Evaluation of developmental toxicity of Methyl Chloride (Chloromethane) in rats, mice, and rabbits.

Methyl Chloride (MeCl; Chloromethane) is a high production volume chemical (>1000 t/a) and is used as an industrial solvent. Based on cardiac lesions reported in developmental toxicity studies in mice, but not in rats, manufacturers decided to classify MeCl as a developmental toxicant, cat. 2. Recently, the European Chemical Agency required a developmental toxicity study in a non-rodent species. No developmental toxicity was observed in rabbits in the recently completed, GLP, OECD 414 guideline study. In view of the absence of cardiac effects in rats and rabbits, the purpose of this review is to consider whether the cardiac effects reported in mice should be considered real effects and, if so, their potential for relevance to humans. This paper provides substantive new evidence with data from a third species and shows that an evaluation of the integrated scientific evidence indicates the reported developmental cardiac effects in mice, if not an artifact, are unlikely to be relevant to humans. As such the classification of MeCl for developmental toxicity was reconsidered.

Adjustment factors for toluene, styrene and methyl chloride by population modeling of toxicokinetic variability.

The availability of experimental data suitable as a basis to quantify human variability in response to chemical exposure has increased in recent years. It has enabled scientifically based, data driven adjustment factors (AF) to be deployed in the risk assessment process. As part of this development, we derive AF for human toxicokinetic variability (HK) for three lipophilic organic solvents; toluene, styrene and methyl chloride using physiologically based pharmacokinetic (PBPK) models in a population framework. The Monte Carlo simulations cover the influence of age and gender on toxicokinetic variability in the general population, as well as workplace ventilation rates and fluctuations in exposure level and workload in adult male and female workers. The derived AFHK are below 2.2 (95th percentile) for all subpopulations, exposure scenarios and chemicals, except for markers of acute effects in workers, where the factors are up to 5.0.

Physical and physicochemical factors effecting transport of chlorohydrocarbon gases from lung alveolar air to blood as measured by the causation of narcosis.

This systematic investigation examines gas transport in the lung for two sets of chlorohydrocarbons (CHCs): the chloromethanes (C1) and chloroethanes (C2). The C1 series includes chloromethane, methylene chloride, chloroform, and carbon tetrachloride, and the C2 series includes chloroethane, 1,2-dichloroethane, 1, 1, 2-trichloroethane, and 1, 1, 2, 2-tetrachloroethane. Most CHC gases cause narcosis. The comprehensive narcosis work of Lehmann and colleagues on CHCs was used as a basis for the narcosis endpoint in the present examination. The sites for narcosis are located in the brain (midline cortex and posterior parietal area), the spine, and at many peripheral nerve sites. Central nervous system (CNS) exposure executes a multisite, neural transmission set of inhibitions that promotes rapid loss of consciousness, sensory feeling, and current and stored memory while providing temporary amnesia. Absorption into the system requires dissolution into many lipid membranes and binding to lipoproteins. Lipophilicity is a CHC property shared with many anesthetics according to the Meyer-Overton Rule. Many structurally different lipid chemicals produce the narcosis response when the lipid concentration exceeds -67 mM. This suggests narcotic or anesthetic dissolution into CNS membranes until the lipid organization is disrupted or perturbed. This perturbation includes loading of Na(+)- and K(+)-channel transmembrane lipoprotein complexes and disrupting their respective channel functional organizations. The channel functions become attenuated or abrogated until the CHC exposure ceases and CHC loading reverses. This investigation demonstrates how the CHC physical and chemical properties influence the absorption of these CHCs via the lung and the alveolar system on route to the blood. Narcosis in test animals was used here as an objective biological endpoint to study the effects of the physical factors Bp, Vp, Kd (oil: gas) partition, Henry's constant (HK), and water solubility (S%) on gas transport. Narcosis is immediate after gas exposure and requires no chemical activation only absorption into the blood and circulation to CNS narcotic sites. The three physical factors Bp, K(d) (oil: air), and S% vary directly with unitary narcosis (UN) whereas Vp and HK vary inversely with UN in linear log-log relationships for the C2 series but not for the C1 series. Physicochemical properties of C1 series gases indicate why they depart from what is usually assumed to be an Ideal Gas. An essential discriminating process in the distal lung is the limiting alveolar film layer (AFL) and the membrane layer of the alveolar acini. The AFL step influences gas uptake by physically limiting the absorption process. Interaction with and dissolution into aqueous solvent of the AFL is required for transport and narcotic activity. Narcotics or anesthetics must engage the aqueous AFL with sufficient strength to allow transport and absorption for downstream CNS binding. CHCs that do not engage well with the AFL are not narcotic. Lipophilicity and amphipathicity are also essential solvency properties driving narcotics' transport through the alveolar layer, delivery to the blood fats and lipoproteins, and into critical CNS lipids, lipoproteins, and receptor sites that actuate narcosis. AFL disruption is thought to be strongly related to a number of serious pulmonary diseases such acute respiratory distress syndrome, infant respiratory distress syndrome, emphysema, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pneumonia, pulmonary infections, and idiopathic pulmonary fibrosis. The physical factors (Bp, Vp, Kd [oil: gas] partition, Henry's constant, and water solubility [S%]) combine to affect a specific transport through the AFL if lung C > C(0) (threshold concentration for narcosis). The degree of blood CHC absorption depends on dose, lipophilicity, and lung residence time. AFL passage can be manipulated by physical factors of increased pressure (kPa) or increased gas exposure (moles). Molecular lipophilicity facilitates narcosis but lipophilicity alone does not explain narcosis. Vapor pressure is also required for narcosis. Narcotic activity apparently requires stereospecific processing in the AFL and/or down-stream inhibition at stereospecific lipoproteins at CNS inhibitory sites. It is proposed that CHCs likely cannot proceed through the AFL without perturbation or disruption of the integrity of the AFL at the alveoli. CHC physicochemical properties are not expected to allow their transport through the AFL as physiological CO(2) and O(2) naturally do in respiration. This work considers CHC inspiration and systemic absorption into the blood with special emphasis on the CHC potential perturbation effects on the lipid, protein liquid layer supra to the alveolar membrane (AFL). A heuristic gas transport model for the CHCs is presented as guidance for this examination. The gas transport model can be used to study absorption for other gas delivery endpoints of environmental concern such as carcinogens.

