Abstract  Five selectively methylated analogs of the flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and of the nematocide 1,2-dibromo-3-chloropropane (DBCP) were synthesized and their relative mutagenicities determined in Salmonella typhimurium TA100 in the presence of rat liver microsomes. In all cases, methylation decreased mutagenicity relative to the parent compound, but the relative degree of reduced mutagenicity varied considerably depending on the position of the methyl substitution. The mutagenicity studies with the selectively methylated analogs and with suspected mutagenic metabolites (2-bromocrotonaldehyde and methyl 1-dibromovinyl ketone) supported earlier work with selectively deuterated analogs of Tris-BP and DBCP. They demonstrated that initial oxidation at C-3, followed by spontaneous dehydrohalogenation and dehydrophosphorylation, was the major route of formation of mutagenic metabolites from Tris-BP. In the case of DBCP, formation of mutagenic metabolites can result following initial oxidation at either C-1 or C-3.

Abstract  HEEP COPYRIGHT: BIOL ABS. RRM HUMAN DI BROMOCHLORO PROPANE TESTICULAR TOXICITY SPERM COUNT

Abstract  Twenty chemicals were tested for their ability to induce sister chromatid exchanges (SCEs) and chromosomal aberrations (ABs) in cultured Chinese hamster ovary cells (CHO). These chemicals were tested with and without an added metabolic activation system (rat liver S9 fraction). Four chemicals were negative in both assays, 1 induced ABs only, and 15 were positive for SCEs; 6 of these 15 also induced ABs. The effect of cell harvest time on the ability to detect the induction of chromosomal aberrations was examined for six chemicals. Five of these had caused at least one of the following: cell cycle delay, aberrations observed in first division metaphase cells in the SCE assay, or a weak response in the standard AB assay (10-12-hr growth period). Three chemicals, chlorinated tri-sodium phosphate, 1, 2-dibromo-3-chloropro-pane, and tetrakis(hydroxymethyl)phosphonium chloride, were positive using both the standard and extended harvest times. N-Nitrosodimethyl-amine and diphenhydramine HCI were only positive using an extended harvest time, and malo-naldehyde was negative using both standard and extended harvest times.

Abstract  The agriculturally important nematocide 1,2-dibromo-3-chloropropane (DBCP) has been implicated as a cause of human male sterility. A survey at the Michigan Division of The Dow Chemical Company included measurements of semen samples, testicular size, and serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone in 232 chemical workers with past potential exposures to DBCP and in 97 nonexposed comparison employees. Potentially exposed groups showed significantly higher, although not abnormal, mean levels of FSH and LH. In the subgroup with the highest potential exposure ending subsequent to 1972, greater duration of exposure correlated with lower sperm count, higher FSH level, and smaller testicular volume. Mean values for this latter time-divided subgroup were not abnormal. The findings are consistent with a testicular effect of DBCP and also with reversibility of that effect over time.

Abstract  Dibromochloropropane (DBCP) was administered orally to 36 sexually mature male Dutch Belted rabbits assigned at random to one of six groups to receive 0, 0.94, 1.88, 3.75, 7.5, or 15.0 mg DBCP per kilogram of body weight daily 5 days per week during a 69-day treatment period. Animals were euthanized and necropsied on Day 70. Body weights and weights of the kidneys, liver, epididymides, and accessory sex glands were not influenced by DBCP treatment. The highest dosage reduced mean paired testes weight to 45% of control values (p less than 0.01). Mean seminiferous tubular diameter was reduced by 15 and 29% with the two highest dosages of DBCP, respectively (p less than 0.01), and the percentage of seminiferous tubules containing elongating and round spermatids, as the most advanced cell type present, was decreased (p less than 0.01). The number of leptotene primary spermatocytes per Sertoli cell, and the numbers of spermatogonia, young and old primary spermatocytes, and Step 1 spermatids per Stage I seminiferous tubular cross section likewise indicated a general depression (p less than 0.01) of spermatogenesis. Based upon the number of Step 1 spermatids per cross section at Day 70, production of sperm at this time by rabbits receiving the highest dose of DBCP was estimated to be less than one-fourth of control values. No significant effect (p greater than 0.05) of 0.94 mg DBCP/kg on any parameters was found, so this could be considered to be the no effect dose. However, the regressions and mean values are interpreted to indicate that the no effect level of DBCP may be less than 0.94 mg DBCP/kg of body weight. It is suggested that the most sensitive tests described here be included in future screening tests for potential effects of agents on male reproduction.

