Some insights into the mode of action of butadiene by examining the genotoxicity of its metabolites.

1,3-Butadiene (BTD) is an important commodity chemical and air pollutant that has been shown to be a potent carcinogen in mice, and to a lesser extent, a carcinogen in rats. To better assess butadiene's carcinogenic risk to humans, it is important to understand its mode of action and how this relates to differences in responses among species. In a series of in vitro experiments, lymphocytes from rats, mice, and humans were exposed to 3,4-epoxy-1-butene (EB) or 1,2:3,4-diepoxybutane (DEB) for 1h at the G(0) stage of the cell cycle, stimulated to divide, and cultured to assess the ability of these metabolites to induce sister chromatid exchange (SCE) and chromosome aberrations (CAs). EB induced no increases in SCEs or CAs in the cells from the three species. DEB was a potent SCE- and CA-inducer, with the results being similar in each rodent species. The response for SCEs seen in the human cells was more complex, with genetic polymorphism for glutathione-S-transferases (GST) possibly modulating the response. The single cell gel electrophoresis assay was used on genetically engineered V79 cell lines to investigate a possible influence of GST status. Experiments were also conducted to investigate the reason for EB's failure to induce SCEs or CAs in G(0) cells. The results indicate that EB-induced DNA damage was repaired before DNA synthesis in unstimulated lymphocytes, but EB caused a large increase in SCEs if actively cycling cells were treated. Thus, the results indicate that DEB damage is persistent in G(0) cells, and DEB is a much more potent genotoxicant than EB. The carcinogenic effect of butadiene will most likely depend on the degree to which DEB is produced and reaches target tissues, and to a lesser extent on the ability of EB to reach actively dividing or repair deficient cells.

Insights into the carcinogenic mode of action of arsenic.

That arsenic can induce cancer in humans has been known since the late 17th century, yet how arsenic induces cancer has been the subject of numerous scientific publications. Various modes of action (MOA) have been proposed for arsenic's carcinogenicity. In this paper we review our previous studies on the ability of arsenicals to cause DNA damage, the relative inability of these arsenicals to induce point mutations, and the involvement of arsenicals in spindle disruption. We present new evidence that shows that reduced glutathione (GSH) can chemically reduce inactive pentavalent arsenicals to trivalent arsenicals which can disrupt tubulin polymerization, and show that reactive oxygen species (ROS) are most likely not involved in tubulin disruption. A hypothesis is also presented on how arsenic may induce stable chromosome aberrations (CAs) that can lead to cancer, thus supporting a role for genetic damage in the MOA for arsenic. We then propose promising areas of research that might give insight into the MOA of arsenic.

Oxidation and methylation status determine the effects of arsenic on the mitotic apparatus.

We investigated the spindle inhibitory properties of six arsenicals differing in their methylation or oxidation state. Human lymphoblasts were exposed for 6 h to either sodium arsenate (NaAs(V)), sodium arsenite (NaAs(III)), monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)), dimethylarsinic acid (DMA(V)), or dimethylarsinous acid (DMA(III)). After exposure slides were prepared, and the mitotic indices (MI) were assessed. We also exposed tubulin directly to each arsenical and spectrophotometrically measured its effect on polymerization. NaAs(V) caused a small but significant increase in MI. MMA(V) also caused only a slight increase in MI that just reached statistical significance. In contrast, DMA(V) caused a significant increase in MI, producing approximately 75% the MI of demecolcine and approximately 4 times the MI of the control. NaAs(III) had no significant effect on MI and was quite toxic. MMA(III) induced more than a twofold increase in MI compared to the control, which was about 40% that caused by demecolcine. On a micromolar basis, MMA(III) was the most potent of the arsenicals tested. DMA(III) gave inconsistent results. None of the pentavalent arsenicals had a substantial effect (either inhibition or enhancement) on GTP-induced polymerization of tubulin. In contrast, NaAs(III) inhibited polymerization at concentrations of 1 mM and above and MMA(III) and DMA(III) at 10 microM and above. Taken together, these results present a complex picture of how arsenicals may affect cells. These studies demonstrate that the metabolites of arsenic are active not only as chromosome breaking and DNA damaging agents but can also interfere with cell division via tubulin disruption.

Cytogenetic studies in mice treated with the jet fuels, Jet-A and JP-8.