Genetic variation in metabolic genes, occupational solvent exposure, and risk of non-hodgkin lymphoma.

Using 1996-2000 data among Connecticut women, the authors evaluated whether genetic variation in 4 metabolic genes modifies organic solvent associations with non-Hodgkin lymphoma and 5 major histologic subtypes. P(interaction) values were determined from cross-product terms between dichotomous (ever/never) solvent variables and genotypes at examined loci in unconditional logistic regression models. The false discovery rate method was used to account for multiple comparisons. Overall associations between the chlorinated solvents dichloromethane (odds ratio (OR) = 1.69, 95% confidence interval (CI): 1.06, 2.69), carbon tetrachloride (OR = 2.33, 95% CI: 1.23, 4.40), and methyl chloride (OR = 1.44, 95% CI: 0.94, 2.20) and total non-Hodgkin lymphoma were increased among women TT for rs2070673 in the cytochrome P4502E1 gene, CYP2E1 (dichloromethane: OR = 4.42, 95% CI: 2.03, 9.62; P(interaction) < 0.01; carbon tetrachloride: OR = 5.08, 95% CI: 1.82, 14.15; P(interaction) = 0.04; and methyl chloride: OR = 2.37, 95% CI: 1.24, 4.51; P(interaction) = 0.03). In contrast, no effects of these solvents were observed among TA/AA women. Similar patterns were observed for diffuse large B-cell lymphoma and follicular lymphoma, as well as marginal zone lymphoma for dichloromethane. The weak, nonsignificant overall association between benzene and diffuse large B-cell lymphoma (OR = 1.29, 95% CI: 0.84, 1.98) was increased among women AA for rs2234922 in the microsomal epoxide hydrolase gene, EPHX1 (OR = 1.77, 95% CI: 1.06, 2.97; P(interaction) = 0.06). In contrast, no effect was observed among AG/GG women. Additional studies with larger sample size are needed to replicate these findings.

An improved system for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay.

