Subcutaneous extraskeletal chondroblastic osteosarcoma in a cat.

CASE SUMMARY: An 11-year-old neutered male cat was presented with a fixed, subcutaneous mass in the left hindlimb. The neoplasm was surgically removed and determined to be a 2 × 2 × 9 cm mass that extended over the plantar surface of the left hindlimb from the tarsus to the phalanges. It was independent from the skeletal system but firmly attached to the adjacent connective tissue. Microscopically, the neoplasm was composed of highly proliferative mesenchymal neoplastic cells that formed both osseous and cartilaginous tissues with associated production of chondroid, osteoid and associated matrixes. This neoplasia was diagnosed as an extraskeletal chondroblastic osteosarcoma. Extraskeletal osteosarcomas, especially the chondroblastic subtype, are extremely rare in cats. Consequently, little is known concerning their course and prognosis. In this case, excision with wide margins appeared to be successful as, at the time of writing, 24 months after limbectomy, the cat is healthy with no evidence of recurrence or metastasis. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this is the first report of an appendicular large extraskeletal chondroblastic osteosarcoma occurring in a domestic cat. As these neoplasms are rare, it should be considered as a less likely cause of soft tissue appendicular neoplasms in domestic cats.

Endocrine-mediated effects of two benzene related compounds, 1-chloro-4-(chloromethyl)benzene and 1,3-diethyl benzene, based on subacute oral toxicity studies using rats.

The purpose of this study was to investigate the endocrine-mediated effects of the benzene-related compounds with reference to Organization for Economic Co-operation and Development (OECD) Test Guideline No. 407. Rats were orally gavaged with 0, 10, 50, and 250 mg/kg/day of 1-chloro-4-(chloromethyl)benzene, and 0, 25, 150, and 1000 mg/kg/day of 1,3-diethyl benzene for at least 28 days, beginning at 8 weeks of age. Thyroid dysfunction was observed in rats given the 1,3-diethyl benzene. Serum T4 values increased in all groups of male rats and in the 1000 mg/kg group of female rats, and TSH values also increased in the 1000 mg/kg groups of both sexes after 28 days' administration. Decreased T3 values were observed in the 1000 mg/kg group of female rats after 28 days' administration, and hormone values increased in the 1000 mg/kg groups of both sexes after the 14-day recovery period. In addition, thyroid weight increased in the 1000 mg/kg groups and thyroid follicular cell hyperplasia was detected in one male rat from the 1000 mg/kg group after 28 days' administration. Endocrine-mediated effects, including thyroid dysfunction were not observed in any groups of rats treated with 1-chloro-4-(chloromethyl)benzene. Our results indicated that endocrine-mediated effects such as thyroid dysfunction were associated with some benzene-related compounds.

Comparison of endocrine-mediated effects of two bisphenol A related compounds, 2,2-bis(4-cyanatophyenyl)propane and 4,4'-cyclohexylidenebisphenol, based on subacute oral toxicity studies using rats.

The purpose of this study was to compare endocrine-mediated effects of bisphenol A related compounds, 2,2-bis(4-cyanatophyenyl)propane and 4,4'-cyclohexylidenebisphenol with reference to OECD Test Guideline No. 407. Rats were orally gavaged with 0, 4, 20, and 100 mg/kg/day of 2,2-bis(4-cyanatophyenyl)propane, and 0, 30, 100, and 300 mg/kg/day of 4,4'-cyclohexylidenebisphenol for at least 28 days beginning at 8 weeks of age. Endocrine-mediated effects were not observed in rats given 2,2-bis(4-cyanatophyenyl)propane. Male accessory sex organ weights decreased in the 4,4'-cyclohexylidenebisphenol 300 mg/kg group and serum T4 values increased in all male groups treated with this compound. Our results suggest that endocrine-mediated changes caused by the present bisphenol related compound can be divided into estrogenic or thyroid hormonal effects, and estrogenic effects observed in the repeated-dose study were related to their estrogenic potency confirmed by uterotrophic assay.

The inhalation exposure of carbon tetrachloride promote rat liver carcinogenesis in a medium-term liver bioassay.