The genotoxic potential of the jet fuels, Jet-A and JP-8, were examined in mice treated on the skin with a single dose of 240 mg/mouse. Peripheral blood smears were prepared at the start of the experiment (t = 0), and at 24, 48 and 72 h following treatment with jet fuels. Femoral bone marrow smears were made when all animals were sacrificed at 72 h. In both tissues, the extent of genotoxicity was determined from the incidence of micronuclei (MN) in polychromatic erythrocytes. The frequency of MN in the peripheral blood of mice treated with Jet-A and JP-8 increased over time and reached statistical significance at 72 h, as compared with concurrent control animals. The incidence of MN was also higher in bone marrow cells of mice exposed to Jet-A and JP-8 as compared with controls. Thus, at the dose tested, a small but significant genotoxic effect of jet fuels was observed in the blood and bone marrow cells of mice treated on the skin.

Induction of genotoxic damage is not correlated with the ability to methylate arsenite in vitro in the leukocytes of four mammalian species.

Arsenic is a natural drinking water contaminant that impacts the health of large populations of people throughout the world; however, the mode or mechanism by which arsenic induces cancer is unclear. In a series of in vitro studies, we exposed leukocytes from humans, mice, rats, and guinea pigs to a range of sodium arsenite concentrations to determine whether the lymphocytes from these species showed differential sensitivity to the induction of micronuclei (MN) assessed in cytochalasin B-induced binucleate cells. We also determined the capacity of the leukocytes to methylate arsenic by measuring the production of MMA [monomethylarsinic acid (MMA(V)) and monomethylarsonous acid (MMA(III))] and DMA [dimethylarsinic acid (DMA(V)) and dimethylarsonous acid (DMA(III))]. The results indicate that cells treated for 2 hr at the G(0) stage of the cell cycle with sodium arsenite showed only very small to negligible increases in MN after mitogenic stimulation. Treatment of actively cycling cells produced induction of MN with increasing arsenite concentration, with the human, rat, and mouse lymphocytes being much more sensitive to MN induction than those of the guinea pig. These data gave an excellent fit to a linear model. The leukocytes of all four species, including the guinea pig (a species previously thought not to methylate arsenic), were able to methylate arsenic, but there was no clear correlation between the ability to methylate arsenic and the induction of MN.

Genotoxicity studies of three triazine herbicides: in vivo studies using the alkaline single cell gel (SCG) assay.

Triazine herbicides are prevalent contaminants of groundwater in the agricultural regions of the United States. The literature on the genotoxicity of triazines is rife with conflicting data, though the general tendency is for most studies to report negative results. In order to investigate further the genotoxicity of triazines, we exposed mice to triazines by intraperitoneal injection up to the maximum tolerated doses. About 24h later, blood was removed, and the leukocytes subjected to DNA damage analysis using the alkaline single cell gel electrophoresis assay (SCG), one of the most sensitive DNA damage assays available. Our results indicate that atrazine induced a small dose-related increase in DNA damage. Simazine did not induce any dose-related increase in DNA damage. Cyanazine induced a marginal increase in DNA damage with dose, but no individual dose was significantly increased compared to the control. These results indicate that these triazines, even at extremely high concentrations, have only marginal DNA-damaging activity in vivo in mouse leukocytes.

Methylated trivalent arsenic species are genotoxic.

The reactivities of methyloxoarsine (MAs(III)) and iododimethylarsine (DMAs(III)), two methylated trivalent arsenicals, toward supercoiled phiX174 RFI DNA were assessed using a DNA nicking assay. The induction of DNA damage by these compounds in vitro in human peripheral lymphocytes was assessed using a single-cell gel (SCG, "comet") assay. Both methylated trivalent arsenicals were able to nick and/or completely degrade phiX174 DNA in vitro in 2 h incubations at 37 degrees C (pH 7.4) depending on concentration. MAs(III) was effective at nicking phiX174 DNA at 30 mM; however, at 150 microM DMAs(III), nicking could be observed. Exposure of phiX174 DNA to sodium arsenite (iAs(III); from 1 nM up to 300 mM), sodium arsenate (from 1 microM to 1 M), and the pentavalent arsenicals, monomethylarsonic acid (from 1 microM to 3 M) and dimethylarsinic acid (from 0.1 to 300 mM), did not nick or degrade phiX174 DNA under these conditions. In the SCG assay in human lymphocytes, methylated trivalent arsenicals were much more potent than any other arsenicals that were tested. On the basis of the slopes of the concentration-response curve for the tail moment in the SCG assay, MAs(III) and DMAs(III) were 77 and 386 times more potent than iAs(III), respectively. Because methylated trivalent arsenicals were the only arsenic compounds that were observed to damage naked DNA and required no exogenously added enzymatic or chemical activation systems, they are considered here to be direct-acting forms of arsenic that are genotoxic, though they are not, necessarily, the only genotoxic species of arsenic that could exist.

