic of)    NPIP           5                   Eisenbrand et al. (1975)
                                  Germany                  NPY            19                  Eisenbrand et al. (1975)
                                  (Federal Republic of)

    smoked raw meat               Germany                  DMN            2 µg/kg             Eisenbrand et al. (1975)
                                  (Federal Republic of)

    smoked ham                    Germany                  DMN            8 µg/kg             Eisenbrand et al. (1975)
                                  (Federal Republic of)

    smoked ham                    USA                      DMN            5 µg/kg             Fazio et al. (1971b)

    smoked ham                    USA                      DMN            5 µg/kg             Fiddler et al. (1974)

    cooked and smoked             Netherlands              DMN            0.4 µg/kg(f)        Stephany et al. (1976)
    ham                                                    DEN            0.6(f)              Stephany et al. (1976)

                                                                                                                       

    Table 2 (Cont'd)
                                                                                                                       

                                                            N-nitroso
    Meat                          Country                  compounds      Levels (c)          Reference
                                  or area                  foundb
                                                                                                                       

                                  Netherlands              NDBA           0.4(f)              Stephany et al. (1976)
                                                           NPY            0.3(f)              Stephany et al. (1976)
                                  Netherlands              NPIP           0.4(f)              Stephany et al. (1976)

    cooked ham                    Netherlands              DMN            6 µg/kg             Groenen et al. (1976)

    Bologna sausage               Canada                   DEN            25 µg/kg            Panalaks et al. (1974)
                                  Canada                   NPY            20,100,105          Panalaks et al. (1974)

    frankfurter sausage           USA                      NPIP           50, 50, 60 µg/kg    Wasserman et al. (1972)

    spiced meat                   Canada                   DMN            5-48 µg/kg          Sen et al. (1976)
    products                                               DEN            6-16
                                  Canada                   NPIP           14-50               Sen et al. (1976)
                                  Canada                   NPY            7-33                Sen et al. (1976)

    fish meal                     Canada                   DMN            0.35-0.5 mg/kg      Sen et al. (1972)

    smoked, nitrate/              USA                      DMN            4-26 µg/kg          Fazio at el. (1971a)
    or nitrite treated
    sable, salmon shad

    fresh, smoked or              UK                       DMN            1-9 µg/kg           Crosby et al. (1972)
    salted fish

    salted white                  Hong Kong                DMN            40-100 µg/kg        Fong & Chan (1973a, 1973b)
    herring

                                                                                                                       

    Table 2 (Cont'd)
                                                                                                                       

                                                            N-nitroso
    Meat                          Country                  compounds      Levels (c)          Reference
                                  or area                  foundb
                                                                                                                       

    salted yellow                 Hong Kong                DMN            10-60 µg/kg         Fong & Chan (1973a, 1973b)
    croakers

    crude salt salted             Hong Kong                DMN            400 µg/kg           Fong & Chan (1973a, 1973b)
    white herring

    crude salt salted             Hong Kong                DMN            200 µg/kg           Fong & Chan (1973a, 1973b)
    yellow croakers

    prime salt salted             Hong Kong                DMN            10 µg/kg            Fong & Chan (1973a, 1973b)
    white herring

    prime salt salted             Hong Kong                DMN            5 µg/kg             Fong & Chan (1973a, 1973b)
    yellow croakers

    salted anchovies              Hong Kong                DMN            20 µg/kg            Fong & Chan (1973a, 1973b)

    whole herring meal            Hong Kong                DMN            300 µg/kg           Fong & Chan (1973a, 1973b)

                                                                                                                       

    a   Adapted from Sen (1974)
    b  DMN --  N-methyl- N-nitrosomethanamine         c  All values confirmed by mass spectroscopy
       DEN --  N-ethyl- N nitrosoethanamine           d  mean of 4 samples
       NDBA -- Nitroso- N-butylamine                 e  mean of 5 samples
       NPY -- Nitrosopyrrolidine                    f  mean of 6 samples
       NPIP -- Nitrosopiperidine                    g  mean of 10 samples
    

        Several authors who detected nitrosamines in foods by screening
    methods but did not confirm these results by mass spectrometry include
    Ender et al. (1964, 1967), Ender & Ceh (1967), Fong & Walsh (1971),
    Freimuth & Glaser (1970), Hedler & Marquardt (1968), Kröller (1967),
    Lembke & Moebus (1970), Möhler & Mayrhofer (1968, 1969), and Sakshaug
    et al. (1965). Nevertheless, results by screening methods should not
    be entirely ignored.

        Considerable variations have been found in the levels of volatile
     N-nitroso compounds in fried and grilled bacon. Attempts to
    correlate these levels with levels of nitrates and nitrites did not
    reveal any definite pattern. Telling et al. (1974) studied the effect
    of various cooking temperatures on the levels of  N-nitroso compounds
    in grilled bacon. The results indicated that the levels of DMN
    remained fairly constant as the cooking temperature was raised but
    those of nitrosopyrrolidine increased.

        Lipid soluble nitrosamines have an affinity for the fatty
    portions of food (Sen et al. 1973a).

    5.2.4  Tobacco and tobacco smoke

        Since precursors for the formation of nitrosamines occur in
    tobacco, Druckrey & Preussman (1962) thought it likely that tobacco or
    tobacco smoke might contain trace amounts of nitrosamines. Initially,
    studies on the nitrosamine content of tobacco products were hampered
    due to interference from other compounds. Later, evidence suggesting
    the presence of DMN, nitrosopyrrolidine, methylbutylnitrosamine, and
    nitrosopiperidine in tobacco smoke was obtained (Kröller, 1967;
    Neurath et al., 1964; Neurath, 1972, Serfontein & Hurter, 1966).
    Although anabasine and nornicotine are constituents of tobacco smoke,
    the corresponding nitroso derivatives were not detected by Neurath
    (1972). Recently, however, Hoffman et al. (1974) reported the presence
    of  N-nitrosonornicotine at levels of up to 88 mg/kg in unburned
    tobacco.

    5.2.5  Estimate of general population exposure

        The  N-nitroso compounds that have been identified and
    determined in meat and fish are listed in Table 2; consumption of
    these foods constitutes a definite exposure of the general population
    to these chemicals. However, at present, no estimate can be made of
    the human exposure from these and other sources because insufficient
    samples have been analysed and because the relevant food consumption
    surveys have not been made.

    5.2.6  Occupational exposure to N-nitroso compounds

        The potential occupational hazards associated with the use or
    manufacture of  N-nitroso compounds in industry have been pointed out
    by Magee & Barnes (1967).

        Only a few quantitative data are available, however, on the
    concentration of  N-nitroso compounds in the work environment. In a
    study by Bretschneider & Matz, (1976) DMN levels in the air in a
    factory manufacturing DMA were roughly estimated to range from 0.001
    to 0.43 µg/m3.

    6.  METABOLISM OF NITRATES, NITRITES, AND N-NITROSO COMPOUNDS

    6.1  Gastrointestinal Absorption

    6.1.1  Nitrates and nitrites

        A part of ingested nitrates is readily absorbed and a part may be
    metabolized by the microflora in the gastrointestinal tract (Ridder &
    Oehme, 1974). Nitrites (NO2-), oxides of nitrogen (N2O5, NO2,
    NO), hydroxylamine (NH2 OH), and ammonia (NH3) can be formed
    depending upon the organisms present, the pH, and the available
    nutrients (trace elements and carbohydrates), and may be absorbed.

        Friedman et al. (1972) gave mice a single oral dose of 150 µg of
    sodium nitrite. Measurement of the rate of disappearance showed that
    the compound was rapidly absorbed and that food in the stomach had
    little effect on absorption. Results using animals with a ligated
    gastro-duodenal junction suggested that the major absorption site was
    the gastric mucosa. Studies by Mirvish et al. (1974) on rats fed a
    diet containing nitrite supported the previous observations on mice.
    Experiments with food containing phenol red showed that a decrease in
    nitrite levels in the stomach contents, especially in the glandular
    part, that occurred within 5 h of feeding, was significantly greater
    than that due to direct faecal elimination. This was attributed to
    decomposition and other acid-catalyzed reactions of nitrite and to
    direct absorption from the stomach.

    6.1.2  N-nitroso compounds

        Although a considerable degree of absorption of nitrosamines can
    be inferred from the nature of their toxic effects following oral
    administration, few reports could be found giving quantitative
    information on absorption. Alarif & Epstein (1974) gave 3H-labelled
    nitrosomethylurea and nitrosomethylurethane by gavage to groups of
    pregnant guineapigs at doses of 2 mg/kg, and 5 mg/kg body weight,
    respectively. The animals were killed 1 h after dosing. When the
    uptake by maternal and fetal tissues was measured by liquid
    scintillation counting and DNA determination, maternal levels were
    generally higher than fetal levels. In studies by Juszkiewicz &
    Kowalski (1974) DMN, DEN, and nitro-propylamine, administered orally
    to goats at 20-30 mg/kg, appeared in the blood within “ h, and later
    in the milk. The concentration in the milk, 2 h after administration
    of DEN at 30 mg/kg, was 14 mg/litre; after 24 h only traces could be
    found. Phillips et al. (1975a) examined the disappearance of DMN from
    the stomach and small intestine of rats as an index of absorption, and
    found that, while very little was absorbed from the stomach, DMN was
    rapidly absorbed from the small intestine.

    6.2  Biotransformation and Elimination

    6.2.1  Nitrates and nitrites

        In a study on 4 rats, 42-90% of nitrates, administered by stomach
    tube, was excreted in the urine within 8 h of administration. Nitrites
    were not detected in the urine either before or after administration
    (Hawksworth & Hill, 1971b). The same authors carried out a study on
    122 samples of human urine, and found that urinary nitrate
    concentration was related to the amount of nitrate ingested.

        The excretion of nitrates and nitrites in the saliva was studied
    by Spiegelhalder et al. (1976) in 11 volunteers given various
    vegetable juices with nitrate concentrations ranging from 30 to
    550 mg/litre. The levels of nitrates and nitrites excreted were
    proportional to the amounts of nitrates ingested. After ingesting
    100 mg of nitrates, nitrite concentrations in the saliva increased, on
    average, by 20 mg/litre.

    6.2.2  N-nitroso compounds

        Magee & Barnes (1967) have reviewed available information on the
    metabolism and elimination of  N-nitroso compounds. Magee (1956)
    measured recovery of DMN from the whole body of the mouse and noted
    that 97% of the total dose (0.05 mg/kg) could be recovered immediately
    after oral administration and that the amounts recovered decreased
    with time until at 4 h no DMN was recovered. Similar results were
    obtained with the rat given DMN orally at 50 mg/kg, the concentration
    fell rapidly with increasing time after injection so that only 30% of
    the dose was recovered at 8 h and none at 24 h.

        Metabolic transformation of DMN was demonstrated by Dutton &
    Heath (1956) using 14C-labelled DMN. In both the mouse and the rat,
    the main radioactive product was expired carbon dioxide. In the mouse,
    65% of the injected 14C was recovered as expired carbon dioxide, 6 h
    after a subcutaneous injection of DMN at 50 mg per kg body weight. In
    the rat, about 40% of the radioactive 14C was recovered as expired
    carbon dioxide, 8 h after the injection. At the end of the experiment,
    the remainder of the 14C was fairly evenly distributed in the
    tissues, apart from about 7% that was excreted in the urine.

        Heath (1962) found that, while part of each of a number of
    nitrosamines studied was excreted unchanged in urine and in expired
    air, the greater part was decomposed. From the rates of expiration of
    labelled carbon dioxide it was shown that decomposition of dimethyl-,
    diethyl-, and  N-butylmethylni-trosamine obeyed Michaelis-Menten
    kinetics. The rate of decomposition was dose-dependent.