A gas exposure system using rotating vessels was improved for exposure of cultured mammalian cells to gaseous compounds in the chromosomal aberration assay. This system was composed of 12 square culture vessels, a device for preparation of air containing test gas, and positive and negative control gases at target concentrations and for supplying these gases to the culture vessels, and a roller apparatus in an incubator. Chinese hamster lung cells (CHL/IU) were grown on one side of the inner surface of the square culture vessel in the MEM medium. Immediately prior to exposure, the medium was changed to the modified MEM. Air in the culture vessel was replaced with air containing test gas, positive or negative control gas. Then, the culture vessels were rotated at 1.0 rpm. The monolayered culture cells were exposed to test gas during about 3/4 rotation at upper positions and alternatively immersed into the culture medium during about 1/4 rotation at lower positions. This system allowed the chromosomal aberration assay simultaneously at least at three different concentrations of a test gas together with positive and negative control gases with and without metabolic activations, and duplicate culture at each exposure concentration. Seven gaseous compounds, 1,3-butadiene, chlorodifluoromethane, ethyl chloride, methyl bromide, methyl chloride, propyne, and vinyl chloride, none of which has been tested to date, were tested on CHL/IU for the chromosomal aberration assay using this gas exposure system. All the compounds except chlorodifluoromethane showed positive responses of the structural chromosomal aberrations, whereas polyploidy was not induced by any of these gases. This improved gas exposure system proved to be useful for detecting chromosomal aberrations of gaseous compounds.

Assessing the reliability of PBPK models using data from methyl chloride-exposed, non-conjugating human subjects.

Physiologically based pharmacokinetic (PBPK) models are often optimized by adjusting metabolic parameters so as to fit experimental toxicokinetic data. The estimates of the metabolic parameters are then conditional on the assumed values for all other parameters. Meanwhile, the reliability of other parameters, or the structural model, is usually not questioned. Inhalation exposures with human volunteers in our laboratory show that non-conjugators lack metabolic capacity for methyl chloride entirely, and that elimination in these subjects takes place via exhalation only. Therefore, data from these methyl chloride exposures provide an excellent opportunity to assess the general reliability of standard inhalation PBPK models for humans. A hierarchical population PBPK model for methyl chloride was developed. The model was fit to the experimental data in a Bayesian framework using Markov chain Monte Carlo (MCMC) simulation. In a Bayesian analysis, it is possible to merge a priori knowledge of the physiological, anatomical and physicochemical parameters with the information embedded in the experimental toxicokinetic data obtained in vivo. The resulting estimates are both statistically and physiologically plausible. Model deviations suggest that a pulmonary sub-compartment may be needed in order to describe the inhalation and exhalation of volatile adequately. The results also indicate that there may be significant intra-individual variability in the model parameters. To our knowledge, this is the first time that the toxicokinetics of a non-metabolized chemical is used to assess population PBPK parameters. This approach holds promise for more elaborate experiments in order to assess the reliability of PBPK models in general.

Glutathione transferase T1 phenotype affects the toxicokinetics of inhaled methyl chloride in human volunteers.

The aim of the present study was to investigate how the genetic polymorphism in glutathione transferase T1 (GSTT1) affects the metabolism and disposition of methyl chloride in humans in vivo. The 24 volunteers (13 males and 11 females) who participated in the study were recruited from a group of 208 individuals previously phenotyped for GSTT1 by measuring the glutathione transferase activity with methyl chloride in lysed erythrocytes ex vivo. Eight individuals with high (+/+), eight with medium (+/0) and eight with no (0/0) GSTT1 activity were exposed to methyl chloride gas (10 p.p.m.) in an exposure chamber for 2 h. Uptake and disposition was studied by measuring the concentration of methyl chloride in inhaled air, exhaled air and blood. A two-compartment model with two elimination pathways corresponding to exhalation and metabolism was fitted to experimental data. The average net respiratory uptake of methyl chloride was 243, 158, and 44 micromol in individuals with high, intermediate and no GSTT1 activity, respectively. Metabolic clearance was high (4.6 l/min) in the +/+ group, intermediate (2.4 l/min) in the +/0 group, and close to zero in 0/0 individuals, while the exhalation clearance was similar in the three groups. No exposure related increase in urinary S-methyl cysteine was detected. However, gender and the GSTTl phenotype seemed to affect the background levels. In conclusion, GSTT1 appears to be the sole determinant of methyl chloride metabolism in humans. Thus, individuals with nonfunctional GSTT1 entirely lack the capacity to metabolize methyl chloride.

Inhibitory action of chloramine on formate-metabolizing system. Studies suggested by an unusual case record.