Cytogenetic studies of three triazine herbicides. II. In vivo micronucleus studies in mouse bone marrow.

Atrazine, simazine, and cyanazine are widely used preemergence and postemergence triazine herbicides that have made their way into the potable water supply of many agricultural communities. Although there are several contradictory genotoxicity studies in the literature, our previous in vitro studies with human lymphocytes showed that atrazine, simazine, and cyanazine did not induce sister chromatid exchanges (SCEs) or chromosome aberrations (CAs) up to the limits of solubility in aqueous medium using 0.5% dimethyl sulfoxide. To expand upon these results and to ensure that our in vitro findings could be replicated in an in vivo system, mice were treated with each triazine by two intraperitoneal injections, 24h apart. The animals were sacrificed and the bone marrow removed for micronucleus (MN) analysis, 24h after the last injection. Two to four independent trials were performed for MN analysis in polychromatic erythrocytes, and in some trials the spleen was removed, cultured, and analyzed for SCEs and CAs. None of the triazines investigated induced MN in the bone marrow, even at doses that caused significant bone marrow suppression and/or death. These results indicate that atrazine, simazine, and cyanazine are not genotoxic as measured by the bone marrow MN assay in mice following high dose exposures.

Cytogenetic studies of three triazine herbicides. I. In vitro studies.

Atrazine, simazine, and cyanazine are widely used pre-emergence and post-emergence triazine herbicides that have made their way into the potable water supply of many agricultural communities. Because of this and the prevalence of contradictory cytogenetic studies in the literature on atrazine, simazine, and cyanazine, a series of in vitro experiments was performed to investigate the ability of these three triazines to induce sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in human lymphocyte cultures. Our results showed that all three triazines failed to produce any significant increases in SCEs or CAs up to the limits of solubility [using 0.5% dimethyl sulfoxide (DMSO)]. Our results are discussed in light of contradictory results in the literature.

Induction of sister chromatid exchanges in human peripheral blood lymphocytes by bromoform: investigation of the role of GSTT1-1 polymorphism.

Brominated trihalomethanes (THMs) are disinfection by-products present frequently in chlorinated drinking water. Brominated THMs are mutagenic in a variety of systems and are carcinogenic in rodents. The metabolism of brominated THMs is thought to involve a GSH conjugation reaction leading either to formaldehyde or DNA-reactive intermediates via glutathione S-transferase-theta (GSTT1-1), which is polymorphic in humans. In the present study, we have determined the genotoxicity of one of the brominated THMs, bromoform (BF), by measuring its ability to induce sister chromatid exchanges (SCEs) in whole-blood (WB) cultures of human peripheral blood lymphocytes from GSTT1-1+ and GSTT1-1- donors. The results showed no differences in SCEs per cell by BF between GSTT1-1+ and GSTT1-1- individuals when the cells were exposed to 5 x 10(-3) M BF at the beginning of cell culturing (10.8+/-0.85 vs. 10.57+/-0.47, respectively), at the 16th (9.66+/-0.91 vs. 9.57+/-0.07), or the 24th h (8.21+/-0.61 vs. 8.29+/-0.24) of cell growth. Although GSTT1-1 is expressed in the erythrocytes, the lack of expression of the GSTT1-1 gene in the target cells (lymphocytes) may account for this observation.

Cell cycle specificity of cytogenetic damage induced by 3,4-epoxy-1- butene.