The potential of carbon tetrachloride (CCl4) to induce pre-neoplastic lesions in rat liver using a medium-term liver assay (Ito method) for the prediction of carcinogenicity was examined by nose-only inhalation exposure of male rats (15/group) to CCl4 vapor at concentrations of 0, 1, 5, 25, 125 ppm for 6h/day, 6 day/week, for a period of 6 weeks. The numbers and area of glutathione S-transferase placental (GST-P) positive foci were then determined. Additionally, other histopathological observations on the livers were recorded and serum chemical parameters and CCl4 concentrations in blood were measured. The areas and numbers of GST-P positive foci significantly increased in the CCl4-exposed rats at 25 and 125 ppm; but not at concentrations of 1 and 5 ppm. CCl4 blood concentration 24h after initiation of exposure in the 125 ppm group remained at about 5% of the 6h maximum concentration. These data from CCl4-exposed rats clearly show that inhalation exposure can be used in the rat medium-term liver assay, the method is available for the screening of volatile chemicals and is therefore a useful tool in cancer risk assessment. This is the first report of the use of inhalation exposure in this medium-term predictive assay.

Relationship between the results of in vitro receptor binding assay to human estrogen receptor alpha and in vivo uterotrophic assay: comparative study with 65 selected chemicals.

For screening chemicals possessing endocrine disrupting potencies, the uterotrophic assay has been placed in a higher level in the OECD testing framework than the ER binding assay to detect ER-mediated activities. However, there are no studies that can demonstrate a clear relationship between these assays. In order to clarify the relationship between the in vitro ER binding and in vivo uterotrophic assays and to determine meaningful binding potency from the ER binding assay, we compared the results from these assays for 65 chemicals spanning a variety of chemicals classes. Under the quantitative comparison between logRBAs (relative binding affinities) and logLEDs (lowest effective doses), the log RBA was well correlated with both logLEDs of estrogenic and anti-estrogenic compounds at r(2)=0.67 (n=28) and 0.79 (n=23), respectively. The RBA of 0.00233% was found to be the lowest ER binding potency to elicit estrogenic or anti-estrogenic activities in the uterotrophic assay, accordingly this value is considered as the detection limit of estrogenic or anti-estrogenic activities in the uterotrophic assay. The usage of this value as cutoff provided the best concordance rate (82%). These findings are useful in a tiered approach for identifying chemicals that have potential to induce ER-mediated effects in vivo.

Subacute oral toxicity study of bisphenol F based on the draft protocol for the "Enhanced OECD Test Guideline no. 407".

Since bisphenol F (4,4'-dihydroxydiphenylmethane) has been reported to exhibit estrogen agonistic properties in the uterotrophic assay, we performed a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) on bisphenol F based on the OECD draft protocols to determine whether it has endocrine-mediated properties. Bisphenol F was orally administered at doses 0, 20, 100 and 500 mg/kg per day for at least 28 days, but no clear endocrine-mediated changes were detected, and it was concluded to have no endocrine-mediated effects in young adult rats. On the other hand, the main effect of bisphenol F was concluded to be liver toxicity based on clinical biochemical parameters and liver weight, but without histopathological changes. The no-observed-effect level for bisphenol F is concluded to be under 20 mg/kg per day since decreased body weight accompanied by decreased serum total cholesterol, glucose, and albumin values were observed in the female rats given 20 mg/kg per day or higher doses of bisphenol F.

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 4,4 -[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol] based on the OECD draft protocols.

We performed an uterotrophic assay, the Hershberger assay, and a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) of 4,4 -[1-[4-[1-(4-hydroxyphenyl)-1-methylethyl]phenyl]ethylidene]bis[phenol] based on the OECD draft protocols. In the uterotrophic assay, female SD rats were subcutaneously injected with the chemical at doses of 0, 100, 300, and 1,000 mg/kg on each of 3 days from postnatal day 20 to day 22, and the uterine weight of rats given the 1,000 mg/kg dose of the test chemical plus ethinyl estradiol decreased. In the Hershberger assay, the test chemical was orally administered at doses of 0, 100, 300, and 1,000 mg/kg day to castrated male SD rats for ten consecutive days beginning on postnatal day 56, and no changes were observed. On the other hand, when the test chemical was orally administered at doses 0, 100, 300, and 1,000 mg/kg day for at least 28 days, a decrease in LH values in rats of both sexes and a decrease in FSH and estradiol values in female rats were detected in the 1,000 mg/kg group, and abnormal estrous cycles, uterine glandular atrophy, persistence of ovarian corpora lutea, vaginal epithelial mucification, and mammary glandular hyperplasia were also observed in one female rat in the 1,000 mg/kg group. Therefore, the uterotrophic assay used in this study showed that the chemical has the estrogen-antagonist properties, and some potentially endocrine-mediated effects were detected in growing rats based on the results of the enhanced OECD test guideline No. 407. However, the changes were observed in rats given a high dose of the chemical, 1,000 mg/kg day.