Abstract  Male pesticide applicators who worked with the nematocide dibromochloropropane (DBCP) were examined to determine the possible testicular toxicity from this exposure. Infertility and azoospermia which were first noted among factory workers exposed to DBCP were not observed among the applicators. Sperm count depression, however, was associated with the duration of exposure in the current year but not with exposure in past years. The extent of exposure to DBCP in the current year was also associated with an elevation of serum follicle stimulating hormone (FSH) but not of luteinizing hormone (LH). Sperm count depression was limited to applicators involved in irrigation setup work and in the calibration of equipment. These results suggest that the testicular toxicity of DBCP for men may occur in a shorter period than was previously reported, that the effect may be reversible in men with mild sperm count depression, and that public health measures might be directed at controlling specific application techniques.

Abstract  The soil fumigant 1,2-dibromo-3-chloropropane (I) undergoes hydrolysis in the rat to a series of epoxide metabolites. Alkylation of glutathione by these epoxides produces 2 urinary metabolites identified as the mercapturic acids VI (R=COCH3) and VII (R-COCH3). Hydrolysis of the epoxides produces the male antifertility agents alpha-chlorohydrin (IX, X-Cl) and alpha-bromohydrin (IX, X-Br) which are oxidatively metabolized to oxalic acid (XII), thus causing renal damage. These metabolic pathways can explain the toxic nature of the fumigant as a carcinogen, a male chemosterilant and as an agent causing kidney damage.

Abstract  A method has been developed for monitoring industrial workers and others exposed to environmental agents which may impair fertility. National birth probabilities specific for maternal birth cohort, age, parity, and race are used to derive expected fertility. Observed fertility is obtained by questionnaire. Standardized fertility ratios are computed for exposure and non-exposure periods and compared. The analytic techniques have been validated by applying the method to a group of 36 male factory employees working in an agricultural chemical division (ACD) where pesticides including the nematocide dibromochloropropane were formulated. Twelve of these employees in mid-1977 had been discovered to have severely depressed sperm counts related to occupational exposure. The standardized fertility ratio (SFR) computed from data available in mid-1977 for the period at risk from employment in the ACD (SFR = 0.75) was significantly lower than those derived for the entire not-at-risk period (SFR = 1.88) and the portion related to employment in other areas of the factory (SFR = 2.16). Similar differences also were evident from data available several years earlier, demonstrating that the surveillance technique would have been capable of detecting occupationally induced infertility among these workers in advance of the actual discovery date.

Abstract  This study represents the largest series to date documenting the gonadotoxic effect in humans of dibromochloropropane, a widely used pesticide. Three semen analyses, serum hormonal determinations (luteinizing hormone, follicle-stimulating hormone and testosterone) as well as genital examinations were completed for 228 workers at 2 chemical production sites and consisting of a dibromochloropropane-exposed and non-exposed cohort. Parameteric and non-parametric statistical analyses of the data sets of the sperm densities from the 2 subpopulations demonstrated statistical significance (p less than 0.10) at the short-term (1.5 years) manufacturing plant. Log transformation of the sperm count and hourly exposure data were necessary to develop meaningful statistical conclusions. The serum concentration of follicle-stimulating hormone as a group mean was significantly greater at both production sites for the exposed cohort when compared to the non-exposed participants but decreased by 10 levels of magnitude when the group demonstraing shorter but more recent exposure was compared to those from the plant with longer chemical production. Finally, a dose-response model suggested significant changes in sperm density at the short-term but more recently operated production site when more than 100 adjusted hours of exposure were exceeded, while the longer operated but longer closed facility demonstrated a significant impairment only when more than 1,000 adjusted hours of dibromochloropropane exposure were surpassed. This difference in exposure data may reflect regenerative changes in the tests once the gonadotoxic substance had been removed but exact nature of the dibromochloropropane effect and the possibility of a "no effect" concentration remain to be defined clearly.

