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 X 2HCl 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.     

Dominant-lethal test results with known mutagens in two laboratories

During investigations of the potential dominant lethal activity of various chemicals, concurrent controls were run to check the sensitivity and the reproducibility of the test systems. These experiments were carried out over an 18-month period in two laboratories using similar protocols.Negative control substances used were maize oil, dispersol, tween 80 and water. Positive control substance used were cyclophosphamide, ethyl methane-sulphonate and mechlorethamine hydrochloride (nitrogen mustard). The substances were administered either intraperitoneally or by gavage.The results were analysed using, principally, a hierarchical analysis of variance of the total implants per pregnancy and the transformed early deaths per pregnancy data.Pregnancy frequency generally did not fall below 80%. The total number of implantations per pregnancy was usually about 11.8 in negative controls and was variably reduced by the mutagens used. With cyclophosphamide or ethyl methanesulphonate (EMS), there were some quantitative variations in the results obtained but qualitative agreement was good in the results both between experiments in the same laboratory and between the two laboratories. It was demonstrated that EMS is a useful positive control substance in experiments with orally administered materials. Sensitivity of the system was indicated by positive effects with a relatively low dose of EMS and consistent positive results with mechlorethamine for which a dominant lethal effect has not been demonstrated previously.It is concluded that the dominant lethal test gives reproducible data and it is, thus, possible to have confidence in the results and to compare findings in different laboratories using similar protocols.     

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.     

Fenitrothion. I. Study of mutagenic activity in rats.

The mutagenic activity of fenitrothion was studied in rats given 0,10,40 or 80 ppm of fenitrothion in the diet. The study combined the dominant lethal test with cytogenetic analysis of chromosomal aberrations. Dominant lethal mutations were investigated: 1. by their so-called tentative determination in single mating in P-to F3 generation males and females following 200 days exposure; 2. by assessing the effect of the agent at individual stages of spermatogenesis, with F2 and F4 generation males having been exposed for 100 days and mated to unexposed females for 10 weeks. Chromosome aberrations were analyzed in the bone marrow of F2 generation males following 200-days exposure and F3 generation (males) following 500-day exposure to a dose of 80 ppm. Negative results were obtained in all experiments in relation both to dose and generation. Hence fenitrothion is not considered to be a substance with a mutagenic activity. The metodical advantages of the proposed combination of reproduction and mutagenic-activity studies of an agent for toxicological evaluation are discussed.     

Female-specific mutagenic response of mice to hycanthone

Male and female gametogeneses differ markedly in all mammals. While male germ cells are continuously being produced from stem cells throughout the reproductive life span, the number of female germ cells is fixed during prenatal development and, soon after birth, all of the oocytes are arrested in a modified diplotene, or dictyate, stage. Following puberty, dictyate oocytes are hormonally triggered to mature either singly or in groups, resulting in ovulation and the completion of the first meiotic division. It has been hypothesized that female mice are more susceptible to dominant lethal effects of intercalating agents than male mice because oocyte chromosomes, which are arrested in a diffuse state, are generally more accessable to intercalation than are the more condensed chromosomes present within most male germ cell stages. This hypothesis was further tested using the intercalating agent hycanthone methane-sulfonate. Effects of hycanthone were studied in maturing and primordial oocytes and in male germ cells throughout spermatogenesis. No induction of dominant lethality was observed for treated males while a significant increase in embryonic death, expressed around the time of implantation, was observed in females that mated within 4.5 days after treatment. These effects were the result of dominant lethal mutations induced in maturing oocytes and not of maternal toxicity as indicated by the presence of chromosomal aberrations observed at first-cleavage metaphase of zygotes obtained from treated females. These results add support to the hypothesis that certain intercalating chemicals, which are not mutagenic to male mice, may be mutagenic to females and point to a need for more in-depth studies of female-specific mutagenesis.     