Effects of in utero and lactational exposure to tamoxifen in SD rats.

Pregnant CD (SD) IGS rats were given tamoxifen (TMX) orally at doses of 0.12, 0.6, or 3 microg/kg/day from gestational day 6 to postnatal day 21, and the effects of TMX exposure on all offspring were examined 10 weeks after birth; the reproductive performance of the offspring was also evaluated. Although the body weights of the dams treated with TMX remained normal from gestational day 6 until the day of autopsy (postnatal day 21), three dams in the 3 microg/kg/day group died during the pregnancy or partum periods. These deaths were regarded as the toxicological effects of TMX. No changes were detected in the reproductive parameters of all the TMX groups except for a decrease in the number of newborns/live newborns in the 3 microg/kg/day group. No abnormal clinical signs, body weight change, ano-genital distance, vaginal opening, or organ weight changes were detected in any of TMX groups, but the day of preputial separation was prolonged in the male offspring of all TMX groups and cleft phallus was detected in the female offspring of the 0.6 and 3 microg/kg/day groups. No abnormalities were detected in the reproductive performance of the male and female offspring.

Comparison of the Hershberger assay and androgen receptor binding assay of twelve chemicals.

We performed the Hershberger assay of 12 chemicals based on the OECD draft protocol. The chemicals tested by the Hershberger assay were phthalic acid di-n-hexyl ester, phthalic acid di-n-amyl ester, phthalic acid di-n-propyl ester, diethylstilbestrol, 17beta-estradiol, tamoxifen, 5alpha-dihydrotestosterone, dichlorodiphenyldichloroethane, cyproterone acetate, 6alpha-methyl-17alpha-hydroxy-progesterone, atrazine, and spironolactone. Phthalic acid di-n-hexyl ester, phthalic acid di-n-amyl ester, and phthalic acid di-n-propyl ester are phthalates; diethylstilbestrol and 17beta-estradiol are estrogenic chemicals; tamoxifen is partial estrogen receptor antagonist with mainly estrogenic properties; 5alpha-dihydrotestosterone is an androgen derivatives; dichlorodiphenyldichloroethane is a reference androgen antagonistic chemical; cyproterone acetate, 6alpha-methyl-17alpha-hydroxy-progesterone, and spironolactone have an androgenic steroid structure and are known as androgen antagonistic chemicals; and atrazine is a reference endocrine disruptor. We also subjected these chemicals to the receptor binding assay for androgen. A clear androgen agonistic effect was detected in 5alpha-dihydrotestosterone, and an androgen antagonistic effect was observed in five chemicals: cyproterone acetate, spironolactone, 6alpha-methyl-17alpha-hydroxy-progesterone, phthalic acid di-n-amyl ester, and dichlorodiphenyldichloroethane. By contrast, diethylstilbestrol, 17beta-estradiol, tamoxifen, 5alpha-dihydrotestosterone, dichlorodiphenyldichloroethane, cyproterone acetate, 6alpha-methyl-17alpha-hydroxy-progesterone, and spironolactone were positive in the receptor binding assay for androgen. Three estrogenic chemicals, diethylstilbestrol, 17beta-estradiol, and tamoxifen, were negative in the Hershberger assay with receptor binding affinity. On the other hand, the Hershberger assays of three phthalates were performed at the same dosages, and the results showed androgen antagonistic affinity only in the assay of phthalic acid di-n-amyl ester without receptor binding affinity.

Comparative study of the uterotrophic potency of 14 chemicals in a uterotrophic assay and their receptor-binding affinity.

