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The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and its metabolite 2-bromoacrolein (2BA) are very potent bacterial mutagens in Salmonella typhimurium (S. typhimurium) TA 100. In this study, we showed that 2BA and Tris-BP are also mutagenic in S. typhimurium TA 104, which detects mutations at AT base pairs, while TA 100 detects mutations at CG basepairs. We also studied the mutagenicity of 2BA in mammalian cells in vitro and in the rat in vivo. Firstly, 2BA was tested in the human lymphoblastoid cell line TK6. The results showed that there was no increase in mutation frequency at the hprt locus, whereas there was a large decrease in cell survival. Secondly, a shuttle vector system was used to study the induction of mutations by 2BA:DNA adducts. The vector was modified by insertion of a single-stranded oligonucleotide containing on average one 2BA:DNA adduct. No increase in mutation frequency above background was detected after replication of this vector in SV40 transformed normal human fibroblasts. Because the liver is a major site for bioactivation of Tris-BP to 2BA in vivo, we tested the initiating capacity of Tris-BP in the rat liver in a modified Solt & Farber initiation and promotion system. Administration of Tris-BP resulted in a small increase in the number of preneoplastic gamma-glutamyl-transpeptidase positive (GGT+) foci in the liver compared to control animals (only significant in the lowest size class). Modification of the experimental protocol by performing partial hepatectomy 24 h after the administration of Tris-BP, did not increase the number of GGT+ or glutathione S-transferase-P (GST-P+) positive foci above the control level. Taken together, these results indicate that, in spite of a high mutagenicity in S. typhimurium, 2BA and Tris-BP have low or negligible mutagenic effects in mammalian systems. The lack of mutagenic activity may explain why Tris-BP is not a carcinogen in the rat liver.

The metabolism of tris(2,3-dibromopropyl) phosphate (Tris-BP) was compared with that of completely deuterated Tris-BP (D15-Tris-BP) in an isolated, recirculating rat liver perfusion system in order to determine the relative quantitative importance of two different biotransformation pathways of Tris-BP: (i) cytochrome P450-mediated metabolism and (ii) GSH S-transferase-mediated metabolism. To accomplish this we quantitated the biliary excretion of S-(3-hydroxypropyl)glutathione (GSOH) as a marker metabolite for cytochrome P450-mediated metabolism and that of S-(2,3-dihydroxypropyl) glutathione (GSOHOH) as a marker metabolite for GSH S-transferase-mediated metabolism. Complete deuterium substitution of Tris-BP significantly decreased the formation of GSOH, whereas there was no effect on the formation of GSOHOH. Because our previous studies showed a large decrease in genotoxicity of D15-Tris-BP compared to Tris-BP, the present results support our hypothesis that cytochrome P450-mediated metabolism is responsible for the genotoxic effects of Tris-BP in the rat liver.

1. The genotoxicity of the flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) was studied in vivo. Results showed that Tris-BP was highly clastogenic, but it could only initiate a low number of preneoplastic foci in the rat liver in vivo. In Drosophila, Tris-BP could be classified as a cross-linking agent, because it was more clastogenic than mutagenic. The use of completely deuterated Tris-BP as a metabolic probe revealed that cytochrome P450 and most likely the formation of 2-bromoacrolein (2BA) from Tris-BP is important for the observed genotoxic effects. 2. In contrast to the high mutagenicity of Tris-BP and 2BA in Salmonella typhimurium, we were unable to detect an increase in mutation frequency of 2BA on the hprt locus of human TK6 cell line. In another system, using a shuttle vector modified with 2BA:DNA-adducts, also no increase in mutation frequency could be detected in human cells. This low mutagenicity of 2BA corresponds with its low mutagenicity in Drosophila and its low induction of preneoplastic foci in the rat liver. 3. Several DNA adducts of 2BA have been identified, including an unstable 3-(bromooxypropyl)thymidine adduct which has the potential to form cross-links and a cyclic 3,N4-(bromo)propeno-deoxycytidine adduct which can possibly be involved in the clastogenicity of Tris-BP. 4. Taken together, these data indicate that Tris-BP and 2BA may not effectively induce gene mutations in eukaryotic systems, but rather be potent clastogens. Risk assessment of these and related compounds should therefore be based on the knowledge of clastogens rather than mutagens.

The bacterium Sphingomonas sp. strain SS33, obtained from parent diphenyl ether-mineralizing strain SS3 (S. Schmidt, R.-M. Wittich, D. Erdmann, H. Wilkes, W. Francke, and P. Fortnagel, Appl. Environ. Microbiol. 58:2744-2750, 1992) after several weeks of adaptation on 4,4'-difluorodiphenyl ether as the new target compound, also utilized 4,4'-dichlorodiphenyl ether for growth. Intermediary halocatechols were also mineralized via the ortho pathway by type I enzymes. 4,4'-Dibromodiphenyl ether was not used as a carbon source although transformation by resting cells yielded mononuclear haloaromatic compounds, such as 4-bromophenol and 4-bromocatechol. The same was true for the conversion of 2,4-dichlorodiphenyl ether, which yielded the respective (halo-) phenols and (halo-) catechols.

