Assessment of in vivo assays for endocrine disruption.

The United States Environmental Protection Agency (USEPA) has developed a screening and testing programme to detect endocrine-disrupting chemicals. This programme is organized into two phases: a tier 1 screening battery and, for those chemicals that are positive in this phase, a tier 2 testing battery. Similarly, the Organization for Economic Cooperation and Development (OECD) has set up a task force to identify, prioritize and validate test methods for the detection of endocrine disrupters. This chapter describes the mammalian in vivo assays recommended by these organizations. The tier 1 screening recommended by both agencies comprises the uterotrophic assay for the detection of oestrogens and anti-oestrogens, and the Hershberger assay for the detection of anti-androgens. Both of these assays are nearing the end of a comprehensive validation process and show consistency across testing laboratories. A further assay recommended by the USEPA is the female pubertal assay, designed to assess steroidogenesis, anti-thyroid activity and anti-oestrogenicity, while the OECD recommends an enhanced version of their standard regulatory test guideline number 407, the 28-day rat toxicity test. The tier 2 test recommended by both agencies is an enhanced version of the two-generation regulatory study. This is a large time- and animal-consuming study, and in order to obtain the maximum information regarding the potential for endocrine-disrupting activity of a chemical, it should be modified depending on the results of the tier 1 assays.

Studies on the mutagenicity of a zinc oxide-hexachloroethane smoke.

1. A suitable method has been developed for generating atmospheres of zinc oxide/hexachloroethane smoke (ZnHCE). 2. The smoke was investigated using the Ames test and the micronucleus assay. 3. It was weakly mutagenic to the bacteria, but in the bone marrow no increases in micronuclei were detected up to toxic levels of the smoke. 4. The method used here could be applied to other pyrotechnic mixtures which give rise to complex mixtures of products.

Germ and somatic cell abnormalities following in vivo administration of thymidine and adenine.

It is known that an excess of or a depletion in bases and nucleosides produce genetic effects in vitro, and a similar effect has been found with the nucleoside thymidine in this laboratory in vivo. To confirm this effect and to see if this occurs with the base adenine, thymidine and adenine were administered to male mice by i.p. injection and the sperm examined for head-shape abnormalities 4 and 5 weeks later. Treated males also were mated to untreated females for the provision of an F1 generation. The F1 males were subjected to the sperm morphology assay when they reached 14 weeks of age. Amongst those F0 males given adenine, there was a dose-related increase in the frequency of abnormal sperm and the group given thymidine also showed increases, confirming the results of previous studies in this laboratory. In the F1 generation, the fraction of mice from treated males showing increases in numbers of abnormal sperm was greater than that of the controls. In a micronucleus test with mice treated with thymidine, mitosis was delayed and there was a marginal increase in micronuclei, suggesting that an imbalance in nucleoside pools may be responsible for chromosomal damage in somatic cells in vivo. Therefore it is considered that similar effects to those produced in vitro can be demonstrated in vivo. Furthermore, the results of the sperm morphology assay show that the damage is transmissible.

High-performance liquid-chromatographic analysis of bases and nucleoside in mouse testes following in vivo thymidine administration.

In order to measure accurately bases and nucleosides in vivo, a method has been developed using high-performance liquid chromatography. This method is reproducible under conditions of constant pressure, temperature and pH and is rapid, requiring approximately 1 h for a complete chromatographic separation of bases and nucleosides. The method has been used to measure quantitatively the bases and nucleosides in mouse testes following in vivo administration of thymidine. Groups of male mice were given thymidine by intra-peritoneal injection and were killed at various time intervals and the testes removed for extraction procedures. There was a significant increase in the thymidine concentration in the testes 1 h after injection, which fairly quickly returned to the control value thereafter. There was a subsequent reduction in uridine concentration. This analytical method allows deoxyribonucleotide (dNTP) precursor pool sizes to be determined in germinal cells. It may also be useful for other tissues in vivo.

Long-term toxicity study of amaranth in rats using animals exposed in utero.

