INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
ENVIRONMENTAL HEALTH CRITERIA 173
Tris(2,3-dibromopropyl) phosphate and
Bis(2,3-dibromopropyl) phosphate.
This report contains the collective views of an international group of
experts and does not necessarily represent the decisions or the stated
policy of the United Nations Environment Programme, the International
Labour Organisation, or the World Health Organization.
First draft prepared by Dr. G.J. van Esch,
Bilthoven, Netherlands
Published under the joint sponsorship of the United Nations
Environment Programme, the International Labour Organisation, and the
World Health Organization
World Health Organization
Geneva, 1995
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WHO Library Cataloguing in Publication Data
Tris(2,3-dibromopropyl) phosphate and Bis(2,3-dibromopropyl)
phosphate.
(Environmental health criteria ; 173)
1.Phosphoric acid esters 2.Environmental exposure
3.Flame retardants I.Series
ISBN 92 4 157173 X (NLM Classification: QP 981.P49)
ISSN 0250-863X
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CONTENTS
ENVIRONMENTAL HEALTH CRITERIA FOR TRIS(2,3-DIBROMOPROPYL)
PHOSPHATE AND BIS(2,3-DIBROMOPROPYL) PHOSPHATE
INTRODUCTION
TRIS(2,3-DIBROMOPROPYL) PHOSPHATE
1. SUMMARY AND EVALUATION; CONCLUSIONS AND RECOMMENDATIONS
1.1. Summary and evaluation
1.1.1. Production and use
1.1.2. Physical and chemical properties
1.1.3. Environmental transport, distribution, and
transformation
1.1.4. Environmental levels and human exposure
1.1.5. Kinetics and metabolism in laboratory animals
and humans
1.1.6. Effects on laboratory mammals and in vitro test
systems
1.1.7. Effects on humans
1.1.8. Effects on other organisms in the laboratory
and field
1.2. Conclusions
1.3. Recommendations
2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL
METHODS
2.1. Identity
2.1.1. Technical product
2.2. Physical and chemical properties
2.3. Analytical methods
2.3.1. General
2.3.2. Urine
3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE
3.1. Natural occurrence
3.2. Anthropogenic sources
3.2.1. Production levels and processes
3.2.2. Uses
3.2.3. Sources of human and environmental exposure
4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION
4.1. Transport and distribution between media
4.2. Transformation
4.2.1. Biodegradation
4.2.2. Abiotic degradation
4.2.3. Bioaccumulation
4.3. Interaction with other physical, chemical, or
biological factors
4.4. Ultimate fate following use
5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
5.1. Environmental levels
5.1.1. Air
5.1.2. Water
5.1.3. Soil
5.1.4. Fish
5.2. General population exposure
5.2.1. Subpopulation at special risk
5.3. Occupational exposure
6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS
6.1. Absorption
6.2. Elimination
6.2.1. Different routes (rat and rabbit)
6.2.2. Dermal exposure (rat and rabbit)
6.2.2.1 TBPP
6.2.2.2 TBPP-treated fibres
6.2.3. Dermal exposure (human)
6.3. Distribution
6.3.1. Rat
6.3.1.1 Oral
6.3.1.2 Intravenous
6.3.2. Dermal (rabbit)
6.4. Metabolic transformation
6.4.1. In vivo studies
6.4.1.1 Oral (rat)
6.4.2. In vitro studies
6.5. Covalent binding to macromolecules
7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS
7.1. Single exposure
7.2. Short-term exposure
7.2.1. Oral exposure (rat)
7.2.1.1 TBPP
7.2.1.2 TBPP-treated fibres
7.2.2. Oral exposure (dog)
7.2.2.1 TBPP
7.2.2.2 TBPP-treated fibres
7.2.3. Dermal exposure
7.2.3.1 Rabbit
7.2.3.2 Dog
7.3. Long-term exposure
7.4. Skin and eye irritation; sensitization
7.4.1. Skin irritation
7.4.2. Eye irritation
7.4.3. Sensitization
7.5. Reproductive toxicity, embryotoxicity, and
teratogenicity
7.5.1. Reproductive toxicity
7.5.2. Teratogenicity
7.6. Mutagenicity and related end-points
7.6.1. DNA damage
7.6.1.1 In vivo
7.6.1.2 In vitro
7.6.2. Mutation assay with Salmonella
typhimurium strains
7.6.3. Mutations by urine of rats treated with TBPP65
7.6.4. Other mutation assays
7.6.5. Chromosomal effects
7.6.6. Cell transformation
7.6.7. Miscellaneous tests
7.6.8. Mechanisms of TBPP genotoxicity
7.7. Carcinogenicity
7.7.1. Oral
7.7.1.1 Mouse
7.7.1.2 Rat
7.7.2. Dermal
7.7.2.1 Mouse
7.8. Special studies
7.8.1. Kidneys
7.9. Factors modifying toxicity; toxicity of metabolites
7.9.1. Toxicity of metabolites
7.9.2. Mutagenicity of metabolites
8. EFFECTS ON HUMANS
8.1. General population exposure
8.2. Occupational exposure
9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD
9.1. Laboratory studies
9.1.1. Microorganisms
9.1.2. Aquatic organisms
9.1.2.1 Invertebrates
9.1.2.2 Vertebrates
9.1.3. Terrestrial organisms
9.1.3.1 Plants
13. PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES
BIS(2,3-DIBROMOPROPYL) PHOSPHATE AND SALTS
A1. SUMMARY AND EVALUATION; CONCLUSIONS AND RECOMMENDATIONS
A2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL METHODS
A2.1 Identity
A2.2 Physical and chemical properties
A2.3 Analytical methods
A3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE
A3.1 Natural occurrence
A3.2 Anthropogenic sources
A3.2.1 Production levels and processes
A3.2.2 Uses
A3.3 Contamination of the environment
A3.4 Environmental transport, distribution,
transformation, and exposure levels
A4. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS
A4.1 Absorption, distribution, elimination,
and biotransformation
A5. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS
A5.1 Single exposure
A5.2 Short-term exposure
A5.3 Long-term exposure
A5.3.1 Mutagenicity and related end-points
A5.3.2 Carcinogenicity
A5.4 Special studies
A5.4.1 Kidneys
A5.5 Effects on humans and other organisms
in the laboratory and field
REFERENCES
RESUME ET EVALUATION, CONCLUSIONS ET RECOMMANDATIONS
RESUMEN
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This publication was made possible by grant number
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WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR TRIS- AND
BIS(2,3-DIBROMOPROPYL)PHOSPHATE
Members
Dr D. Anderson, BIBRA Toxicology International, Carshalton,
United Kingdom
Dr D. Osborn, Institute of Terrestrial Ecology, Monks Wood,
Huntingdon, United Kingdom
Dr E. Soderlund, National Institute of Public Health, Oslo,
Norway (Rapporteur)
Dr B. Jansson, Institute of Applied Environmental Research,
Stockholm University, Solna, Sweden
Dr J. Kielhorn, Fraunhofer Institute for Toxicology and
Aerosol Research, Hannover, Germany
Dr R.D. Kimbrough, Institute for Evaluating Health Risks,
Washington DC, USA (Vice-chairman)
Dr Wai-On Phoon, Department of Occupational Health,
University of Sydney, Sydney, Australia (Chairman)
Dr R. Benson, Drinking Water Branch, US EPA, Denver, USA
Dr J. Sekizawa, National Institute of Health Sciences, Tokyo,
Japan (Rapporteur)
Observers
Dr M.L. Hardy, Toxicology Advisor, Albemarle Corporation,
Baton Rouge, USA
Dr D.L. McAllister, Director, Quality, Environment, Health
and Safety, and Research Support, Great Lakes Chemical
Corporation, West Lafayette, USA
Secretariat
Dr K.W. Jager, International Programme on Chemical Safety,
World Health Organization, Geneva, Switzerland (Secretary)
ENVIRONMENTAL HEALTH CRITERIA FOR TRIS- AND BIS(2,3-DIBROMOPROPYL)
PHOSPHATE
A WHO Task Group on Environmental Health Criteria for tris- and
bis(2,3-dibromopropyl) phosphate met at BIBRA Toxicology
International, Carshalton, United Kingdom, from 6 to 11 June 1994.
Dr K.W. Jager, IPCS, welcomed the participants on behalf of Dr M.
Mercier, Director of the IPCS, and the three IPCS cooperating
organizations (UNEP/ILO/WHO). The Group reviewed and revised the
draft and made an evaluation of the risks for human health and the
environment from exposure to tris- and bis(2,3-dibromopropyl)
phosphate.
The first draft was prepared by Dr G.J. van Esch, the
Netherlands, who also prepared the second draft, incorporating
comments received following circulation of the first drafts to the
IPCS Contact Points for Environmental Health Criteria monographs.
Dr K.W. Jager of the IPCS Central Unit was responsible for the
scientific content of the monograph and Mrs M.O. Head of Oxford for
the technical editing.
The efforts of all who helped in the preparation and finalization
of the monograph are gratefully acknowledged.
INTRODUCTION
The IPCS is preparing several EHC monographs on Flame Retardants,
which will provide additional information relevant to TBPP.
There will be one monograph, "Flame Retardants - A General
Introduction" (in preparation), giving a general introduction to the
use, the mode of action, and the potential risks of flame retardants,
and listing the substances used as flame retardants with a general
indication of the data available on them.
Flame retardants in wide use are discussed in separate
monographs, e.g., EHC 162: Brominated Diphenyl Ethers (IPCS, 1994a)
and EHC 172: Tetrabromobisphenol-A (IPCS, 1995).
Some flame retardants considered hazardous for humans and the
environment have been reviewed in separate monographs including EHC
152: Polybrominated Biphenyls (IPCS, 1994b), and EHC 173: Tris- and
Bis(2,3-dibromopropyl) phosphate (this monograph).
Because of the possibility of the formation of halogenated
dibenzodioxins and dibenzofurans under certain circumstances, such as
pyrolysis, the following monographs have been developed: EHC 88:
Polychlorinated Dibenzodioxins and Dibenzofurans (IPCS, 1989) and
Polybrominated Dibenzodioxins and Dibenzofurans (in preparation).
The reader should consult these monographs for further
information.
Tris(2,3-dibromopropyl) phosphate was an important commercial
flame retardant ("TRIS"), especially for children's sleepwear. In
1977, the US Consumer Product Safety Commission banned children's
clothing treated with tris(2,3-dibromopropyl) phosphate. Since then,
in several other countries, the use of this compound as a flame
retardant has been severely restricted in consumer products and
prohibited in textiles.
Because tris(2,3-dibromopropyl) phosphate can also be used for
other applications, the information available on physical and chemical
properties, behaviour in the environment, occurrence in the
environment and humans, kinetics and metabolism, toxicity for
laboratory animals and in the field, and the exposure of the general
population and workers, is summarized in this Environmental Health
Criteria monograph. General properties and uses of brominated flame
retardants are given in "Flame Retardants - A General Introduction"
(in preparation).