On the genotoxicity of the pesticides Endodan and Kilacar in 6 different test systems

Two pesticides, the fungicide Endodan (ethylene thiuram monosulphide) and the insecticide-acaricide Kilacar (bis(parachlorophenyl)cyclopropyl methanol), produced or used in the neighbouring countries of Bulgaria and Greece were investigated in a coordinated research programme for their genotoxic effects in a variety of test systems. This included the Ames test, Aspergillus nidulans for mitotic segregation, in vitro human lymphocyte cell cultures for SCE and chromosomal aberrations, in vivo bone marrow cells in hamsters and rats and the dominant lethal test in rats. The genotoxicity of Endodan was found to range from negative to slightly positive in different test systems. At concentrations of 7.5 and 12.0 micrograms/plate together with S9 mix it induced base-pair substitutions in the TA100 strain of Salmonella typhimurium at a rather low level. At a dose of 93 mg/kg b.w. it also caused chromosomal aberrations in acutely treated hamster bone marrow cells. A significant increase of SCE was also found in human lymphocyte cultures at a concentration of 20.0 micrograms/ml. Endodan was found to be negative in A. nidulans for somatic segregation, lymphocyte cultures for chromosomal aberrations and mitotic activity and in rats for dominant lethals and chromosomal aberrations. Kilacar was found to be a weak mutagen in the TA97 strain of S. typhimurium at concentrations of 2.5 and 5.0 micrograms/plate together with S9 mix. At concentrations of 1.0, 1.5 and 2 micrograms/ml Kilacar increased the number of mitotic segregants in A. nidulans by 160%, 220% and 156% respectively over the control. In Syrian hamster bone marrow cells after acute administration at concentrations of 0, 40, 80 and 160 mg/kg, the MI was 5.50, 4.30, 3.10 and 1.30 respectively, and an increase in chromosomal aberrations of about 300% over the control was observed with a concentration of 80 mg/kg. In human lymphocytes no significant changes were observed in either MI or SCE. In the dominant lethal test after chronic treatment of male rats at doses of 5.1, 10.2 and 102.0 mg/kg b.w. no significant mutagenic effect was found although a decrease was shown in the percentage of females with implants mated with treated males in the first week.     

Genotoxicity of lomefloxacin--an antibacterial drug in somatic and germ cells of Swiss albino mice in vivo

The in vivo genotoxicity of lomefloxacin, a diflourinated antibacterial drug, was evaluated by employing mouse in vivo chromosomal aberration test in bone marrow cells and dominant lethal mutation assay in germ cells. Statistically significant reduction in mitotic index, increase in chromosomal aberrations (CAs)/cell and percent abnormal metaphase was observed only at the highest dose (160 mg/kg b.w.) of the drug. In the dominant lethal mutation assay, a statistically significant decrease in the number of implants/female, compared to vehicle control, was noticed only in the females mated with males treated with 32 mg/kg b.w. during the third week of mating, while statistically significant reduction in live implants/female was noticed at both the doses during the second and third weeks of mating. Nevertheless, no significant change in the number of dead implants/female was observed after lomefloxacin treatment. These results seems to indicate that lomefloxacin is a weak clastogen in the bone marrow cells and non-mutagenic in the germ cells of mouse in vivo.     

Exploring Extra-Binomial Variation in Teratology Data Using Continuous Mixtures

Discrete data from animal teratology experiments are known to exhibit extra-binomial variation. For example, we discuss a dominant lethal assay experiment in which male mice are exposed to various levels of radiation and are then mated to females. The response of interest is the number of resorptions out of the number of implantations. Most statistical work on analyzing such data has focused on modeling response rates as a function of dose of a suspected teratogen (radiation in this case) while accounting for the extra-binomial variability when calculating standard errors of the regression coefficients. Sometimes, however, when an unobserved genetic or exposure variable is suspected, the shape of the mixing distribution is of interest. We propose a mixture of beta-binomials (MBB) family of distributions that includes the non-parametric mixture of binomials model of Laird (1978) as a special case. The MBB family can accommodate a mixing distribution with one or more modes, and we develop a bootstrap test for multimodality. We apply the method to data from a dominant lethal teratology experiment.     

Studies on the mutagenic effect of contraceptive drugs. I. Induction of dominant lethal mutations in female mice

30 adult virgin female mice (2 strains) received either high or low doses of Anovlar or Lyndiol oral contraceptives and were tested for induction of dominant lethal mutations. The pregnant mice were dissected on Day 14 of pregnancy and total implantations, early deaths, late deaths, and corpora lutea were counted in each pregnancy. A significant reduction in fertile mating (p .025) was found in 1 strain of those who received the high dose of Lyndiol (10 times that of the low dose, which is physiologically equivalent to the human dose). This dose also increased the number of dead implants in both strains which resulted in higher estimates of dominant lethal mutations. It is concluded that when Lyndiol and Anovlar were given at the physiological dose level to control ovulation in mice, the frequency of dominant lethal mutations was not increased above the control level.     