3,4-epoxy-1-butene (EB), a primary metabolite of butadiene, is a direct-acting "S-dependent" genotoxicant that can induce sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) in cycling cells in vitro. However, EB is almost inactive when splenic or peripheral blood lymphocytes are exposed at the G(0) stage of the cell cycle. To investigate whether repair of DNA lesions is responsible for the lack of cytogenetic responses seen after G(0) treatments, we used cytosine arabinoside (ara-C) to inhibit DNA polymerization during DNA repair. If enough repairable lesions are present, double-strand breaks should accumulate and form chromosome-type ("S-independent") deletions and exchanges. This is exactly what occurred. EB induced chromosome deletions and dicentrics at the first division following treatment, when the EB exposure was followed by ara-C. Without ara-C treatment, there was no induction of CAs. These experiments indicate that the relatively low levels of damage induced by EB in G(0) lymphocytes are removed by DNA repair prior to DNA synthesis and thus, before the production of SCEs or chromatid-type aberrations.

An evaluation of the feasibility of using cytogenetic damage as a biomarker for alachlor exposure.

Alachlor is a widely used herbicide for which there is significant human exposure, principally through groundwater contamination and inhalation. Because alachlor is purported to be carcinogenic and mutagenic, we initiated studies to determine if induced cytogenetic damage could be used as a biomarker for exposure to this herbicide. Both isolated and whole blood human lymphocytes were exposed to alachlor using several protocols. The lymphocytes were cultured for analysis of sister chromatid exchange (SCE), chromosome aberrations (CAs), micronuclei (MN) in cytochalasin B-induced binucleated cells, and proliferation kinetics using the replicative index (RI). In addition, CD rats were injected with either 10 or 50 mg kg-1 of alachlor, 2-chloro-N-(2,6-diethylphenyl) acetamide (CDEPA) or 2, 6-diethylanaline (DEA). After 24 h, the peripheral blood lymphocytes were removed and cultured for SCE and RI analysis. Alachlor did induce a concentration-related increase in SCE in vitro, but neither it nor its metabolites (CDEPA or DEA) induced a significant increase in SCEs or an alteration of RI in vivo. At the highest in vitro concentration tested, alachlor induced a statistically-significant increase in MN, but no concomitant increase in CAs was seen. From analyses of our data and the literature on alachlor clastogenicity and exposure levels, we concluded that cytogenetic damage may not be an adequately sensitive marker for evaluating human exposure to alachlor.

Comparison of cytogenetic effects of 3,4-epoxy-1-butene and 1,2:3, 4-diepoxybutane in mouse, rat and human lymphocytes following in vitro G0 exposures.

To understand better the species differences in carcinogenicity caused by 1,3-butadiene (BD), we exposed G0 lymphocytes (either splenic or peripheral blood) from rats, mice and humans to 3, 4-epoxy-1-butene (EB) (20 to 931 microM) or 1,2:3,4-diepoxybutane (DEB) (2.5 to 320 uM), two of the suspected active metabolites of BD. Short EB exposures induced little measurable cytogenetic damage in either rat, mouse, or human G0 lymphocytes as measured by either sister chromatid exchange (SCE) or chromosome aberration (CA) analyses. However, DEB was a potent inducer of both SCEs and CAs in G0 splenic and peripheral blood lymphocytes. A comparison of the responses among species showed that the rat and mouse were approximately equisensitive to the cytogenetic damaging effects of DEB, but the situation for the human subjects was more complex. The presence of the GSTT1-1 gene (expressed in the erythrocytes) reduced the relative sensitivity of the lymphocytes to the SCE-inducing effects of DEB. However, additional factors also appear to influence the genotoxic response of humans to DEB. This study is the first direct comparison of the genotoxicity of EB and DEB in the cells from all three species.

Cytogenetic effects of butadiene metabolites in rat and mouse splenocytes following in vitro exposures.

As a first step in investigating the genotoxic effects of the principal metabolites of 1,3-butadiene (BD) in both rats and mice, splenocytes (which have little mixed function oxidase activity) from each specimen were exposed to a series of concentrations of either 3,4-epoxy-1-butene (EB) (20 to 931 microM) or 1,2:3,4-diepoxybutane (DEB) (2.5 to 160 microM) for 1 h. The splenocytes were then washed, cultured, and stimulated to divide with concanavalin A, and metaphases were analyzed for the induction of sister chromatid exchanges (SCEs) and chromosome aberrations (CAs). In addition, cells from some experiments were taken after exposure but before culture, and subjected to the single cell gel (SCG) assay to measure DNA damage in the form of DNA strand breakage and/or alkaline-labile sites. Initial studies indicate that EB does not induce cytogenetic damage in either rat or mouse G0 splenocytes. However, DEB was an extremely potent SCE- and CA-inducer in both species with no species differences apparent. Neither DEB nor EB produced any statistically significant DNA-damaging effects as measured by the SCG assay.

