Dead Implants Research Articles

Incidence of spontaneous post-implantation deaths in CFT-Swiss mice: its suitability to assess dominant lethal mutations.

The primary objective of the study was to determine the incidence of spontaneous post-implantation deaths (DI) in our randomly-bred closed colony of CFT-Swiss mice. These data are required mainly to ascertain the suitability of the strain for their use in dominant lethal (DL) mutation assays, a well validated in vivo mammalian germ cell assay. To achieve the first goal, randomly drawn healthy adult males were sequentially mated to adult virgin females (1:2 ratio) for a period of 8 weeks. Male-based analysis of the 3 major variables viz., implantations, live embryos, and dead implants (DI) were carried out. Absence of outliers and low variation in spontaneous DI, as well as the other variables associated with DL assay were observed in all matings. Secondly, the degree of induction of DI were studied following administration (i.p.) of an acute dose of ethyl methane sulphonate (EMS). EMS induced nearly a 5-fold increase in the DI during the first 2 weeks and a 3-fold during the third week suggesting its DL-type effect only in specific post-meiotic stages of spermatogenesis as reported earlier.

Use of cyclophosphamide as a positive control in dominant lethal and micronucleus assays

Analyses of dominant lethal (DL) mutations and micronuclei (MN) are 2 important and widely used genotoxicity assays to measure drug-induced chromosome damage in germ cells and somatic cells, respectively. Cyclophosphamide (CP) has been widely used as a positive control in the single-dose mouse MN assay; however, its utility as a positive control for the DL assay has not been fully studied. In the present study, CP was tested in both assays under similar experimental conditions and MN seen in somatic tissue (bone marrow) were correlated with DL mutations seen in germinal tissue. In a dose-range finding study, groups of 5 male mice were dosed i.p. daily for 5 days at 0, 30 or 40 mg/kg CP and bone marrow was harvested 24 h later for MN assay. CP induced a dose-related increase (7- and 11-fold over controls at 30 and 40 mg/kg) in micronucleated polychromatic erythrocytes (MNPCEs) and decreased %PCEs (to 60% and 54% of controls at 30 and 40 mg/kg, respectively). Based on this, a definitive DL and MN study was conducted using separate groups of 30 male mice at 0 and 40 mg/kg CP with a daily times 5 dosing regimen. For the MN assay, bone marrow was collected 24 h after the last dose from 5 animals and evaluated for MNPCEs and %PCEs. For the DL assay, each male was caged with 2 untreated females per week for 8 weeks to cover the postmeiotic germ cell stages. On day 17 after the initiation of breeding, the females were evaluated for the number of implantation sites and live, dead and resorbed implants. The results indicated that CP induced about a 17-fold increase in MNPCEs and a 46% decrease in PCEs in relation to controls. In the DL assay, CP produced a slight (13%) but statistically significant reduction in fertility index at week 7 of mating. Also, the total number of implants was significantly lower during weeks 1, 2, 3, 6 and 7 and the numbers of dead implants and postimplantation loss (PIL) were increased for weeks 1, 2 and 3 (55%, 71% and 34% PIL, respectively) over controls. These data clearly show that CP produced clastogenicity and some toxicity in both somatic tissue and germinal tissue. It was concluded that a dose of 40 mg/kg CP can be used as a positive control compound in the DL assay and in the multiple-dose bone marrow MN assay.

Dominant lethal studies in male mice after exposure to a 50 Hz magnetic field

The potential mutagenicity of power frequency magnetic fields was investigated using a dominant lethal assay in mice. A total of 42 male mice were exposed for 8 weeks to a 50 Hz sinusoidal magnetic field at 10 mT (rms) and 47 males acted as simultaneous cage controls. Each male was subsequently mated with two females on weeks 1, 3, 5, 7, and 9 post-exposure. The numbers of pregnant females, corpora lutea, and live and dead implants were recorded. Multiple logistic regression analyses examined the effects of exposure on pregnancy rate, pre-implantation survival and post-implantation survival. There were no statistically significant differences in overall response between exposed and control groups, nor was there any significant effect of exposure in any post-exposure week. Thus, exposure to power frequency magnetic fields at 10 mT for the approximate period of spermatogenesis did not appear to induce dominant lethal mutation in the germ cells of male mice.

