Histopathological Analysis of Brains from Dogs Infected with Canine Distemper Virus.

We describe the use of conventional histology and immunohistochemistry against canine distemper virus (CDV) to examine the brains of domestic dogs with a confirmed diagnosis of CDV infection. Histologically, to identify the main typical lesions, we used conventional H&E stain; to evaluate the progressive demyelination, we used Luxol Fast Blue stain; and to identify the presence of viral particles in these affected regions, we used immunohistochemistry against CDV. We confirm that the histopathological analysis of brains of distemper-infected dogs is a powerful tool to evaluate the typical brain lesions and could be used as an interesting natural model to continue studying the pathogenesis of canine distemper in different species and/or other morbillivirus infections, like measles.

Central nervous system lesions caused by canine distemper virus in 4 vaccinated dogs.

We examined the cerebellum and cerebrum of 4 vaccinated dogs, 3-60-mo-old, that displayed clinical signs of canine distemper virus (CDV) infection, and died 7-40 d after developing neurologic signs. The main histologic lesions were demyelination, gliosis, meningitis, perivascular lymphocytic cuffing, and inclusion bodies. These lesions were similar in all 4 cases regardless of the time since vaccination, except that meningoencephalitis and gliosis were subacute in 3 dogs and chronic in 1 dog. However, these differences did not appear to be related to their vaccination status. Immunohistologically, a CDV-positive immunoreaction was seen mainly in astrocytes, neurons and their axons, lymphocytes around and in the blood vessels of the pia mater and choroid plexus, ependymal cells of each ventricle, and the cells of the choroid plexus. The histologic and immunohistologic changes were similar in the cerebellum and cerebrum. The genetic characterization of the virus strains in 2 of these naturally occurring canine distemper cases confirmed that they were South American wild-type strains (Kiki and Uy251) belonging to the EU1/SA1 lineage. These strains are not included in the commercial CDV vaccines available in Uruguay.

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.

Risk assessment study of dioxins in sanitary napkins produced in Japan.

A risk assessment study of dioxins in sanitary napkins produced in Japan was performed. The daily estimated exposure volume to dioxins was compared with the tolerable daily intake (TDI). The concentrations of dioxins such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (DL-PCBs) in seven sanitary napkins were measured using gas chromatography and mass spectroscopy analytical methods. Among the seven napkins, a range of 0.0044-0.076pg TEQ/g dioxins was measured. Daily estimated exposure volume from sanitary napkins was calculated as follows: (dioxin volumes in a sanitary napkin (0.0044-0.076pg TEQ/g)×pulp weight in a sanitary napkin (11.2g)×used napkin numbers/d (7.5)×the number of days/month that women use sanitary napkins (7)×skin absorption rate (0.03)×used years (40))/(average body weight of women (50kg)×the number of days in the month (30)×life years (86)). Daily exposure volumes were estimated to be 0.000024-0.00042pg TEQ/kg/d. For hazard assessment, we used 0.7pg TEQ/kg/d which was the lowest level of TDI among TDI values reported by international agencies. When the daily exposure volume was compared with the TDI, the former was approximately 1666-29,166 times less than the latter. This fact indicated that the risk of exposure to dioxins from sanitary napkins produced in Japan was negligible.

Correlation study in skin and eye irritation between rabbits and humans based on published literatures.

The purpose of this study was to investigate the correlations in skin and eye irritations between rabbits and humans using published international databases. We selected 60 and 56 compounds for skin and eye irritation, respectively. When the reactions were divided into irritation-negative or irritation-positive, including corrosion, similar reactions between rabbits and humans were detected for 53 compounds in skin irritation and 54 compounds in eye irritation, showing rates of agreement in skin and eye as 88% and 96%, respectively. These findings revealed that correlation in skin and eye irritations between rabbits and humans were high. However, corrosion was observed in rabbit skin treated with 14 compounds, 4 of which showed similar changes in humans, and in rabbit eyes treated with 9 compounds, 1 of which revealed similar changes in humans. These findings indicated that the incidence of corrosion was higher in rabbits than in humans. Our results showed that the data on rabbit irritations in the skin and eye were useful for identifying risk of irritation in human.

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.

Endocrine-mediated effects of 4,4'-(hexafluoroisopropylidene)diphenol in SD rats, based on a subacute oral toxicity study.