ABBREVIATIONS
BA 2-bromoacrolein
BBPP bis(2,3-dibromopropyl) phosphate
DBCP 1,2-dibromo-3-chloropropane
DBP 2,3-dibromopropanol
DMBA dimethylbenzanthracene
mono-BPP mono(2,3-dibromopropyl) phosphate
TBPP tris(2,3-dibromopropyl) phosphate
TPA tetradecanoyl phorbolacetate
TRIS-(2,3-DIBROMOPROPYL) PHOSPHATE
1. SUMMARY AND EVALUATION; CONCLUSIONS AND RECOMMENDATIONS
1.1 Summary and evaluation
1.1.1 Production and use
Tris(2,3-dibromopropyl) phosphate (TBPP) was first produced in
about 1950; commercial production was reported in 1959. Production of
TBPP, in the USA, in 1975, was estimated to be between 4100 and
5400 tonnes. As far as is known, TBPP is not produced or used
currently in the world as a flame retardant in textiles, but may be
added to polymers used for other purposes. TBPP was an important
flame retardant for cellulose and tri-acetate and polyester fabrics,
especially in children's sleepwear, but has been banned for these
applications in several countries in Europe, the USA (1977), and Japan
(1978).
TBPP exists both in, and on, the fabric. When it is in the
fabric, it is not extractable with solvents and, therefore, probably
not available for dermal absorption. However, when it is on the fibre
surface, it can be extracted during laundering, and by acetic acid,
other solvents, and saliva, and is available for dermal absorption. In
this case, substantial losses of surface TBPP from fabrics during use
and/or laundering of the finished products, will occur, and will
contaminate the environment. Furthermore, release of TBPP into the
environment has been reported from textile-finishing plants and the
ultimate disposal of solid wastes, containing TBPP.
1.1.2 Physical and chemical properties
TBPP is available in at least two grades. The high-purity grade
is a clear, pale-yellow, viscous liquid, with up to 1.5% volatiles.
The low-purity grade may contain up to 10% volatiles.
TBPP (purity > 97%), has a boiling point of 390°C, a melting
point of 5.5°C, and a vapour pressure of 1.9 × 10-4mmHg at 25°C.
The solubility of TBPP in water is low (8 mg/litre).
When heated to decomposition, above 260-300°C, TBPP emits
compounds containing bromine and phosphorus. The n-octanol/water
partition coefficient (log Pow) is 3.02.
Analytical methods to determine TBPP and its metabolites in
biological samples and other matrices are available.
1.1.3 Environmental transport, distribution, and transformation
The limited information available suggests that TBPP is
relatively persistent in the environment. Oxidation and
photodegradation are not likely to be significant fate processes.
However, hydrolysis involving the bromine atoms on the propyl group
may occur, especially under basic conditions. Volatilization from
water may occur, but no actual data are available. Although
biodegradation of TBPP (half-life 19.7 h) in activated sewage is
reported to occur, it is not thought to be an important process in
natural soils and waters. In sterilized sludge, almost no breakdown
takes place. Bis(2,3-dibromopropyl) phosphate (BBPP) was found as a
major breakdown product. Because TBPP is virtually insoluble in
water, adsorption on particulate matter and sediment may be an
important process.
An estimated log Koc (3.29) suggests strong adsorption on soil.
On the basis of this Koc value and the low measured water
solubility, TBPP is expected to leach only slowly to groundwater. TBPP
will tend to accumulate in rubbish dumps and other disposal sites,
which may result in biological accumulation. A bioaccumulation study
with fathead minnow showed a bioconcentration factor of 2.7, which is
low, while the n-octanol/water partition coefficient (Log Pow) was
3.02. Because of its low vapour pressure, TBPP is expected to be
mostly sorbed on particulate matter in air. Thermal oxidative
degradation of TBPP at 370°C showed that hydrogen bromide and
C3-brominated compounds, such as bromopropenes, dibromopropenes, and
diand tribromopropanes, are formed.
1.1.4 Environmental levels and human exposure
Data on environmental levels and human exposure are limited.
Studies carried out in Japan in 1975 showed that 20 samples of water,
soil, and fish did not contain TBPP. TBPP was identified, but not
quantified, in air particulates in the surroundings of an industry.
Children wearing TBPP-treated sleepwear were the group of the
general population particularly exposed to this flame retardant. The
estimated intake via the skin of children wearing TBPPtreated
sleepwear in the USA was calculated to be 9 µg/kg body weight per day.
The Consumer Product Safety Commission of the USA calculated that,
over a 6-year period, a child wearing TBPP-treated clothing could
absorb a total of 2-77 mg TBPP/kg body weight or more.
1.1.5 Kinetics and metabolism in laboratory animals and humans
TBPP is absorbed readily from the gastrointestinal tract and at a
moderate rate via the skin in rats and rabbits. In rats, TBPP or its
metabolites are eliminated within 5 days. Approximately 50% is
eliminated via the urine, 10% via the faeces, and 10-20% is exhaled as
CO2.
One day after oral administration of labelled TBPP to rats,
radioactivity was found in the blood, liver, kidneys, muscles, fat,
and skin, in a range of 0.2-6.6%. The half-life of clearance of
radioactivity from these organs was approximately 2-4 days. After 8
h, only bis(2,3-dibromopropyl) (BBPP) phosphate was still present in
substantial concentrations in most tissues.
After oral administration of TBPP to rats, six metabolites
were identified in the urine and bile. The main metabolite in
the urine, faeces, bile, and tissues was BBPP. The metabolite
2,3-dibromopropanol (DBP) was also identified in rats and in children
wearing TBPP-treated clothing.
Liver microsomes metabolize TBPP in the presence of NADPH and
oxygen. The main metabolites are BBPP and 2,3-dibromopropanol (DBP).
It has been shown that BBPP is formed by oxidation at the C3 and,
possibly, also at the C2 position of TBPP. In addition to BBPP,
2-bromoacrolein, 2-bromoacrylic acid, and propyl-hydroxylated
compounds and metabolites conjugated with glutathione have been found.
S-(2,3-dihydroxypropyl) glutathione was identified in the bile
of rats, and, it was suggested that TBPP and/or BBPP are conjugated
directly with glutathione by glutathione S-transferase with the
formation of episulfonium ion metabolites.
TBPP has been shown to be activated to form products that bind
covalently to proteins and DNA in vivo and in vitro. After
intraperitoneal injections of tritiated-TBPP, male mice, hamsters, and
guinea-pigs, which are less sensitive to TBPP-induced nephrotoxicity
than rats, showed similar levels of covalent binding to proteins in
the liver and kidneys. In the male rat, which is highly susceptible to
TBPP-induced nephrotoxicity, much higher amounts of radiolabel were
bound to kidney proteins than to liver proteins.
Liver microsomes from mice, guinea-pigs, hamsters, and humans all
metabolized TBPP to genotoxic intermediates. However, the rate of
formation of reactive TBPP metabolites with human liver microsomes was
lower than with liver microsomes from the rodents.
The binding of labelled TBPP and analogues in rats at a
nephrotoxic dose showed that the covalent protein binding was highest
in the kidneys followed by the liver and testes. The results of
comparative in vitro and in vivo studies on renal DNA damage
suggested that BBPP is formed in the liver by P450-mediated oxidation
at either C2 or C3 of TBPP. BBPP is transported to the kidneys, where
it is metabolized to reactive intermediates that cause DNA damage and
bind to kidney proteins. The activation occurring in the kidney
appears not to involve P450 but seems to be mediated by GSH-dependent
metabolism. In vitro studies with labelled TBPP and analogues
showed that oxidation of TBPP incorporates one atom of oxygen from
water. This implies that oxidation at C2 of the propyl moiety yields
a reactive alphabromoketone that can alkylate protein directly or
hydrolyse to BBPP and a reactive bromo-alpha-hydroxyketone.
1.1.6 Effects on laboratory mammals and in vitro test systems
The acute and short-term oral, and the acute dermal, toxicities
of TBPP are low. The oral LD50 for the rat > 2 g/kg and the dermal
LD50 for the rabbit > 8 g/kg body weight. Extensive kidney damage
(necrosis of renal proximal tubular cells) was noted in male rats
following a single ip injection of 100 mg TBPP/kg body weight.
Four-week, and 90-day, oral toxicity tests with TBPP (by gavage
or in the diet) in rats showed a dose-related increase in the
incidence and severity of chronic nephritis at dose levels of 25 mg/kg
body weight or more.
In rabbits, daily dermal applications of 2.2 g TBPP/kg body
weight or more, for 4 weeks, resulted in degenerative changes in the
liver and kidneys. All rabbits died within four weeks. No deaths
occurred in another study with dose levels of up to 250 mg/kg body
weight.
In a 90-day test on rabbits, weekly application of 2.27 g/kg body
weight to the skin resulted in kidney changes, testicular atrophy, and
aspermatogenesis.
No skin or eye irritation was observed in rabbits with dose
levels of 1.1 g or 0.22 g TBPP and no skin sensitization was observed
in guinea-pigs.
Two teratogenicity studies were carried out on rats. In one
study with dose levels of up to 125 mg/kg body weight, no
teratogenicity was observed. In another study with a dose level of
200 mg/kg body weight, a significant increase in skeletal variations
in the fetuses was observed, and, with 50 and 100 mg/kg body weight, a
significantly lower viability index was found. The authors concluded
that the observed effect resulted from maternal toxicity.
Extensive DNA damage was found in various organs of rats
administered TBPP. In vitro, TBPP has been shown to induce DNA
strand breaks in human KB cells. It induced unscheduled DNA synthesis
in rat liver hepatocytes, but not in human foreskin epithelial cells.
TBPP was mutagenic in several studies on Salmonella typhimurium,
especially in base-pair substituting strains with, and without,
metabolic activation.
Forward gene mutation assays using Chinese hamster V79 cells,
with, and without, metabolic activation were negative. However, a
positive effect in the presence of liver microsomes of rats pretreated
with phenobarbital was obtained. A weak positive effect was obtained
with mouse lymphoma cells (L5178YTK locus).
TBPP increased the number of sister chromatid exchanges (SCEs) in
Chinese hamster V 79 cells, but no chromosomal aberrations were
induced in Chinese hamster cells, mouse bone marrow cells, or in
cultured human lymphoid cells. SCEs but no chromosomal aberrations
were found with diploid human fibroblastic cells (line HE 2144)
without metabolic activation. However, in an in vitro chromosome
aberration test with the Chinese hamster cell line (CHL), TBPP was
positive.
A positive result was obtained with TBPP in a micronucleus test
on Chinese hamster bone marrow cells. Another micronucleus study with
mice showed a weak positive effect.
Studies with Drosophila melanogaster showed that TBPP increased
sex-linked recessive lethals in male germ cells and in adult males,
reciprocal translocations were induced. TBPP showed a strong positive
response in the w/w+ eye mosaic assay.
Several studies have been directed towards the elucidation of the
mechanisms involved in TBPP-induced mutagenicity and/or genotoxicity.
Bacterial mutagenicity of TBPP is mediated by the microsomal
monooxygenase system. TBPP is activated by cytochrome P450 in a
reaction depending on NADPH and oxygen. Microsomes prepared from
livers of animals treated with phenobarbital or PCBs give increased
mutagenicity. The mono-and bis(2,3-dibromopropyl) phosphates are less
mutagenic than TBPP. In vitro studies have shown that oxidation at
C3 of the TBPP molecule yields the potent direct acting mutagen
2bromoacrolein that also binds to DNA.
Species differences in the bioactivation of TBPP to metabolites
mutagenic to Salmonella typhimurium TA 100 have been reported. Liver
microsomes from mice were more effective than those from guinea-pigs,
hamsters, and rats.