Abstract  Although the mutagenic effect of Dibromochloropropane (DBCP) on experimental mammal systems has been described, its possible effect on the human genome has not yet been investigated. The present study describes the results of chromosomal analysis and health evaluation of offspring conceived to families during and after paternal exposure to DBCP. Ten children conceived during or following severe exposure and four who were conceived prior to DBCP exposure were evaluated. The chromosomal constitution of peripheral lymphocytes was normal in all cases. The mode of delivery, birth weight, physical examination and growth pattern were normal. No congenital malformations were detected. One spontaneous abortion out of 23 pregnancies was recorded. These results suggest that paternal exposure to DBCP, severe enough to cause azoospermia or oligozoospermia did not alter the paternal sperm genome or the chromosomal constitution of offspring conceived during or after exposure. This is further supported by the excellent health and lack of malformations among the children, along with the low rate of spontaneous abortions in the families studied.

Abstract  Groups of 10 male New Zealand white rabbits were exposed by inhalation to 0, 0.1, 1.0 or 10 ppm of 1,2-dibromo-3-chloropropane (DBCP) vapor for 6 hours/day, 5 days/week for 14 weeks, except that the 10 ppm group was exposed for only 8 weeks due to mortality. The semen of rabbits was evaluated on a weekly basis during the exposure period and at periodic intervals during a recovery period (32 weeks for all groups except the 10 ppm groups which was for 38 weeks). In order to assess the fertility of the exposed rabbits, each male was allowed to mate with an unexposed female at the 14th and 41st week of the study. Exposure of rabbits to 1 and 10 ppm of DBCP by inhalation produced adverse reproductive effects as well as decreases in sperm count, motility and viability. Rabbits treated at 1 and 10 ppm had decreased sperm counts between the 8th and 14th weeks of the study. All of the 10 ppm rabbits were infertile when mated during the 14th week. The effects of DBCP on spermatogenesis were shown to be essentially reversible in rabbits exposed to 1 ppm; however, at 10 ppm, recovery was not complete under the conditions of the test. Rabbits exposed to 10 ppm had severe testicular alterations as early as 4 weeks into the study and these progressed to severe testicular atrophy by 8 weeks. Those exposed to 1 ppm for 14 weeks developed moderate testicular atrophy (approximately 50% reduction in size). Following the recovery period, the rabbits in the 10 ppm group had evidence of partial reversibility of the testicular atrophy. Electron microscopic evaluation of testicular tissue confirmed findings by light microscopy effects and also indicated increased numbers of abnormal sperm within the seminiferous tubules of rabbits at both the 10 and 1 ppm exposure levels. Those exposed to 0.1 ppm had an equivocal increase in abnormal sperm after the 14-week exposure period but not after the recovery period. Based on these results 0.1 ppm level of DBCP is considered as a no effect level for reproductive parameters.

Abstract  The nematocide DBCP (1,2-dibromo-3-chloropropane) produced negative results in a specific-locus test for gene-mutation induction in the germline of male (101 X C3H)F1 mice, most of which were treated with 5 daily intraperitoneal injections of 80 mg/kg (total exposure, 400 mg/kg); a few received lower exposures. For treated spermatogonial stem cells, the finding of 2 mutations among 39519 offspring--a rate almost identical to the control rate--rules out (at the 5% significance level) an induced mutation frequency greater than 2.0 times the historical control rate. From treated poststem-cell stages, no mutants were found among 6240 offspring, ruling out (at the 5% significance level) a multiple of 8.0 times the control for these cell types. A multiple rearrangement (7 chromosomes involved in 3 translocations) found in one of the mutants probably arose as a postmeiotic event not associated with the DBCP treatment. The fertility of DBCP-treated males was not disturbed, in keeping with the absence of germ-cell toxicity and dominant lethals found by other investigators in these mice, and in contrast to results in certain other species. While the treated (101 X C3H)F1 mice are Ah-responsive, other findings make it questionable whether biotransformation of DBCP to reactive intermediates is accomplished via the Ah-receptor system.