The purpose of this study was to investigate the endocrine-mediated effects of 4,4'-(hexafluoroisopropylidene)diphenol according to OECD test guideline no. 407. The estrogenic properties of this chemical have already been shown on uterotrophic assay, and this chemical is classified as a low-production volume chemical in REACH program. Rats were orally gavaged with 0, 10, 30, and 100 mg/kg/day of test chemical for at least 28 days, beginning at 8 weeks of age. In the 100 mg/kg group of male rats, endocrine-mediated effects, atrophic changes in the mammary glands and testicular Leydig cells, decreased accessory sex organ weights, and hypertrophy of the adrenal zona fasciculata with increased organ weights were seen; there was dysfunction of the estrous cycle in the 30 and 100 mg/kg groups, and increased serum T4 values were observed in the 100 mg/kg groups of both sexes. In addition, we also noted other findings, such as reduced body weight gains in the 30 and/or 100 mg/kg groups of both sexes, dilatation of the large intestinal lumen in the 100 mg/kg groups of both sexes, decreased hematopoiesis in the bone marrow and spleen, and decreased white blood cell counts in the 100 mg/kg group of male rats. Our results demonstrate that in a repeated-dose toxicity study, 4,4'-(hexafluoroisopropylidene)diphenol has various endocrine-mediated effects and its NOAEL (no observed adverse effect level) is 10 mg/kg/day.

Enhanced OECD TG 407 in detection of endocrine-mediated effects of 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol.

The purpose of this study was to investigate whether the estrogenic effects were detected in the enhanced TG 407 if the estrogenic property was not so strong in the uterotrophic assay. The estrogenic property of 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol in the uterotrophic assay was slightly stronger than that of genistein or nonylphenol, but weaker than that of ethinyl estradiol. We performed a 28-day repeated-dose toxicity study (enhanced OECD test guideline No. 407) on 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol based on the OECD draft protocol. The test chemical, administered orally at doses of 0, 10, 50, and 250 mg/kg per day for at least 28 days, caused such estrogenic effects as abnormal estrous cycle, increased ovarian follicles, increased uterine epithelial height, and vaginal mucification in the 50 and/or 250 mg/kg groups. Moreover, follicular epithelial cell hyperplasia of the thyroid was detected in all male rats given the test chemical and in female rats in the 250 mg/kg group. It was concluded that the estrogenic effects were detected in growing rats given 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, and thyroid dysfunction was also observed as the endocrine-mediated effects.

Effects of in utero through lactational exposure to dicyclohexyl phthalate and p,p'-DDE in Sprague-Dawley rats.

Anti-androgenic chemicals alter sexual differentiation by a variety of mechanisms, and the mechanisms between phthalate esters and p,p'-DDE are considered to be different. We performed an in utero through lactational exposure assay using dicyclohexyl phthalate and p,p'-DDE to investigate the sexual differentiation of these chemicals. Pregnant CD (SD) IGS rats were given dicyclohexyl phthalate or p,p'-DDE orally from gestational day (GD) 6 to postnatal day (PND) 20, and the endocrine-mediated effects in dams and their offspring were examined. The reproductive performance of offspring was also examined. The doses of dicyclohexyl phthalate were 0, 20, 100, and 500 mg/kg/day, and those of p,p'-DDE were 5, 15, and 50mg/kg/day. Using the dicyclohexyl phthalate, a dam in the 500 mg/kg group showed dystocia and died. The viability index of offspring on PND 4 decreased in the 500 mg/kg group. Prolonged preputial separation, reduced ano-genital distance, increased areolas/nipple retention, hypospadia, decreased ventral prostate and levator ani/bulbocavernosus muscle weights and decreased testicular germ cells were observed in male offspring in the 500 mg/kg group. In the assay using p,p'-DDE, decreased viability index of offspring on PND 21, prolonged preputial separation in male offspring and early vaginal opening in female offspring were observed in the 50mg/kg group. The copulation and fertility indices decreased in the reproductive performance of offspring in the 50mg/kg group. The endocrine-mediated effects were detected in offspring of dams given 100mg/kg dicyclohexyl phthalate, and in offspring of dams given 20mg/kg p,p'-DDE. Our results suggest that the in utero through lactational exposure assay is a useful method to detect endocrine-mediated effects and that further comparative study between this assay and two-generation reproductive test are necessary when this assay becomes one of the definitive tests.

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 3-amino-1,2,4-triazole based on the Organization for Economic Co-operation and Development draft protocols.

We performed a uterotrophic assay, the Hershberger assay, and the 28-day repeated-dose toxicity study (enhanced OECD test guideline no. 407) of 3-amino-1,2,4-triazole 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 no changes were observed. In the Hershberger assay, the test chemical was orally administered at doses of 0, 40, 200, and 1,000 mg/kg/day to castrated male Wistar rats for 10 consecutive days beginning on postnatal day 56, and no androgen agonistic and antagonistic changes were observed. Alternatively, when the test chemical was orally administered at doses 0, 5, 25, and 125 mg/kg/day for at least 28 days in the subacute oral toxicity study, thyroid follicular epithelial cell hypertrophy with increased thyroid weights was detected in the male and female rats in 25 and/or 125 mg/kg groups, and hypertrophy of the anterior pituitary cells with increased pituitary weights in male and female rats was also observed in the 125 mg/kg group. Furthermore, serum T3 and T4 values decreased and serum TSH values increased in male and female rats in the 125 mg/kg group. Therefore, 3-amino-1,2,4-triazole was concluded to have anti-thyroid acting as endocrine-mediated effects, but no estrogenic or androgenic effects. In addition, decreased body weight, and abnormal biochemical parameters attributed to thyroid, liver or kidney dysfunction were observed in male and female rats in the 25 and/or 125 mg/kg groups.