Three studies in which C3H/10T1/2 cells were used to study cell
transformation were carried out. In one study, a positive effect was
noted, but, in the other two studies, the results were negative.
TBPP was tested on mice and rats by oral administration and on
female mice by skin application in long-term studies. In mice,
following oral administration, TBPP produced tumours of the
fore-stomach and lung in the animals of both sexes, benign and
malignant liver tumours in females, and benign and malignant tumours
of the kidneys in males. In rats, TBPP produced benign and malignant
tumours of the kidneys in males and benign kidney tumours in females.
After skin application to female mice, TBPP produced tumours of the
skin, lung, fore-stomach, and oral cavity. From these studies, it can
be concluded that TBPP has carcinogenic potential in mice and rats.
When the TBPP metabolite BBPP was administered to rats orally, it
caused tumours in both sexes in the digestive system. The tumours
found included papillomas and adenocarcinomas of the tongue,
oesophagus, and forestomach, adenocarcinomas of the intestine, and
hepatocellular adenomas and carcinomas.
Another metabolite of TBPP, DBP, was tested on rats and mice by
dermal application. In male rats, there was an increased incidence of
neoplasms in skin, nose, oral mucosa, oesophagus, forestomach, small
and large intestine, Zymbal's gland, liver, kidney, tunica vaginalis,
and spleen. In female rats, there was an increased incidence of
neoplasms of the skin, nose, oral mucosa, oesophagus, forestomach,
small and large intestine, Zymbal's gland, liver, kidney, clitoral
gland, and mammary gland. In male mice, there was an increased
incidence of neoplasms in the skin, forestomach, liver, and lung, and
in female mice, there was an increased incidence of neoplasms of the
skin and the forestomach.
1.1.7 Effects on humans
Limited data are available regarding the effects of TBPP on
humans.
TBPP has been tested for skin sensitization potential in a few
studies on humans. The results of these studies indicate that TBPP
has a low sensitization potential and no skin irritation was reported.
However, persons who showed a positive sensitization response to pure
TBPP also reacted when exposed to fabrics
treated with TBPP.
1.1.8 Effects on other organisms in the laboratory and field
There are very few data on the effects of TBPP on other
organisms. All 6 goldfish (Carassius auratus), exposed to 1 mg
TBPP/litre, died within 5 days.
The EC50 for growth inhibition in oat seed was 1000 mg/kg soil.
This concentration caused a 100% inhibition of growth in turnip seed
(Brassica rapa sp.).
1.2 Conclusions
TBPP has been used as a flame retardant in fabrics, particularly
in children's sleepwear, but there is inadequate information on its
use in other applications. Exposure of the general population was
primarily through contact with fabrics treated with TBPP.
There is little information on the exposure of, and hazards to,
workers from the commercial production of TBPP and its use in a
variety of products.
Because of the paucity of data, no firm conclusions can be drawn
as to the exposure levels and hazards of TBPP for organisms in the
environment, other than humans.
Animal studies have shown that TBPP can be absorbed from the
gastrointestinal tract and, to a lesser extent, from the skin. TBPP
can also be absorbed through the skin of humans. In the rat, TBPP
appears to be extensively metabolized in the liver to BBPP, which is
the major metabolite detected in the urine and, to a lesser extent, to
DBP. In addition, other brominated metabolites of TBPP have been
found in small amounts. DBP has also been detected in humans wearing
TBPP-treated fabrics. The main route of elimination is the urine and
very little is excreted as the parent compound. The main metabolic
pathway seems to be through metabolism by cytochrome P450 and
glutathione S-transferases.
From the available data, it can be concluded that TBPP has a low
acute toxicity for experimental animals. Repeated dose studies with
relatively high doses of TBPP have revealed kidney and liver damage in
rats and also testicular toxicity in rabbits. TBPP has elicited a
clear genotoxic effect in several test systems, both in vitro and
in vivo. Carcinogenic effects were found in rats and mice. The
metabolites BBPP and DBP have also been shown to produce carcinogenic
effects in experimental animals. No irritation effects were found in
animals and a low sensitization potential in humans was noted.
In 1977, the US Consumer Product Safety Commission banned
children's clothing treated with TBPP, because of concerns that the
chemical might be a human carcinogen, and, because of the possibility
of significant human exposure through contact with treated fabrics.
Since then, the use of this substance as a flame retardant in consumer
products has been severely restricted in several other countries and
it has been prohibited in textiles.
1.3 Recommendations
Because of its toxic effects, TBPP should no longer be used
commercially.
If uses are identified for which there are no less hazardous
alternatives to TBPP, studies to demonstrate the absence of exposure
of, and hazards for, humans and the environment should be conducted.
2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, AND ANALYTICAL
METHODS
2.1 Identity
Chemical formula C9H15Br6O4P
Chemical structure
BrCH2-CHBr-CH2O
\
BrCH2-CHBr-CH2O - P = O
/
BrCH2-CHBr-CH2O
Relative molecular mass 697.7
Synonyms tris(2,3-dibromopropyl) phosphate;
tris(2,3-dibromopropyl) phosphoric
acid ester; phosphoric acid, tris(2,3-
dibromo-propyl) ester;
tris(dibromopropyl) phosphate
CAS registry number 126-72-7
CAS chemical name 2,3-dibromo-1-propanol-phosphate (3:1)
RTECS registry number UB0350000
Trade names T 23 P; TP-69; DBP-TP; Apex
(emulsion) 462-5; Hamcogard FR;
Fyrol 59; Tanotard PN-2; Cav Gard
FR 1811 and FR 1812; Pyrosan 497;
Firemaster LV-T23P and T23P-LV;
Firemaster 200; Glotard PE-2; PE 10;
Anfram 3PB; Bromkal P 67-6HP; ES
685; Firemaster T23 and T23P;
Flacavon R; Flamex T23P; Flammex
AP; Zetofex ZN; Fyrol HB-32; NCI-
CO3270; Phoscon PE60; Phoscon UF-
S; RCRA waste number U 235;
USAF-DO-41 (LeBlanc, 1976; IARC,
1979; Ulsamer et al., 1980; IRPTC,
1987). FR 2406; Berkflam T23 P;
Flammex LVT 23P; 3PBR; TDBP;
TDBPP; TRIS; TRIS-BP; Zetifex ZN;
(Andersen, 1977).
2.1.1 Technical product
Commercial TBPP contains up to 0.2% of the following impurities:
2,3-dibromopropanol, 1,2,3-tribromopropane, and 1,2-dibromo-
3-chloropropane (DBCP) (Blum & Ames, 1977; Van Duuren et al., 1978;
Ulsamer et al., 1980).
2.2 Physical and chemical properties
Two grades of TBPP were available in the USA. The highpurity
grade had the following typical properties: a clear, pale-yellow,
viscous liquid; relative density at 25°C, 2.20-2.26; refractive index
at 25°C, 1.576-1.577; viscosity at 25°C, 3900-4200 centistokes; acid
number (mg KOH/g), 0.05 max; volatiles, 1.5% max; bromine content,
68.7%, and phosphorus content, 4,0%. Typical properties for a lower
grade are as follows: density at 25°C, 2.2-2.3; viscosity at 25°C,
1400-1700 centistokes; acid number (mg KOH/g), 0.05 max; and
volatiles, 10% max. (US EPA, 1976; IARC, 1979).
Osterberg et al. (1977) reported a viscosity of 9200 cP (25°C)
for TBPP of a purity of 99.76%. Firemaster LVT 23P has a viscosity of
9200 cP (Kerst, 1974).
Specific gravity 2.27 (2.2-2.3) g/ml at 25°C
(density) (Kerst, 1974)
Boiling point: 390°C (Dybing et al., 1989)
Melting point: 5.5°C (Dybing et al., 1989)
Vapour pressure: 1.9 × 10-4 mmHg at 25°C
1.2 × 10-3 mmHg at 45°C
4.8 × 10-3 mmHg at 65°C
(Kerst, 1974)
Solubility: Virtually insoluble in water
(6.3 mg/litre at 20°C) and hexane;
miscible in organic solvents, such
as carbon tetrachloride, acetone,
chloroform, methylene chloride,
dimethyl formamide, methanol,
xylene, benzene, toluene, and ethyl
acetate (Kerst, 1974)
Stability:
Heat stability: Major decomposition begins at
about 260-300°C; when heated to
decomposition, TBPP emits toxic
fumes of Br- and POx (Sax, 1984)
Light stability: Stable in sunlight
Hydrolytic stability: Hydrolysed by acids and bases
(IRPTC, 1987)
n-Octanol/water partition
coefficient (log Pow): 3.02 (IARC, 1979)
2.3 Analytical methods
2.3.1 General
TBPP is determined using a gas chromatograph equipped with a
flame photometric detector with possible cleaning processes. Direct
mass spectrometry, GC-MS, and HPLC are also used for the analysis of
biological samples containing TBPP and its metabolites (Cope, 1973;
Lynn et al., 1980, 1982; Pearson et al., 1993a).
Recovery and limits of determination vary, depending on sampling
procedures and matrices. GC analysis shows that TBPP can be
determined at the 10 ng level by using a column packed with a high
liquid loaded support. In an indirect analytical method, TBPP is
determined by spectrophotometry, by complexing phosphor with
molybdenum blue after hydrolysis of the TBPP by hydrobromic acid
(Nakamura, 1980; Gutenmann & Lisk, 1975).
Gardner (1979) described a densitometric method using thin-layer
chromatography. TBPP was chromatographed on silicagel thin-layer
plates, using ethyl acetate hexane (30:70) as a developing solvent.
TBPP was visualized by spraying the chromatograms with 1% aqueous
silver nitrate followed by exposure to UVR for 40 min. The spots were
quantified by densitometry at 600 nm. The lower level of sensitivity
was 50 ng; calibration plots were linear from 50 to 800 ng. The
recovery of TBPP from sewage sludge samples fortified at the 1.0 ppm
level was 97%.
Techniques for the qualitative detection of TBPP in textiles have
been described, including thin-layer chromatography, HPLC, and NMR
(Iliano et al., 1982).
2.3.2 Urine
In mammalian species, organophosphates undergo enzymatic or
chemical hydrolysis to form the corresponding acids and alcohols. The
alcohols are often excreted in the urine as soluble conjugates. Since
the hydrolysis of TBPP yields 2,3-dibromopropanol (DBP), an analytical
method has been developed to determine free, and conjugated, DBP.
Extraction of urine by diethylether/hydrochloric acid, followed by
methylation with diazomethane gives the methylether of DBP.
Determination is by electron affinity gas chromatography. The limits
of determination in rat and human urine were 0.4 and 0.2 mg/litre,
respectively (St. John et al., 1976).
3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE
3.1 Natural occurrence
TBPP is not known to occur naturally.
3.2 Anthropogenic sources
3.2.1 Production levels and processes
It is estimated that TBPP was first produced in 1950, when it was
prepared by the addition of bromine to a solution of triallyl
phosphate in benzene. However, it is synthesized in the USA by a
two-step process in which bromine is added to allyl alcohol to give
2,3-dibromopropanol (DBP). This is then reacted with phosphorus
oxychloride, in the presence of a Lewis acid such as, aluminum
chloride or stannium chloride as a catalyst (Overbeek & Nametz, 1962).