Abstract  The use of indirect standardization in the assessment of the fertility of occupationally exposed workers is briefly reviewed and critiqued. The calculation of expected births in the method of Levine et al. (J Occup Med 1980;22:781-91) is modified to eliminate negative bias. An example is given using data from a 1977 survey of 60 male workers at a chemical manufacturing plant in Denver, Colorado, who were exposed to dibromochloropropane. The example illustrates how in-plant nonexposed reproductive experience provides a valuable supplement to US fertility tables which are specific only to race, birth cohort, age, and parity. It is also shown, however, that explicit control for potential confounding factors not included in the tables, such as marital status and surgical sterilization, can actually create rather than alleviate confounding error. This occurs when the additional factors co-vary in the reference population with the factors already included in the tables. For martial status, the control-induced error was readily minimized by restricting analysis to married experience at parity one or greater. For surgical sterilization, the corresponding error could not be reduced without severely compromising sample size, and hence control of this potential confounder in similar circumstances is not recommended.

Abstract  The risk assessment process is an imprecise procedure aimed at determining a toxicant exposure level with an acceptable risk to the human population. The lack of precision is due to the uncertainties in the assumptions that must be made due to the lack of specific scientific information or knowledge of how to use certain types of data. Unfortunately, every necessary piece of information cannot be obtained for every chemical requiring a risk assessment. In order to better identify and understand some of the assumptions that are made in the risk assessment of reproductive and developmental toxicants, a workshop was organized to specifically define the assumptions underlying the risk assessments for seven specific toxicants (dibromochloropropane, dioxin, glycol ethers, heptachlor, lead, tetrahydrocannabinol, and vitamin A) and to determine the potential research which would reduce the uncertainty associated with making those assumptions. The major assumptions discussed centered around the topics of heterogeneous populations, thresholds, safety factors, exposure assessment, quantitative structure-activity relationships, and mechanisms. This report is the summary of the workshop discussions.

Abstract  Testicular cells prepared from human organ transplant donors or from Wistar rats were used to compare 15 known reproductive toxicants with respect to their ability to induce DNA damage, measured as single-strand DNA breaks and alkali labile sites (ssDNA breaks) with alkaline filter elution. The compounds tested included various categories of chemicals (i.e., pesticides, industrial chemicals, cytostatics, and mycotoxins) most of which are directly acting genotoxicants (i.e., reacting with DNA either spontaneously or via metabolic activation). In addition, a few indirect genotoxic and nongenotoxic reproductive toxicants were included. Six of the chemicals induced no significant levels of ssDNA breaks in human and rat testicular cells: methoxychlor (10 to 100 ÁM, human and rat), benomyl (10 to 100 ÁM, human and rat), thiotepa (10 to 1000 ÁM, human and rat), cisplatin (30 to 1000 ÁM, human; 100 to 1000 ÁM, rat), Cd2+ (30 to 1000 ÁM, human; 100 to 1000 ÁM, rat), and acrylonitrile (30 to 1000 ÁM, human; 30 to 300 ÁM, rat). Four chemicals induced significant levels of ssDNA breaks in testicular cells from both species: styrene oxide (>/= 100 ÁM, rat and human), 1,2-dibromoethane (EDB) (>/= 100 ÁM, rat; 1000 ÁM human), thiram (>/= 30 ÁM, rat; >/= 100 ÁM, human), and chlordecone (300 ÁM, rat; >/=300 ÁM, human). Finally, five chemicals induced ssDNA breaks in one of the two species. Four chemicals induced significant ssDNA breaks in rat testicular cells only: 1,2-dibromo-3-chloropropane (DBCP) (>/= 10 ÁM), 1,3-dinitrobenzene (1,3-DNB) (>/= 300 ÁM), Cr6+ (1000 ÁM, and aflatoxin B1 (>/= 100 ÁM), the last two of these produced only a minor positive response. One chemical, acrylamide, induced a marginal increase in ssDNA breaks in human at 1000 ÁM but not in rat testicular cells. Although based on a limited number of donors, the data indicate a close correlation between the induction of DNA damage in human and rat testicular cells in vitro. For some chemicals, however, there appears to be differences in the susceptibility to chemically induced ssDNA breaks of isolated testicular cells from the two species. The data indicate that the parallel use of human and rat testicular cells provides a valuable tool in the assessment of human testicular toxicity.