        When the metabolic transformation of dialkylnitrosamines,
    especially of the bladder carcinogen di- N-butylnitrosamine was
    investigated, major urinary metabolites with retained  N-nitroso
    structure were identified (Blattmann & Preussman, 1973, 1974;
    Blattmann et al., 1974; Okada & Suzuki, 1972; Okada et al., 1975).
    Hydroxylation, particularly at the terminal CH3 group, has been
    observed as well as chain shortening. Butyl (3-carboxypropyl)
    nitrosamine (BCPN), a major metabolite of butyl (4-hydroxy butyl)-
    nitrosamine (BBN) was an equally potent and selective bladder
    carcinogen (Okada & Suzuki, 1972). Ring-opening was observed during
    metabolism of nitrosomorpholine (Stewart et al., 1974).

    7.  EXPERIMENTAL STUDIES ON THE EFFECTS OF NITRATES, NITRITES, AND
        N-NITROSO COMPOUNDS

    7.1  Nitrates and Nitrites

    7.1.1  Acute and subacute toxicity studies

        Acute nitrate poisoning was first recognized in cattlea by Mayo
    as early as 1895 (Wright & Davidson, 1964), while Comly (1945) was the
    first to report nitrate poisoning from well water in infants in the
    USA. As a result of these and other reports, several studies have been
    conducted on the toxicity of nitrates in a wide variety of animal
    species. They are mainly centred on the formation of methaemoglobin
    that accompanies excessive exposure to nitrates and nitrites. The
    acute toxicity of nitrates and nitrites was recently reviewed by the
    Committee on Nitrate Accumulation (1972).

        Although the outstanding feature of nitrate toxicity is the
    development of methaemoglobinaemia, nitrates may also cause
    vasodilation which aggravates the effects of the methaemoglobinaemia.
    The nitrite ion formed by reduction of nitrates, oxidizes the iron in
    the haemoglobin molecule from the ferrous to the ferric state. The
    resultant methaemoglobin is incapable of reversibly binding oxygen
    (Bosch et al., 1950). Clinical signs of nitrate toxicity, attributable
    to hypoxia appear when methaemoglobin values exceed about 20% (section
    8.1). Oxidation of haemoglobin to methaemoglobin by the nitrite ion
    occurs at different rates for each animal species, but there is little
    difference between individuals of the same species (Smith & Beutler,
    1966). Similarly, the reduction of methaemoglobin in erythrocytes
    mainly by the enzyme system, NADH--methaemoglobin reductase, is
    characteristically different for each animal species. The chemical
    induction of methaemoglobinaemia has recently been reviewed by Smith
    (1969, 1975). These physiological processes appear to be related, even
    though there is a large variation in the rate of formation of
    methaemoglobin and its subsequent reduction. This may help to explain
    the difference in species susceptibility and the variation in signs
    seen in nitrate poisoning.

        In an effort to develop sensitive tests for the detection of the
    possible effects of subclinical methaemoglobinaemia, behavioural
    studies with mice were undertaken by Behroozi et al., 1971. Groups of
    57 black, 6J, male mice were given nitrites in their drinking water at
    doses aimed at producing methaemoglobin levels varying from slightly
    above normal to 15%, which can be considered to be in the subclinical

              

    a  For further discussion of nitrate intoxication in livestock,
       that may involve significant economic loss, see Oehme (1975).

    range. Sodium nitrite doses in water were 100, 1000, 1500, or
    2000 mg/litre. The results showed a significant reduction of overall
    motor activity in the groups receiving the highest levels of
    nitrites. There was a significant inverse relationship between the
    methaemoglobin level and motor activity, with a coefficient of
    correlation of 0.65. An effort to counteract the methaemoglobinaemia
    was made by giving ascorbic acid to the group receiving the highest
    level of nitrites (2000 mg/litre). (See section 7.1.5). The effect
    was to reduce the methaemoglobin levels to almost normal but the
    motor activity level of the group so treated remained low and about
    equal to the equivalent group that had not received an antidote.
    These experiments seemed to indicate that the nitrites had some form
    of sedative effect on the treated mice, that was not necessarily
    associated with the development of methaemoglobinaemia.

        Experiments on rabbits have shown that methaemoglobinaemia caused
    by nitrates in water also affects cardiac activity by increasing the
    number of cardiac contractions to an extent directly proportional to
    the increase in methaemoglobin levels. At the 10-15% methaemoglobin
    level, the electrocardiogram shows a shortening of the Q-T interval
    and a reduction in the T wave, which may even become negative (Garbuz,
    1968, 1971).

    7.1.2  Chronic toxicity and carcinogenicity studies

        In a study conducted by Shuval & Gruener (1972), groups of 8 male
    rats (3 months old) were given tap water (control) or 100, 1000, 2000,
    or 3000 mg of sodium nitrite per litre of drinking water. After 24
    months, there were no significant differences in growth and
    development, mortality, and total haemoglobin levels between the
    control and treated groups. However, the methaemoglobin levels in the
    groups receiving sodium nitrite at 1000, 2000, or 3000 mg/litre were
    raised significantly throughout the study and averaged 5%, 12%, and
    22% of total haemoglobin respectively. The methaemoglobin levels in
    the group receiving 100 mg/litre were slightly above those of the
    control group for the first 60 days only. There were some changes in
    the liver and spleen of treated animals but the main pathological
    changes occurred in the heart and lungs. In the heart, small foci of
    cells and fibrosis were seen in some animals with pronounced
    degenerative foci in animals receiving the highest concentrations of
    nitrites. The coronary arteries were thin and dilated. The bronchi
    were frequently dilated with the walls infiltrated by lymphocytes and
    the mucosa and muscle were often atrophied. Emphysema was the rule.
    These changes, which were present in 1 or 2 control rats and in a
    small number of those receiving nitrite levels of 100 mg/litre, were
    found with increasing frequency and severity in the 3 highest dose
    groups.

        Druckery et al. (1963a) did not observe extensive methaemoglobin
    formation in rats given 100 mg sodium nitrite per kg body weight in
    the drinking water, but there was a slight reduction in the life span.
    Studies by Van Logten et al. (1972) in which groups of 30 male and 30
    female rats received sodium nitrite at concentrations of 0, 0.02, or
    0.05% with or without glucono--lactone (GDL) in the diet for 29
    months did not demonstrate any significant haematological or
    biochemical effects. Carcinogenic action, which could be related to
    the administration of sodium nitrite with or without DEA or GDL, was
    not observed in either of these studies.

        It has been reported that prolonged administration to rats of
    1/20 of the LD50 of calcium and sodium nitrates disturbed the energy
    conversion processes such as glycolysis and the pentose phosphate
    cycle, changed the activity of the glutathione-ascorbic acid system in
    the blood and in the hepatic and cerebral tissues, raised the levels
    of methaemoglobin and of NADH-methaemoglobin reductase activity, and
    reduced the haemoglobin levels (Diskalenko & Dobrjanskaja, 1972;
    Diskalenko & Trofimenko, 1972).

        Greenblatt & Mirvish (1972) gave 3 groups of about 40, 7-9 week
    old, male, strain A mice, 1 or 2 g of sodium nitrite or 12.3 g of
    sodium nitrate/litre of water respectively, for 20 weeks and did not
    observe any increase in lung tumour incidence in comparison with
    untreated controls. Lijinsky et al. (1973a) reported similar negative
    results when groups of 30 rats were given sodium nitrate at 5 g/litre
    or sodium nitrite at 2 g/litre in their drinking water at a daily rate
    of 20 ml/rat throughout most of their lifetime. Other studies by
    Lijinsky et al. (1973c) on groups of 30 rats given 20 ml of sodium
    nitrite at 2 g/litre daily, for 5 days a week, also produced negative
    results. Taylor & Lijinsky (1975) reported that tumour formation in 57
    Sprague-Dawley rats, exposed for 2 years to a drinking solution
    containing sodium nitrite at 2 g/litre, was no greater than in the
    controls.

    7.1.3  Embryotoxicity

        A study has been reported by Shuval & Gruener (1972) in which 2
    groups of 12 pregnant rats were given 2000 or 3000 mg of sodium
    nitrite per litre of drinking water, respectively. A control group did
    not receive any treatment. Pregnant rats developed anaemia and had
    higher methaemoglobin levels than nonpregnant rats receiving similar
    doses. There was a pronounced increase in mortality among the newborn
    rats of treated dams compared with those of untreated controls,
    particularly in the 3-week period before weaning. Mortality in the
    offspring was 6% in controls, 30% in those given 2000 mg/litre and
    53% in those given 3000 mg/litre. Birthweights were similar in all
    groups but growth was markedly reduced in pups of treated dams. Such
    pups had thin hair coats. Treatment of 2 groups of 10 and 15 pregnant
    rats with 1% and 0.3%, respectively, of sodium nitrate in the diet did


    not result in any embryotoxic or teratogenic effects on the 9th and
    10th days of gestation (Alexandrov & Jänisch, 1971).

        Sleight & Atallah (1968) conducted a study in which 46 female
    guineapigs, divided into 12 groups each containing at least 1 male,
    were given potassium nitrate in doses ranging from 300 to 10 000 mg/kg
    body weight in the water and potassium nitrite in amounts ranging from
    300 to 10 000 mg/kg for periods ranging from 100-240 days.
    Reproduction in the female was grossly impaired in the high nitrate
    group. Fetal losses were 100% in females given 5000 or 10 000 mg/kg of
    the nitrite and one female died. Reproduction was maintained at lower
    levels of treatment. Apparently male fertility was not impaired, since
    conception took place at all levels of treatment. Food and water
    consumption and weight gains of treated animals were normal except for
    a diminished rate of gain at a nitrite level of 10 000 mg/kg body
    weight. Uterine and cervical inflammatory lesions and degenerative
    placental lesions were present in females in which the fetuses had
    been aborted, mummified, or absorbed. Sinha & Sleight (1971) reported
    studies in which 4 pregnant guineapigs, given sodium nitrite at 50 mg
    per kg of body weight, subcutaneously, underwent normal parturition.
    However, fetal deaths followed by abortion, occurred in 3 guineapigs
    given sodium nitrite at 60 mg/kg. The fetal deaths occurred
    approximately 1 h after nitrite administration, when the maternal and
    fetal methaemoglobin levels were highest. At the time of death, there
    were no noticeable changes in the placenta; pathological changes
    developed after the death of the fetuses. There were lower blood pO2
    values in the fetuses of the guineapigs treated with nitrite at
    60 mg/kg than in those of the controls. Fetal death did not occur in
    pregnant animals given sodium nitrite at 60 mg/kg combined with
    simultaneous intraperitoneal treatment with 10 mg of methylene blue
    per kg body weight. The data suggest that fetal death resulted from
    hypoxia, mainly induced by maternal methaemoglobinaemia.

        Studies by Shuval & Gruener (1972) indicated that
    methaemoglobinaemia might be induced transplacentally and that the
    observed limit for transplacentally induced methaemoglobinemia was a
    sodium nitrite dose of 2.5 mg/kg body weight. A steep increase in
    effect occurred with increasing doses of sodium nitrite.

    7.1.4  Mutagenicity

        The mutagenic potential of nitrates and nitrites has not been
    studied extensively; in fact, no data are available on their mutagenic
    action in the mammalian systems. The mutagenicity of nitrates and
    nitrites with respect to the transformation of DNA has been reported
    (Bressler et al., 1968; Horn & Herriot, 1962; Strack et al., 1964) and
    nitrous acid has been shown to be mutagenic in bacterial systems such
    as  Escherichia coli (Kaudewitz, 1959; Verly et al., 1967) and
     Salmonella typhimurium (de Serres et al., 1967). Positive results in
    mutagenic studies have been reported with the yeast  Saccharamyces

    cerevisiae (Nashed & Jabbur, 1966; Zimmerman & Schwaier, 1967;
    Zimmerman et al., 1966), as well as with  Aspergillus nidulans
    (Siddiqi, 1962),  A. niger, and  A. amstelodami (Steinberg & Thom,
    1940), and tobacco mosaic Virus (Sehgal & Krause, 1968).