The commercial production of TBPP was reported in 1959 and US
production in 1975 has been estimated to have been between 4100-5400
tonnes (US EPA, 1976). Prior to 1977, 4500 tonnes of TBPP were
produced annually in the USA by 6 manufacturers. There was no
evidence of production of TBPP in the USA in 1986.
Production of TBPP in Japan in 1976 and 1977 is estimated to have
been 100 and 300 tonnes per year, respectively, made by one
manufacturer. No TBPP is produced in Japan at present.
It has not been possible to assess whether TBPP is currently
produced. However, no reports are available that describe any
production of TBPP.
3.2.2 Uses
TBPP has been used as a flame retardant for cellulose and
triacetate and polyester fabrics, which are widely used in children's
sleepwear. It has also been used as a flame retardant in other
materials, such as urethane foam and acrylic carpets and sheets,
polyvinyl- and phenolic resins, polystyrene foam, paints, lacquers,
paper coatings and styrene-butadiene rubber, latexes, and cured
unsaturated polyesters products. Rigid foams containing TBPP were
used in insulation, furniture, automobile interior parts, and water
flotation devices. About 65% of the 4500 tonnes of TBPP that were
produced annually in the USA by 6 manufacturers was applied to fabrics
used for children's clothing. TBPP was added to these children's
garments to an extent of 5-10% by weight (US EPA, 1976; Kirk-Othmer,
19781984).
TBPP was applied to cellulose acetate and triacetate by addition
to the melt prior to spinning. The process involved the thermal
diffusion of TBPP by driving it into the fibre under pressure dying.
For materials such as, polyesters, nylons, and acrylics, the TBPP was
either "padded on" at 5-10% by weight with heat fixation to the woven
or knitted material or applied via emulsion from conventional batch
dying equipment (Prival, 1975).
Fire-retarded polyurethane required about 0.5% phosphor and 4-7%
bromine; being equivalent to about 10% TBPP by weight in the product
(US EPA, 1976).
By actions taken on 8 April and 1 June 1977, on the basis of the
genotoxic and possible carcinogenic effects of TBPP, the US Consumer
Product Safety Commission banned children's clothing treated with
TBPP, the chemical itself when used or intended to be used in
children's clothing, and fabric, yarn, or fibre containing it, when
intended for use in such clothing (US Consumer Product Safety
Commission, 1977a,b; US Consumer Product Safety Commission, 1977a,b).
In March 1978, The Consumer Product Safety Commission listed 22
products that contained TBPP and were available to USA consumers.
These included children's clothing, industrial uniforms, draperies,
tent fabric, automobile headliners, epoxy resins for the electronics
industry, Christmas decorations, and polyester thread (IARC, 1979).
In Japan, the use of TBPP as a fire-retardant in textile products
was banned in 1981, because the chemical might be a human carcinogen
and genotoxicant.
As from December 1987, TBPP could not be used in the EC in
textile articles such as, garments, under-garments, and linen intended
to come into contact with the skin (EEC, 1976, 1979).
Several other countries including Finland, New Zealand, and
Sweden have also banned, or severely restricted, the use of TBPP in
textiles and textile articles (UN, 1991).
3.2.3 Sources of human and environmental exposure
Potential sources of human exposure and environmental
contamination include: the manufacturing of the flame retardant, its
application to materials, leaching out of the flame retardant during
use and/or washing, and ultimate disposal of the material.
Studies indicated substantial losses of surface TBPP from fabrics
after laundering, but TBPP was not completely removed after repeated
laundering. For example, acetate fabrics (65-600 mg TBPP/kg) showed
up to 85% reduction in surface concentration after one laundering,
and, polyester fabrics (260-37 500 mg TBPP/kg), from 21 to 82%
reduction after one laundering. A significant portion, approximately
10% of the total production reached the environment from
textile-finishing plants and laundries. Most of the rest will find
its way into solid wastes (US EPA, 1976).
Surface TBPP can be extracted from treated fabric by saliva (up
to 3%) as well as by water, acetic acid, sodium bicarbonate, and salt
(Ulsamer et al., 1980).
Gutenmann & Lisk (1975) heated polyester flannel material,
treated with TBPP, in distilled water at 60°C for 20 min, simulating
a laundering operation. It was calculated from the extraction rate
that laundering of flame-retarded sheets could result in a
concentration of 6 mg/litre in combined washing and rinsing water.
This release was maintained during several subsequent launderings.
The presence of detergents may increase the extraction rate.
TBPP exists both in, and on, the fabric. In the fabric fibres,
it is not extractable with a benzene/hexane mixture and, therefore,
is probably not available for dermal absorption. However, when it is
on the fibre surface, it is extractable and is available for dermal
absorption (Morrow et al., 1976; Ulsamer et al., 1980).
While most of the TBPP is within the fabric in both polyester and
acetate, polyester contains considerably more surface TBPP as a result
of differences in methods of addition. Concentrations of surface
bromine in polyester fabric ranged from 2000 to 37 500 mg/kg with the
actual TBPP content ranging from 20 to 90% of the bromine value. The
non-TBPP organic bromides have not yet been identified (Ulsamer et
al., 1980).
4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION
4.1 Transport and distribution between media
An estimated log Koc (3.29) suggests strong adsorption on soil.
On the basis of this Koc value and the low measured water solubility
of the technical chemical (8.0 mg/litre), TBPP is expected to leach
only slowly into groundwater. The water solubility of pure TBPP may
be lower than the solubility of the technical grade chemical and so
the extent of leaching of the pure chemical may be even lower than the
Koc above suggests (Kenaga, 1980; Lyman, 1982; Verschueren, 1983; US
EPA, 1985).
Although hydrolysis of the phosphate ester is not expected to be
significant, hydrolysis involving the bromine atoms on the propyl
groups may occur, especially under basic conditions. Direct
photolysis is not expected to be a major process, since TBPP should
not absorb light of wavelengths found in sunlight (> 290 nm) (Mabey &
Mill, 1978).
No data on volatilization from water or soil are available. Using
measured water solubility (8.0 mg/litre) and vapour pressure of 1.9 ×
10-4 mmHg, volatilization half-life values were estimated. The
half-life values for TBPP volatilization from streams, rivers, and
lakes were 3.64, 4.66, and 392 days, respectively, assuming current
velocities of 3, 1, and 0.01 m/second, respectively. The river and
stream depths were assumed to be 1 m, while the lake was assumed to be
50 m deep (Verschueren, 1983).
4.2 Transformation
4.2.1 Biodegradation
The biodegradability of TBPP was determined following a
shake-flask test. TBPP was incubated with a microbial inoculum of raw
sewage. Samples of the test solutions were taken at 0, 5, 10, and 15
days for final analysis using neutron activation to determine the
bromine content of the liquid. Assuming the increased bromide content
of the inoculated samples relative to the blank samples is due to
biodegradation, and the solubility of TBPP is 1.6 mg/litre, an amount
of TBPP equal to 2.4 times the dissolved TBPP was degraded in 5 days
(Kerst, 1974).
Activated return sludge (at 21°C), used within 1 h of
collection, diluted with a basal medium, with an added 2 mg
14C-labelled TBPP/kg, showed that 6% of the added radio-activity was
evolved as 14CO2. A major metabolite bis(2,3-dibromopropyl)
phosphate (BBPP) was identified, but neither dibromopropanol (DBP) nor
dibromopropionic acid was detected. The half-life of TBPP was 19.7 h
(by least squares regression analysis). In a sterilized sludge
control study, 93% of the added TBPP was found and metabolites were
not identified (Alvarez et al., 1982).
A biodegradation study on TBPP (100 mg/litre) was carried out
under sewage treatment condition with sludge (30 mg/litre). The
degree of biodegradation, as measured by BOD, was 1.8% of TBPP after a
2-week incubation period (Chemicals Inspection & Testing Institute,
1992).
4.2.2 Abiotic degradation
No data available.
4.2.3 Bioaccumulation
Tissue residue analysis of rats fed TBPP for a period of 28 days
at levels of 100 or 1000 mg/kg diet has shown dose-related residue
levels (measured as total bromine) in the muscle, liver, and body fat,
of the treated animals (see section 7.2.1.).
Groups of 30 adult fathead minnow (Pimephales promelas) (six
months old), were exposed to 47.7 µg TBPP/litre for 2-32 days in a
flow-through system. The temperature of the water was 25°C, pH 7.49,
dissolved oxygen > 5 mg/litre, and hardness
45.5 mg/litre. The bioconcentration factor determined was 2.7 (Veith
et al., 1979).
Bioconcentration of TBPP (0.1 mg/litre, 0.03 mg/litre) from water
to carp was estimated to be between < 0.7 to 1.9, and < 2.2 to 4.3,
respectively, after 6 weeks of exposure (Chemicals Inspection &
Testing Institute, 1992).
4.3 Interaction with other physical, chemical, or biological factors
The thermal oxidative degradation at 370°C of TBPP produced
hydrogen bromide and the C3-brominated species - bromopropenes,
dibromopropenes, dibromopropanes and tribromopropanes, accounting for
87% of the volatiles. The detection of chlorinated species can only
be explained by the presence of chlorinated impurities in the original
ester. The residue (ether soluble aliquot) was composed mainly of
1,2,3-tribromopropane, whereas the aqueous layer contained the
phosphoric acid produced. The gas chromatographic analyses of the
volatiles showed a number of isomeric dibromopropenes. It was
established that 1,3-dibromopropene was the major dibromopropene
formed (Paciorek et al., 1978).
4.4 Ultimate fate following use
It is to be expected that TBPP would be released into the
environment in wastewater after laundering articles coated with TBPP
flame retardant.
With regard to disposal, it must be assumed that clothes and
other products containing TBPP ultimately end up in landfills, which
may result in some biological accumulation. Incineration should be
carried out at high temperature with scrubbers or the equivalent.
5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE
5.1 Environmental levels
5.1.1 Air
TBPP was identified, but not quantified, in Arkansas air
particulates (DeCarlo, 1979).
5.1.2 Water
In 1975, 20 water samples were collected at different places in
Japan and analysed for the presence of TBPP. None of the samples
contained the compound (limit of determination 1 µg/litre)
(Environment Agency Japan, 1978, 1987).
5.1.3 Soil
In 1975, 20 sediment samples were collected at different places
in Japan and analysed for the presence of TBPP. None of the samples
contained TBPP (limit of determination 0.4-10 mg/kg) (Environment
Agency Japan, 1978, 1987).
TBPP was identified, but not quantified, in Arkansas soil
(DeCarlo, 1979).
5.1.4 Fish
In 1975, 20 fish samples, collected at different places in Japan,
were analysed for the presence of TBPP. None of the samples contained
TBPP (limit of determination 1 mg/kg) (Environment Agency Japan, 1978,
1987).
5.2 General population exposure
5.2.1 Subpopulation at special risk
Tests for the extraction of TBPP from fabrics by water at various
pH values and by a simulated saliva solution failed to reveal any TBPP
in the extracts, but sodium bromide and hydrobromic acid were detected
(limits of determination not mentioned) (Prival, 1975). However,
surface TBPP can be extracted from treated fabric by saliva (up to 3%)
as well as by water, acetic acid, sodium bicarbonate, and salt
(Ulsamer et al., 1980).
In the USA, the estimated intake via the skin of children,
wearing sleepwear treated with the compound, was estimated to be
9 µg/kg body weight (Blum et al., 1978).