We performed an immature rat uterotrophic assay of 14 chemicals having various receptor-binding affinities in order to assess the relationship between their uterotrophic potency and receptor-binding affinity. The chemicals tested were phthalic acid di-n-hexyl ester, phthalic acid di-n-amyl ester, phthalic acid di-n-propyl ester, 2-ethylhexyl-p-hydroxybenzoate, 4,4'-biphenol, 4,4'-sulfonyldiphenol, 4,4'-dihydroxydiphenylmethane, 2,4-dihydroxybenzophenone, 4,4'-cyclohexylidenebisphenol, 4-t-butylpyrocatechol, clomiphene citrate, 4,4'-(1,3-phenylenediisopropylidene)bisphenol, p-t-butylphenol, and diallylterephthlate. Two of the 14 chemicals, phthalic acid di-n-propyl ester and diallylterephthlate, exhibited no receptor-binding affinity, and the receptor-binding affinity of phthalic acid di-n-hexyl ester and phthalic acid di-n-amyl ester was lower than that of the other chemicals. Ten of the chemicals showed uterotrophic potency, the exceptions being phthalic acid di-n-propyl ester, diallylterephthlate, phthalic acid di-n-hexyl ester, and phthalic acid di-n-amyl ester. The results of the present study demonstrate that the affinity of the chemicals in the receptor-binding assay correlated well with their potency in the uterotrophic assay except for a few chemicals with very low receptor-binding affinity.

In utero through lactational exposure to ethinyl estradiol induces cleft phallus and delayed ovarian dysfunction in the offspring.

Most of the attention currently focused on endocrine-active chemicals is directed to their effects on the development of offspring exposed to them in utero or during the neonatal period. Pregnant Crj:CD(SD)IGS rats were given ethinyl estradiol (EE) orally in doses of 0.5-50 microg/kg/day from gestational day 7 to postnatal day 18, and their offspring were examined for its effects. Our previous study according to a similar protocol demonstrated the occurrence of cleft phallus in the female offspring exposed to 50 microg/kg of EE in utero and during the lactation period. The present study was designed to assess (1) the reproducibility of the induction of cleft phallus, (2) the fertility of female rats with cleft phallus, and (3) whether any delayed effects, possibly delayed anovulation, were induced. At 50 microg/kg cleft phallus was observed in almost all of the female offspring, and slight retardation of body weight gain was detected in both sexes. At 15-17 weeks of age the animals with cleft phallus could copulate and had fertility comparable to the control group. At 6 months of age, on the other hand, 6/8 of the female offspring at 50 microg/kg exhibited abnormal cyclicity, including persistent estrus, and histological examination revealed follicular cysts and absence of corpora lutea in the ovaries of the rats with persistent estrus. These findings are consistent with delayed anovulation syndrome. The results suggest that observation of cyclicity at 6 months old is able to detect possible delayed ovarian dysfunction induced by perinatal exposure to chemicals.

Comparison of the reporter gene assay for ER-alpha antagonists with the immature rat uterotrophic assay of 10 chemicals.

We performed a reporter gene assay for estrogen receptor (ER)-alpha agonists and antagonists of 10 chemicals that showed both estrogen agonistic and reduced the estrogenic effect of ethinyl estradiol in a rat uterotrophic assay. The chemicals tested by the immature uterotrophic assay were p-(tert-pentyl)phenol, 4,4'-thiobis-phenol, 4,4'-(hexafluoroisopropylidene)diphenol, 2,2-bis(4-hydroxyphenyl)-4-methyl-n-pentane, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, 4-(phenylmethyl)phenol, 4,4'-dihydroxybenzophenone, 2,2',4,4'-tetrahydroxybenzophenone, 4-hydroxybenzophenone and 2,4,4'-trihydroxybenzophenone. Although all chemicals examined in this study were positive in the reporter gene assay for ER-alpha agonists, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol was only positive in the reporter gene assay for ER-alpha antagonists. These findings demonstrate that results of the reporter gene assay for ER-alpha agonists correlated well with those of the uterotrophic assay, but antagonistic change of 9 of 10 chemicals in the uterotrophic assay was not detected by the reporter gene assay for ER-alpha antagonists.