    7.1.5  Interaction with nutritional factors

        Studies by Kociba & Sleight (1970) showed that maternal blood
    levels of methaemoglobin were significantly higher in 12 ascorbic
    acid-deficient, pregnant guineapigs, following the subcutaneous
    administration of sodium nitrite at 40 mg/kg body weight, than in
    those on a normal diet. Following subcutaneous administration of
    sodium nitrite at 50 mg/kg, there was a higher percentage of fetal
    death in the ascorbic acid-deficient guineapigs.

        Experiments were conducted by Stoewsand (1973) with young, male,
    guineapigs (number of animals not stated) to investigate the influence
    of feeding beets with naturally occurring low and high amounts of
    nitrates and nitrites, and the influence of dietary supplementation
    with ascorbic acid and methionine on methaemoglobinaemia, induced by
    orally administered sodium nitrite at doses of 25 or 50 mg/kg body
    weight. Low-nitrate beet diets seemed to "protect" guineapigs from
    nitrite intoxication. In addition, a 1% diet containing L-ascorbic
    acid and 1% methione reduced nitrite-induced methaemoglobin blood
    levels.

        Sell & Roberts (1963) showed that  ad libitum feeding of diets
    containing 0.4% potassium nitrite to 6 groups of 50 chicks depressed
    growth, irrespective of the amount of vitamin A administered. Also,
    all the test animals, except those given massive injections of vitamin
    A, exhibited reduced liver stores of the vitamin and enlarged thyroid
    glands.

        Studies by Phillips (1966) demonstrated that the liver vitamin A
    contents of rats fed 1% potassium nitrite in diets containing carotene
    or vitamin A, were less than those of control rats. It was suggested
    that the dietary nitrite degraded the carotene and vitamin A in the
    digestive tract before their absorption.

    7.2.  N-nitroso Compounds

    7.2.1  Acute and subacute toxicity studies

        The acute toxicity of  N-nitroso compounds is not of great
    toxicological significance because there is no relationship between
    acute toxicity and the carcinogenic potential of this class of
    compounds (Druckrey et al., 1967, Magee & Barnes, 1967). Because DMN
    was reported to cause cirrhosis and other toxic effects in industrial
    workers, Barnes & Magee (1954) examined the toxicity of this compound.
    Doses of 20-40 mg/kg body weight given to rats, dogs, rabbits, and

    guineapigs produced severe hepatic damage. A single dose of DMN given
    to rats orally or by intravenous, intraperitoneal, or subcutaneous
    injection, produced centrilobular necrosis accompanied by haemmorhages
    in the liver. In rats given 20 mg DMN/kg body weight the liver cells
    in the centrilobular and mid-zonal regions became pale and, after
    18 h, the cytoplasm was amorphous and vacuolated; the nuclei were pale
    and irregular in outline. By 24 h, the cells were necrotic and
    confluent areas became haemorrhagic. Haemorrhage was usually more
    pronounced after 48 h but after 72 h the recovery process had begun
    and was almost complete in 3 weeks (Barnes & Magee, 1954; Magee &
    Barnes, 1962). A detailed study by light and electronmicroscopy of
    changes in the liver cytoplasm of rats treated with
     N-nitrosomorpholine has been reported by Bannasch (1968).

        The acute toxicity of acyl-alkyl-nitrosamides and diazoalkanes
    was reported by Druckery et al. (1967), Magee & Barnes (1967), and
    Shank (1975). The acute toxicity (LD50) of the nitroso compounds
    varied widely; some were only mildly toxic while others produced
    highly destructive lesions.  N-nitroso- N-methylurethane, for
    example, when given orally, produced severe necrotic lesions in the
    stomach, congestion of the lungs, and periportal necrosis of the liver
    (Schmähl & Thomas, 1962; Schoental, 1960).  N-nitroso- N-methylurea
    also produced inflammatory haemorrhagic lesions of the stomach,
    intestine, and pancreas and a reduction in the bone marrow when given
    orally to rats, (Druckrey et al., 1961, 1967).

        The acute toxicity of some  N-nitroso compounds, e.g. nitroso-
    piperidine and nitroso-morpholine, was not manifested as liver damage
    but as neurotoxic effects, e.g. convulsions (Lee & Lijinsky, 1966).

        The acute toxicity of nitrosomines formed  in vivo was studied
    by Asahina et al. (1971). Sodium nitrite was administered by gavage to
    mice at 100 or 150 mg/kg bodyweight alone or in combination with DMA
    and methylbenzylamine at doses of 500-2500 mg/kg and 800-1600 mg/kg,
    respectively. Combinations of the amines and nitrite produced hepatic
    lesions similar to those produced by DMN or nitrosome-thylbenzylamine.
    Similar effects were noted when nitrite was administered up to 3 h
    after DMA but these effects were markedly reduced if the nitrite was
    given prior to the amine. Lijinsky & Greenblatt (1972) reported
    hepatic necrosis following coadministration of aminopyrine and sodium
    nitrite.

        In a number of studies, ascorbic acid has been shown to have an
    inhibitory effect on: a) nitrosamine formation in the stomach and
    small intestine of rats treated with 7-chloro-2-methylamino-5-phenyl-
    3H-1, 4-benzodiazepine-4-oxide, (chlordiazepoxide, Librium) and sodium
    nitrite (Preda et al., 1976); b) hepatotoxicity induced by the
    combined administration of sodium nitrite and aminopyrine in rats
    (Kamm et al., 1973) and mice (Greenblatt, 1973); c) liver necrosis

    produced by DMA and nitrite administered to rats by gavage (Cardesa et
    al., 1974); d) the teratogenic and transplacental carcinogenic effects
    produced in rats by treatment with alkylurea and nitrite (Ivankovic et
    al., 1973); and e) the induction of lung adenomas in mice by prolonged
    treatment with morpholine, piperazine, and methylurea plus nitrite
    (Mirvish et al., 1975).

        Species differences exist with respect to the toxic effects of
     N-nitroso compounds. Mink appear to be especially sensitive to DMN,
    and as in other species, the effects are seen primarily in the liver.
    Carter et al. (1969) noted widespread liver degeneration and necrosis
    of hepatocytes in mink given DMN at 2.5 or 5.0 mg/kg in the diet for
    7-11 days. The liver lesions were accompanied by bile-duct
    proliferation, ascites, and haemorrhage of the gastrointestinal tract.
    Sheep and cattle are also more sensitive to nitrosamines than
    laboratory animals (Sakshaug et al, 1965; Koppang, 1964). Sheep, given
    a single dose of DMN at 5 mg/kg body weight or 12 doses of 0.5 mg/kg,
    died or were severely affected displaying anoxia, lack of rumination,
    ataxia, and respiratory difficulties, while cattle given DMN at
    0.1 mg/kg body weight showed pronounced hepatotoxic effects in 1-6 months.

        Pathological and biochemical effects, observed in the liver of a
    number of animal species including the rat following continuous
    administration of nitrosamides, have been reviewed by Magee & Barnes
    (1967) and Magee et al. (1976); the main effect is inhibition of
    protein synthesis which might be a result of an accelerated breakdown
    of messenger ribonucleic acid (RNA).

    7.2.2  Carcinogenicity

        The carcinogenic activity of  N-nitroso compounds has been
    summarized in several reviews (Druckrey, et al., 1967; Magee & Barnes,
    1967; Magee et al., 1976). Various animal species including mammals,
    birds, fish, and amphibia have been shown to be susceptible to the
    carcinogenic action of nitrosamines. At present, some 80 nitrosamines
    and 23 nitrosamides have been tested in rats and about 80% of the
    nitrosamines and practically all the nitrosamides have proved to be
    carcinogenic (Montesano & Bartch, 1976). These carcinogens show a
    marked organ specificity as shown in Table 3.

        Nitrosamines produce a carcinogenic effect in the liver,
    oesophagus, respiratory system, and kidney, whereas nitrosamides
    affect the peripheral and central nervous systems, and the gastro-
    intestinal tract organs.

        The dose schedule seems to play an important role in this organ
    specificity. For example, in rats, long-term exposure to relatively
    low doses of DMN induced mainly liver tumours, whereas a single or a
    few high doses over a short period induced mainly kidney tumours
    (Magee & Barnes, 1962). Similar responses in relation to the liver and

    oesophagus were observed with DEN in rats (Druckey et al., 1967). The
    route of administration does not seem to play an important role in the
    carcinogenicity of this chemical. It is worthwhile pointing out that
    many of these chemicals were carcinogenic following a single
    administration and that, furthermore, exposure of rats to a single
    dose during pregnancy resulted in carcinogenesis in the immediate
    descendants and also in the two succeeding generations (Tomatis et
    al., 1977).

        Nitrosamines exert their adverse biological effects after being
    metabolically activated by microsomal mixed function oxidases to form
    reactive intermediates. On the other hand, nitrosamides decompose
    enzymatically to reactive, and in most cases, alkylating derivatives.
    The importance of hydroxilation of the alpha-hydrogen atoms of the
    nitrosamines is demonstrated by the lack of carcinogenicity of
    compounds, such as diphenylnitrosamine, which do not have this alpha-
    hydrogen (Magee et al., 1976).

    Table 3.  Localization of tumours induced by  N-nitroso
              compounds in ratsa
                                                                   

                                    Number of  N-nitroso compounds
                                           affecting target organ

    Target organ                  Nitrosamines          Nitrosamides
                                                                   

    liver                              35                     2
    oesophagus-pharynx                 32                     3
    nasal cavities                     18                     -
    respiratory tract                  10                     1
    kidney                              8                     9
    tongue                              8                     -
    forestomach                         7                    11
    bladder                             4                     1
    central and peripheral              2                     9
      nervous system
    ear duct                            2                     1
    testis                              1                     -
    ovary                               1                     2
    mammary glands                      1                     1
    sites of injection                  3                     4
    intestine                           -                     7
    glandular stomach                   -                     6
    skin                                -                     3
    jaw                                 -                     1
    uterus                              -                     2
    vagina                              -                     1
    haemopoietic system                 -                     2
                                                                   

    a  From: Montesano & Bartch (1976)

    7.2.2.1  Interspecies variation in response

        Several  N-nitroso compounds have been tested in different
    animal species. DEN, tested in more than 20 species including
    primates, induced tumours of the liver in all of them, together with
    various tumours of other organs. In some cases, there have been marked
    differences between species in response to  N-nitroso compounds. For
    example, nitrosoheptamethyleneimine produced squamous carcinoma of the
    lung in rats (Lijinsky et al., 1969), the same effects in European
    hamsters, but tumours of the forestomach and the lung in Syrian
    hamsters (Lijinsky et al., 1970). In all three species, this compound
    also induced oesophageal tumours. Nitrosomethylurethane induced
    tumours of the pancreas in guineapigs (Druckrey et al., 1968) and
    forestomach carcinomas in rats. Bis-2-hydroxypropyl-nitrosamine
    produced pancreatic tumours in Syrian hamsters. These results show
    the difficulty of attributing, with certainty, any particular tumour
    response of man to a particular  N-nitroso compound.

    7.2.2.2  Intraspecies variation in response

        Kuwahra et al. (1972) administered DMN orally or by subcutaneous
    or intraperitoneal injections, to 8 to 12-week-old, male and female
    mice of the DDD, BALB/C, and SJL/J strains. Tumours were found mainly
    in the retroperitoneum and abdominal cavity and the incidence and
    distribution were little affected by the strain of mouse but markedly
    by the route of administration. Clapp et al. (1971) noted that DEN
    induced forestomach and oesophageal squamous cell carcinomas in both
    BALB/C and RF/Un strains of mice but induced liver haemangiosarcomas
    in the former and hepatomas in the latter. In addition, DEN induced a
    high incidence of lung tumours in RF mice but a very low incidence in
    BALB/C mice. In both strains, DMN induced lung adenomas and liver
    haemangiosarcomas. Tissue sensitivity did not appear to be related to
    the spontaneous tumour incidence of the strain.