Chemical and radiation induced late dominant lethal effects in mice.

Although theoretically expected, experimental data to date have not shown dominant lethal expression to occur throughout the developmental period. Specifically, late post-implantation effects have not been demonstrated. We routinely use an experimental technique in which parental females mated to mutagenically treated males are allowed to give birth and wean their litter, and their uterine horns are then inspected for uterine scars indicative of live and dead embryos. In a number of experiments in which males were mutagenically treated with either chemicals or X-irradiation, a discrepancy was observed between the number of live embryos as determined by the scar technique and the number of live observed at birth, suggesting the possibility of embryonic losses at a late stage in development. Initial analyses showed that mutagenic treatment increased the percentage of these late losses. These differences were statistically significant in 2 of 3 analyses. Factors affecting statistical significance and an understanding of dominant lethal mutations are discussed.     

Lack of dominant lethality in mice following 1-bromopropane treatment

1-Bromopropane (1-BP) is widely used in spray adhesives, precision cleaner, and degreaser. This study was conducted to investigate the potential of 1-BP to induce dominant lethality in mice. 1-BP was orally administered to males at doses of 300 and 600 mg/kg for 10 days before mating. Cyclophosphamide was used as a positive control (PC), which was administered intraperitoneally to males at 40 mg/kg for 5 days. The vehicle control (VC) group received corn oil only. Thereafter, males were mated with untreated females during six sequential mating periods of a week each. Males were sacrificed at the end of mating and so were the pregnant females on days 15-17 of gestation. Clinical signs, gross findings, mating index, gestation index, the numbers of corpora lutea, implantations, live fetuses, resorptions and dead fetuses, pre- and post-implantation losses, and dominant lethal mutation rate were examined. There were no treatment-related changes in clinical signs, gross findings, mating index, gestation index, number of corpora lutea and implantations, pre-implantation loss, live fetuses, resorptions, dead fetuses, post-implantation loss at any 1-BP doses tested. In the PC group, there were no treatment-related changes in mating index, gestation index, number of corpora lutea, and dead fetuses. However, a decrease in the number of implantations and an increase in pre-implantation loss were observed during the first 2 weeks as compared to those of the VC group. No treatment-related changes were observed in the third to sixth weeks. Increases in resorptions, fetal deaths and post-implantation loss, and a decrease in the number of live fetuses were observed in the first 3 weeks of the PC group compared to those of the VC group. However, no treatment-related changes were observed during the forth to sixth weeks. An increase in dominant lethal mutation rate was observed in 1-3 weeks of mating of the PC group, but there was no significant difference in 1-6 weeks of mating of the 1-BP treatment groups. In conclusion, 1-BP did not induce dominant lethality in mice. These results are in agreement with the report of Saito-Suzuki et al., demonstrating that no dominant lethality of 1-BP was observed in Sprague-Dawley rats.     

Dominant lethal assay of some hair-dye components in random-bred male rats.

Male rats were exposed to maximally tolerated doses of 5 hair-dye components in a dominant lethal test. Each component was tested at 3 dosage levels with 15 random-bred male rats per level. The highest dose, selected on the basis of subacute toxicity testing, generally reduced weight gains without being lethal. Freshly prepared solutions were injected i.p. at 1 ml/kg 3 times a week for 10 weeks. Rats injected with dimethylsulfoxide and triethylenemelamine served as solvent and positive controls, respectively. A majority of rats survived the treatment at the levels tested and were mated to two virgin females each per week for 2 weeks. The females were sacrificed at midterm of pregnancy and examined for live and dead implants. Dominant lethality was evaluated on the basis of 4 criteria: dead implants per pregnant female, dead implants per total implants, proportion of females with one or more dead implants, and proportion of females with two or more dead implants. 2-Nitro-p-phenylenediamine, 2,4-diaminoanisole sulfate and 2,5-diaminoanisole sulfate produced negative responses, whereas m-phenylenediamine and 4-nitro-o-phenylenediamine induced weak dominant lethality in the first trial. On retesting these weakly positive components, both m-phenylenediamine and 4-nitro-o-phenylenediamine produced negative responses.     