Groups of 90 (control) and 54 (treated) rats of each sex were given amaranth in their diet to provide daily intakes of 0 (control), 50, 250 or 1250 mg/kg for 111 wk (male) and 112 wk (female) after weaning. The rats had also been exposed to the same dose levels in utero, and their parents were exposed for 60 days before mating. The colouring had no adverse effects on fertility, haematological parameters, serum chemistry or incidence of tumours. All treated animals showed contamination of the fur and red colouring of the faeces and at the high dose only the faecal pellets were poorly formed. Rats in the high-dose group produced more pups, and the average pup weight was lower than that of the controls. Rats of the F1 generation given the highest dose level were slightly lighter than the controls despite a small increase in food and water intake. Both sexes given the highest dose level and males given 250 mg/kg/day had increased caecal weight. High-dose females excreted more protein in the urine after 18 months and on histopathological examination females in all treated groups showed an increased incidence of renal calcification and pelvic epithelial hyperplasia with degenerative changes. It is concluded that amaranth fed to rats at dose levels of up to 1250 mg/kg/day in the diet did not have any carcinogenic effect. However, because of the effect on the kidneys of the females it was not possible to establish a no-untoward-effect level in this study.

Short-term toxicity study of Green S in rats.

Groups of 15 rats of each sex were fed Green S at dietary concentrations to provide dose levels of 0 (control), 250, 500 or 1500 mg/kg body weight/day for 13 wk. Additional groups of five animals of each sex were given the same treatments for 2 or 6 wk. There was a marked excretion of green colour in the faeces and some green colouring of the urine, although the latter may have been due to contamination. The males showed increased water and food intakes associated, particularly at the highest dose, with a higher rate of body-weight gain. Haematological examination revealed a transitory mild anaemia at the highest dose level, whilst no findings indicative of a toxic effect were found in the renal concentration tests or the serum analyses. With a dose of 1500 mg Green S/kg a greater proportion of the rats showed higher urinary protein, protein casts, increased caecal weight, thyroid degeneration in female animals and enlargement of the lymph nodes in the intestine wall. The no-effect dose level for Green S in this study was considered to be 500 mg/kg.

Teratogenicity and embryotoxicity study of Green S in rats.

Daily oral doses of 0 (control), 250, 500 or 1000 mg Green S/kg body weight were given to groups of 30 pregnant rats on days 0-19 of pregnancy. This treatment did not adversely influence maternal body weight, the numbers of implantations, of pre- or post-implantation losses or of live foetuses, the sex ratio or the weight of the litters or foetuses. No definite abnormalities were seen and the only finding in the examination of stained skeletons was a slightly more advanced ossification of the forelimbs of the offspring from females given 500 or 1000 mg Green S/kg/day. More foetuses with mucus in the trachea were found in the treated groups than in the controls but this was not considered to be a teratogenic effect. Thus no embryotoxic or teratogenic effects were detected with doses of up to 1000 mg Green S/kg/day throughout pregnancy.

Effect of ethylene glycol monomethyl ether on spermatogenesis, dominant lethality, and F1 abnormalities in the rat and the mouse after treatment of F0 males.

Adult male CD rats and CD-1 mice were given a single oral dose of ethylene glycol monomethyl ether (EGM) at 0, 500, 750, 1,000, or 1500 mg/kg. Groups of 10 were killed at weekly intervals after dosing for analysis of sperm counts and morphology or testicular histology; further groups of 10 were sequentially mated to pairs of virgin females to test for dominant lethality or gross foetal malformations in the F1 generation (F1 abnormalities). EGM was found to deplete the spermatocytes of both species severely, principally pachytene cells, but with other stages affected with increasing dose. A delay in the progression of spermatogenesis may account for a discrepancy between the apparent stage-specificity of damage deduced from lowered sperm counts and that observed histologically. In the rat, morphological abnormalities were observed in sperm that had been exposed as spermatocytes; in the mouse, however, the sensitive cells were the late spermatocytes and early spermatids. In all these parameters there was an indication of a dose-response relationship in both rats and mice. In the mating studies EGM induced a dose-related decrease in fertility 5 weeks after dosing in the rat, but complete sterility in all but the lowest dose after 6 weeks. In contrast, EGM had no effect on the reproductive capacity of the mouse. There was no statistically significant evidence for the induction of dominant lethal mutations or F1 abnormalities in either species. A single oral dose of cyclophosphamide (CTX) at 100 mg/kg induced a significant increase in dominant lethality in both species. CTX reduced the number of total implants in the rat and induced a nonsignificant increase in the number of abnormal offspring sired by treated male mice.