The Consumer Product Safety Commission of the USA stated that,
over a 6-year period, a child wearing TBPP-treated clothing could
absorb a total of 2-77 mg TBPP/kg body weight and there are
indications that this may be even higher (IRPTC, 1987).
5.3 Occupational exposure
There are no data on levels of exposure to TBPP during
manufacture or further processing.
6. KINETICS AND METABOLISM IN LABORATORY ANIMALS AND HUMANS
6.1 Absorption
TBPP is absorbed readily by the gastrointestinal tract and at a
moderate rate via the skin in rats and rabbits. Studies on children
revealed that TBPP is dermally absorbed from TBPP-treated sleepwear
(Kerst, 1974; Blum et al., 1978; Ulsamer et al., 1978, 1980).
Following the dermal application of 14C-TBPP to the clipped
backs of New Zealand White rabbits (2-3 kg), 3.5-3.8% of the 0.9 ml/kg
dose and 15.2% of the 0.05 ml/kg dose were absorbed over 96 h.
Osborne Mendel rats (200-250 g) absorbed approximately 1/6 as much
14C-TBPP at each dose, when TBPP was applied to an equivalent area
of skin/kg. The dermal uptake of 14C-TBPP by rats and rabbits
showed that the primary elimination was via the kidneys (Ulsamer et
al., 1980).
6.2 Elimination
6.2.1 Different routes (rat and rabbit)
Four male Sprague-Dawley rats (290-310 g) were administered
14C-TBPP (98%) intravenously. The animals were housed in metabolism
cages for 5 days. Urine, faeces, and air samples were collected for
5 days, and bile for 1 day. In 5 days, 58% of the administered
radioactivity was found in the urine; 9% in the faeces and 19% in the
air as CO2. In 24 h, bile contained 34% of the radioactivity while
9% was found in the bodies of the rats. In three additional rats,
it was found that biliary excretion and enterohepatic recirculation
was a major route in the disposition of TBPP. Bis(2,3-dibromopropyl)
phosphate (BBPP) was detected in the urine of male rats (290-310 g)
dosed iv with 25 mg 14C-TBPP (98%)/animal (in Emulphor) in
amounts of 7.8% of the dose during 5 days following administration.
BBPP was identified in the urine, faeces, bile, and tissues.
2,3-Dibromopropanol (DBP) was found in tissues and DBP and a few other
metabolites were found in urine, but TBPP was not detected (Lynn et
al., 1980, 1982).
An adult male Sprague-Dawley rat (150-200 g) was administered
(iv or orally) 1.39 mg 14C(propyl)-TBPP (99%)/kg body weight. One
day after iv administration, 17% of the administered radioactivity was
found in the urine, 7.4% in the faeces and 20% in the air (as CO2).
One day after oral administration of TBPP, the concentrations were 24%
in the urine and 11.5% in the faeces, but no radioactivity was
detected in the air. Mainly metabolites were excreted in the urine
and bile (Nomeir & Matthews, 1983).
Small amounts of DBP and conjugates appeared in urine, when the
rat was allowed to chew on TBPP-finished polyester fabric (St. John et
al., 1976).
Radiolabel from 14C-TBPP, applied to the skin, was excreted
primarily in the urine (70% for rabbits and 50% for rats) with lesser
amounts appearing in the faeces and 12 and 18% exhaled as CO2,
respectively. TBPP itself did not appear in the urine, but a number
of metabolites including DBP were found (section 6.4) (Ulsamer et al.,
1980).
6.2.2 Dermal exposure (rat and rabbit)
6.2.2.1 TBPP
One hundred mg of TBPP was spread over the surface of a gauze pad
(one square inch) bandage and pressed tightly against the shaved skin
of a rat. Urine was assayed for free and conjugated (released by acid
hydrolysis) DBP. By day 7, the total concentrations of free and
conjugated DBP in the urine were 17.61 and 23.58 mg/litre,
respectively (St. John et al., 1976).
6.2.2.2 TBPP-treated fibres
TBPP has been shown to penetrate rabbit skin from 14C-TBPP
labelled polyester cloth containing 15 000 mg TBPP/kg of surface (4.3%
of the radioactivity in 96 h) (Ulsamer et al., 1980).
A shaved rat wore a garment made of 100% polyester flannel
(4 × 6 inches), treated with TBPP, for 9 days. No DBP could be
detected in the urine (limit of determination 0.4 mg/litre) (St. John
et al., 1976).
6.2.3 Dermal exposure (human)
The skin of a 7-year-old child was exposed on days 1, 2, and
8-12, by wearing repeatedly washed sleepwear that may have been
TBPP-treated. On days 3-7, she wore new TBPP-treated pyjamas. Urine
samples were collected daily from the child. In the urine, a maximum
concentration of DBP of 29 µg/litre was found 2 days after wearing the
new treated pyjamas. DBP at a concentration of 0.4 µg/litre was
present in the urine, prior to wearing the new treated pyjamas. DBP
was still excreted 5 days after the child stopped wearing the new
TBPP-treated pyjamas. Urine samples were collected from 10 other
children and one adult. All samples were analysed for DBP; it was not
found in the urine of one child and one adult (who had never used
washed TBPP-treated sleepwear). Seven children had levels of about
0.5 µg DBP/litre in the urine and one child had a level of 5 µg/litre.
Approximately 180 µg/day (9 µg/kg body weight) was absorbed through
the skin of children wearing pyjamas treated with TBPP (Blum et al.,
(1978).
No DBP could be detected in the urine of an adult or in the urine
of a 5-year-old boy who wore 100% polyester knit pyjamas, treated with
TBPP, for 7 nights. Morning urine samples were collected daily
throughout this period and up to 8 days thereafter (limit of
determination 0.2 mg/litre) (St. John et al., 1976).
6.3 Distribution
6.3.1 Rat
6.3.1.1 Oral
Male adult Sprague-Dawley rats (150-200 g) were administered
1.39 mg 14C(propyl)-TBPP (99%) orally. The percentages of the total
dose of radioactivity, found after one day, in the blood, liver,
kidneys, lung, muscles, fat, and skin, were 6.6, 3.4, 0.7, 0.2, 5.5,
1.3, and 3.4%; 24 and 11.5% of the total dose were found in the urine
and faeces, respectively. The terminal clearance of TBPP-derived
radioactivity from most of the tissues was described by a single
component exponential decay with a half-life of 2.5 days. The
half-life of TBPP in the liver and kidneys was 3.8 days (Nomeir &
Matthews, 1983).
Dose-related bromine concentrations were detected by neutron
activation analysis in the muscles, liver, and fat of male rats fed
TBPP for 28 days. The levels decreased to control levels during the
six-week withdrawal period (Kerst, 1974).
6.3.1.2 Intravenous
Eight male Sprague-Dawley rats (290-310 g) were administered
14C-TBPP (98%) by the iv route and the distribution was studied.
All tissues contained TBPP-derived radioactivity. The concentrations
of TBPP-derived radioactivity declined rapidly in most tissues, but
the concentration of radioactivity in kidneys was 11 times the average
body concentration, five days after dosing. No TBPP was detected,
though bis(2,3-dibromopropyl) phosphate (BBPP) was still present in
substantial concentrations. By day five, only small quantities of
this metabolite were detected. The concentration of TBPP increased
in the fat during the first 5-30 min, but, after 8 h, TBPP was no
longer detectable. In contrast to the rapid disappearance of TBPP,
the half-life of BBPP was relatively long in most tissues. BBPP
represented a major portion of the radioactivity in several tissues
including the lung, muscles, fat, and blood. In blood, it accounted
for 90% of the radioactivity at 30 min and 8 h. By 5 min, 75% of the
radioactivity in plasma was BBPP. The initial plasma half-life of
this metabolite was 6 h. For 5 days it was 36 h. TBPP was not
detectable in plasma after 1 h (Lynn et al., 1982).
6.3.2 Dermal (rabbit)
Substantially more TBPP-derived radiolabel was detected in the
kidneys and liver than in other organs of New Zealand rabbits,
dermally treated with polyester fabrics containing 14C-TBPP (Ulsamer
et al., 1978).
6.4 Metabolic transformation
6.4.1 In vivo studies
6.4.1.1 Oral (rat)
TBPP was readily metabolized in rats. The main metabolite found
in the urine, faeces, bile, and tissues of rats was BBPP.
2,3-Dibromopropanol (DBP) was also identified in tissues and urine.
Only small amounts of unchanged TBPP were found in the excreta (Lynn
et al., 1982; Nomeir & Matthews, 1983).
Male adult rats (150-200 g) were administered 1.39 mg
14C(propyl)-TBPP (99%) orally (by intubation), and the urine and
bile were analysed for metabolites. Six metabolites were identified
in urine and bile, respectively:
- 2,3-dibromopropanol; 1.0 and 1.1%;
- bis(2,3-dibromopropyl) phosphate; 2.8 and 25.8%;
- 2-bromo-2-propenyl 2,3-dibromopropyl phosphate; 4.8 and 13.8%;
- bis(2-bromo-2-propenyl) phosphate; 10.3 and 5.2%;
- 2,3-dibromopropyl phosphate; 4.1 and 2.6%;
- 2-bromo-2-propenyl phosphate; 9.5 and 2.4%
and TBPP was found in concentrations of 0.8 and 2.0%, respectively.
These data are expressed as a percentage of total radioactivity
excreted in the urine in 24 h, and, bile in 3 h. The total quantity
of metabolites eliminated in the urine and bile were, in these
periods, 33.3 and 52.9% of the radioactivity administered,
respectively (Nomeir & Matthews, 1983).
The formation of BBPP has been studied using selectively
deuterated analogues of TBPP. Plasma concentrations of BBPP in rats
dosed with either C2-D1- or C3-D2-TBPP were substantially lower than
levels obtained with TBPP up to 4-6 h after administration. This
indicates that oxidative metabolism of TBPP to form BBPP is important
in vivo. Furthermore, in addition to oxidation at C3, BBPP
formation may result from oxidation at C2. This latter reaction may
be of particular importance with phenobarbital-pretreated microsomes
(Pearson et al., 1993a; Dybing et al., 1989).
In addition to these TBPP metabolites, 2-bromoacrolein,
2-bromoacrylic acid, bis(2,3-dibromopropyl)-3-hydroxypropyl phosphate,
S-(2,3-dihydroxypropyl) glutathione, S-(3hydroxypropyl)
glutathione and S-(2-carboxyethyl) glutathione have been detected
in vitro and/or in vivo (Marsden & Casida, 1982; Nelson et al.,
1984).
2-Bromoacrylic acid has been detected in the urine of rats
administered TBPP. It was suggested that 2-bromoacrylic acid is an
oxidation product of 2-bromoacrolein and that 2-bromoacrolein is
formed spontaneously from DBP generated via initial cytochrome
P450-mediated oxidation of TBPP (Marsden & Casida, 1982; Soderlund et
al., 1984).
Recent data indicate that the formation of 2-bromoacrolein occurs
mainly from oxidative dehalogenation at the C3 position (Pearson et
al., 1993a).