Metabolism studies were conducted using Fischer 344 and Sprague-Dawley rats following inhalation exposure to 1.0% (v/v) air atmospheres of 1,1-dichloro-2,2,2-trifluoroethane (HCFC-123), 2-chloro-1,1,1,2-tetrafluoroethane (HCFC-124), 1-chloro-1,1-difluoroethane (HCFC-142b), bromochlorodifluoromethane (Halon 1211), and perfluorohexane (PFH) for 2 h. There were no remarkable differences in results between the two strains of rats. Animals exposed to HCFC-123 or HCFC-124 excreted trifluoroacetic acid in their urine. Urinary fluoride concentrations were increased in rats exposed to HCFC-124, and urinary bromide levels were increased in rats exposed to Halon 1211. Small quantities of volatile metabolites 2-chloro-1,1,1-trifluoroethane (HCFC-133a) and 2-chloro-1,1-difluoroethylene were observed in the livers of rats exposed to HCFC-123. Rats exposed to HCFC-142b excreted chlorodifluoroacetic acid in their urine; no volatile metabolites were detected in tissue samples. For PFH studies, no metabolites were detected in the urine or tissues of exposed animals. These results are consistent with proposed oxidative and reductive pathways of metabolism for these chemicals. Pharmacokinetic studies were carried out in rats exposed by inhalation to 1.0%, 0.1%, or 0.01% of HCFC-123. Following exposure, blood concentrations of HCFC-123 fell sharply, whereas trifluoroacetic acid levels rose for approx. 5 h and then declined gradually. Using a physiologically based pharmacokinetic model, saturation of HCFC-123 metabolism was estimated to occur at approx. 0.2% (2000 ppm) HCFC-123.

Previous studies indicate that tris(2-chloroethyl)phosphate (TCP) preferentially produces hippocampal brain lesions in female versus male rats, and the expression of these lesions is inversely related to the in vivo rate of TCP metabolism. In the present studies, TCP (0.17 mM in all incubations) was metabolized in vitro by liver slices and microsomes from human and Fischer 344N rat liver to bis(2-chloroethyl) hydrogen phosphate (BCP), 2-chloroethanol (CE), and three unidentified metabolites. The rate of TCP metabolism by male rat liver microsomes and liver slices was 0.049 nmol/min/mg protein and 2.53 nmol/min/g liver, respectively. TCP metabolism by male rat liver microsomes was inhibited by 10 microM diisopropyl fluorophosphate, 10 microM paraoxon and carbon monoxide. TCP did not appear to be metabolized by female rat liver microsomes, but female rat liver slices metabolized TCP at a rate of 1.51 nmol/min/g liver. TCP was metabolized by male and female rat plasma at a rate of 0.156 and 0.169 nmol/ml plasma, respectively. TCP was metabolized by male and female human liver microsomes at a rate of 0.027 and 0.031 nmol/min/mg protein, respectively. TCP was metabolized by male and female human liver slices at a rate of 1.37 and 1.82 nmol/min/g liver, respectively. BCP and CE were the major metabolites formed in all studies, except for liver slices and microsomes from two human male subjects in which an unidentified metabolite constituted 29 to 38% of the total TCP metabolism. TCP was not metabolized by plasma or whole blood from male or female human subjects. These results support the previously reported sex-specific difference in TCP metabolism by male and female Fischer 344N rats. However, no sex-specific difference in rates of TCP metabolism by male and female human liver microsomes or slices was observed.

Tris(2-chloroethyl) phosphate (TRCP), a flame retardant, produces a dose-, sex-, and species-dependent lesion in the hippocampal region of the brain following subchronic oral administration. This lesion is more common and more severe in female F344 rats than in male F344 rats, and is not observed in B6C3F1 mice. The present investigation of the metabolism of TRCP was designed to detect sex and species variations that might account for differences in toxicity. Elimination of TRCP-derived radioactivity was more rapid in mice, which excreted greater than 70% of an oral dose of 175 mg/kg in urine in 8 hr vs. approximately 40% for male or female rats. However, the metabolic profile of TRCP-derived radioactivity in urine was similar for both species. The major metabolite in female rat urine was identified as bis(2-chloroethyl) carboxymethyl phosphate. This metabolite co-chromatographed with the major metabolite found in both male rat and mouse urine. Two additional metabolites identified in female rat urine were bis(2-chloroethyl) hydrogen phosphate and the glucuronide of bis(2-chloroethyl) 2-hydroxyethyl phosphate. These metabolites also cochromatographed with metabolites found in male rat and mouse urine. TRCP metabolism in rats was not induced or inhibited by nine daily 175 mg/kg doses. Toxicity, as evidenced by seizures, was potentiated in male rats pretreated with inhibitors of aldehyde dehydrogenase.