    7.2.2.3  Dose-response relationships of N-nitroso compounds

        Druckrey et al. (1963b) were the first to study the dose-response
    relationships of  N-nitroso compounds in relation to minimum effect
    doses. They concluded, from studies in rats given DEN, that the
    carcinogenic effect was related to dose and induction time in such a
    way that  d.t2.3 = constant, where  d represents the daily dose and
     t the induction time. Even a low dose of 0.15 mg/kg body weight
    resulted in liver carcinomas in 27 out of 30 surviving animals. The
    average induction time was 609 ± 38 days. The lowest dose studied
    (0.075 mg/kg body weight) produced liver and oesophageal tumours in
    all four surviving animals with an induction time of 830 days.

        Mohr & Hilfrich (1972) gave a single intravenous injection of DEN
    to rats at 8 dose levels between 1.25 and 160 mg/kg body weight. A
    dose-response relationship was noted; at the lowest dose of 1.25
    mg/kg, only one kidney tumour was observed in 20 treated rats, but, at
    higher doses, the incidence of these tumours increased. Single dose
    experiments with ethylnitrosourea in a transplacental carcinogenesis
    experiment also showed that the incidence of induced tumours (mainly
    of the brain and nervous system in the progeny) was directly
    proportional to the dose of the carcinogen. Four doses between 1 and
    50 mg/kg were used and even in the lowest dose group 36/41 animals in
    the progeny died with tumours (Swenberg et al., 1972). Terracini et
    al. (1967) fed rats concentrations of DMN ranging from 2 to 50 mg/kg
    diet. At 2 and 5 mg/kg the incidence of liver tumours in the survivors
    was 1/26 and 8/74, respectively, at 60 weeks. At 20 and 50 mg/kg,
    liver tumours were observed in more than 60% of the test animals.
    From this study, it can be concluded that a dietary level of DMN of
    5 mg/kg is still carcinogenic in the rat.

        A current dose-response study on rats receiving oral doses of 
     N-nitrosopyrrolidine has been reported by Preussmann (unpublished
    data).a Liver tumours were induced in groups receiving 10, 3, and
    1 mg/kg body weight per day, respectively, but not in a group
    receiving 0.3 mg/kg per day.

        Clapp & Toya (1970) gave male, RF mice DMN in the drinking water
    for various lengths of time, with cumulative doses ranging from 87 to
    243 mg/kg body weight. The incidence of lung adenomas in all treated
    groups was higher than that in the untreated control groups. Whereas,
    apparently, the incidence of hepatocellular tumours was not affected,
    the incidence of liver haemangiosarcomas increased in the higher dose
    groups and reached a maximum of 96%. Bertram & Craig (1973) using 2
    groups of 100, C57BL/6 mice found that the incidence of bladder
    tumours fell from 80% in animals given 30 mg of nitrosodi- N-
    butylamine per kg/body weight per day to 36% at 7.5 mg/kg body weight
    per day. Sander & Schweinsberg (1973) noted that an increasing
    incidence of tumours of the oesophagus and forestomach was induced in
    NMRI mice by adding a given dose of methylbenzylnitrosamine to the
    drinking water at various times to provide total doses ranging from
    1.4 to 44 mg/kg body weight.

              

    a  Paper presented at the Proceedings of the Second International
       Symposium on Nitrite in Meat Products, Zeist, September 1976.

        In studies conducted by Vesselinovitch (1969), DMN was
    administered repeatedly by intraperitoneal injection to 73 male and 54
    female C57BL x C3H mice starting at 7 days of age. The injections were
    given at 3 day intervals for a total of six doses of 1, 2, or 4 mg/kg
    body weight. Mice killed at 66 weeks of age showed an increase in the  
    incidence of hepatomas, hepatocarcinomas, lung adenomas, and
    haemangiomas as the dose increased. The incidence of liver tumours was
    higher in males (75%) than in females (28%).

        Tomatis & cefis (1967) gave a dose of 3.6 mg of DMN by stomach
    tube to one group of 30 Syrian golden hamsters in 3 administrations of
    1.6 mg, 1.0 mg, and 1.0 mg over a 5-week period. A second group
    received a single dose of 1.6 mg. Both dosing regimes produced liver
    cell carcinomas and a few cholangiomatous lesions but no kidney
    tumours. Montesano & Saffiotti (1968) administered 12, weekly,
    subcutaneous injections of DEN at 0.5, 1.0, 2.0, or 4.0 mg to 4 groups
    of 36 Syrian golden hamsters. The results demonstrated a positive
    dose-response relationship for tumour induction in the upper
    respiratory tract (nasal cavities, larynx, and trachea). The incidence
    of nasal cavity tumours, which developed early, varied from 6/35 to
    27/36. The incidence of tumours of the larynx varied from 6/35 to
    26/36 and that of tumours of the trachea from 29/35 to 35/36.

    7.2.2.4  Tumour induction by combined administration of nitrites
             and amines or amides

        Carcinogenic effects following the combined administration of
    secondary and tertiary amines or amides with nitrite have been
    reported. Tumours were not observed when sodium nitrite or the amine
    or amide were given singly. Tumours of the same type occurred at the
    same site when the  N-nitroso compound, believed to be formed from
    the nitrite and amine or amide, was given as a positive control. In
    some of the experiments, the nitrite was given in the drinking water
    and the amine or amide in the food. In others, the compounds were
    combined in the same medium (drinking water or food).

        Preussman (1975) reviewed studies in which tumours that had been
    produced at certain sites by the oral administration of combinations
    of amines and nitrite, were compared with the tumours induced by the
    corresponding  N-nitroso compounds. Table 4 has been adapted from
    Preussman (1975) and updated.



        Table 4.   In vivo formation of  N-nitroso compounds following oral administration of sodium nitrite and amino
              compounds as demonstrated by specific carcinogenesisa

                                                                                                                 

    sodium nitrite + amine                                      Expected tumour          Reference
    oral administration           Observed tumour               site for
    amino compounds               site                          corresponding
                                                                 N-nitroso compound
                                                                                                                 

     Rat

    diethylamine                  --                            liver                    Druckrey et al.
                                                                                         (1963b)
                                                                                         Sander et al.
                                                                                         (1968)
                                                                                         Sander (1971a)

    triethylamine                 --                            liver (for               Schweinsberg &
                                                                diethylnitrosamine       Sander (1972)

    morpholine                    liver, kidney, lung           liver                    Sander & Burkle
    N-methylbenzylamine           oesophagus                    oesophagus               (1969), Sander
                                                                                         (1971c)

    piperidine                    --                            oesophagus
                                                                liver

    N-methylanaline               oesophagus, nasal             oesophagus
                                  cavity

    N-methylcyclohexylamine       oesophagus                    oesophagus               Sander (1971a)
                                                                                                                 

    Table 4 (Cont'd)
                                                                                                                 

    sodium nitrite + amine                                      Expected tumour          Reference
    oral administration           Observed tumour               site for
    amino compounds               site                          corresponding
                                                                 N-nitroso compound
                                                                                                                 

    N-methylbenzylamine           oesophagus                    oesophagus

    phenylbenzylamine             negative                      untested

    N,N'-dibenzylethylenediamine  negative                      untested

    indole                        negative                      untested

    morpholine                    liver (kidney)                liver (kidney)           Shank & Newberne
                                                                                         (1972)

    aminopyrine                   liver                         liver (for               Lijinsky et al.
       (Pyramidon)                                              dimethylnitrosamine)     (1973c)

    heptamethyleneamine           lung                          lung
                                  oesophagus                    oesophagus

    aminopyrine                   liver                         liver                    Taylor &
                                  (for DMN)                                              Lijinsky
                                                                                         (1975)

    oxytetracycline               liver (for DMN)               liver                    Greenblatt et al. (1973)

    proline                       negative                      untested
                                                                                                                 

    Table 4 (Cont'd)
                                                                                                                 
    sodium nitrite + amine                                      Expected tumour          Reference
    oral administration           Observed tumour               site for
    amino compounds               site                          corresponding
                                                                 N-nitroso compound
                                                                                                                 

    hydroxyproline                negative                      untested

    arginine                      negative                      untested

    N-methylacetamide             negative                      forestomach

    N-methylurethane              negative                      forestomach

    N-ethylurethane               negative                      (carcinogenic            Sander (1971b)
                                                                following
                                                                i.v. administration)

    acetanilide                   negative                      untested

    glycylglycine                 negative                      untested

    N-methyluracil                negative                      untested

    N-methylguanidine             negative                      untested

    N-phenylurea                  negative                      (carcinogenic            Sander (1971b)
                                                                following
                                                                s.c. administration)

    N-methylthiourea              negative                      untested

    N-methylurea                  brain, nervous                brain, nervous
                                  system, kidney                system, kidney
                                                                                                                 

    Table 4 (Cont'd)
                                                                                                                 
    sodium nitrite + amine                                      Expected tumour          Reference
    oral administration           Observed tumour               site for
    amino compounds               site                          corresponding
                                                                 N-nitroso compound
                                                                                                                 

    N-ethylurea                   brain, nervous                brain, nervous
                                  system, kidney                system, kidney

    N'N'-dimethylurea             brain, nervous                brain, nervous           Sander (1971b)
                                  system, kidney                system, kidney

    imidazolidinone               kidney                        (carcinogenic            Sander & Burkle
                                                                following                (1971)
                                                                s.c. administration)

    ethylurea (during             brain, nervous                brain, nervous           Ivankovic &
    pregnancy,                    system                        system (in               Preussmann
    transplacental)               (in descendants)              descendants)             (1970); Osske
                                                                                         et al. (1972)
    Mouse

    dimethylamine                 lung (adenoma)

    piperazine                    lung (adenoma)                lung (adenoma)           Greenblatt et al. (1971)

    morpholine                    lung (adenoma)                lung (adenoma)

    N-methylaniline               lung (adenoma)                lung (adenoma)

    morpholine                    lung (adenoma)                lung (adenoma)

                                                                                                                 
    Table 4 (Cont'd)
                                                                                                                 
    sodium nitrite + amine                                      Expected tumour          Reference
    oral administration           Observed tumour               site for
    amino compounds               site                          corresponding
                                                                 N-nitroso compound
                                                                                                                 

    N-methylbenzylamine           oesophagus                    oesophagus               Sander (1971a)
                                  forestomach                   forestomach

    piperazine                    lung (adenoma)                lung (adenoma)           Greenblatt &
                                                                                         Mirvish (1972)

    N-methylurea                  lung (adenoma)                lung (adenoma)           Mirvish et al. (1973b)

    N-ethylurea                   lung (adenoma)                lung (adenoma)
                                                                                                                 

    a  Adapted from Preussmann (1975)
    

    7.2.2.5  Dose-response relationship for combinations of nitrite
             and amines

        Greenblatt & Mirvish (1972) conducted a study in which groups of
    40, male, strain A mice, given 0.69-18.75 g of piperazine per kg of
    food and 0.05-2.0 g of sodium nitrite per litre of drinking water for
    20-25 weeks, were killed 10-13 weeks later. The yield of lung adenomas
    was statistically significantly greater than in untreated controls,
    when doses as low as 0.69 g piperazine/kg plus 1.0 g sodium nitrite/
    litre, or 6.25 g piperazine/kg plus 0.25 g sodium nitrite/litre were
    given. In the animals given 6.25 g piperazine per kg of food, plus
    2.0 g sodium nitrite per litre of water, 39 out of 40 had adenomas,
    in contrast with 5 out of 39 controls. A progressive decrease in the
    incidence of adenomas was seen with reduction in the nitrite level.
    On the other hand, when the nitrite level was maintained at
    1.0 g/litre, the tumour incidence increased marginally when the dose
    of piperazine was increased from 0.69 g to 18.75 g/kg of food.
    Nitrite and piperazine administered alone yielded negative results.