Evaluation of an in utero through lactational exposure protocol for detection of estrogenic effects of ethinyl estradiol on the offspring of rats: preliminary trial.

As a preliminary trial of an in utero through lactational exposure protocol, ethinyl estradiol, 0, 0.5, 5, or 50 micro g/kg/day, was administered by gavage to pregnant Crj: CD (SD) IGS BR rats from gestational day (GD) 7 to day 18 after delivery to evaluate the efficacy of this protocol and to estimate optimal endpoints. The dams showed no abnormalities. Cleft phallus was observed in female offspring at 50 micro g/kg. Other than a retardation of body weight gain in both sexes at 50 micro g/kg/day, no other abnormal findings were detected. The fact that cleft phallus was the only change induced suggests that the protocol is applicable to the detection of effects of estrogenic chemicals given to pregnant rats on offspring development and that the morphology of the female external genitalia may be a useful endpoint.

Development of a high-performance reporter plasmid for detection of chemicals with androgenic activity.

A number of chemicals are present in the environment, and some synthetic chemicals may disrupt endocrine function of wild animals and humans. An effective procedure to screen chemicals for endocrine modulating activity has been needed to ensure the safety of chemicals, and the reporter gene assay technique may provide a powerful tool for screening endocrine-disrupting chemicals. We have developed a high-performance reporter plasmid that can trigger high androgen-dependent induction with high selectivity by using mouse mammary tumor virus (MMTV) androgen-responsive elements and a partial fragment of the rat alpha(2u)-globulin promoter region. This new type plasmid can induce higher transcriptional activation than a commercial PGV-P-based construct bearing the SV40 promoter fragment, and the basal induction level of this plasmid is much lower than that of the PGV-P-based construct. Moreover, only androgen derivatives could selectively induce a high response in the reporter gene assay with the new reporter plasmid. This new type of reporter plasmid, ARE-AUG- Luc+, should be of value in endocrine research and in screening to identify endocrine-modulating chemicals.

Immature rat uterotrophic assay of 18 chemicals and Hershberger assay of 30 chemicals.

We performed an immature rat uterotrophic assay of 18 chemicals and Hershberger assay of 30 chemicals to assess the relationship between the results of two assays. The chemicals tested by the uterotopic assay were 4-n-amylphenol, p-dodecyl-phenol, p-(tert-pentyl)phenol, 4-cyclohexylphenol, 4-(1-adamantyl)phenol, 4,4'-thiobis-phenol, diphenyl-p-phenylenediamine, 4-hydroxyazobenzene, 4-(phenylmethyl)phenol, 4,4'-(hexafluoroisopropylidene)diphenol, 2,2-bis(4-hydroxyphenyl)-4-methyl-n-pentane, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, 4,4'-dihydroxybenzophenone, 2,2',4,4'-tetrahydroxybenzophenone, 4-hydroxybenzophenone, 2,4,4'-trihydroxybenzophenone, testosterone enanthate, and methyltestosterone. The chemicals tested by the Hershberger assay were the 18 chemicals tested in the uterotrophic assay plus the following: 17 alpha estradiol, estrone, equilin, norethindrone, norgestrel, ethynyl estradiol, bisphenol A, bisphenol B, bisphenol F, 4-tert-octylphenol, p-cumyl phenol, and nonylphenol. All chemicals examined in this study were positive in a reporter gene assay for ER-alpha. In the immature rat uterotrophic assay, all chemicals induced uterotrophy and p-(tert-pentyl)phenol, 4,4'-thiobis-phenol, 4-(phenylmethyl)phenol, 4,4'-(hexafluoroisopropylidene)diphenol, 2,2-bis(4-hydroxyphenyl)-4-methyl-n-pentane, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, 4,4'-dihydroxybenzophenone, 2,2',4,4'-tetrahydroxybenzophenone, 4-hydroxybenzophenone, and 2,4,4'-trihydroxybenzophenone exerted both estrogen agonistic effect and reduced the estrogenic effect of ethynylestradiol. In the Hershberger assay, a clear androgen agonistic effect was detected in the androgen derivatives testosterone enanthate and methyltestosterone.