Although glutathione acts as a detoxifying agent for reactive
TBPP metabolites (Soderlund et al., 1984), conjugation could also
result in the formation of reactive episulfonium ion intermediates
(Pearson et al., 1993b). Van Beerendonk (1994) noted that there is
S-(2,3-dihydroxypropyl) glutathione in the bile of Sprague-Dawley
rats. They suggested that TBPP and/or BBPP are conjugated directly
with glutathione by glutathione S-transferases, with subsequent
formation of episulfonium ions.
6.4.2 In vitro studies
TBPP is readily metabolized by microsomal and cytosolic rat liver
fractions. Liver microsomes metabolized TBPP in the presence of NADPH
and oxygen, as evidenced by the release of bromine and the formation
of BBPP (Kerst, 1974; Nomeir & Matthews, 1983).
The role of debromination in the formation of reactive
metabolites was demonstrated in a series of TBPP analogues (Soderlund
et al., 1984). The rate of NADPH-dependent metabolism was increased
5-10 times with microsomes from phenobarbital-pretreated rats compared
with control microsomes and was reduced in the presence of cytochrome
P450 inhibitors, indicating that cytochrome P450 is responsible for
microsomal TBPP biotransformation (Soderlund et al., 1979, 1981, 1984;
Nomeir & Matthews, 1983).
Liver microsomes from mice, guinea-pigs, hamsters, and humans all
metabolized TBPP to reactive intermediates. However, the rate of
formation of reactive TBPP metabolites with human liver microsomes was
lower than with liver microsomes from rodents (Soderlund et al.,
1982a).
In addition, a 1.5 to 2-fold increase in the rate of TBPP
metabolism occurred when phenobarbital-pretreated microsomes were
fortified with GSH, indicating that microsomal GSH- S-tranferases are
able to conjugate TBPP with GSH. Dialysed rat liver cytosolic
fractions, supplemented with GSH, metabolized TBPP at rates that were
3 times higher than those observed with control microsomes and NADPH
(Nomeir & Matthews, 1983; Soderlund et al., 1981, 1984). Thus, in
animals, GSH-dependent metabolism may be an important route in the in
vivo biotransformation of TBPP to more water-soluble products.
Soderlund et al. (1984) detected the in vitro formation of
2-bromoacrolein, by a reaction catalysed by cytochrome P450, in a
process liberating bromide ions with subsequent formation of BBPP
using rat liver microsomes (Soderlund et al., 1984). Mass spectral
analysis of 2-bromoacrolein, formed from selectively deuterated
analogues of TBPP, revealed that the primary mechanism for the
formation of 2-bromoacrolein involves the initial oxidative
dehalogenation at C-3 followed by a betaelimination reaction (Nelson
et al., 1984).
In vitro studies were carried out with deuterated analogues of
TBPP, or, analogues labelled at specific positions with carbon-14,
phosphorus-32, or oxygen-18, or dual-labelled with both deuterium and
tritium. These were used as metabolic probes to study the chemical
and metabolic events in the bioactivation of TBPP to chemically
reactive metabolites in the liver microsomal preparations of male
Sprague-Dawley rats. Studies with deuterated analogues of TBPP
implicated oxidation at C-2 of the propyl moiety as a major pathway
that leads to protein binding, which is enhanced by phenobarbital
pretreatment of rats. Investigations with 18O-TBPP and H218O
showed that the BBPP that is formed from the oxidation of TBPP
incorporates one atom of oxygen from water. These results imply that
oxidation at C-2 yields a reactive alpha-bromoketone that can alkylate
proteins directly, or, hydrolyse to BBPP and a reactive alpha-
bromoalpha'-hydroxyketone that alkylates microsomal proteins (Pearson
et al., 1993a). These studies also showed that TBPP is oxidized at
C-3, yielding the direct acting mutagen 2-bromoacrolein as the major
metabolite that binds to DNA. This is consistent with earlier studies
that indicate that 2-bromoacrolein is the major reactive metabolite
formed in in vitro microsomal incubations (Nelson et al., 1984;
Dybing et al., 1989).
6.5 Covalent binding to macromolecules
TBPP has been shown to be activated to products that bind
covalently to proteins (total macromolecules) and DNA in vitro and
in vivo (Soderlund et al., 1981, 1984; Pearson et al., 1993a,b).
The covalent binding of radiolabel TBPP to macromolecules was
dependent on microsomes and NADPH, and was reduced by carbon monoxide,
inhibitors of P450, and glutathione (Soderlund et al., 1981). The
extent of TBPP covalent binding in vivo was five times higher in the
kidneys than in the liver, whereas the rate of in vitro covalent
binding was much higher with liver microsomes than with kidney
microsomes. The low levels of TBPP binding in the liver in vivo may
be the result of an extensive detoxification of TBPP to non-reactive
metabolites or to low tissue concentrations of the proximate
metabolite(s).
Male NMRI and female B6C3F1 mice (20-25 g), male F344 rats
(200-250 g), and guinea-pigs (80-100 g) were injected ip once with
250 mg 3H-TBPP/kg body weight in DMSO. The animals were killed 9 h
after injection. All species showed similar levels of covalent
binding to proteins in the liver and kidneys except for the rat which
had much higher amounts of radiolabel bound to kidney proteins
(Soderlund et al., 1982a).
The binding of TBPP and analogues has also been studied in
vivo. Analogues of TBPP either labelled at specific positions with
carbon-14, and phosphorus-32 or dual-labelled with both deuterium and
tritium were administered to male Wistar rats at a nephrotoxic dose of
360 µmol/kg body weight. The covalent binding of TBPP metabolites to
rat hepatic, renal, and testicular proteins was determined after 9 and
24 h. The covalent protein binding was 5 times higher in the kidneys
than in the liver and approximately 25 times higher than that in the
testes. The results of comparative studies on renal DNA damage
induced by TBPP and BBPP labelled with deuterium at C-2 or C-3
suggested that BBPP is formed in the liver by P450-mediated oxidation
at either C-2 or C-3 of TBPP. BBPP is then transported to the
kidneys, where it is subsequently metabolized to reactive
intermediates that cause DNA damage and bind to kidney proteins in a
process, independent of cytochrome P450, involving activation by
conjugation with glutathione (Pearson et al., 1993b).
Van Beerendonk et al. (1992) studied the formation of thymidine
adducts and the cross-linking potential of 2-bromoacrolein (BA), a
reactive metabolite of TBPP. In this study, [3-3H]BA was reacted
with single-stranded (ss) DNA or double-stranded (ds) DNA and
subsequently incubated with methoxylamine to covert the reaction
product to an unstable BA:thymidine adduct. Because the unstable
BA:thymidine adduct may have the potential to form cross-links, the
reaction with various nucleophiles in vitro was studied. A reaction
occurred between the adduct and cystein, but not with lysine or
desoxynucleosides. Reaction of BA with ssDNA in the presence of
[3H]glutathione also resulted in the binding of radiolabelled GSH to
DNA. The results indicated that the reactive aldehyde group of the
adduct can react with thiol groups in proteins to form protein-DNA
cross-links. When the possibility that tris- and bis-(2,3-
dibromopropyl) phosphates form such cross-links was examined in vivo
in Drosophila, it was found that TBPP was a cross-linking agent,
whereas BBPP was not.
7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS
7.1 Single exposure
The oral LD50 for TBPP was calculated to be 5.24 g/kg body
weight, when administered as a suspension in propyleneglycol to male
albino Spartan rats with a weight of 202-250 g. The observation
period was 14 days (Kerst, 1974). In another study, TBPP dissolved in
propyleneglycol or in ethanol was given to Osborn-Mendel rats. Oral
LD50s of 1.88 and 3.12 g/kg body weight, respectively, were obtained
(Ulsamer et al., 1980).
A dermal toxicity study showed an acute LD50 for rabbits of
17.6 g/kg body weight (Ulsamer et al., 1980). In another study, TBPP
was applied once to the back of four groups of two male and two female
New Zealand white rabbits (2.56-2.96 kg) in concentrations of 1, 2, 4,
or 8 g/kg body weight. The application area was wrapped with a gauze
bandage and occluded; after 24 h, the bandages were removed and the
skin washed with water. The observation time was 14 days. An LD50
of > 8 g/kg body weight was found (Kerst, 1974).
A dose of 2 g TBPP was applied to the intact and abraded skin of
10 albino rabbits. No deaths were observed during a 14-day
observation period (Moldovan, 1972).
7.2 Short-term exposure
7.2.1 Oral exposure (rat)
7.2.1.1 TBPP
Groups of male rats received daily doses of 250 mg TBPP/kg in
either propyleneglycol or saline, by gavage, and were sacrificed after
1, 2, 4, 6, 8, or 10 days. Liver and testes were unaffected by any
treatment, but nephrotic changes were observed to commence on day 2
and to become progressively more severe with time. In addition to the
tubular lesions, the glomeruli were adversely affected, an observation
not seen in the 13-week study (Osterberg et al., 1979).
In a pilot study, groups of 10 non-pregnant rats were
administered TBPP for 10 days at dose levels of 100, 150, 500, or
1000 mg/kg body weight per day. Mortality rates were 0, 0, 70, and
100%, respectively (Seabaugh et al., 1981).
Male weanling rats were fed TBPP at concentrations of 100 or
1000 mg/kg diet for 28 days. The animals were then sacrificed
immediately or after 2 or 6 additional weeks of recovery. The results
showed a decrease in food efficiency (approximately 10% at the
highest dose), decreased body weight gain (approximately 20% at the
highest dose), and decreased organ to body weight ratios for heart,
liver, spleen, kidney, and gonads (approximately 20% for each organ at
the highest dose). Haematology, blood chemistry, urinalysis, and
histopathology did not differ from the control values. In the
recovery period, the body weight gain became normal. The authors
suggested that the effect might be because of the palatability of the
substance. Tissue residues (measured as bromine) increased 40-50
times in the first 4 weeks of treatment in the fat, liver, and
muscles. By the end of the 6-week withdrawal period, the residues
were at control levels (Kerst, 1974).
Groups of rats were gavaged with TBPP in corn oil at 10, 50, or
100 mg/kg per day for 4 weeks. One half of each group was sacrificed
at 4 weeks and the remainder at 6 weeks. While no adverse responses
were observed, elevated bromine levels in blood were reported (Brieger
et al., 1968).
A 90-day study was carried out on rats administered TBPP in
propylene glycol, daily (by gavage), at 25, 100, or 250 mg/kg body
weight. The control groups received either the vehicle, normal
saline, or no treatment. Weight gain for males was 34-50% less and,
for females, 40% less in the test groups and vehicle group compared
with the control values. Liver/body weight ratios were lower for both
sexes in the low TBPP group, but higher in females in the highest dose
group, compared with those in the control group. Kidney/body weight
ratios were 18% lower than in controls. Testes/body weight ratios in
the TBPP groups were 25% lower. There was an increased incidence and
severity of chronic nephritis associated with regenerative epithelium,
hypertrophy, and dysplasia of renal tubular epithelial cells in all
TBPP-treated rats. The complex of changes was more severe with higher
dose, and among males (Osterberg et al., 1978).
7.2.1.2 TBPP-treated fibres
The results of a 2-week study on rats fed 15% shredded
TBPP-treated acetate fibres in their food (3 times/week) showed no
changes in blood-bromine levels and no adverse effects (Ulsamer et
al., 1980).
7.2.2 Oral exposure (dog)
7.2.2.1 TBPP
In a study on dogs, doses of 50 or 100 mg TBPP/kg body weight
were given in the diet for four weeks. A decrease in body weight was
noted in the treated dogs as well as increased blood-bromine levels.