        When various concentrations of nitrite and morpholine (up to
    1000 mg/kg) were fed to groups of Sprague-Dawley rats, the
    development of hepatocellular carcinomas and angiosarcomas identical
    to those produced by  N-nitrosomorpholine was noted (Newberne &
    Shank, 1973).

        A 2-3% incidence of hepatomas was induced with only 5 mg of
    morpholine plus 1000 mg of sodium nitrite per kg of food, or 1000 mg
    of morpholine plus 50 mg sodium nitrite/kg. The tumour incidence was
    98% when the concentration of both components was 1000 mg/kg, and 0%
    when diet alone was given.

    7.2.2.6  Transplacental carcinogenesis

        Induction of neoplasms in offspring as a result of prenatal
    exposure to various  N-nitroso compounds and related substances has
    been reported in different animal species including the rat, mouse,
    golden hamster, guineapig, rabbit, dog, and monkey (Magee et al.,
    1976). The various routes of administration (subcutaneous,
    intraperitoneal, intravenous, oral, and inhalation) were equally
    effective. However, a critical factor was the time of treatment during
    gestation.

        Since many of these substances appeared to be metabolically
    activated to exert their carcinogenic action, the lack of an adequate
    metabolic system during the first period of pregnancy may explain the
    failure to observe tumours, when exposure was limited to this period.
    DMN induced tumours in the offspring only when administered during the
    last days of pregnancy (Alexandrov, 1968a). The data of Magee (1972)
     

    are in keeping with these findings; the formation of 7-methylguanine
    in the nucleic acids of fetal rat tissues was detected following
    treatment with DMN on the 21st day of pregnancy but not on the
    15th day.

        When considering the effect of different acyl alkyl nitrosamides,
     N-ethyl- N-nitrosourea was found to be more active than its methyl
    analogue (Alexandrov, 1969b; Ivankovic & Druckrey, 1968).

        The sensitivity of the nervous system at various stages of
    prenatal development was examined in rats and golden hamsters by
    Ivankovic & Druckrey (1968) by means of single intravenous injections
    of  N-ethyl- N-nitrosourea on different days during gestation. A
    high incidence of tumours was observed after treatment on the 18th day
    or shortly before delivery (21st day) but none developed when the
    mother animals were treated before the 12th day. A positive dose-
    response relationship was obtained with a single dose in the range of
    5 to 80 mg/kg bodyweight on the 15th day. A single dose as low as
    2 mg/kg was sufficient to produce malignant, neurogenic tumours in
    2/25 newborn whereas in adult animals an effect was produced in 50%
    of the animals by a single dose at 160 mg/kg. Thus a 50-times higher
    sensitivity was demonstrated for the fetal nervous system. Similar
    results were obtained by Swenberg et al. (1972) in Sprague-Dawley and
    Fisher rats using a dose of  N-ethyl- N-nitrosourea as low as
    1 mg/kg. In a recent study on rats, Maekawa & Odashima (1975) explored
    the effects of subcutaneous injections of  N-butyl-1-nitrosourea on
    embryonal, fetal, and newborn nervous systems. Treatment during early
    gestation led only to the death of the embryo. When the compound was
    given in the middle of the pregnancy (8-14 days), a high incidence of
    nervous system tumours and pituitary tumours was found in the
    offspring. Treatment late in pregnancy (15-21 days) led to the
    development of nervous system tumours in 33/36 offspring.

        When lactating animals were given the compounds, tumours were
    induced in 19/39 of the offspring, the target organs being mainly the
    testes and uterus.

        Tomatis (1977) reported an increased incidence of cancer in the
    descendants (second and third generation) of transplacentally treated
    rats. In certain cases, the tumour-producing doses were lower than
    those for adult animals. The target organs were usually similar in
    fetal, newborn, and adult animals but the nervous system has been
    shown to be highly sensitive to certain compounds, notably the
    nitrosoureas. The induction time for transplacental tumours may be
    shorter than that in adults.

    7.2.2.7  Morphological studies

        Morphological events associated with the development of hepatic
    tumours, following exposure to  N-nitroso compounds, were studied by
    Rabes et al. (1970), Scherer et al. (1972), and Schmitz-Moorman et al.
    (1972). Schmitz-Moorman et al. (1972) followed the carcinogenesis
    induced by DMN in rat liver, using histological and histochemical
    procedures. In the initial phase, there were vacuolar changes
    accompanied by a decrease in the RNA and glycogen contents. In the
    second phase, glycogen storage was noted while in the third phase,
    basophilic cells with atypical nuclei were observed and could not be
    distinguished from microcarcinomas. The RNA content of these cells was
    substantially higher. The activities of acid phosphatase (3.1.3.2),
    succinic dehydrogenase (1.3.99.1) and glucose-6-phosphatase (3.1.3.9)
    substantially decreased. In the last carcinogenic stage, dramatic
    changes were noted in several enzymes (Jennissen et al., 1971).

        Early changes in the lung tissue of mice exposed to carcinogenic
    doses of DMN were reported by Calafat et al. (1970) and DEN-induced
    alterations in the respiratory tract of hamsters were reported by
    Althoff et al. (1971). Greenblatt & Rijhsinghani (1969) compared the
    cytopathological changes induced by DEN and DMN in the nasal
    epithelium of hamsters; other light microscopic studies have been
    published by Bertram & Craig (1972), Boyland et al. (1968), Hicks et
    al. (1973), and Terracini et al., (1967). The histological appearance
    of tumours under the electron microscope has been described by
    Kirkland & Pick (1973). Veno-occlusive disease in the liver of rats
    given DMN was reported by Butler & Hard (1971) who had also studied
    the effects of this compound on rat testes (Hard & Butler, 1970c).

        Other studies, by light microscopy, to observe early changes
    related to the formation of neoplasms induced in the kidneys of rats
    treated with DMN, were reported by Benemanski & Litvinov (1969), Hard
    & Butler (1970a, 1970b), and Hard et al. (1971).

        Electron microscopic studies of DMN-induced liver and kidney
    tumours have been reported in rats (Bhathal & Hurley, 1973; Geil et
    al., 1968; Hard & Butler, 1971a, 1971b, 1971c; Ireton et al., 1972;
    Jasmin & Cha, 1969; Svoboda & Higginson, 1968) and in mice (Takayama,
    1968). Treatment with DEN resulted in ultrastructural changes in the
    lungs of hamsters (Stracks & Feron, 1973) and in the liver of monkeys
    (Williams, 1970) and rats (Bader et al., 1971; Bruni, 1973). Early
    morphological changes in the bladder of rats exposed to  N-butyl- N-
    butanol-4-nitrosamine were reported by Riedel & Piper (1973).

    7.2.2.8  Biochemical mechanisms

        Extensive work on the biochemical mechanisms of carcinogenesis
    produced by  N-nitroso compounds has been reviewed by Magee & Barnes
    (1967) and more recently by Magee et al. (1976). Alkylation of nucleic
    acids by  N-nitroso compounds or their metabolites has been
    investigated extensively and has been suggested as the mechanism of
    carcinogenicity (Krüger, 1972, 1973; Lijinsky et al., 1973b; Swann &
    Magee, 1971; Takayama & Muramatsu, 1969). In early studies, it was
    thought that the 7 position of guanine was the significant site of the
    reaction.

        Following the important discovery by Loveless and Hampton (1969)
    of the  O,6-alkylation of deoxyguanosine by nitrosomethylurea (NMU)
    and the implications of this reaction in terms of carcinogenicity,
    O'Connor et al. (1973) estimated the amount of methylation at the
     O,6-position of guanine DNA isolated from animals treated with DMN.
    This base accounted for 4-6% of the methylation after DMN treatment.
     O,6-methylguanine was lost (by "excision") from DNA with a half life
    of approximately 13 h. The excision of the abnormal components of DNA,
     O,6-methylguanine, and the unstable acid-labile products, may be
    important processes in liver carcinogenesis. O'Connor et al. (1973)
    suggested that events leading to the development of tumours may be
    related to the efficiency of the cellular excision system for certain
    products of alkylation rather than to the level of alkylation obtained
    at a particular site.

    7.2.2.9  Interaction with various chemical factors

        Several studies are available on the combined effects of
     N-nitroso compounds and other carcinogens. Schmähl et al. (1963)
    showed that combined oral administration of the hepatocarcinogens DEN
    and  N,N-dimethyl-4-(phenylazo)benzenamine (4-dimethyl-
    aminoazobenzene) significantly reduced the time for tumour induction
    and that only 66% of the total dose administered when single compounds
    were used, was necessary for tumour induction. Takayama & Imaizumi
    (1969) also demonstrated synergism with the combined administration of
    DMN and 4-dimethylaminoazo-benzene. Liver tumours were induced in rats
    by sequential administration of the two carcinogens in doses that did
    not induce tumours when each carcinogen was given alone and the tumour
    induction time was reduced. The syncarcinogenic effects of a single
    dose of a combination of DEN and carbon tetrachloride were observed by
    both Pound et al. (1973) and Schmähl et al. (1965). The incidence of
    liver cancer increased and the induction time decreased in combined
    treatments compared with treatment with the individual compounds.
    Schmähl (1970) administered hepatocarcinogens (DMN, DEN,
    nitrosomorpholine, and 4-dimethylaminoazobenzene) to rats in such low


    daily doses that the administration of any one of the compounds alone
    did not lead to tumours during the lifetime of the animals. However,
    combined administration induced tumours in 43% of the treated animals.
    Combined administration to rats of DMN plus 1,2-dihydro-3-methyl-
    benz[j]aceanthrylene (3-methylcholanthrene) did not increase the
    number of liver tumours compared with that induced by DMN alone but
    resulted in tumours in the lungs, that were not seen in the treatment
    with DMN alone (Hoch-Ligeti et al., 1968).

        1-Isothiocyanatonaphthalene (1-napthyl-isothiocyanate) and
    3-methyl-cholanthrene failed to inhibit the hepatocarcinogenic
    effects of DEN (Makiura et al., 1973). Similarly, local treatment of
    the glandular mucosa of the stomach of rats with 4-nitroquinoline-
    1-oxide or  p-dimethylaminoazobenzene did not have any effect on
    the production of liver or oesophagal tumours due to oral
    administration of DEN (Odashima, 1969). It has been reported that
    ionizing radiation did not increase tumour incidence in animals
    exposed to nitrosamines (Flaks et al., 1973; Schmähl et al., 1966).

        The effect of many other substances on nitrosamine carcinogenesis
    has been studied. For example, various dusts including aluminium (III)
    oxide, magnesium (II) oxide, and carbon had little effect on the
    respiratory carcinogenesis of DEN in hamsters (Stenback et al., 1973)
    but other studies on hamsters demonstrated that iron (III) oxide
    markedly increased the incidence of respiratory tract tumours induced
    by DEN (Feron et al., 1972). The synergistic effects of various
    substances on respiratory carcinogenesis in hamsters given  N-nitroso
    compounds were reviewed by Montesano (1970).

        The effect of noncarcinogenic chemicals on the incidence of other
    tumours induced by  N-nitroso compounds has also been studied. In
    rats given DEN, liver tumour incidence was decreased by the
    administration of calcium heparin (Platt & Hering, 1973),
    aminoacetonitrile (Hadjiolov, 1971), and reserpine (Lacassagne et al.,
    1968). Lactoflavin, nicotinamide, or 2,6-bis-(diethanolamino)-4,8-
    dipiperidino-pyrimido [5,4-d] pyrimidine (dipyridamole) (Schmähl &
    Stackelberg, 1968) and hydrocortisone (Schmähl et al., 1971) did not
    influence the incidence of liver tumours induced in rats by DEN.
    Tryptophan was shown to inhibit the production of liver tumours, but
    not bladder tumours in rats exposed to  N-nitrosodibutylamine
    (Okajima et al., 1971).  N-(2-chloroethyl)- N (phenylmethyl)
    benzenemethanamine (dibenamine) did not have any effect on the
    incidence of oral, pharyngeal, or oesophageal tumours in DEN-treated
    rats, but did significantly reduce the number and severity of hepatic
    neoplasms (Weisburger et al., 1974). In mice, treatment with
    phenobarbital decreased the toxicity and carcinogenicity of DEN (Kunz
    et al., 1969).