The metabolism, distribution, and excretion of the flame retardant, tris(1,3-dichloro-2-propyl) phosphate (TDCP), were studied in the rat. Five days after iv administration of 14C-TDCP, 92% of the administered radiolabel had been excreted in the urine (54%), feces (16%), and expired air (22% as 14CO2); 4% was recovered in the body. The major urinary, fecal, and biliary metabolite was identified as bis(1,3-dichloro-2-propyl) phosphate (BDCP). In disposition studies performed 5 days after iv administration of TDCP, 63% of the administered phosphate was recovered as BDCP. Other metabolites included the mono ester, 1,3-dichloro-2-propyl phosphate, and the bacterial mutagen, 1,3-dichloro-2-propanol. Less than 0.1% of the administered radiolabel was recovered as TDCP in the excreta. Kinetic and distribution studies demonstrated that TDCP was eliminated primarily by rapid metabolism whereas the slowly metabolized BDCP was eliminated by excretion. A major portion of the radiolabel excreted in bile underwent enterohepatic recirculation. In the presence of phenobarbital-induced liver homogenates, TDCP was mutagenic to Salmonella typhimurium (TA100), whereas its major metabolite, BDCP, and a minor metabolite, 1,3-dichloro-2-propyl phosphate, were nonmutagenic. Another metabolite, 1,3-dichloro-2-propanol, was mutagenic in the same system without liver homogenate activation.

Radiolabelled 2,2',4,4',5,5'-hexabromobiphenyl was metabolized in vitro by rat liver microsomal enzymes to give more polar ether soluble lipophilic metabolites, trichloroacetic acid soluble conjugates and a macromolecular adduct fraction. The rates of formation of the three metabolic fractions were significantly enhanced using Firemaster BP-6 and 2,2',4,4',5,5'-hexabromobiphenyl induced microsomal enzymes. Comparative metabolic studies with the 4-brombiphenyl substrate showed that the lower brominated biphenyl substrate was more readily metabolized and the rate of metabolism was enhanced only with the Firemaster BP-6 induced microsomal enzymes and not the 2,2', 4,4',5,5'-hexabromobiphenyl induced enzyme system.

The metabolism and disposition of the flame retardant, tris(2,3-dibromopropyl)phosphate (Tris-BP), were studied after po and iv administration of the 14C-labeled compound to the male rat. Tris-BP was readily absorbed from the gastrointestinal tract and rapidly distributed throughout the body. The distribution and excretion of Tris-BP derived radioactivity were similar after either po or iv administration. The only effects of route of administration on tissue distribution were slightly higher concentrations in liver after po administration and in lung after iv administration. The initial elimination of Tris-BP derived radioactivity in urine, feces, and as CO2 accounted for approximately 50% of the dose in 24 hr. An analysis of Tris-BP derived radioactivity remaining in the tissues one day after administration indicated that most of the radioactivity in all tissues was in the form of various metabolites rather than the parent compound. The terminal clearance of Tris-BP derived radioactivity from most of the tissues studied was best described by a single component exponential decay with a half-life of approximately 2.5 days. Clearance from liver and kidney was somewhat slower having a half-life of approximately 3.8 days. Approximately 33% of the radioactivity excreted in urine and approximately 50% of the radioactivity excreted in bile were identified by cochromatography with synthesized standards on high performance liquid chromatography (HPLC). Six metabolites and a trace of the parent compound were identified in urine and bile by this method. The six metabolites products of dealkylation and dehydrobromination of the parent compound. The metabolites of Tris-BP isolated from urine and bile were also formed in vitro by NADPH-dependent microsomal enzymes from rat liver. The soluble enzymes from liver metabolized Tris-BP to at least three unidentified polar metabolites.