Abstract  A series of halogenated compounds was tested by oral intubation in 200 Osborne-Mendel rats and 200 B6C3F1 mice of both sexes. Carbon tetrachloride, used as a positive control, induced liver and adrenal tumors in mice and neoplastic nodules in the livers of rats. 1,2-Dibromoethane and 1,2-dibromo-3-chloropropane caused stomach tumors with many metastases in both rats and mice. Chloroform, known to cause hepatocellular carcinomas in mice, led in addition to kidney tumors in male rats. 1,2-Dichloroethane was much weaker than the analog, 1,2-dibromoethane, and induced only a few stomach tumors in rats. It increased liver and lung tumors in mice. Most of the compounds, namely, trichloroethylene, 1,1-dichloroethane, 1,1,2-trichloroethane, hexachloroethane, and tetrachloroethylene, increased hepatocellular carcinomas in mice but had little or no action in rats. Iodoform tended to increase thyroid tumors in male rats and hepatocellular carcinomas in male mice. The action of 3-chloropropene was questionable. No tumors could be attributed to 1,1,1-trichloroethane (methylchloroform).

Abstract  #Recently, we inserted the plasmid vector pKK233-2 containing rat GSH S-transferase (GST) 5-5 cDNA into Salmonella typhimurium TA1535 and found that these bacteria (GST 5-5(+)) expressed the protein and produced mutations when ethylene or methylene dihalides were added (Thier, R., Taylor, J. B., Pemble, S. E., Ketterer, B., Persmark, M., Humphreys, W. G., and Guengerich, F. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8576-8580). After exposure to the known GST 5-5 substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane, the GST 5-5(+) strain showed fewer mutants than the bacteria transfected with the cDNA clone in a reverse orientation (GST 5-5(-)), suggesting a protective role of GST 5-5. However, mutations were considerably enhanced in the GST 5-5(+) strain (as compared to GST 5-5(-)) when 1,2,3,4-diepoxybutane (butadiene diepoxide) or 1,2-epoxy-4-bromobutane was added. The GST 5-5(+) and GST 5-5(-) bacterial stains showed similar responses to 1,2-epoxypropane, 3,4-epoxy-1butene, and 1,4-dibromobutane. The results suggest that some bifunctional activated butanes are transformed to mutagenic products through GSH conjugation. We also found that the GST 5-5(+) strain showed enhanced mutagenicity with 1,4-dibromo-2,3-epoxybutane, 1,2-epoxy-3-bromopropane (epibromohydrin), and (+-)-1,4-dibromo-2,3-dihydroxybutane. The possibility was considered that a 5-membered thialonium ion may be involved in the mutagenicity. Model thialonium compounds were rather stable to hydrolysis in aqueous solution at pH 7.4 and slowly alkylated 4-(4-nitrobenzyl)pyridine. The presence of ahydroxyl group beta to the sulfur did not enhance reactivity. Mechanisms involving episulfonium ions are considered more likely. Potential oxidation productsof the toxic pesticide 1,2-dibromo-3-chloropropane (DBCP) were also consideredin this system. DBCP itself gave rather similar results in the two strains. Others have reported that oxidation of DBCP is required for mutagenicity, along with GSTcatalyzed GSH conjugation (Simula, T. P., Glancey, M. J., Soderlund, E. J., Dybing, E., and Wolf, C. R. (1993) Carcinogenesis 14, 2303-2307). The putative oxidation product 1,2-dibromopropional did not show a difference between the two strains. However, 1,3-dichloroacetone, a model for the putative oxidation product 1-bromo-3-chloroacetone, was considerably more mutagenic in the GST 5-5(+) strain.