Immature uterotrophic assay of estrogenic compounds in rats given diets of different phytoestrogen content and the ovarian changes with ICI 182,780 or antide.

To investigate the influence of phyotestrogens in the diet, an immature uterotrophic assay of ethinylestradiol, bisphenol A, 4-nonylphenol or genistein was performed in rats given the formula MF diet, modified NIH-07 open formula diet, or modified NIH-07 phytoestrogen-lowered-diet (study 1). The chemicals were administered subcutaneously from 20 days of age for 3 days. Doses of ethinylestradiol, bisphenol A, 4-nonylphenol or genistein were 0.06-0.6 micro g/kg per day, 1-10 mg/kg per day, 10-100 mg/kg per day or 1-20 mg/kg per day, respectively. In another study, an immature uterotrophic assay of genistein and ethinylestradiol together with ICI 182,780 or antide was performed to compare the ovarian changes with these chemicals (study 2). Doses of genistein or ethinylestradiol were 30 mg/kg per day or 0.6 micro g/kg per day, respectively, and these chemicals were injected subcutaneously from 20 days of age for 3 days. In study 1, there were no essential differences in the uterus weights among the various phytoestrogen-content diets. In study 2, the ovary weights in rats given genistein were significantly higher than in the controls, whereas the ovary weights in rats given ethinylestradiol were lower than in the controls. The ovary weights in the ICI 182,780 plus genistein group were significantly higher than in the genistein group, but decrease of the ovary weights was detected in the antide plus genistein group. There was no significant difference in ovary weights between the ICI 182,780 plus ethinylestradiol group and the ethinylestradiol group, but decrease of ovary weights was detected in antide plus ethinylestradiol group. In a histological examination of the ovary, fluid-filled follicles in the genistein group were more numerous than in other groups and increase of granulosa cell fragmentation was seen in the ethinylestradiol and other groups with the exception of the genistein group. The present findings demonstrate that the sensitivity of the immature rat uterotrophic assay is not influenced by the relatively low level of phytoestrogen in diets and that the ovarian changes occurring with genistein and ethinylestradiol are different.

Effect of gonadotropin-releasing hormone antagonist on ovarian and uterine weights in immature female rats.

The immature rat uterotrophic assay has been proposed as a screening test method for detecting estrogenic and antiestrogenic chemicals. Although the immature rat uterotrophic assay is advantageous because the test animals are not traumatized by the ovariectomizing process, the effect of endogenous estrogen on ovarian and uterine weight in the immature animals that are used in immature rat uterotrophic assay has not received much attention. In this study, 19-day-old rats were treated with antide, a gonadotropin-releasing hormone antagonist, antide, to block gonadal production of endogenous estrogen. Uterine and ovarian weights of the antide-treated animals were markedly lower than those of control animals. This finding suggests that endogenous gonadal estrogen may already be acting on the uterus and ovaries in immature rats. Blocking endogenous estrogen with a gonadotropin-releasing hormone antagonist may enhance the sensitivity of the immature rat uterotrophic assay; however, the possibility that this protocol may interfere with the ability of the immature rat uterotrophic assay to detect centrally-mediated effects can not be discounted.

Age-related changes of genital systems in the female Crj:CD (SD) IGS rats during sexual maturation.

The age-related changes of vaginal opening, body weight, the weights of the uterus and ovary, together with histological examination, serum 17beta-estradiol (E2) and progesterone levels were examined in intact female Crj:CD (SD) IGS rats between 21 and 36 days of age to understand the basic biological profile of changes of the female genital system during sexual maturation in the rat for female pubertal assays. With the beginning of the elevation of serum E2 level from 28 days of age, all parameters except body weight started to show drastic change until 31 days of age. The highest incidence of vaginal opening was recorded at 34 days of age. On macroscopic examinations, a number of rats showed uterine imbibition but vaginal opening. Immediately after the confirmation of the vaginal opening, the genital systems of three rats were observed microscopically. Both ovaries already had multiple corpora lutea, and degeneration of endometrial epithelial cells was observed. In conclusion, we obtained essential data on genital tract development of female Crj:CD (SD) IGS rats for in vivo screening assays that will contribute to detect potential endocrine active chemicals. In addition, it is assumed that the first ovulation precedes or occurs simultaneously with vaginal opening.