We previously reported on a patient exposed simultaneously to methyl chloride and chloramine gas who developed metabolic acidosis and permanent blindness [M. Minami et al., Hum Exp Toxicol 11: 27-34, 1992]. The case report suggested the possibility of potentiation of methyl chloride toxicity by chloramine. The potentiating mechanism was investigated by exposing mice to methyl chloride followed by ammonia chloramine, and then the level of formate in urine samples was measured with an enzyme coupling method to detect disturbance of formate metabolism. Mice dosed with 0.05 mL 1.0 mM chloramine after methyl chloride exposure excreted a significantly larger amount of urinary formate than mice treated with only methyl chloride. There was no difference in urinary formate levels between mice treated with only 0.05 mL 1.0 mM chloramine and those given only the vehicle (0.1 M phosphate buffer pH 6.0) for chloramine. The underlying biochemical mechanism of deterioration of formate metabolism was found to be the inhibition of the enzyme, N10-formyl tetrahydrofolate (N10-f-THF) dehydrogenase by 0.56-3.35 microM chloramine in the in vitro experiment using the purified enzyme. Positive control mice, given orally 0.1 mL 10% methanol in 0.1 M phosphate buffer (pH 6.0) excreted the same amount of urinary formate as those receiving 0.05 mL 1.0 mM chloramine after methanol administration. This was ascribed to the inhibitory effect of chloramine on formaldehyde dehydrogenase and depletion of substrate for further metabolism. The inhibition of the enzyme by chloramine (2.7-100.8 microM) was confirmed by in vitro experiments, using the purified enzyme, formaldehyde dehydrogenase.

[DNA methylation of monohalogenated methanes of F344 rats]. / DNA-Methylierung von monohalogenierten Methanen in F-344 Ratten.

Monohalogenated methanes (methyl chloride, methyl bromide and methyl iodide) are mutagenic and carcinogenic. The possible mechanism of these effects, DNA methylation, was studied. DNA adducts from organs of F344 rats exposed to these chemicals were separated and identified with high performance liquid chromatography (HPLC) and gas-chromatography/mass-spectrometry (GC/MS). DNA adducts, 7-methylguanine (7-MeG) and O6-Methylguanine(0(6)-MeG), incorporation of 14C into de novo synthesis of nucleobases could be observed in enzymatic DNA hydrolysates by HPLC and determination of the radioactivity in the fractions. The formation of DNA adducts in the studied organs was only quantitatively different. The formation of O6-MeG was further proved by analysing the acidic hydrolysates using HPLC with non-radioactive O6-MeG as internal standard. 7-MeG and 3-MeA were identified with GC/MS analysis.

Mechanisms of carcinogenicity of methyl halides.

Methyl chloride, bromide, and iodide are used as methylating agents. These compounds are mutagenic in short-term tests and do not require activation by exogenous S9 mix. In DNA-binding studies performed in rats and mice, 14C-labeled methyl chloride was given by inhalation, and methylation of DNA bases was examined. The compound did not lead to specific DNA adducts. In particular, methylation of DNA bases was not observed. In contrast, methyl bromide and methyl iodide, upon oral and inhalation administration to rats and mice, caused systemic DNA methylation. Specifically, 3-methyl-adenine, 7-methyl-guanine, and O6-methyl-guanine were formed. Long-term inhalation bioassays have been performed in rats and mice with methyl chloride and methyl bromide. Methyl chloride induced renal tumors, but only in male mice at the highest concentration tested (1000 ppm). Under these special conditions, a number of secondary effects occur subsequent to glutathione depletion in the target tissue, resulting in DNA damage (DNA-protein cross-links and probably DNA single-strand breaks). The particular coincidence of secondary high-dose effects precludes a risk extrapolation to man. Methyl bromide did not induce tumors in rats and mice when administered by inhalation. However, experimental data point to a possible local carcinogenic effect on the rat forestomach when the compound is given by gavage. A factor that accounts for the discrepancy between systemic DNA methylation and apparent noncarcinogenicity upon inhalation might be the preference of 7-N over O6 methylation of guanine. An extrapolation of the negative rodent inhalation bioassay of methyl bromide to man might be problematic because rodents metabolize methyl bromide very quickly whereas in humans there is a particular subpopulation that only poorly metabolizes the compound ("nonconjugators"). Such individuals can be characterized by incubation of erythrocytes with methyl chloride or methyl bromide and measurement of the substrate decline. Methyl iodide has been tested, with positive outcome, in early carcinogenicity bioassays not based on modern methodology. However, these results, along with the proven systemic methylating potency of methyl iodide, argue in favor of a carcinogenic effect of the compound.

Formation and repair of DNA lesions in kidneys of male mice after acute exposure to methyl chloride.