    7.2.2.10  Miscellaneous modifying factors

        Nasal infection of mice with the influenza viruses PR8/FIK and
    A2/Bethesda 10/63 followed by treatment with DEN significantly
    increased lung tumour incidence in comparison with that in mice
    treated only with DEN (Schmidt-Ruppin & Papadoupulu, 1972). The Motol
    virus has been shown to increase hepatic carcinoma in mice given DEN
    (Kordac et al., 1969). Rats with chronic respiratory disease showed an
    increased lung tumour incidence when given nitrosoheptamethylenemine
    compared with germ-free or specific pathogen-free animals (Schreiber
    et al., 1972).

        Feeding with a protein-deficient diet protected rats against the
    lethal and hepatotoxic effects of DMN (McLean & Verschuuren, 1969) but
    increased the incidence of renal carcinomas (McLean & Magee, 1970).
    Low protein diets and low zinc intake failed to influence the
    incidence of oesophageal tumours in rats treated with  N-methyl- N-
    nitroso-'pentyl' amine (Van Rensburg, 1972). A single intraperitoneal
    injection of DMA induced nasal tumours in rats, previously starved for
    48 h (Noronha & Goodall, 1972).

        The carcinogenic activity of nitroso- N-(hydroxybutyl)
    butanamine (butyl-( N-hydroxybutyl) nitrosamine) appears to be
    influenced by sex hormones (Bertram & Craig, 1972). Tumours developed
    much earlier in male, than in female mice. This difference was
    abolished, however, if the males were castrated or, conversely, if the
    females were treated with testosterone.

        Surgical manipulation of experimental animals may influence the
    induction of tumours by  N-nitroso compounds. This has been shown for
    partial hepatectomy (Craddock, 1971; Grunthal et al., 1970; Rabes et
    al., 1971) unilateral nephrectomy (Ito et al., 1969) and ureter
    ligation (Ito et al., 1971).

    7.2.3  Embryotoxicity and teratogenicity

        Whereas nitrosamines are reported to have toxic and lethal
    effects on the embryo, usually at dose levels that are toxic to the
    mother animals, nitrosamides ( N-ethyl-and N'methyl urea) bring about
    malformations of several of the organs and systems of the developing
    organisms at levels not toxic to the pregnant animal. Animal
    experiments have shown that immature tissues are especially sensitive
    to these compounds. Dose-response relationships have been established
    and the existence of a noneffect level has been indicated.

        DMN, administered orally at 30 mg/kg to pregnant rats, produced
    increased prenatal mortality (Alexandrov, 1967). Toxic effects on the
    embryo were noted following intravenous or intraperitoneal
    administration of DMN at various times during gestation but no

    teratological abnormalities were observed. Similar results were
    observed with DEN and with nitroso- N,N-bis butanamine (nitrosodi-
     N-butylamine) in rats (Alexandrov 1967), and with DEN in hamsters
    (Pielsticker, 1967). The aromatic nitroso compounds such as
    nitrosomethylbenzenamine (nitrosomethylaniline) were toxic to the
    embryo and had a mild teratogenic action when given to rats at the
    maximum tolerated doses (Alexandrov, 1968a, 1968b).

        Treatment of pregnant rats with a single dose of 10-30mg of
    nitrosomethylurea per kg body weight on day 9 of gestation produced
    anophthalmia, hydrocephaly, exencephaly, and occasionally spina bifida
    while treatment on days 12-15 produced microcephaly (Alexandrov,
    1969a; Koyama et al., 1970; Napaikov, 1971; Von Kreybig, 1965a, 1965b;
    Von Kreybig & Schmidt, 1966, 1967).

        The teratogenic action of nitrosoethylurea in rats was described
    by Druckrey et al. (1966) and Ivankovic & Druckrey (1968) who noted
    that oligodactylia and syndactylia of the fore and hind limbs were
    dose-related. Von Kreybig & Schmidt (1967) and von Kreybig (1968)
    confirmed these effects and also found brain anomalies. Napalkov
    (1971) and Wechsler (1970, 1971) reported similar results. The
    teratogenic and embryotoxic effects of  N-nitroso compounds including
    the pathogenesis of brain lesions were reviewed by Wechsler (1973).

    7.2.4  Mutagenicity

        The mutagenicity of  N-nitroso compounds was reviewed by Magee &
    Barnes (1967) and more recently by Montesano & Bartch (1976). Until
    recently, the data available were mainly concerned with the
    nitrosamides, the data on nitrosamines being limited to test systems
    which do not take into consideration the metabolic activation of these
    compounds by mammalian enzymes. As with other biological effects,
    there is a clear distinction between the mutagenic action of
    nitrosamides and nitrosamines. Nitrosamides were found to be mutagenic
    to almost all genetic indicators; this was attributed to the
    nonenzymatic formation of alkylating reactants.

        On the other hand, nitrosamines have been reported, prior to
    1970, to have a much more limited range of mutagenic activity; they
    were found to be mutagenic in tests with  Drosophila melanogaster
    (Pasternak, 1964) but no such activity was observed in assays in
    which bacteria, yeast, or fungi were used. Malling (1966) showed that
    DMN and DEN were mutagenic for  Neurospora crassa when the conidia
    were suspended in Udenfriend's hydroxylating model system and the
    treatment carried out under conditions believed to give rise to the
    same metabolic products as those formed by the action of liver enzymes


    in rat and mouse. Garbidge & Legator (1969), using the host-mediated
    assay, were the first to show that DMN, administered to mice, induced
    mutations in  Salmonella O,phimurium, which had been injected
    beforehand and was then reisolated from the peritoneal cavity. Most of
    the mutagenicity assays were carried out using bacteria or fungi as
    genetic indicators in the presence or absence of a microsomal
    activation system. Only a few data are available on the mutagenicity
    of DMN in mammalian systems such as the dominant lethal test or
    cytogenetic studies. However, in general, the last two systems have a
    very low sensitivity and false negatives can easily result with
     N-nitroso compounds. The early observation of Pasternak (1962)
    that DMN induced lethal mutations in  Drosophila melanogaster
    demonstrated the possible value of this system for testing the
    mutagenicity of compounds that require metabolic activation.

        The mutagenicity of certain nitrosated pesticides, herbicides,
    and primary amines has been examined. The  N-nitrosated derivatives
    of the pesticides propoxur and carbaryl, which are aryl- N-methyl
    carbamates, and of the herbicide benzothiazuron, a methylurea
    derivative, induced mitotic gene conversion in  Saccharomyces
     cerevisiae (Sibert & Eisenbrand, 1974).  N-nitrosocarbaryl was
    also found to be mutagenic in  Haemophilus infiuenzae (Elespuru
    et al., 1974). Endo et al. (1973) screened a number of
     N-nitrosated guanidine derivatives for their ability to induce
    base pair substitutions in  Salmonella typhimurium and a mutagenic
    effect was observed for many of these compounds; nitrosated
    methylguanidine was the most potent.

        The mutagenicity of sodium nitrite, DMA, methylurea, and
    ethylurea given orally to mice, has been demonstrated in a host-
    mediated assay using a strain of  Salmonella typhimurium as an
    indicator (Couch & Friedman, 1975). When combined with sodium nitrite,
    both ethylurea and methylurea had a greater effect than DMA.

        Other properties of  N-nitroso compounds such as cell
    transformation  in vitro and their influence on DNA repair mechanisms
    are being investigated, but as yet, there are not sufficient data for
    their evaluation.

    8.  EFFECTS OF NITRATES, NITRITES AND N-NITROSO COMPOUNDS ON MAN

    8.1  Nitrates and Nitrites

        In the erythrocytes of healthy individuals, the process of
    methaemoglobin formation and reduction is continuous. The mean content
    of methaemoglobin in healthy populations is usually reported to be
    below 2% of the total haemoglobin concentration (Committee on Nitrate
    Accumulation, 1972; Gobbi et al., 1974; Smith, 1972). However,
    Goldsmith et al. (1975) recently found mean levels in Californian
    populations ranging up to 2.11% with 1% of the adults and 8% of
    infants having methaemoglobin levels exceeding 4%. Higher values are
    found in premature than in full-term infants and levels in infants are
    higher than those in older children and adults (Kravitz et al., 1956).
    At a level of about 10%, methaemoglobinaemia may produce symptomless
    cyanosis, whereas levels of 20-50% are associated with conspicuous
    cyanosis accompanied by hypoxic signs and symptoms, such as weakness,
    exertional dyspnoea, headaches, tachycardia, and loss of consciousness
    (Arena, 1970; Committee of Nitrate Accumulation, 1972; Jaffé & Heller,
    1964). The lethal concentration of methaemoglobin is not known, but
    death may occur at levels exceeding 50% (Committee of Nitrate
    Accumulation, 1972).

    8.1.1  Epidemiologicai studies

        The National Academy of Sciences, USA (Committee on Nitrate
    Accumulation, 1972) recently reviewed about 350 cases of
    methaemoglobinaemia in the USA and about 1000 cases in Europe that
    were reported to be associated with the intake of nitrates in well
    water or in food. There were 41 fatalities in the USA and about 80 in
    Europe. Only one case of infant methaemoglobinaemia resulting from the
    consumption of water from a municipal water supply was reported in the
    USA (Vigil et al., 1965).

    8.1.1.1  Exposure through water

        The toxicity to man of nitrates in water was first reported by
    Comly (1945). He noted high levels of methaemoglobin and the
    associated signs of nitrate toxicity in 2 infants who had consumed
    water containing high concentrations of nitrates (619 and
    388 mg/litre). Since then, several epidemiological and case studies
    have been carried out in various parts of the world, particularly in
    areas with naturally high nitrate levels in water.

        Robertson & Riddell (1949) reported 10 cases in which infants
    receiving powdered milk preparations made with well waters with
    nitrate concentrations exceeding 75 mg/litre had blood levels of
    methaemoglobin ranging from 5 to 50% of total haemoglobin. Two of

    the infants, whose dried milk preparations had been reconstituted
    with well waters containing nitrate levels of 1200-1300mg/litre, had
    methaemoglobin levels of 25% and 44% of total haemoglobin,
    respectively; both cyanosed rapidly and died before therapy could be
    applied.

        In the state of Minnesota (USA), Bosch et al. (1950) reported 139
    cases of infant methaemoglobinaemia due to the ingestion of well water
    with a high nitrate content (over 89 mg/litre); the mortality rate was
    10%. In Kansas, 13 cases of infant methaemoglobinaemia including 3
    deaths caused by drinking well water, were reported between the early
    1940s and 1950 (Walton, 1951). Many other cases in the USA have been
    reported by this author. On the other hand, infant methaemoglobinaemia
    was absent in urban areas of New York and the province of Ontario,
    Canada where nitrate levels in water were low (Ciavaglia & Thompson,
    1969). Knotek & Schmidt (1964) reported 115 cases of methaemo-
    globinaemia from a total of 5800 children born in central
    Czechoslovakia between 1953 and 1960. Of these, 8% were fatal, 52%
    were severe, and 40% were mild. Most deaths were associated with the
    consumption of water containing nitrate concentrations ranging from
    70 to 250 mg/litre. In these cases, methaemoglobinaemia was
    invariably associated with the consumption of infant milk
    preparations, which contained microorganisms such as  B. subtills,
    capable of reducing nitrates to nitrites. These workers reported the
    inhibitory effect of buttermilk on this conversion. They attributed
    this effect to the presence of  Streptococcus lactis which
    produces the antibiotic nisin and is capable of preventing the growth
    of the spores of  B. subtilis.