Cytogenetic effects in mice of divinylbenzene-55 inhalation.

Male B6C3F1 mice (8 weeks of age) were exposed by inhalation to divinylbenzene-55 (DVB-55), at target concentrations of 0, 25, 50 and 75 ppm for 6 h per day for 3 days. Following exposure the animals were killed blood smears were prepared for micronucleus (MN) analysis, and the spleens were removed and cultured for sister chromatid exchange (SCE) and chromosome aberration (CA) analyses. DVB-55 induced a dose dependent increase in SCE with the two highest doses reaching statistical significance. Similarly, there was a statistically significant although less pronounced increase in the frequency of CAs in splenocytes and MN in polychromatic erythrocytes. There was no indication of toxicity as measured by cell cycle kinetics in the splenocytes or the percentage of polychromatic erythrocytes in the peripheral blood smears. Thus, DVB-55 appears to be a weak genotoxicant in vivo.

Frequency of micronucleated-binucleated lymphocytes is not significantly affected by the harvest time following G0 exposure to X-radiation.

Whole blood from two male individuals was X-irradiated using a linear accelerator at 200 cGy/min to give a total exposure of 300 cGy. Lymphocytes were cultured using standard techniques with the addition of 3 micrograms/ml cytochalasin B at 26 h to produce binucleation through the inhibition of cytokinesis for the scoring of micronuclei after the first nuclear division. Replicate cultures from each individual were harvested at 48, 68, 70, 72, 74, 76, 92, 94, 96, 98 and 100 h postinitiation using a cytocentrifuge. Slides were stained with acridine orange, and binucleated cells were scored for the presence of micronuclei. There were no statistically significant differences in the frequency of micronucleated binucleates between replicate cultures, between individuals, or among cultures harvested from 48 to 100 h postinitiation. This indicates that the phytohaemagglutinin-stimulated lymphocytes are a relatively homogeneous population of cells with respect to X-radiation-induced chromosome damage. In addition, these data show that for determining the frequency of micronuclei in lymphocytes irradiated in G0, the harvest time (up to at least 100 h postinitiation) is not critical as long as analysis is confined to the first mitosis after irradiation (i.e. the binucleated cells).

Cytogenetic analyses of the in vitro and in vivo responses of murine cells to peroxyacetyl nitrate (PAN).

Peroxyacetyl nitrate (PAN) is one of a class of common air pollutant formed by the action of sunlight on volatile organic compounds and nitrogen oxides. PAN has been shown to be a bacterial mutagen. To determine if PAN can cause DNA damage in mammalian cells, we exposed murine peripheral blood lymphocytes (PBLs) to various volumes of PAN in vitro and analyzed the cells for chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and DNA damage using the single cell gel (SCG) assay. At in vitro concentrations of PAN that were cytotoxic (inhibited cell division), an increase in DNA damage was noted in the SCG assay. At lower exposure levels that permitted cell division, no increases in SCEs, CAs, or DNA damage were evident. For in vivo studies, male mice were exposed nose-only by inhalation for 1 h to 0, 15, 39 or 78 ppm PAN, and their lung cells removed and cultured for the scoring of SCEs and CAs. In addition, PBLs and lung cells were analyzed by the SCG assay. No dose-related effects were found in any of the assays. These data indicate that PAN does not appear to be a potent clastogen or DNA damaging agent in mammalian cells in vivo or in vitro.

Bleomycin sulfate-induced micronuclei in human, rat, and mouse peripheral blood lymphocytes.