Statistical tests for recessive lethal-carriers.

This paper presents a statistical method for testing whether a male mouse is a recessive lethal-carrier. The analysis is based on a back-cross experiment in which the male mouse is mated with some of his daughters. The numbers of total implantations and intrauterine deaths in each litter are recorded. It is assumed that, conditional on the number of total implantations, the number of intrauterine deaths follows a binomial distribution. Using computer-simulated experimentation it is shown that the proposed statistical method, which is sensitive to the pattern of intrauterine death rates, is more powerful than a test based only on the total number of implant deaths. The proposed test requires relatively simple calculations and can be used for a wide range of values of total implantations and background implant mortality rates. For computer-simulated experiments, there was no practical difference between the empirical error rate and the nominal error rate.     

Dominant lethal study of ribavirin in male rats

Ribavirin, a new synthetic antiviral agent, was studied for dominant lethal effects in male CD rats. The drug was administered intraperitoneally at doses of 50, 100 and 200 mg/kg/day for 5 days. Males were mated weekly with 8 consecutive batches of female rats. Marginal increase in early foetal death detected in Assessment Weeks 3 and 8 in females mated with the low-dose and high-dose males were not dose-related and were most probably chance events caused by the particularly low vehicle control frequencies for these 2 weeks. Also, the slightly reduced pregnancy proportion among females mated with the high-dose treated males was to a substantial extent the effect of a single male rate which failed to fertilize any females. Ribavirin was, therefore, regarded as being devoid of any mutagenic potential demonstrable by a rat dominant lethal assay.     

Reproductive capacity and dominant lethal mutations in female guinea-pigs and Djungarian hamsters following X-rays or chemical mutagens.

The reproductive capacity and induction of dominant lethal mutations in adult female guinea-pigs and Djungarian hamsters were tested following treatment with 400 rad X-rays, 1.6 mg/kg triethylenemelamine (TEM) or 75 mg/kg isopropylmethanesulphonate (IPMS). A fairly high level of dominant lethals were observed in female guinea-pigs mated at the first oestrus after irradiation (23.4 +/- 6.4%) with a lower yield at 3 months (9.6 +/- 8.2%). Neither of the chemicals caused any significant induction of dominant lethals at either mating time. In the reproductive capacity experiments, the mean litter size of irradiated female guinea-pigs was reduced for about 12 months and this was especially marked in the first 6 months following treatment. Neither of the chemicals caused any significant differences in early litter sizes but there was a noticeable reduction in the litter sizes of TEM-treated females in the 18--24 month interval. With Djungarian hamsters a marked effect of X-rays on reproductive capacity was apparent. After 400 rad a smaller proportion of irradiated females littered in the first 25-day interval than after the other treatments, and no irradiated females produced more than one litter. Neither of the chemicals caused such a drastic reduction in fertility but TEM-treated females produced fewer litters and became sterile at an earlier age than control or IPMS-treated females. With IPMS, the number of litters produced was similar to the controls. Both chemicals caused a significant reduction in litter-size but further work is needed to establish whether this was due to induction of dominant lethals. No translocations were observed in the sons of treated female guinea-pigs or hamsters, but the numbers of animals studied were too small for any conclusions to be drawn.     

Male-mediated dominant lethal mutations in mice following prooxidant treatment.

This study's primary aim is to examine if prooxidant treatment has the propensity to induce dominant lethal (DL) type mutations in a randomly bred closed colony of CFT-Swiss mice. Initially, graded doses of both organic hydroperoxides viz., t-butyl hydroperoxide (tbHP), and cumene hydroperoxide (cHP) were administered (i.p.) to adult males and the mortality data was analysed to determine the LD(50) values. cHP was relatively more toxic compared to tbHP. The computed LD(50) values were 1500 and 3000 micromol (kg body weight)(-1) for cHP and tbHP, respectively. Subsequently, adult males were administered (i.p.) with 1/10 LD(50) doses of hydroperoxide (HP) (tbHP--30 micromol (100 g body weight)(-1) and cHP - 15 micromol (100 g body weight)(-1)) on 5 consecutive days and were mated with virgin females for a period of 5 weeks to characterise the male-mediated DL mutations. Male-based analysis of the three major variables viz., implantations, live embryos and dead implants (DI) were carried out to assess the DL-type response induction. While tbHP induced significant increases (2- to 5-fold) in the incidence of DI during the first 4 weeks, cHP induced a marginal increase only during the first week. These results suggest that prooxidants induce DL-type effect only in specific post-meiotic stages of spermatogenesis and stress the need to further investigate the implications of chronic oxidative stress on the male reproductive system.     