Studies on the effect of ethanol on dominant lethal mutations in Swiss, C57BL6 and CBA mice

Swiss, C57BL6 and CBA males were given 0.1 ml of 40% ethanol per mouse per day for three consecutive days intraperitoneally. These males were mated with untreated virgin Swiss females employing a 4-day mating schedule and three consecutive matings were carried out. In other study, C57BL6 males were given an ascending gradient of 5% to 40% ethanol in drinking water for a total period of 11 weeks. These males were mated with C57BL6 females for 2 weeks. Females were dissected at mid-term pregnancy for the examination of uterine contents including total, live and dead implants. All the investigations comprised at least two or three independent experiments which were evaluated independently as well as after pooling the data. Swiss, C57BL6 and CBA males given 0.1 ml of 40% ethanol, intraperitoneally, gave no evidence of any significant increase in post-implication lethality in the postmeiotic phase of spermatogenesis attributable to ethanol treatment. A moderate but significant reduction in mean total implants indicating pre-implantation losses was seen in Swiss but not in CBA mice. Prolonged feeding of ethanol up to 40% in drinking water failed to provide any evidence of dominant lethal mutations in C57BL6 males at the pre-implantation level and the post-implantation lethals were also not significantly higher than in controls. In Swiss mice, however, the mutagenic index based on both pre- and post-implantation lethality was consistently positive.

Dose-Response Assessment for Developmental Toxicity: II. Comparison of Generic Benchmark Dose Estimates with No Observed Adverse Effect Levels

Dose-Response Assessment for Developmental Toxicity. II. Comparison of Generic Benchmark Dose Estimates with No Observed Adverse Effect Levels. Allen, B. C., Kavlock, R. J., Kimmel, C. A., and Faustman, E. M. (1994). Fundam. Appl. Toxicol. 23, 487-495. Developmental toxicity risk assessment currently relies on the estimation of reference doses (RfD DTs) or reference concentrations (RfC DTs) based on the use of no observed adverse effect levels (NOAELs) divided by uncertainty factors (UFs). The benchmark dose (BMD) has been proposed as an alternative basis for reference value calculations. A large database of 246 developmental toxicity experiments representing 1825 endpoints related to dead implants or malformed fetuses has been compiled for use in evaluating alternative approaches to developmental toxicity risk assessment. Using this database we have compared two approaches for BMD estimation with each other and with corresponding statistically derived NOAELs. Comparisons have been based on proportion of affected litters (litters with one or more affected offspring, a quantal response variable) and on the proportion of affected offspring within each litter (a continuous response variable). A quantal Weibull model was used to calculate generic BMDs for the quantal response variable (QBMDs) and a continuous power model was used to calculate generic BMDs for the continuous response variable (CBMDs) at three levels of additional risk (10, 5, and 1%). CBMD 05s (continuous benchmark doses for 5% risk) and CNOAELs (statistically derived NOAELs based on the continuous response variable) were similar, with over 98% of the data subsets having CBMD 05 and CNOAEL values that differed by less than an order of magnitude. In contrast, QNOAELs tended to be greater than corresponding QBMD 10s. The observed conservatism of the QBMD values relative to the corresponding CBMD values was attributed to two factors, lower maximum likelihood estimates for the quantal model and wider confidence intervals around the maximum likelihood estimates, compared to the continuous model. Comparisons of different quantitative dose-response assessments for developmental toxicity experiments should help to identify appropriate risk assessment approaches for developmental toxicity risk assessment.

Evaluation of spermatogenic response of mice to the induction of mutations by combined treatment with X rays and antineoplastic drugs.