The sensitivity to micronucleus (MN) induction of human, mouse, and rat peripheral blood lymphocytes (PBLs) exposed to bleomycin sulfate (BLM) in vitro was compared in cytochalasin B-induced binucleated (BN) cells. For the PBLs of each species, either 0, 5, 10, 20, 40, 60, 80, or 160 micrograms/ml BLM was added to 5 ml aliquots of whole blood for 4 hr at 37 degrees C in a 5% CO2 atmosphere. Leukocytes were isolated on a density gradient and cultured in the presence of phytohemagglutinin to stimulate blastogenesis, and cytochalasin B was added to each culture at 21 hr postinitiation to prevent cytokinesis. A total of 4,000 BNs/concentration/species was analyzed for MN in two independent experiments. In addition, multiple-MN-BNs were quantitated, and the nucleation index was determined. Significant increases both in total MN-BNs and multiple-MN-BNs were observed at all concentrations in all species. All three species' concentration-response curves gave good fits (r2 values from 0.87 to 0.95) to either a linear or a square root model (y = mx + b or y = m[x]0.5 + b, respectively; where y = the percentage of MN-BN, m is the slope, and b is the y-intercept). The MN induction in the human and rat PBLs was not statistically different, but both were significantly less sensitive than the response shown by the BLM-exposed mouse PBLs. This difference in MN susceptibility was observed only at BLM test concentrations > or = 20 micrograms/ml. The nucleation index was significantly decreased in all species at either 80 or 160 micrograms/ml.

Inhalation studies of the genotoxicity of trichloroethylene to rodents.

Trichloroethylene (TCE) (CAS No. 79-01-6) is an industrial solvent used in degreasing, dry cleaning, and numerous other medical and industrial processes. Controlled inhalation studies were performed using male C57BL/6 mice and CD rats to determine if TCE can induce cytogenetic damage in vivo. Animals were exposed in groups of five to target concentrations of either 0, 5, 500, or 5000 ppm TCE for 6 h. Tissue samples were taken between 18 and 19 h post exposure. Peripheral blood lymphocytes (PBLs) in rats and splenocytes in mice were cultured and analyzed for the induction of sister-chromatid exchanges, chromosome aberrations, and micronuclei (MN) in cytochalasin B-blocked binucleated cells. Bone marrow polychromatic erythrocytes (PCEs) were analyzed for MN. The only positive response observed was for MN in rat bone marrow PCEs. TCE caused a statistically significant increase in MN at all concentrations, inducing an approximate fourfold increase over control levels at 5000 ppm. TCE was also cytotoxic in rats, causing a significant concentration-related decrease in the ratio of PCEs/normochromatic erythrocytes. This study indicates that there may be species-specific cytogenetic effects attributed to TCE inhalation exposure. In follow-up studies, CD rats were exposed for 6 h/day over 4 consecutive days to either 0, 5, 50 or 500 ppm TCE. No statistically significant concentration-related increases in cytogenetic damage were observed. While the MN frequencies in the 4-day study were comparable to those at the equivalent concentrations in the 1-day study, they were not significantly elevated due to an unusually high MN frequency in the controls. A subsequent replication of the 1-day 5000 ppm TCE exposure with rats again showed a highly significant increase in MN frequencies compared to concurrent controls.

Cytogenetic and germ cell effects of phosphine inhalation by rodents: II. Subacute exposures to rats and mice.

Phosphine (PH3) is a highly toxic grain fumigant to which there is significant human workplace exposure. To determine the in vivo cytogenetic effects of inhalation of PH3, male F344/N rats and B6C3F1 mice were exposed to target concentrations of 0, 1.25, 2.5, or 5 ppm PH3 for 6 hr/day for 9 days over an 11-day period. Approximately 20 hr after the termination of exposures, blood was removed from the mice and rats by cardiac puncture and the lymphocytes cultured for analyses of sister chromatid exchanges and chromosome aberrations in rats and mice, and micronuclei (MN) in cytochalasin B-induced binucleated lymphocytes from mice. In addition, bone marrow (rats) and peripheral blood (mice) smears were made for the analysis of MN in polychromatic and normochromatic erythrocytes. No significant increase in any of the cytogenetic endpoints was found at any of the concentrations examined. These results indicate that concentrations of PH3 up to 5 ppm are not genotoxic to rodents when administered by inhalation for 9 days during an 11-day period as measured by several cytogenetic assays. To evaluate the effects of PH3 on male germ cells, a dominant lethal test was conducted in male mice exposed to 5 ppm PH3 for 10 days over a 12-day period and mated to groups of untreated females (2 females/male) on each of 6 consecutive 4-day mating intervals. None of the 6 groups of females exhibited a significant increase in percent resorptions. These results indicate that exposure to 5 ppm PH3 by inhalation does not induce dominant lethality in male mouse germ cells at steps in spermatogenesis ranging from late differentiating spermatogonia/early primary spermatocytes through mature sperm.