The time-course of DNA lesions (DNA-protein cross-links, single-strand breaks) induced by high concentrations of methyl chloride (1000 ppm) was measured in renal tissue of male mice by means of the alkaline elution assay in order to gain an insight into repair processes. DNA-protein cross-links were removed at a fast rate, whereas single-strand breaks appeared to accumulate, even during repair of DNA-protein cross-links. However, 48 h after exposure to methyl chloride, neither of these lesions were detectable in mouse kidney. Both types of DNA damage were ascribed to the action of formaldehyde, an intermediate in methyl chloride metabolism which may react with nucleic acids and protein and interfere with DNA repair.

Ethyl chloride: 11-day continuous exposure inhalation toxicity study in B6C3F1 mice.

Groups of seven B6C3F1 mice per sex were exposed for 23 hr/day to 0, 250, 1250, or 5000 ppm ethyl chloride (EtCl) for 11 consecutive days to evaluate the potential toxicity of EtCl under near-continuous exposure conditions. On the day following the last exposure, a neurobehavioral observation battery was performed, samples were obtained for clinical chemistry and hematology, and necropsies were conducted. Histopathologic examination was subsequently performed. The only observed effects were increased relative liver weights and a slight increase in hepatocellular vacuolation (glycogen or fat) in 5000 ppm-exposed mice. Exposures to EtCl were well tolerated despite the unusually long exposure periods.

The use of multiple endpoints to define the mechanism of action of reproductive toxicants and germ cell mutagens.

Although a variety of endpoints are routinely assessed in reproductive toxicity studies, the inclusion of additional (and potentially more sensitive) endpoints may increase our ability to detect adverse effects on male or female reproduction and also provide information pertaining to the mechanism of action of the reproductive toxicant. Methyl chloride (MeCl) is a well characterized reproductive toxicant in the male rat, and can serve as a model to illustrate the importance of using multiple endpoints to determine the biological basis of chemically induced toxicity in the reproductive system. Exposure of male rats to MeCl results in bilateral testicular degeneration and epididymal inflammation and sperm granuloma formation. Females bred to these males in a dominant lethal assay exhibit elevated rates of postimplantation embryonic death during the first 2 weeks after treatment and increased preimplantation embryonic loss during weeks 2 to 8 post-exposure. Since the chemical is known to be a direct-acting mutagen in vitro and a kidney carcinogen in vivo, the increased embryo death rate observed might reasonably be considered good evidence that MeCl is a direct-acting germ cell mutagen. However, subsequent investigations revealed that MeCl-induced preimplantation loss was a result of cytotoxic rather than genotoxic effects on sperm, with a significant decrease in the count of motile sperm of normal morphology in exposed males during weeks 2 to 8 after treatment. In fact, examination of the fertilization rate during these weeks using a system of embryo recovery and culture revealed that the entire elevated rate of preimplantation loss detected in the dominant lethal assay was the result of failure of fertilization; it had no genetic component at all. Postimplantation death is considered a more reliable indicator of dominant lethality than is preimplantation loss. In the MeCl dominant lethal assay, such increased postimplantation loss was detected only when the fertilizing sperm had been present at the site of MeCl-induced acute inflammation in the cauda epididymis. Inflammatory cells, such as those in the MeCl-exposed epididymis, are known to produce a variety of genetic lesions in the DNA of neighboring cells. Therefore, male rats were concurrently exposed to MeCl and treated with an anti-inflammatory agent (BW755C) to inhibit the epididymal inflammation.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemical effects of methyl chloride in relation to its tumorigenicity.