Combined treatment with low doses of X-rays plus cyclophosphamide (0.25 Gy + 25 mg/kg body weight) or X-rays plus mitomycin C (0.25 Gy + 1.75 mg/kg body weight) did not induce significant dominant lethal effects in any stage of spermatogenesis when a parameter representing pre- and postimplantation loss, such as the decrease of live implants per female, was applied. After combined exposure to high dose of X-rays plus cyclophosphamide (1.00 Gy + 100 mg/kg body weight) an increase of dominant lethal mutations (DLMs) was observed in differentiating spermatogonia, spermatids, and spermatozoa with the same parameter. Combined treatment with high doses of X-rays plus mitomycin C (1.00 Gy + 5.25 mg/kg body weight) produced DLMs in differentiating spermatogonia and late spermatocytes. A calculation of "enhanced risk" was applied to the data of DLMs from the combined treatment regimen and was based on the proportion of dead implants (postimplantation loss only). Enhanced risk could be shown not only after high but also after low combined exposure to X-rays plus cyclophosphamide and X-rays plus mitomycin C. With low doses this enhanced risk was observed in spermatids for X-rays plus cyclophosphamide and in differentiating spermatogonia to early spermatocytes for X-rays plus mitomycin C.

Summary report of the working group on mammalian germ cell tests

The two tests considered by the Working Group were the mammalian germ cell cytogenetic assay and the rodent dominant lethal test. It was agreed that both tests were mainly used for identification of germ cell hazards, however, that the commonly applied protocol of the dominant lethal assay often supplied information for hazard characterization such as sensitivity of particular developmental stages of male germ cells. No particular species or strains were indicated. Concurrent solvent controls were regarded as indispensable for both tests. In the discussion of the mammalian germ cell cytogenetic assay, harmonization was obtained to a large extent with the cytogenetic bone marrow assay regarding the number of animals (5), the number of cells analyzed per animal (200), the highest exposure dose (MTD) and sampling times (twice within 24 and 48 h after dosing). However, it was pointed out that only the single acute exposure was adequate for the mammalian germ cell cytogenetic assay. Furthermore, it was stated that only structural chromosome aberrations could be analyzed and that it was not informative to score polyploidies or aneuploidies. In the discussion of the rodent dominant lethal test, it was stated that the assay was generally performed with treated males, however, increasing concern about female specific effects required that a protocol for female dominant lethal testing should be developed and validated. Acute and subacute treatment schedules were considered equally acceptable. It was regarded as highly important that the entire male germ cell development from meiosis to mature sperm was covered in the test protocol either by the appropriate mating schedules after single dosing or by subchronic dosing during the respective period. Postimplantation loss, preimplantation loss and fertility rate were the main parameters to be assessed in the rodent dominant lethal tests. It was agreed that the size of the experiment depended on the spontaneous frequency of dead implants, the mating scheme and the statistical design of the experiment.

Dose—Response Assessment for Developmental Toxicity

Developmental toxicity risk assessment currently relies on the estimation of reference doses (RfDDTs) or reference concentrations (RfDDTs) based on the use of no observed adverse effect levels (NOAELs) divided by uncertainty factors (UFs). The benchmark dose (BMD) has been proposed as an alternative basis for reference value calculations. A large database of 246 developmental toxicity experiments representing 1825 endpoints related to dead implants or malformed fetuses has been compiled for use in evaluating alternative approaches to developmental toxicity risk assessment. Using this database we have compared two approaches for BMD estimation with each other and with corresponding statistically derived NOAELs. Comparisons have been based on proportion of affected litters (litters with one or more affected offspring, a quantal response variable) and on the proportion of affected offspring within each litter (a continuous response variable). A quantal Weibull model was used to calculate generic BMDs for the quantal response variable (QBMDs) and a continuous power model was used to calculate generic BMDs for the continuous response variable (CBMDs) at three levels of additional risk (10, 5, and 1%). CBMD05s (continuous benchmark doses for 5% risk) and CNOAELs (statistically derived NOAELs based on the continuous response variable) were similar, with over 98% of the data subsets having CBMD05 and CNOAEL values that differed by less than an order of magnitude. In contrast, QNOAELs tended to be greater than corresponding QBMD10s. The observed conservatism of the QBMD values relative to the corresponding CBMD values was attributed to two factors, lower maximum likelihood estimates for the quantal model and wider confidence intervals around the maximum likelihood estimates, compared to the continuous model. Comparisons of different quantitative dose-response assessments for developmental toxicity experiments should help to identify appropriate risk assessment approaches for developmental toxicity risk assessment.