Tris(2,3-dibromopropyl)phosphate (Tris-BP) was found to be metabolized by liver microsomes obtained from untreated and phenobarbital-pretreated rats. Metabolites of Tris-BP, whose formation was dependent on NADPH and oxygen, included bromide ion and bis(2,3-dibromopropyl)phosphate (Bis-BP). The rates of formation of these metabolites were markedly increased in liver microsomes isolated from phenobarbital-pretreated rats compared to microsomes from untreated rats. In the presence of either SKF 525-A or metyrapone, the formation rates of bromide ion and Bis-BP were decreased, whereas alpha-naphthoflavone had no effect. The effects of the various treatments on bromide release and Bis-BP formation paralleled those that have been previously observed with respect to the activation of Tris-BP to mutagenic and covalently protein bound metabolites. Furthermore, rates of oxidative debromination of several Tris-BP analogs directly correlated with their respective mutagenicities. Addition of glutathione (GSH) to microsomal incubations of Tris-BP increased bromide release substantially over control, values but had no effect on Bis-BP formation. On the other hand, the addition of GSH to microsomes decreased covalent binding and mutagenicity of Tris-BP with increased formation of water soluble metabolites. GC/MS analysis of ethyl acetate extracts from incubations of rat liver microsomes with Tris-BP identified 2-bromoacrolein (2-BA) as a metabolite. Introducing deuterium at the carbon atom number 1 of the propyl moiety of Tris-BP had no effect on either bromide release or mutagenicity, whereas the analog labelled at carbon atom 3 showed significant isotope effects on both activities. In contrast, deuterium substitution at carbon atom 2 gave a significant isotope effect on bromide release, but not on mutagenicity. The data indicate that Tris-BP can be metabolized by rat liver microsomes to Bis-BP and 2-bromoacrolein catalyzed by cytochrome P-450 in a process liberating bromide ions. Further, the results are consistent with oxidation at the terminal carbon atom of Tris-BP thereby forming 2-bromoacrolein, which is postulated to be the metabolite mainly responsible for Tris-BP mutagenicity.

Surface tris-(2,3-dibromopropyl) phosphate (TRIS) was removed from flame-retarded polyester fabric by benzene-hexane extraction and replaced with 14C-TRIS. Sections of the radiolabeled fabric (10 X 12 cm) were placed in contact with the clipped skins of rabbits, and urine and feces were collected over a 96 hr period. The cloths were allowed to remain dry or were moistened with either urine or simulated sweat. A similar collection of urine and feces was performed following i.v. injection of 14C-TRIS. From the results of these experiments, it can be calculated that up to 17 percent of the radiolabel on the cloth penetrated rabbit skin over the 96 hr period of exposure. Most of the excreted radiolabel appeared in the urine, with the kidney being the organ of highest specific radioactivity. The highest absorption of radiolabel occurred in the urine-moistened cloth group. Moistening the cloth with simulated sweat produced no increase in absorption over that seen with dry cloth.

The potent mutagen 2- bromoacrolein is formed from the carcinogenic flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) on incubation with hepatic microsomes. Substitution of deuterium for hydrogen at the terminal carbon atoms (C-3) of Tris-BP significantly decreased both the mutagenic response and the formation rate of 2- bromoacrolein . Mass spectral analysis of the 2- bromoacrolein that was formed from the selectively deuterated analogs of Tris-BP revealed that the primary mechanism for the formation of 2- bromoacrolein involves an initial oxidative dehalogenation at C-3 followed by a beta-elimination reaction.

The concentration of polybrominated biphenyl (PBB) in serum in a large number of organs was determined in a population of rats for 36 weeks a single dose of PBB. Groups were killed at 6, 12, 24 and 36 weeks after exposure to PBB (1 mg/100g body wt, i.p.). Growth, weight gain and appearance of the rats and their internal organs were normal. Complex and varied relationships were found in tissue concentrations with time after PBB administration. Serum and fat had apparent first-order elimination kinetics with calculated half-times of 23.1 and 69.3 weeks, respectively. For five other tissues, apparent t 1/2s ranged from 9.0-63 weeks, while for four others, kinetics could not be determined from these 4 time points. It is likely that a substantial residue of PBB will still remain in the body of the rat at the end of its life span because of the persistence of PBB in lipid-rich tissues (adipose, adrenal, and brain).

The flame retardant, tris(2,3-dibromopropyl)phosphate (tris-BP), which is a mutagen and causes cancer and sterility in animals is absorbed from fabric by people. 2,3-Dibromopropanol, a metboloite of tris-BP and a mutagen itself, has been found in the urine samples of ten children who were wearing or who had worn tris-BP-treated sleepwear. Eight of these children were wearing well-washed sleepwear and the possibility of absorption of tris-BP from well-washed sleepwear discussed. 2,3-Dibromopropanol was not found in the urines of one child and one adult who had never worn tris-BP-treated garments.

The intestinal absorption, distribution, and excretion of the major component of Firemaster BP-6,2,4,5,2',4',5'-hexabromobiphenyl, has been studied in the male rat. This polybrominated biphenyl was readily absorbed from the intestine, initially distributed throughout the body, and eventually stored primarily in the adipose tissue, was not subject to appreciable metabolism, and was excreted almost exclusively in the feces and at a very slow rate. Approximately 90% of an oral dose was absorbed from the intestine, and extrapolation of the rate of excretion to infinity indicates that less than 10% of the total dose would ever be excreted.