The biochemical effects of methyl chloride were investigated in tissues of F-344 rats and B6C3F1 mice (both sexes). Activities of GST were 2-3 times higher in livers of male B6C3F1 mice, compared with those of female mice, and with rats of both sexes. In kidneys GST activities of (male) mice were about 7 times lower than those found in livers. The activity of FDH was higher in livers of mice (both sexes) than in those of rats. No obvious sex difference was found in livers of rats and mice with respect to FDH. In kidneys, however, (minor) differences in FDH activities occurred between male and female B6C3F1 mice (4.7 vs. 3.1 nmol/min per mg). Sex differences of FDH activity in kidneys were not observed in F-344 rats. The microsomal transformation (by cytochrome P-450) of methyl chloride and S-methyl-L-cysteine to formaldehyde in tissues of B6C3F1 mice occurred preferentially in the liver. More formaldehyde was produced in liver microsomes of male, compared to those of female mice. Kidney microsomes metabolized methyl chloride to formaldehyde much less than liver microsomes. After a single exposure of mice of both sexes to 1000 ppm methyl chloride no elevation in formaldehyde concentrations was observed in livers and kidneys ex vivo. The determination of DNA lesions, using the alkaline elution technique, revealed no DNA-protein crosslinks in kidneys of male B6C3F1 mice after exposure to methyl chloride (1000 ppm, 6 h day-1, 4 days) and gave only minor evidence of single-strand breaks. Lipid peroxidation (production of TBA reactive material), induced by single exposure to methyl chloride (1000 ppm, 6 h), was very pronounced in livers of male and female mice. Smaller increases in peroxidation were observed in the kidneys of exposed mice. The theory that renal tumors observed in male mice after chronic exposure of the test animals to high (1000 ppm) concentrations of methyl chloride, are evoked by intermediates and in situ produced formaldehyde is proven unlikely by our results.

Role of testicular versus epididymal toxicity in the induction of cytotoxic damage in Fischer-344 rat sperm by methyl chloride.

Exposure of male Fischer-344 (F-344) rats to methyl chloride (MeCl) results in testicular and epididymal toxicity and the induction of both pre- and postimplantation embryonic loss; the preimplantation loss is caused by cytotoxic damage to sperm that leads to failure of fertilization (Toxicol Appl Pharmacol 1986; 86:124-130). The present study examined whether the cytotoxicity of MeCl to sperm is due to the testicular or epididymal toxicity of MeCl. Groups of 18 males were exposed to 3000 ppm MeCl 6 h/day for 5 days, with and without concurrent treatment with the anti-inflammatory agent 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW755C; 10 mg/kg, i.p. 1 h pre- and postexposure); BW755C was used to inhibit the epididymal toxicity of MeCl. Control groups were untreated or injected as described above with BW755C. Six males from each group were killed weekly for 3 weeks. Toxic effects of MeCl on the testis were demonstrated by decreased relative organ weight (week 3), testicular histopathology (weeks 1-3) and decreased daily sperm production (weeks 1-3); these effects were not prevented by BW755C. In both the MeCl and the MeCl + BW755C treatment groups, tubules devoid of sperm were observed in regions 4 and 5 of the epididymis at week 2, and in regions 6A and 6B at week 3. Sperm were present in the vas deferens of both groups at week 3 in decreased numbers and had decreased motility and more frequent morphologic abnormalities compared to untreated controls. In conjunction with known epididymal transit times for F-344 rat sperm, these data indicate that the induction of preimplantation loss by MeCl at weeks 2 and 3 postexposure is likely to result from cytotoxic effects on sperm located in the testes at the time of exposure.

Role of epididymal inflammation in the induction of dominant lethal mutations in Fischer 344 rat sperm by methyl chloride.

This study assessed the possible relationship between methyl chloride (MeCl)-induced epididymal inflammation and the formation of dominant lethal mutations in sperm of Fischer 344 rats. Groups of 40 males were exposed to MeCl (3000 ppm 6 hr/day for 5 days), with or without concurrent treatment with the anti-inflammatory agent 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW 755C; 10 mg/kg, i.p. 1 hr pre- and postexposure); BW 755C was shown previously to inhibit MeCl-induced epididymal inflammation. Control groups (n = 20) were either untreated, injected as described above with BW 755C, or injected on the afternoon of day 5 with triethylenemelamine (0.2 mg/kg), a known dominant lethal mutagen. Each male was caged with one female weekly for 3 weeks; 12-18 days after mating, females were killed to assess dominant lethal parameters. In females bred to MeCl-exposed males, significant increases were observed in postimplantation loss at postexposure week 1 (0.84 dead implants per female vs. 0.29 in untreated controls) and in dead implants/total implants at both week 1 (0.10 vs. 0.04 control) and week 2 (0.24 vs. 0.06 control). These increases were not observed in females bred to males treated with BW 755C during MeCl exposure. Coadministration of BW 755C to males along with MeCl also reduced the percentage of mated females with two or more postimplantation losses from 31% to 8% (week 1) and 30% to 12% (week 2). Therefore, the dominant lethal mutations induced by MeCl appear to be a consequence of its induction of inflammation in the epididymis. These data demonstrate the potential genotoxicity of inflammatory processes in vivo.