Cholinesterase activity was reported to be unaffected (Brieger et al.,
1968). No further details were available for this study.
7.2.2.2 TBPP-treated fibres
In a 2-week study on dogs fed 15% shredded TBPP-treated acetate
fibres in their food (3 times/week), no changes in blood-bromine
levels or adverse effects were seen. Two additional, 3-week studies
on dogs using TBPP-treated shredded rayon and acetate fibres added to
foods did not show any detectable changes in health or in
blood-bromine levels (Brieger et al., 1968).
7.2.3 Dermal exposure
7.2.3.1 Rabbit
Short-term dermal studies have been performed using groups of
clipped rabbits dosed with 2.2, 4.4, or 8.8 g TBPP/kg body weight,
daily, for 4 weeks. A dose-related increase in bromine was found in
the blood and urine. All rabbits died within 4 weeks. Significant
degenerative changes in the kidneys and the liver were found. Slight
decreases in cholinesterase activity were recorded (Brieger et al.,
1968). In another study in which the animals were administered dose
levels of 50 and 250 mg/kg body weight, bromide levels in the blood
and urine were increased, but no deaths occurred (Ulsamer et al.,
1980).
A 13-week study was carried out on 12 young (3 months old) New
Zealand white rabbits, 6 with intact, and 6 with abraded, dorsal skin.
They were treated with a weekly application of 2.27 g TBPP (99.76%)/kg
body weight for 13 weeks. In a third group, 6 rabbits were initially
clipped and maintained untreated as controls. The TBPP-treated sites
were not occluded with a patch, but the animals were fitted with a
collar. Besides a statistically significant increase in relative
liver weights in the rabbits with intact and abraded skin (53% and
59%, respectively), a significant decrease in testes weight (54% and
40%, respectively) was observed. Microscopically, chronic
interstitial nephritis (in 6/8 males) with tubule involvement and
bizarre nuclei as well as testicular atrophy and aspermatogenesis
(spermatogonia were present in seminiferous tubules, and also
secondary spermatocytes, but no spermatozoa) were observed in 7/8
males of the test groups. Female rabbits did not exhibit any adverse
responses. No histopathological changes were seen in the liver
(Osterberg et al., 1977, 1978).
In a study in which TBPP-treated rayon cloth was applied to the
clipped skin of rabbits for 4 weeks, no significant effects were found
(bromine levels were not increased) in treated animals (Ulsamer et
al., 1980). No further details were available for this study.
7.2.3.2 Dog
When TBPP-treated rayon cloth was applied to the clipped skin of
dogs for 4 weeks, no significant effects (no increased bromine levels)
were found in the treated animals (Brieger et al., 1968). No further
details were available for this study.
7.3 Long-term exposure
Apart from carcinogenicity studies, no long-term toxicity studies
are available (see section 7.7).
7.4 Skin and eye irritation; sensitization
7.4.1 Skin irritation
TBPP (1.1 g) was applied to the abraded or intact skin of six
albino rabbits. The animals were fitted with collars for 24 h. After
this period, the coverings were removed and the test material washed
off. The extent of erythema and oedema was determined after 24 and
72 h. No signs of irritation were observed (Kerst, 1974).
7.4.2 Eye irritation
Administration of 0.22 g TBPP to the eyes of 6 adult rabbits did
not cause noticeable irritation or damage to the cornea, iris, or
palpebral conjunctiva during a 72-h observation period (Kerst, 1974;
US EPA, 1976).
7.4.3 Sensitization
TBPP was tested for skin sensitization in groups of 5-10
guinea-pigs using a modified Landsteiner method and the footpad
technique. No sensitization was noted in either test (no details
given) (Morrow et al., 1976).
7.5 Reproductive toxicity, embryotoxicity, and teratogenicity
7.5.1 Reproductive system
Groups of 6 adult male Sprague-Dawley rats (56-60 days of age)
were used in a study to investigate the effects of TBPP on the
reproductive system. Six rats were injected with 0.1 ml
propyleneglycol intraperitoneally, three times/week, and, six rats
were untreated controls. Nine groups of 6 rats were given (ip
injection), three times/week, 0.4, 0.9, 1.8, 3.5, 7.1, 14.2, 28.4,
56.8, or 113.5 mg TBPP in propyleneglycol for a period of 72 days. The
four highest dose levels of TBPP did not dissolve completely and were
injected as an emulsion. The rats were treated for a minimum of 72
days (6 cycles of the germinal epithelium) before being killed. The
three highest dose levels (28.4-113.5 mg/injection) caused significant
dose-related declines in the weights of the testes and prostate,
epididymides, and seminal vesicles. Sperm production of testes and
sperm storage in the epididymides were reduced, and the percentage of
the motile sperm and the motility index were decreased. Histological
examination of the testes revealed that the seminiferous tubules were
affected. The affected tubules contained very few germinal cells and
the macrophages in the interstitium of the affected testes appeared to
be phagocytically active. The Leydig cells were normal. TBPP did
not have any significant effects on the serum concentration of
testosterone or on the in vitro testicular capacity for testosterone
secretion (Cochran & Wiedow, 1986).
The effects on the testes were also reported in a 13-week study
on New Zealand white rabbits, treated with weekly dermal applications
of 2.27 g TBPP on the intact or abraded skin. Decreased testes
weights and, microscopically, testicular atrophy and aspermatogenesis
were found in male rabbits (Osterberg et al., 1977).
B6C3F1 mice (15 weeks old) were administered (ip) TBPP in corn
oil at dose levels of 0, approximately 200, 400, 600, 800, and
1000 mg/kg body weight daily, for 5 days. The mice were killed 35
days after the fifth treatment. Their epididymides were removed and
abnormal sperm heads determined. The frequency of abnormal sperm
heads in TBPP-treated mice was significantly greater than in controls,
predominantly at dose levels of 800 mg/kg body weight or more
(Salamone & Katz, 1981).
7.5.2 Teratogenicity
In a pilot study on groups of ten pregnant Sprague-Dawley rats,
orally intubated with 0, 250, or 1000 mg TBPP/kg body weight on days
6-15 of gestation, an increase in maternal mortality was observed.
The mortality rates were 0, 10, and 100% respectively. The rats given
1000 mg/kg died on days 9-11 of gestation (Seabaugh et al., 1981).
Sexually mature, timed-pregnant Sprague-Dawley rats, 30 animals
per group, were intubated on days 6-15 of gestation with TBPP (99.7%
TBPP, 0.14% 1,2,3-tribromopropane, and 0.17% 2,3-dibromopropanol) in
undiluted propyleneglycol at levels of 0, 5, 25, or 125 mg/kg body
weight per day. Maternal body weight gain was decreased at the
highest dose level. No effects of treatment were apparent on the
number of corpora lutea, implantations, or early or late deaths.
Furthermore, the percentage of females with resorptions, the number of
viable fetuses, the percentage of resorptions, and the percentage of
pre-implantation losses, did not show compound-related changes. Fetal
body weight and crown-rump length were not affected. Some fetal soft
tissue and skeletal variations found were not dose-related or
statistically significant. It was concluded that TBPP was not
teratogenic in this study (Seabaugh et al., 1981).
Female Wistar rats were exposed orally to 25, 50, 100, or 200 mg
TBPP in olive oil/kg body weight on days 7-15 of gestation. A
significant increase in skeletal variation was found in the fetuses at
200 mg/kg. A significantly lower viability index was observed in the
50 and 100 mg/kg groups. The authors concluded that TBPP did not
produce teratogenic effects in rats. A dose of 200 mg/kg elicited
maternal toxicity (Kawashima et al., 1983).
7.6 Mutagenicity and related end-points
7.6.1 DNA damage
7.6.1.1 In vivo
When male Wistar rats (250-320 g) were given a single ip
injection of 350 µmol TBPP/kg (250 mg/kg) body weight and assayed for
DNA damage 2 h later, single strand breaks/alkali labile sites were
found in the DNA from nuclei isolated from several organs. DNA damage
was detected using an automated alkaline elution system. Extensive
DNA damage was detected in the liver, kidneys, and small intestines.
In addition, substantial DNA damage was found in the brain and lungs;
less DNA damage was detected in the testes, spleen, and large
intestines (Holme et al., 1983; Soderlund et al., 1992). DNA damage
was clearly detectable in the kidneys 20 min after a single ip dose of
36 µmol TBPP/kg (25 mg/kg) body weight (Pearson et al., 1993b).
7.6.1.2 In vitro
Monolayer cultures of human (KB) cells were grown with
[3H]-thymidine for 30 h, and without, for another 17 h. The cells
were then exposed to TBPP (2 µl/ml of growth medium devoid of serum)
for 4.5 h and processed for analysis of the DNA on alkaline-sucrose
gradients. They were re-incubated for various intervals to permit DNA
repair. TBPP was shown to have induced DNA repair, which indicated a
specific action on human cellular DNA. TBPP was found to damage human
DNA in vitro and to cause unscheduled DNA synthesis in human cells
in tissue culture (Gutter & Rosenkranz, 1977; Blum & Ames, 1977).
A semiquantitative, in vitro method for measuring unscheduled
DNA synthesis (UDS) was developed by Lake et al. (1978). Normal
foreskin epithelial cells from a cryopreserved skin pool were grown
from explants and replanted in replicate culture wells. Cultures were
then treated for 3 days in an arginine-deficient medium and further
inhibited in S-phase DNA-synthesis by a 2-h (10 mmol/litre)
hydroxyurea treatment. 3H-Thymidine and TBPP were added
simultaneously and the UDS, accumulated over a 24-h incubation period,
was determined by direct scintillation counting of acid-precipitable
whole-cell radioactivity. TBPP did not induce an UDS response in this
assay, with input dose ranges of 10-99 and 100-400 µg/ml.
UDS was detected in rat liver hepatocytes, grown as monolayer
cultures, exposed to 0.01-0.1 mmol TBPP/litre for 18-19 h in the
presence of [3H]-thymidine and hydroxyurea. UDS was determined by
scintillation counting (Holme et al., 1983; Holme & Soderlund, 1984;
Gordon et al., 1985; Soderlund et al., 1985).
In in vitro test systems, DNA damage was detected in isolated
rat hepatocytes exposed to concentrations as low as 5 µmol TBPP/litre,
while a 10-fold higher concentration was necessary to induce DNA
damage in testicular cells (Soderlund et al., 1992). No DNA damage
was found in cultured Reuber rat hepatoma cells, without the addition
of an exogenous metabolism system (Gordon et al., 1985).
7.6.2 Mutation assay with Salmonella typhimurium strains
Species differences in the bioactivation of TBPP to metabolites,
mutagenic to Salmonella typhimurium TA 100, have been reported.
Liver microsomes from mice (NMRI strain) were more effective in
activating TBPP to mutagenic intermediates than those from
guinea-pigs, hamsters, and rats. Phenobarbitalinduced liver
microsomes from NMRI mice were especially effective (Soderlund et al.,
1982a).
TBPP was activated to mutagens in the Salmonella/microsome
test. S9-fractions from rats pretreated with phenobarbital increased
the mutagenicity of 0.05 mmol TBPP/litre in TA 100 strain compared
with liver microsomes from untreated rats (Holme et al., 1983).