Studies on the genotoxicity of asataf (acephate), an organophosphate insecticide, in a mammalian in vivo system

The genotoxic potential of asataf (acephate) was evaluated by a battery of in vivo tests: bone marrow chromosome aberrations, micronucleus, sperm-shape abnormality and dominant lethal tests in mice. A significant enhancement in the percentage of chromosome aberrations was noticed in 3 doses, 3 routes and 3 h after asataf treatment of groups of mice as well as in chronic (sub-acute) treatment. A significant difference in the occurrence of micronuclei was found only at the highest dose whereas all the results of the sperm-shape abnormality test were highly significant. In the dominant lethal mutagenicity assay only the result (dead implants) of a single week (3rd) with the higher dose differed significantly from control. On the basis of the present in vivo results in mouse test systems asataf may be considered to be a potential mutagen.

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.

The effects of love canal soil extracts on maternal health and fetal development in rats

The effects of a solvent extract of the surface soil of the Love Canal chemical dump site, Niagara Falls, New York, and of a natural extract, or leachate, which is drained from the canal for treatment, on the maternal health and fetal development were determined in rats. The solvent extract, which was contaminated with 2,3,7,8-tetrachlorodibenzo- p-dioxin (2,3,7,8-TCDD) at 170 ppb and numerous other chlorinated organic compounds with the primary identified components being the isomers of benzenehexachloride (BHC), was dissolved in corn oil and administered by gavage to pregnant rats at 0, 25, 75, or 150 mg crude extract/kg/day on Days 6–15 of gestation. A 67% mortality was observed at the highest dose. The rats were sacrificed on Day 20. Dose-related increases in relative liver weight accompanied by hepatocyte hypertrophy were observed at all dose levels. Fetal birthweight was decreased at 75 and 150 mg extract/kg/day. No major treatment-related soft tissue or skeletal malformations, except for delayed ossification, were observed. Based on literature values for BHC, all of the observed toxicity could be accounted for by the BHC contaminants of the extract. The crude organic phase of the leachate was administered to pregnant rats at 0, 10, 100, or 250 mg/kg/day as described above. Maternal weight gain decreased at 100 and 250 mg/kg/day, accompanied by 5 and 14% maternal mortality, and 1 and 3 dead fetuses, respectively. Early resorptions and the percentage of dead implants increased whereas fetal birthweights were decreased at 250 mg/kg/day. No major treatment-related soft tissue or skeletal malformations, except for delayed ossification, were observed. The primary components of the complex leachate by mass were tetrachloroethanes; however, 2,3,7,8-TCDD, which was present at 3 ppm, probably accounted for all the observed toxicity.