Subacute oral toxicity study of ethynylestradiol and bisphenol A, based on the draft protocol for the "Enhanced OECD Test Guideline no. 407".

We performed a 28-day repeated-dose toxicity study of ethynylestradiol (EE) and bisphenol A (BPA) based on the draft protocol of the "Enhanced OECD Test Guideline no. 407", and assessed the sensitivity of a list of parameters for detecting endocrine-related effects of endocrine disruption. Doses of EE at 0, 10, 50 or 200 microg/kg per day, or BPA at 0, 40, 200 or 1000 mg/kg per day were orally administered to Sprague-Dawley rats. The highest dose of BPA was decreased to 600 mg/kg per day from the second week of administration because a male rat given 1000 mg/kg BPA had died within 1 week with toxic clinical signs. In the assay using EE, the decrease of prostate, seminal vesicle and pituitary weights, increase of the testis weight, atrophic changes of the prostate, seminal vesicle and mammary gland, and degenerative changes in the testes were detected in male rats in the 50 and/or 200 microg/kg groups. In females of the 200 microg/kg group, decrease of the ovary weight, increase of the uterine weight, atrophy of the ovary, hypertrophy or squamous metaplasia of the uterine epithelial cells and mucification in the vagina were observed. Furthermore, diestrous, estrous or the unknown stage was prolonged in the 50 and 200 microg/kg groups of rats. Endocrine-mediated effects of EE were not detected in general observations, hematology, serum biochemistry, or hormonal or spermatological examinations. In the assay using BPA, the diestrous stages were prolonged at the highest dose, but changes related to endocrine effects were not detected in other examinations. Thus, among the parameters tested, the weight of endocrine-linked organs and their histopathological assessment and estrous cycle stage allowed the detection of the endocrine-related effect of EE, whereas the estrous cycle stage was only a useful parameter to detect the effect of BPA.

The efficacy of endocrine disruptor screening tests in detecting anti-estrogenic effects downstream of receptor-ligand interactions.

Several predictive test methods for endocrine disrupters have been evaluated by international organizations. In this study, we performed a series of predictive tests for endocrine disrupters, i.e. the receptor binding assay, reporter gene assay, and immature rat uterotrophic assay, on all-trans retinoic acid (tRA), which may cause antiestrogenic activity via their receptors, interfere with estrogenic action at estrogen responsive element level, and we examine the efficacy of endocrine disruptor screening tests in detecting anti-estrogenic effects downstream of receptor-ligand interactions. Despite showing complete lack of binding affinity to ER in the receptor binding assay, tRA exhibited clear antagonist activity without any agonist activity in the reporter gene assay. In the in vivo test, tRA was subcutaneously administered to immature Crj:CD (SD) IGS rats at doses of 5 and 25 mg/kg per day for 3 days, beginning at 20 days of age. Additional groups of rats given tRA at the above doses were also subcutaneously injected with ethinyl estradiol (EE) at a dose of 0.6 microg per rat per day. A vehicle control group given olive oil alone and a positive control group given EE alone were also established. Although no uterotrophic activity was detected in any of the rats given only tRA, co-treatment with 5 and 25 mg/kg tRA and EE reduced the EE-induced increases in uterine weight. We confirmed that the ER antagonist activity of tRA may be mediated by transcriptional interference after ER-ligand complex binding to an estrogen responsive element of the gene by the gel mobility shift analysis. These findings suggest the reporter gene assay and uterotrophic assay can detect anti-estrogenic effects downstream of receptor-ligand interactions, but the receptor binding assay can not detect this type of interference. In any case, a screening strategy for endocrine disrupters, especially the primary screening battery for prioritizing the chemicals to be tested in the higher screening stages, should be designed to detect various kinds of chemicals possessing endocrine modulating activity including a retinoid-like endocrine modulator. Accordingly, reporter gene assay or uterotrophic assay should be conducted in the early stage of screening process for endocrine disrupting chemicals, because they can detect antagonist activity caused by both inhibition of receptor-ligand interaction and transcriptional interference. Particularly, the reporter gene assay may be a promising prescreening procedure, because it can be adopted in the high throughput screening process for thousands of chemicals and it requires no use of experimental animals.