It was demonstrated that the metabolic activation is dependent on
the presence of NADPH and oxygen, which indicates that TBPP is
metabolized by cytochrome P450 enzymes to mutagenic products. In
studies conducted in an anaerobic atmosphere or in the presence of
GSH, the mutagenicity of TBPP was significantly decreased (Soderlund
et al., 1979, 1984).
TBPP (97%) in DMSO was tested in concentrations of 0.0110 µlitre
on Salmonella typhimurium TA 100, TA 1535, TA 1537, and TA 1538,
using the plate assay, in the absence, and presence, of a metabolic
activation system from rat liver. A mutagenic effect was found with
TA 100 and TA 1535 with, and without, metabolic activation. TA 1537
and TA 1538 gave negative results (Blum & Ames, 1977; Brusick et al.,
1978; Prival et al., 1977).
TBPP was tested on Salmonella typhimurium tester strains
TA 1535 and TA 1538 in the absence, and presence, of metabolic
activation derived from Aroclor-induced rat liver. Dose levels of 0,
0.1, and 1.0 µlitre/plate were used. Weak mutagenic activity was
observed in TA 1535 without activation, but a strong effect was seen
with microsomal activation. TA 1538 gave negative results (Carr &
Rosenkranz, 1978).
MacGregor et al. (1980) confirmed the mutagenicity of TBPP in the
Salmonella typhimurium strains TA 100, TA 98, and TA 1535, with dose
levels ranging from 10 to 1000 µg/plate, with metabolic activation.
Without activation, no mutagenicity was found. A negative result was
obtained in strain TA 1537 with, and without, activation.
Nakamura et al. (1979) tested TBPP on Salmonella typhimurium
strains TA 100 and TA 1535 with, and without, metabolic activation at
dose levels of 0.3-100 µmol/plate. A positive effect was seen in both
strains, without and with S9 mix. McCann & Ames (1977) found a
mutagenic effect in Salmonella typhimurium TA 100 with dose levels
up to 100 µg/plate, in the presence of liver S9 fraction of rats
treated with Aroclor.
TBPP at dose levels of 0, 112, 224, 2240, 4480, and
11 200 µg/plate was tested on Salmonella typhimurium strain TA100
with, and without, metabolic activation by Aroclor 1254-induced rat
liver S9 fraction. With the S9 fraction, all dose levels showed a
mutagenic effect. Without the S9 fraction, TBPP showed direct-acting
properties only at dose levels of 2240 µg/plate or more (Salamone &
Katz, 1981).
In an interlaboratory study, TBPP and 62 other chemicals were
tested for mutagenic activity. TBPP was tested on the Salmonella
typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, and on
Escherichia coli WP2uvrA. The dose levels were between 0.3 and
10 000 µg/plate. TBPP was tested without metabolic activation and
with liver S9 fractions from uninduced and Aroclor 1254-induced F344
rats, B6C3F1 mice, and Syrian hamsters. TBPP tested positive in all
four laboratories involved in this study (Dunkel et al., 1985).
Results obtained by Prival et al. (1977) indicated that TBPP
induces mutations of the base-pair substitution type in Salmonella
typhimurium TA100. Although, at higher concentrations
(> 1 µl/plate), TBPP behaves as a direct acting mutagen not requiring
metabolic activation, at a much lower concentration (0.01 µl/plate) it
demonstrates significant genetic activity only with metabolic
activation.
Brusick and coworkers demonstrated that amounts of 50 µg/plate or
more were clearly mutagenic for Salmonella typhimurium TA 100
(Brusick et al., 1980). When tested for bacterial mutagenicity in
Salmonella typhimurium TA 100, a 4-fold interindividual variation in
the capability to activate TBPP was noted with human liver microsomes
prepared from 5 liver donors (Soderlund et al., 1982a).
The CASE structure-activity method was applied to a Gene-Tox
derived Salmonella mutagenicity data base. Strains TA 97, TA 98, TA
100, TA 1535, TA 1537, and TA 1538 with, or without, exogenous
metabolic activation, were used. TBPP was found to be positive
(Klopman et al., 1990).
7.6.3 Mutations by urine of rats treated with TBPP
The urine was collected of rats exposed to TBPP directly by
either the oral or dermal route, or from treated fabric. Salmonella
typhimurium TA 1535 was used as indicator organism. TBPP was
dermally applied at doses of 5, 50, 500, or 5000 mg/kg body weight or
given orally at 5, 50, or 500 mg/kg body weight. In the oral study,
only 500 mg/kg produced a positive response. In the dermal studies, a
dose of 500 mg/kg produced a weak positive response, while 5000 mg/kg
produced a definitive positive response. When fabrics with surface
TBPP levels of 3000, 28 000, and 67 000 mg/kg product were applied
dermally, no mutagenic responses were detected in the urine of the
rats over the 5-day period (data were lacking on whether or not
metabolic activation was used) (Brusick et al., 1978; Ulsamer et al.,
1980).
TBPP at 500 mg/kg body weight in corn oil was applied dermally to
CD-1 mice. Urine was collected over approximately 16 h and the
bacterial mutagenicity of 0.3 ml urine samples was assayed in
Salmonella typhimurium TA1535, TA1537, and TA100. A positive
response was found only with TA100 (Brusick et al., 1982).
7.6.4 Other mutation assays
TBPP was tested in the forward mutation assay with mouse-lymphoma
cells (L5178YTK locus). While the results at lower doses were
inconclusive, a 2 to 3-fold increase in mutations was consistently
produced at 5 mg/litre (Brusick et al., 1978; Ulsamer et al., 1980).
TBPP has been reported to induce increased mutation frequencies
(6-TG resistance) in V79 Chinese hamster cells incubated with
0.02 mmol TBPP/litre in the presence of liver microsomes of rats
pretreated with phenobarbital as an exogenous metabolism system (Holme
et al., 1983; Soderlund et al., 1985). However, in a similar study,
concentrations of TBPP up to 150 µg/ml did not increase the frequency
of 6-TG resistance, both with, and without, an exogenous metabolism
system (Sala et al., 1982).
7.6.5 Chromosomal effects
Using Chinese hamster V79 cells, TBPP severely inhibited the
colony-forming activity and significantly increased sisterchromatid
exchanges, but no significant increase in chromosome aberrations was
found (Furukawa et al., 1978). Interestingly, chromosomal aberrations
were not significantly increased in Chinese hamster cells, in mouse
bone-marrow cells, or in cultured human lymphoid cells. The lack of a
TBPP effect on rat bone-marrow chromosomes was also observed after
rats received 25, 250, or 2500 mg TBPP/kg body weight, by gavage,
after either a single dose, or, 5 daily doses/week for 13 weeks
(Osterberg, 1977; Nakanishi & Schneider, 1979).
TBPP was tested for the induction of chromosome aberrations, and
sister chromatid exchanges in the diploid human fibroblastic cell line
HE 2144 (from a 10-week-old male embryo) without metabolic activation
(Sasaki et al., 1980). The dose levels used were 0.349, 0.070, and
0.035 mg/ml. Sister chromatid exchanges were induced with 0.070 mg
TBPP/ml in the human HE 2144 cell line. No chromosomal aberrations
were found.
In a comparative study, Brusick and coworkers found that TBPP
gave a positive response in tests for sister chromatid exchanges and
chromosomal aberrations in the mouse lymphoma L5178Y cell line at
concentrations of 0.005 and 0.01 µlitre/ml, respectively (Brusick et
al., 1980).
TBPP was tested in an in-vitro test for sister chromatid
exchanges in Chinese hamster V79 cells with, and without, S9 fraction
of livers of Wistar rats administered (ip) methylcholanthrene.
Acetone was used as solvent. The dose levels 17.2, 35, 100, and
200 µg/ml were tested only without S9 fraction, while levels of 24.5
and 50 µg/ml were tested with, and without, metabolic activation. A
significant increase in sister chromatid exchanges was found at dose
levels of more than 35 µg/ml (Sala et al., 1982).
Two male and two female Chinese hamsters per group were used in a
micronucleus test. The dose levels were 200, 400, and 800 mg TBPP/kg
body weight administered by ip injection. The solvent was DMSO.
Bone-marrow samples were obtained after 24 h. Two thousand
polychromatic erythrocytes/animal were analyzed for the presence of
micronuclei. Levels of 400 and 800 mg/kg body weight showed a
positive effect (Sala et al., 1982).
Salamone & Katz (1981) studied the clastogenic effect of TBPP in
a bone marrow micronucleus test. B6C3F1 mice (15 weeks old) were
given two ip treatments of TBPP in corn oil. Dose levels of 0, 204,
408, 612, 816, 1020, 1275, and 1530 mg/kg body weight were tested. In
this test, TBPP showed a weak clastogenic effect.
An in vitro chromosome aberration test was carried out with
TBPP, using a Chinese hamster CHL cell line of lung fibroblast origin.
CHL cells cultured in plates were exposed to different dose levels of
TBPP including the 50% growth inhibition dose. The number of
polyploid cells and cells with structural aberrations, such as
chromatid-type gaps, breaks, exchanges, and rings, were scored. A
microsome fraction (S9-mix) from the liver of Wistar rats, pretreated
with the PCB; KC-400, was used. TBPP was positive in this test. A
dose level of 0.25 mg/ml showed chromosomal aberrations in 20% of the
metaphases (Ishidate et al., 1981).
Vogel & Nivard (1993) studied the effects of TBPP in the
(white/white+) (w/w+) eye mosaic assay, and an in vivo, short-term
test measuring genetic damage in the somatic cells of Drosophila
melanogaster, after treatment of the larvae. The genetic principle
of this system is the loss of heterozygosity for the wild-type
reporter gene w+, an event predominantly resulting from homologous,
interchromosomal, mitotic recombination between the two X-chromosomes
of female genotypes. The w/w+ eye mosaic test detects a broad
spectrum of DNA modifications. Between 12 and 15 pairs of flies were
permitted to lay eggs for three days on food supplemented with 0.25,
0.5, or 1.0 mmol TBPP/litre (dissolved in 3% ethanol). TBPP gave a
strong positive response in the w/w+ bioassay.
7.6.6 Cell transformation
TBPP was tested for its ability to induce malignant
transformation in vitro using mouse BALB/3T3 cells. The results of
this test showed that TBPP can transform mammalian cells in vitro,
perhaps indicating a potential for the induction of carcinogenic
responses (Brusick et al., 1978; Ulsamer et al., 1980).
C3H/10T1/2 cells were treated with TBPP, with or without S9 mix
from the liver of Wistar rats administered methylchloanthrene
intraperitoneally. Some cell samples were additionally treated
several times with tetradecanoyl phorbolacetate (TPA) (0.1 µg/ml).
The TBPP concentrations tested were 40 µg/ml (with and without S9
fraction) and 80 µg/ml (without S9 fraction). A very low frequency of
transformed type 3 foci was obtained and the authors considered the
results of this study to be negative (Sala et al., 1982). Dunkel et
al. (1988) also found a negative result for TBPP in the C3H/10T1/2
cell transformation assay. The dose levels tested were between 0.16
and 20 µg/ml.
7.6.7 Miscellaneous tests
TBPP induced a significant increase in sex-linked recessive
lethal mutations in male germ-cell stages of Drosophila melanogaster
at a dose of 1000 mg/kg. The spermatids were