Studies on germinal effects of quercetin, a naturally occurring flavonoid

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is one of the most widely occuring flavonoids ingested by man in food. It has been showen to be mutagenic in prokaryotes as well as in in vitro mammalian cell lines. In view of the unavoidability of ingesting them via a normal diet, there is a need to assess the potential genetic risk to man, due to flavonoid ingestion, using whole animal assays. Dominant lethal studies have been carried out in adult Swiss male mice and Wistar male rats to investigate the germinal effects of quercetin. Adult Swiss males were treated with 200, 300 or 400 mg/kg of quercetin dissolved in 60% dimethyl sulfoxide, intraperitoneally. In the rat study, 200 and 300 mg/kg of quercetin were used. Individually caged males were paired with untreated females for a week and six sequential matings were carried out. Two independent experiments in mice and a single experiment in rats constituted the study. Females were evaluated for inducted dominant lethality during the midterm of pregnancy. At 200 mg/kg dose of quercetin, there were no significant differences between control and the test group in pregnancy, total or live implantations in mice or rats during the whole test period that could be attributed to the flavonoid exposure. In mice at 300 and 400 mg/kg of quercetin, there was a profound reduction in fertility of the males during all six matings. The number of total and live implantations also decreased, particularly at the 400 mg/kg dose, although the sample size was too small to be statistically significant. Contrary to this, the rat study did not show any impairment of fertility, nor was there any substantial suppression of total and live implantations at the highest (300 mg/kg) dose tested. There were no significant differences between control and treated groups with regard to the number of dead implantations at any dosage level at any stage of the study in mice and rats. Thus, no post-implantation losses — a reliable measure of dominant lethal mutations — were induced by quercetin in mice or rats. The loss of fertility could be due to germinal cytotoxicity, oligospermia or impairment of fertilizing ability of the treated animals. Similarly, several non-genetic factors which can contribute to reduction of live implantations would have to be excluded before considering induction of preimplantation lethality due to quercetin ingestion. In view of the small sample size and the 7-day mating schedule used in the present study, dominant lethal effects of quercetin require investigation using a 4-day mating schedule.

Dominant lethal testing of theobromine in rats

Theobromine (TBR) 3,7-dimethylxanthine, the major purine alkaloid present in chocolate and cocoa products, was evaluated for dominant lethality in male Sprague-Dawley rats after administration of subacute oral doses of 0, 50, 150 and 450 mg/kg. Dominant lethal mutations were assessed weekly for a 10-week period by killing the females 13 days after the midweek of presumptive mating with treated males and counting total implants (living and dead), corpora lutea and number of pregnant females. Dead implants per total implants and preimplantation loss per total corpora lutea were evaluated statistically. No significant dose-dependent effects were observed in the % dead implants of preimplantation loss throughout the study; pregnancy rate averaged 94%. These results indicated no induction of dominant lethal mutations or adverse effects on pregnancy rate after TBR doses equivalent to 25–225 times the maximum human consumption level.

Dominant Lethal Induction and Testicular Uptake of Iodine-125 in Mice

The mutagenic potential of 125I was studied using dominant lethal (DL) and testicular uptake studies. Dominant lethality (DL) represents embryonic death resulting from the chromosomal breakage in gametes of parents. When compared to controls, mice treated with different doses of 125I showed significant levels of induced DL. Significant pre-implantation losses were observed and variations in live implantations indicated total losses by the isotope. Dead implantations per pregnant female in the isotope treated groups showed a significant increase from controls indicating induced levels of post-implantation losses. All the stages of spermatogenesis, i.e., spermatozoa, spermatid, spermatocyte and spermatogonia were found to be sensitive to the induction of post-implantation losses, the spermatid stage being the most sensitive. Testicular uptake was measured from 1/2 hour to 72 hours after injection and maximum uptake was recorded at 1/2 hour, indicating the permeability of the blood/testis barrier.

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA · 2HC1 of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.

N-Butyl acrylate: Prenatal inhalation toxicity in the rat

n-Butyl acrylate was examined for its prenatal toxicity in Sprague-Dawley rats. Inseminated rats were exposed to concentrations of 0, 25, 135 and 250 ppm for a duration of 6 hours per duy from the 6th to 15th day post coitum. Subsequently the animals were held to the 20th day post coitum. During the inhalation period concentrations of 135 and 250 ppm n-butyl acrylate led to maternal toxicity (delayed body weight gain depending on the concentration, clinical signs of irritation). At the end of the exposure period the signs subsided. The same concentrations induced embryolethality (increased number of dead implantations depending on the concentration). 25 ppm of n-butyl acrylate did not lead to maternal toxicity or embryolethality. Under the conditions chosen, a teratogenic effect of the test substance was not detected at any concentration.