        Shuval & Gruener (1972) studied communities with various
    concentrations of nitrates in the drinking water, in Israel. Infants
    from rural communities with reported nitrate levels of 50 --
    90 mg/litre in the water supplies were compared with controls where
    the average level was 5 mg/litre. They did not find any definite cases
    of methaemoglobinaemia nor were there any significant differences in
    the methaemoglobin levels. However, there was widespread consumption
    of citrus juices and the water consumption in infant milk preparations
    was low (94% of the infants studied were breast fed or received whole
    cow's milk). Soviet literature contains information concerning a
    comparatively small number of cases of symptomless methaemoglobinaemia
    caused by nitrates in water (Diskalenko, 1969; Motylev, 1969). In
    children whose drinking water contained a high level of nitrates, the
    methaemoglobin level did not usually exceed 10% although higher levels
    were found occasionally. Sattelmacher (1962) and Simon et al. (1964)
    compiled 1060 and 745 cases, respectively, of infant
    methaemoglobinaemia due to nitrate-contaminated water in the Federal
    Republic of Germany. Most of the cases were associated with water
    from private wells and in 84-90% of the cases the water contained

    nitrate concentrations exceeding 100 mg/litre (although a few cases
    of methaemoglobinaemia were reported with water containing less than
    50 mg/litre).

        Commoner et al. (1972) found small, but statistically
    significant, subclinical elevations in methaemoglobin levels in adults
    exposed to high intakes of nitrates in a rural area when compared with
    an urban control population. There is also a small amount of data
    showing that under similar conditions, pregnant women in rural areas
    have higher methaemoglobin levels than those in urban areas. This is
    of particular interest since previous workers have reported an
    increased susceptibility to nitrates in pregnant women. (Skrivan,
    1971). Thus, there is concern over the effects on the fetus of the
    general lowering of oxygen tension. Gelperin et al. (1971) recently
    reported the presence of methaemoglobinaemia in a newborn infant,
    presumably exposed to nitrates transplacentally. In this study, 72
    mothers and infants tested were exposed to water with nitrate
    concentrations ranging from 28 to 45 mg/litre over a 2-month period.
    During the 2 weeks of maximum concentration (45 mg/litre), the average
    methaemoglobin levels were 1.18% for mothers and 1.91% for newborns
    with those of one mother and one infant rising to 6.39% and 5.87%,
    respectively.

        Children aged 12-14 years who drank water with a nitrate level of
    105 mg/litre were noted to have slightly delayed reactions to light
    and sound stimuli combined with a mean methaemoglobin level of 5.3% in
    comparison with control children drinking water with a nitrate level
    of 8 mg/litre whose methaemoglobin levels average 0.75% (Petukhov &
    Ivanov, 1970).

    8.1.1.2  Exposure through vegetables

        Cases of methaemoglobinaemia, some resulting in death, have been
    observed following the consumption of spinach. Hölscher & Natzschka
    (1964) reported 2 cases in young infants (aged 2 and 3.5 months) who
    had eaten spinach purée. Fresh spinach from the same source contained
    only traces of nitrates but had nitrite ion levels of 2180 mg/kg.
    Fourteen further cases of methaemoglobinaemia in infants (aged 2-10
    months) were reported from the Federal Republic of Germany (Sinios &
    Wodsak, 1965). Unprocessed spinach was used almost exclusively in the
    preparation of the infants' meals and preparation took place at least
    24 h before the mealtime. Since in most cases some of the same spinach
    had been eaten 24 and 48 h before without causing illness, the authors
    assumed that the nitrites were formed within the final 24 h of
    storage. Information on 7 cases showed that the mother tasted the
    spinach before feeding the child and no change in taste was apparent.
    None of the children refused the meal which caused the poisoning.
    Conversion of nitrates to nitrites in fresh spinach was demonstrated
    by Schuphan (1965) (section 4.2.1). Keating et al. (1973) reported a
    case of methaemoglobinaemia in an infant given carrot juice. Other
    cases of food-induced methaemoglobinaemia have recently been reviewed
    by Luhrs (1973).

    8.1.1.3  High accidental exposures through food

        Certain meat products contain nitrates and nitrites. Normally, no
    adverse effects result from consumption of such products. However, in
    1955, an outbreak of 10 cases of methaemoglobinaemia in children
    occurred in New Orleans, USA, attributed to the consumption of large
    amounts of nitrites in sausage meats (Orgeron et al., 1957). Further
    studies revealed that the meats had nitrite concentrations of more
    than 200 mg/kg and that some had levels as high as 6570 mg/kg. Singley
    (1962) reported 3 cases of methaemoglobinaemia resulting from the
    consumption of fish, that had been adulterated with sodium nitrite.
    One patient died and it was assessed that he had consumed
    approximately 33 mg sodium nitrite/kg body weight. Other cases of
    poisoning involving the consumption of nitrite-treated sausage and
    frankfurters were reported by Bakshi et al. (1967) and Henderson &
    Raskin (1972).

    8.1.1.4  Ambient air exposures

        Effects on man of nitrate aerosols in ambient air were not
    considered by the Task Group. However, because of the recent concern
    with the role of nitrates in urban air pollution, it may be of
    interest to briefly summarize the current information on this problem.
    Airborne nitrates may act as respiratory irritants (Knelson & Lee,
    1977) and a recent study conducted by the US Environmental Protection
    Agency in the New York-New Jersey metropolitan area showed that
    increased asthmatic attacks were significantly associated with
    elevated levels of suspended nitrates in six of the seven communities
    studied. No such effect was observed with nitrogen dioxide. Another
    study in two south-eastern communities in the USA showed some evidence
    that a combination of suspended nitrates and suspended sulfates
    increased the risk of asthma attacks more than either pollutant did
    alone. Because of the present difficulties in measuring suspended
    nitrates, these results should be considered as qualitative instead of
    quantitative (French et al., unpublished data)a.

    8.1.2  Factors involved in susceptibility to nitrates

        The work of Marriott et al. (1933), who investigated the acidity
    and bacterial flora of 200 infants suffering from diarrhoea, supports
    the hypothesis that lack of acidity in the gastric juices of newborn
    infants might permit the growth of nitrate-reducing organisms in the
    upper gastrointestinal tract and, thus, the reduction of nitrates to
    nitrites before the former could be completely absorbed. The pH of the

              

    a  French, J. G., Hasselblad, V., & Johnson, R. Aggravation of
       asthma by air pollutants. 1971-72 Southeastern CHESS studies.

    stomach contents of healthy infants varied from 2.0 to 5.0 (average
    3.7) and that for infants suffering from bacillary dysentery ranged
    from 2.0 to 5.0 (average 3.0). However, for infants with nonspecified
    diarrhoea the pH varied from 4.6 to 6.5 (average 5.6).

        According to estimates made by Burden (1961), the water intake of
    young infants is nearly 10 times higher than that of adults on a per
    unit body weight basis.

        The susceptibility of infants to methaemoglobinaemia during the
    first six months of life, and especially during the first trimester
    (Bailey, 1966; Kübler, 1965) can be explained by various mechanisms,
    none of which is fully understood at present. It has been reported
    that fetal haemoglobin, which in newborn infants makes up to 60-80% of
    the total haemoglobin decreasing to about 20-30% in 3 months (British
    Medical Journal, 1966; Kübler, 1965), is more readily oxidized to
    methaemoglobin than adult haemoglobin (Betke, 1953; Künzer &
    Schneider, 1953). This might explain why premature infants, who
    frequently have a higher percentage of fetal haemoglobin than full-
    term infants are more susceptible to methaemoglobinaemia. Keohane &
    Metcalfe (1960) reported that the sensitivity of erythrocytes to
    oxidation to methaemoglobin on exposure to nitrites gradually declined
    during childhood until the age of puberty, after which it decreased
    rapidly. This decline in sensitivity was not related to the
    disappearance of fetal haemoglobin and the authors suggested that some
    other factor might be responsible. The susceptibility of newborn and
    young infants to develop methaemoglobinaemia could also be attributed
    to the incomplete development of the NADH methaemoglobin reductase
    system. Several studies have shown that the erythrocytes of newborn
    and young infants have a lower capacity to reduce methaemoglobin than
    those of older children and adults and that the erythrocytes of
    premature newborns have a lower reduction capacity than those of full-
    term infants (Bartos et al., 1966; Ross, 1963; Ross & Desforges,
    1959). Except in rare cases of hereditary enzyme deficiency (Balsamo
    et al., 1964), this deficiency in the NADH methaemoglobin reductase
    system seems to disappear after the first 3-4 months of life (Bartos
    et al., 1966: Künzer & Schneider, 1953).

        The haemoglobin of pregnant women and that of patients suffering
    from carcinomata has also been reported to be sensitive to oxidation
    to methaemoglobin. However, this sensitivity disappeared in the first
    instance after delivery and in the second, after radical extirpation
    of the neoplasms (Metcalf, 1961).

    8.1.3  Dose-response relationships for nitrates and nitrites

        Estimates of the effective exposure of the general population to
    nitrates and nitrites have been discussed briefly in section 5.1.4.
    However, this information is not sufficient to relate environmental
    concentrations to actual intake of nitrates or nitrites in cases of
    observed methaemoglobinaemia. Retrospective epidemiological studies
    discussed in section 8.1.1 provide little information on intake

    because in most cases either the data concerning concentrations in
    water or food were not reliable enough, or the amounts of water or
    food consumed had not been measured. However, a study reported by
    Winton et al. (1971) considered the nitrate intake from water in some
    detail. The study was conducted in southern California and central
    Illinois, USA, and involved 111 infants whose ages ranged from less
    than two weeks to six months. The mother of each infant was asked
    about the fluid intake of the infant in the previous 24 h, the method
    of formula preparation, and the possible inclusion of any other source
    of nitrate in the diet (e.g. vegetables) or administration of
    methaemoglobin-forming medicines. The variables that determined the
    nitrate dose were the daily fluid intake per body weight (which might
    increase in hot and arid climates, or with fever), the fraction of the
    total daily fluid intake taken in the form of water, and the
    concentration of nitrates in the water. Using this method, it was
    possible to estimate that a daily dose of 10 mg/kg body weight could
    be obtained from water containing a nitrate concentration of
    50 mg/litre. The daily water intake varied from 10% of the total daily
    fluid intake for breast-fed infants or for those receiving ready-to-
    feed preparations to 90% for infants who were fed preparations made
    with powder. This shows that no generalization is possible, at
    present, about the relationship between the nitrate concentration in
    drinking water and the dose of nitrate, and that estimates of nitrate
    dose from nitrate concentrations in water or food would have to be
    made for individual cases, taking into account local conditions and
    dietary habits.