Ethylene thiourea (ETU). A review of the genetic toxicity studies

Ethylene thiourea (ETU) is a common contaminant, metabolite and degradation product of the fungicide class of ethylene bisdithiocarbamates (EBDCs); as such, they present possible exposure and toxicological concerns to exposed individuals. ETU has been assayed in many different tests to assess genotoxicity activity. While a great number of negative results are found in the data base, there is evidence that demonstrates ETU is capable of inducing genotoxic endpoints. These include responses for gene mutations (e.g. Salmonella), structural chromosomal alterations (e.g. aberrations in cultured mammalian cells as well as a dominant lethal assay) and other genotoxic effects (e.g. bacterial rec assay and several yeast assays).It is important to consider the magnitude of the positive responses as well as the concentrations/doses used when assessing the genotoxicity of ETU. While ETU induces a variety of genotoxic endpoints, it does not appear to be a potent genotoxic agent. For example, it is a weak bacterial mutagen in the Salmonella assay without activation in strain TA1535 at concentrations generally above 1000 μg/plate. Weak genotoxic activity of this sort is usually observed in most of the assays with positive results. Since ETU does not appear very potent and is not extremely toxic to test cells and organisms, it is not surprising to find that ETU does not produce consistent effects in many of the assays reviewed. Consequently, in many instances, mixed results for the same assay type are reported by different investigators, but as reviewed herein, these results may be dependent upon the test conditions in each individual laboratory. A primary shortcoming with many of the reported negative results is that the concentrations or doses used are not high enough for an adequate test for ETU activity. There are also problems with many of the negative assays generally in protocol or reporting, particularly with the in vivo studies (e.g. inappropriate sample number and/or sampling times; inadequate top dose employed).Overall, while ETU does not appear to be a potent genotoxic agent, it is capable of producing genotoxic effects (e.g. gene mutations, structural chromosomal aberrations). This provides a basis for weak genotoxic activity by ETU. Furthermore, based on a suggestive dominant lethal positive result, there may be a concern for heritable effects. Due to the many problems with the conduct and assessment of the in vivo assays, it is worth repeating in vivo     

Induced chromosomal aberrations in pronuclei, 2-cell stages and morulae of mice

C₃H female mice, 10–12 weeks old, were treated in the pre-ovulatory phase of oogenesis or in the first mitotic interphase after fertilization with amethopterin (M) at 200 mg/kg body weight. The females were mated with untreated males of the same age group and strain and were checked hourly for vaginal plugs. At different times after fertilization the oviducts of the females of experiment and control were flushed out and the cleavage stages obtained were examined cytologically. Chromosome analysis was performed in the pronucleus stage, in the 2-cell stage and in morulae. Separate analyses of maternal and paternal chromosomes of the pronucleus stage can show whether the spontaneous or induced aberrations are derived from oogenesis or spermatogenesis. After treatment the frequency of numerical and structural aberrations increased compared with the matched control. During the early prenatal development at least 2 steps seem to be important for the elimination of chromosomal aberrations: 1, from the unfertilized mII oocyte to the pronucleus stage; and 2, from early morulae to the registration of dominant lethal mutations.     

A review and analysis of the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase assay to determine the mutagenicity of chemical agents. A report of phase III of the U.S. Environmental Protection Agency Gene-Tox Program

Published literature on the Chinese hamster ovary cell/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT) assay from mid-1979 through June 1986 was reviewed and evaluated. Data from the papers considered acceptable include test results on 121 chemicals belonging to 25 chemical classes. A total of 87 chemicals were evaluated positive, 3 negative, and 31 inconclusive. Mutagenicity data on 49 of the 121 chemicals evaluated could also be compared with in vivo animal carcinogenicity data. 40 of the 43 reported animal carcinogens were considered mutagenic. Caprolactam, the only definitive noncarcinogen in the group of 49, was not mutagenic. The CHO/HGPRT assay was concluded to be an appropriate assay system for use in the screening of chemicals for genotoxicity.