Evaluation of the mutagenic effects of phenobarbital by dominant lethal assay in Swiss mice

Phenobarbital, a potent antiepileptic drug, was tested for the induction of dominant lethal mutations in male Swiss mice. The drug was administered in sterile distilled water at doses of 42, 84 or 126 mg/kg body weight by intubation over five consecutive days. Controls were given distilled water alone. The males were then mated for 1 wk sequentially with pairs of untreated virgin females for a total of 8 wk. The females were killed 17 days after presumed mating and the uterine contents were examined. There was a significant reduction in the total number of implantations and an increase in the number of dead implantations per pregnant female in the treated groups compared with the controls. The results from sequential matings showed that the pre-meiotic stages of spermatogenesis were more affected than the post-meiotic stages.

Detection of dominant lethal mutation in mice after repeated low-dose administration of 6-mercaptopurine.

Detection of dominant lethality after repeated low-dose administration was investigated, using the base analog 6-mercaptopurine (6-MP). The compound was administered to groups of 30 outbred CD-1 male mice by i.p. injection at dosage levels of 12.5, 25.0, or 50.0 mg/kg/day 5 days a week for 8 weeks. At the end of treatment, each male was cohoused for 1 week with two untreated females of different strains: one CD-1 and one (C3H x C57BL/10)F1. Negative (solvent) and positive (triethylenemelamine, TEM) control groups were included. Implant data were analyzed statistically. Exposure of male mice to 6-MP at 50 mg/kg/day resulted in 93% mortality and severe weight loss of the survivors. Body weights were also reduced in the group given 25 mg/kg/day. At the lowest dose level of 12.5 mg/kg/day, 6-MP had no noticeable toxic effect on the treated males. Dominant lethal analysis of the implant data showed that a statistically significant increase in dead implantations was induced in CD-1 but not in (C3H x C57BL/10)F1 females. The dominant lethal effect of TEM, the positive control, was detected in both strains of females tested.

N-Butyl Acrylate: Prenatal Inhalation Toxicity in the Rat

n-Butyl Acrylate: Prenatal Inhalation Toxicity in the Rat. Merkle, J. and Klimisch, H.-J. (1983). Fundam. Appl. Toxicol. 3:443–447. n-Butyl acrylate was examined for its prenatal toxicity in Sprague-Dawley rats. Inseminated rats were exposed to concentrations of 0, 25, 135 and 250 ppm for a duration of 6 hours per day from the 6th to 15th day post coitum. Subsequently the animals were held to the 20th day post coitum. During the inhalation period concentrations of 135 and 250 ppm n-butyl acrylate led to maternal toxicity (delayed body weight gain depending on the concentration, clinical signs of irritation). At the end of the exposure period the signs subsided. The same concentrations induced embryo-lethality (increased number of dead implantations depending on the concentration). 25 ppm of n-butyl acrylate did not lead to maternal toxicity or embryolethality. Under the conditions chosen, a teratogenic effect of the test substance was not detected at any concentration.

Dominant-lethal study of technical-grade hexachlorocyclohexane in Swiss mice

Male Swiss mice, 6–8 weeks old, were given a diet containing technical-grade hexachlorocyclohexane (BHC) at 500 ppm continuously for 4,6 and 8 months. After the completion of the scheduled exposure period, the males were sequentially mated with 2–3 untreated virgin females at weekly intervals for 8 weeks. The females were autopsied at mid-term pregnancy for evaluation of dominant-lethal mutation. The number of dead implants, including deciduomas and dead embryos, showed a significant increase. Similarly, the percentage fertility and live embryos per female showed a decline when compared with the control.