        The relationship between the doses of nitrate and methaemoglobin
    levels is even more difficult to establish because of large individual
    differences in response, depending on age and many other host
    variables. For example, using the method described in the previous
    paragraph, Winton et al. (1971) found that in a group of 111 infants,
    63 received a nitrate dose of less than 1 mg/kg body weight, 23 were
    exposed to 1-4.9 mg/kg, 20 to 5.0-9.9 mg/kg, and 5 infants to
    10-15.5 mg/kg. However, only 3 infants appeared to have methaemoglobin
    levels above normal (0-2.9%) and they were the youngest of the five
    who had received more than 10 mg/kg. The highest methaemoglobin level
    (5.3%) was found in 30-day-old baby who had received 15.5 mg/kg.
    Another possible approach is to analyse the available retrospective
    epidemiological data. This has been done by the Committee on Nitrate
    Accumulation (1972) and by Diskalenko (1968) who concluded that only
    very crude estimates of correlations between nitrate concentration in
    water and methaemoglobin levels could be obtained, probably because of
    the delay between the analyses of water and blood, difficulties in
    identifying the sources of water consumed, and the heterogeneity of
    the population samples studied, particularly with respect to age. The
    analyses performed by the Committee on Nitrate Accumulation (1972)
    showed, for example, that an increase in the nitrate concentration in
    water from 0-49 mg/litre to 49-98 mg/litre, increased the
    methaemoglobin level, on average, from 1.0% to 1.3% in infants aged
    0-3 months, but did not affect the methaemoglobin level (0.8%) in

    infants aged 3-6 months (Simon et al., 1964). Methaemoglobin and
    haemoglobin levels, measured in 96 infants from 22 localities in
    Rheinhessen, Federal Republic of Germany, during official maternal
    counselling, were correlated with the nitrate concentrations of the
    drinking water in the localities concerned by Würkert (1974,
    unpublished data)a. Nitrate concentrations of 0-5 mg/litre,
    31-50 mg/litre, and over 100 mg/litre corresponded to methaemoglobin
    levels of 1.65%, 2.44%, and 6.59%, respectively. Higher methaemoglobin
    levels were found in the presence of infections and in infants given
    tea to drink or vegetable nutrients.

        The last question pertains to the relation between the level of
    methaemoglobin and clinical signs and symptoms of methaemoglobinaemia.
    Normally, methaemoglobin is present in the blood at a concentration of
    less than 2% of total haemoglobin.

        Subclinical methaemoglobinaemia (less than 10% methaemoglobin)
    has not been considered to be of direct health significance, but
    Petukhov & Ivanov (1970) reported behavioural effects at these levels.

        Clinical signs of methaemoglobinaemia such as cyanosis become
    apparent at a methaemoglobin level of about 10%. Hypoxic signs and
    symptoms may develop at levels exceeding 20% and death may occur at
    levels of 50% or more.

        In conclusion, the available information does not permit the
    establishment of a quantitative dose-response relationship for human
    exposure to nitrates in water or food.

    8.2  N-nitroso Compounds

        Freund (1937) first described acute intoxication by DMN. Barnes &
    Magee (1954) described 2 cases of industrial intoxication due to DMN
    in which one individual had hepatic cirrhosis at death; the other
    survived but, 6 months later, was shown to have a hard liver suspected
    of being cirrhotic. Watrous (1947) and Wrigley (1948) reported cases
    of accidental exposure to  N-nitroso-methylurethane. Reddening of the
    conjunctiva and erythema of the face and feet developed quickly, and a
    respiratory disorder developed later.

        No reports are available concerning carcinogenesis in industrial
    or other workers exposed to  N-nitroso compounds, nor have
    relationships been established, from epidemiological and analytical
    data, that link cancer in man with exposure to  N-nitroso compounds or
    their possible precursors such as nitrates, nitrites, and compounds

              

    a  Thesis reported in the contributed of the Federal Republic of
       Germany to the WHO environmental health critera document on
       Nitrates, nitrites and  N-nitroso compounds.


    that can be nitrosated, occurring as food components, drugs, and
    pesticides. A recent review of these data is available (Mirvish,
    1976).

        Some reports have been concerned with the possible etiological
    role of  N-nitroso compounds in nasopharyngeal cancer in south-east
    Asia (Clifford, 1970; Fong & Chan, 1973a) and oesophageal cancer in
    South Africa, Iran, and China (Burrell et al., 1966; Coordinating
    Group for Research on the Etiology of Esophageal Cancer of North
    China, 1974; Day, 1975; Harmozdian et al., 1975). However, no
    relationship has been established; nitrosamines were detected in food
    from these areas but this was not confirmed by mass spectroscopy
    (Eisenbrand et al., 1976; Purchase et al., 1975).

        The epidemiology of stomach cancer has been discussed from a
    similar point of view by Correa et al. (1975), Endo et al. (1973),
    Haenzel & Correa (1975), Hill et al. (1973), Mirvish (1971), and
    Weisburger & Raineri (1975). It was suggested that nitrosamides might
    be formed in the stomach from amides occurring in the diet, and might
    then act locally on this organ. Relationships have been sought between
    the occurrence of stomach cancer and the nitrate contents of the soil
    or water in Chile, Colombia, and the United Kingdom (Hawksworth et
    al., 1974; Hill et al., 1973; Zaldivar & Wetterstrand, 1975) but none
    was established.

    9.  EVALUATION OF HEALTH RISKS TO MAN FROM EXPOSURE TO NITRATES,
        NITRITES, AND N-NITROSO COMPOUNDS

    9.1  Nitrates and Nitrites

    9.1.1  General considerations

        Man is exposed to nitrates and nitrites mainly through water and
    food. Nitrate concentrations may be particularly high in drinking
    water derived from dug wells. Nitrates in food may occur naturally or
    may be added for various technological or even public health reasons
    (e.g. addition of nitrates and nitrites to certain meat products to
    protect against botulism). Although intake of very large doses of
    nitrates can be fatal to man, such intake is not likely to occur
    through environmental exposure, except in the case of infants and very
    young children who are high risk groups because of their
    susceptibility to nitrates and nitrites. Weekly intakes of nitrates by
    members of the general population are difficult to evaluate but rough
    estimates are available for England and the USA giving values of about
    400-450 mg/week (85-105 mg from water; 210-225 mg from vegetables, and
    about 110 mg from meat products).

        These figures cannot be applied generally and a separate
    estimation of the nitrate intake from food and water should be made
    for each case, especially when the subjects are infants or young
    children (section 8.1.3). Exposure to nitrates can also occur through
    the inhalation of polluted air.

        The assessment of health risks to man (section 9.1.2) has been
    based on epidemiological studies and clinical evidence. The animal
    data discussion in section 7.1, confirm the findings in man that
    methaemoglobinaemia is the main toxic effect of nitrate and nitrite
    ingestion. Methaemoglobinaemia is caused by nitrites, the reduction
    products of nitrates. The reduction usually occurs through microbial
    action either in the environment or in the body. The health risks from
    exposure to nitrates is therefore related not only to their
    concentration in drinking water and food, but also to the presence or
    absence of conditions conducive to their reduction to nitrites. Young
    infants constitute the most vulnerable group for the following
    reasons:

         (1)  Lower acidity in their stomach allows the growth of certain
              microbes that contain enzymes capable of reducing nitrates
              to nitrites;

         (2)  Fetal haemoglobin, which constitutes a considerable
              proportion of the haemoglobin of the young infant, and the
              erythrocytes during childhood may be more susceptible to
              conversion to methaemoglobin by the action of nitrites;

         (3)  The enzyme system capable of reducing methaemoglobin to
              haemoglobin is deficient in the young infant; and

         (4)  The fluid intake of the young infant is higher than that of
              the adult in relation to the body weight.

    9.1.2  Assessment of health risks

        Precise dose-response relationships could not be established by
    the Task Group because of the existence of various strongly modifying
    factors and the lack of accurate quantitative data. However, on the
    basis of the available information, the Task Group reached the
    following conclusions:

         (a)  General population -- The prevailing levels of nitrates and
         nitrites in water and food do not seem to have any harmful
         effects in adults and older children, although there are reports
         of susceptible individuals who have been affected by meat treated
         with nitrites, and of cases of poisoning resulting from the
         ingestion of certain foods accidently containing excessive
         amounts of nitrites (8.1.1.3). Subclinical methaemoglobinaemia
         may also be found in individuals consuming water containing high
         levels of nitrates (8.1.3).

         (b)  Susceptible group -- Infants less than 6 months old and
         especially those under 3 months of age are particularly
         susceptible to methaemoglobinaemia caused by intake of water
         containing elevated levels of nitrates, especially when they are
         fed with preparations made from dried milk of low acidity. While
         a few cases of methaemoglobinaemia have been reported associated
         with water nitrate levels of less than 50 mg/litre, most cases
         occur with nitrate levels of 90 mg/litre or more. Nitrates in
         water may cause death of the infant, but the lowest level that
         may be fatal cannot be estimated at present.

        The ingestion of vegetables (e.g. spinach, carrots) containing
    elevated nitrate and/or nitrite levels may also cause
    methaemoglobinaemia in infants, especially in those aged between 6
    months and 1 year. Storage of vegetables, other than in the frozen or
    canned state, is likely to increase the nitrite level and hence the
    risk.

         (c)  Effects of airborne nitrates -- A few recent studies have
         indicated that airborne nitrates may act as respiratory irritants
         but, at present, adequate quantitative data are not available and
         the Task Group did not consider this exposure in their health
         risk evaluation.

    9.2  N-nitroso Compounds

    9.2.1  General considerations

        Nitrites (and indirectly nitrates) can react with amines and
    amides to form nitrosamines and nitrosamides. The precursors of these
     N-nitroso compounds are widely distributed in various environmental
    media. Information concerning the presence of  N-nitroso compounds
     per se is limited although they have been identified in certain
    foods such as luncheon meats and in air and water samples. The
    conditions under which  N-nitroso compounds can be formed are
    outlined in sections 4.2-4.6.

        More than 80% of over one hundred  N-nitroso compounds tested
    proved to be carcinogenic in animal experiments giving rise to tumours
    in many organs and also producing tumours transplacentally.
     N-nitroso compounds are carcinogenic in a wide range of animal
    species; most are mutagenic in test systems and some have been shown
    to be teratogenic to animals.

        The possible health hazard from  N-nitroso compounds is not
    confined to those present in the environment. Their formation, from a
    variety of precursors in the body of animals, has been demonstrated,
    and this may also occur in man.

    9.2.2  Assessment of health risks

        A dose-response relationship has been shown to exist in different
    species of rodents for some carcinogenic  N-nitroso compounds. As the
    dose is reduced, the tumour incidence decreases and the time for
    tumour induction increases and may exceed the life span of the animals.

        Although there is no clinical or epidemiological evidence, it is
    highly probable that these compounds are also carcinogenic to man.
    However, present limitations concerning available dose-response data
    in animals and their interpretation, and inadequate knowledge of the
    biomechanism of cancer induction preclude a quantitative estimation of
    the carcinogenic risk to man that may be associated with different
    exposures to  N-nitroso compounds.

    9.3  Reduction of Exposure

        The assessments of health risk given in sections 9.1.2
    [(a) and (b)] and 9.2.2 lead to a number of practical conclusions
    concerning the need to reduce the exposure to nitrates, nitrites, and
     N-nitroso compounds.

        As regards the exposure to nitrates and nitrites, the Task Group
    made the following specific recommendations:

        (a) Infant dried milk preparations should be reconstituted only
        with water containing low levels of nitrates. If such water is
        not available, breast feeding or the use of cow's milk should be
        encouraged.

        (b) Only vegetables with a low nitrate content should be used in
        the preparation of baby foods. If vegetables known to contain
        high levels of nitrates are used, appropriate food processing
        precautions should be instituted. Nitrates and nitrites should
        not be added to baby foods.

        (c) The use of nitrates and nitrites in foods as preservatives
        should be reduced to the minimum level that provides protection
        against botulism. This applies particularly to cured and canned
        meats and to fish. The use of nitrates and nitrites on fresh
        meats or fish should be avoided.

        (d) Nitrate levels in public drinking water should comply with,
        or preferably be lower than, the tentative limit of
        45 mg/litre recommended in the International Standards for
        Drinking Water (WHO, 1971).

        With respect to the carcinogenic risk from exposure to
     N-nitroso compounds, it is prudent to assume that any exposure may
    involve some degree of risk, and that exposure should therefore be
    kept as low as practically achievable. This may not be an easy task in
    many instances, since these compounds may occur in the environment in
    concentrations of the order of parts per billion, as a result of a
    variety of natural and technological processes, and, moreover, they
    may be formed  in vivo from nitrates, amines, and amides, which
    are ubiquitous. Obviously the recommendations (a) to (d) will also
    contribute to the reduction of carcinogenic risk related to
     N-nitroso compounds.

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    See Also:
       Toxicological Abbreviations