Abstract  This study was set up to determine if there are any age-dependent differences in the adverse effects of DBCP on the reproductive system of male rats. Groups of male rats were injected at the ages of 7,30 or 90 days with a single (50 mg kg-1 body weight) or repeated (20 mg kg-1 body weight once a week for 3 weeks) dose of DBCP, dissolved in DMSO. Ninety days after the last injection the males' fertility was estimated and the animals were killed. Blood was collected for future hormone assays, organs were weighed, testes were then taken for histological studies and sperm counts. The results were compared with those of control peers. The results showed that animals injected at the ages of 7 or 90 days under both regimens of treatment were adversely affected. The damage was noted in their fertility rate, sperm production, testicular histology and hormonal profile. Those injected at the age of 30 days showed only an insignificant variation compared with controls.

Abstract  1,2,3-Tribromopropane, 1,2,3-trichloropropane, and 1,2-dibromo-3-chloropropane are mutagenic in strains TA1535 and TA100 of Salmonella typhimurium, but only in the presence of rat liver S9 mix. This requirement for metabolic activation was unexpected for an alkyl halide and thus suggested the metabolic formation of the 2-keto derivatives (di-haloacetone). The 2-methyl derivatives of the halopropane compounds did not induce a doubling of revertants compared to controls. It was demonstrated that none of these compounds is converted into a secondary material that could be determined as structurally different by gas chromatography. These observations suggest that lack of mutagenicity of the methylated compounds is a manifestation of a steric effect.

Abstract  Studies of the sodium-cholate extraction of rat liver nuclear xenobiotic metabolizing enzymes were described. Male Sprague-Dawley-rats were treated with three daily intraperitoneal injections of 50mg/kg phenobarbital (50066) or 3-methylcholanthrene (56495) (3MC), single oral doses of 1.0 milliliter per kilogram (ml/kg) carbon-tetrachloride (56235) or 0.1ml/kg 1,2-dibromo-3-chloropropane (96128) (DBCP), or a protocol in which partially hepatectomized rats were initiated with 10mg/kg diethylnitrosamine (55185) followed by 32 weeks promotion with 0.05 percent phenobarbital. Liver nuclei were isolated by sedimentation through a sucrose buffer cushion. Proteins were extracted with a buffer solution containing the detergent sodium-cholate. After the nuclei were detergent extracted, they and the microsomes were assayed for cytochrome-P450, cytochrome-b5, NADPH-cytochrome-P450-reductase (NADPH-cytP450-red), NADH-cytochrome-b5-reductase (NADH-cytb5-red), and epoxide-hydrolase (EH). Responses of the nuclear and microsomal enzymes to phenobarbital, 3MC, DBCP, and carbon-tetrachloride were qualitatively similar. Liver nuclei and microsomes were prepared as before and assayed for cytochrome-P450, cytochrome-b5, NADPH-cytP450-red, NADH-cytb5-red, and EH. All microsomal enzymes were induced by the initiation/promotion protocol. Of the nuclear enzymes, only EH was induced. The authors conclude that detergent extraction of nuclei is an effective means of recovering nuclear xenobiotic metabolizing enzymes. The significance of the differential response of nuclear and microsomal enzymes to carcinogen initiation and promotion is unclear.