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.

Uterotrophic assay, Hershberger assay, and subacute oral toxicity study of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol) and 3-(dibutylamino)phenol, based on the OECD draft protocols.

We performed a uterotrophic assay, the Hershberger assay, and a 28-day repeated-dose toxicity study [enhanced Organization for Economic Co-operation and Development (OECD) test guideline No. 407] of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol) and 3-(dibutylamino)phenol, based on the OECD draft protocols. In the uterotrophic assay of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol), 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 no changes were observed. In the Hershberger assay of 4,4'-butylidenebis(2-tert-butyl-5-methylphenol), the test chemical was orally administered to castrated male SD rats at doses of 0, 50, 200, and 1,000 mg/kg/day for ten consecutive days beginning on postnatal day 56, and no changes were observed. When this chemical was orally administered at doses 0, 5, 25, and 125 mg/kg/day for at least 28 days in the subacute oral toxicity study, an increase in thyroid weight was observed in the female rats in the 125 mg/kg group, an increase in serum thyroid-stimulating hormone (TSH) values in the male and female rats in the 125 mg/kg group, and a decrease in serum T3 and T4 values in the male rats in the 125 mg/kg group, and thyroid follicular epithelial cell hypertrophy was observed in some of the female rats in the 125 mg/kg group. These findings were concluded to be the result of endocrine-mediated effects of the chemical on thyroid function. In addition, increased liver weight, abnormal histological findings in the liver, and abnormal biochemical parameters related to liver function were observed in male and/or female rats in 5 mg/kg group and higher dose groups. The no-observed-effect level for 4,4'-butylidenebis(2-tert-butyl-5-methylphenol) was concluded to be <5 mg/kg/day. In the uterotrophic assay of 3-(dibutylamino)phenol, 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 no changes were observed. In the Hershberger assay of 3-(dibutylamino)phenol, the test chemical was orally administered at doses of 0, 50, 200, and 400 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 this test chemical was orally administered at doses 0, 30, 100, and 300 mg/kg/day for at least 28 days in the subacute oral toxicity study, thyroid weight increased in the male rats in the 300 mg/kg group, thyroid follicular epithelial cell hypertrophy was observed in a small number of male rats in the 300 mg/kg group, serum T3-values decreased in the female rats in the 300 mg/kg group, and a tendency for TSH-values to increase was observed in the male and female rats in the 300 mg/kg group. Therefore, 3-(dibutylamino)phenol was also concluded to have slight anti-thyroid acting effects as the endocrine-mediated effects. On the other hand, increased hemosiderin deposition in the spleen, increased spleen weight, hematological abnormalities, and squamous epithelial hyperplasia of the forestomach were detected in male and/or female rats in the 100 and/or 300 mg/kg groups, and thus the no-observed-effect level for 3-(dibutylamino)phenol was concluded to be 30 mg/kg/day.

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.

The OECD program to validate the rat Hershberger bioassay to screen compounds for in vivo androgen and antiandrogen responses: phase 2 dose-response studies.

OBJECTIVE: The Organisation for Economic Co-operation and Development (OECD) has completed phase 2 of an international program to validate the rodent Hershberger bioassay. DESIGN: The Hershberger bioassay is designed to identify suspected androgens and antiandrogens based on changes in the weights of five androgen-responsive tissues (ventral prostate, paired seminal vesicles and coagulating glands, the levator ani and bulbocavernosus muscles, the glans penis, and paired Cowper's or bulbourethral glands). Protocol sensitivity and reproducibility were tested using two androgen agonists (17alpha-methyl testosterone and 17beta-trenbolone), four antagonists [procymi-done, vinclozolin, linuron, and 1,1-dichoro-2,2-bis-(p-chlorophenyl)ethylene (p,p'-DDE)], and a 5alpha-reductase inhibitor (finasteride). Sixteen laboratories from seven countries participated in phase 2. RESULTS: In 40 of 41 studies, the laboratories successfully detected substance-related weight changes in one or more tissues. The one exception was with the weakest antiandrogen, linuron, in a laboratory with reduced sensitivity because of high coefficients of variation in all tissue weights. The protocols performed well under different experimental conditions (e.g., strain, diet, housing protocol, bedding, vehicle). There was good agreement and reproducibility among laboratories with regard to the lowest dose inducing significant effects on tissue weights. CONCLUSIONS: The results show that the OECD Hershberger bioassay protocol is reproducible and transferable across laboratories with androgen agonists, weak androgen antagonists, and a 5alpha-reductase inhibitor. The next validation phase will employ coded test substances, including positive substances and negative substances having no androgenic or antiandrogenic activity.

Advantage of using CBA/N strain mice in a non-radioisotopic modification of the local lymph node assay.

The murine local lymph node assay (LLNA) is currently recognized as a stand-alone test method for determining the skin sensitizing potential of chemicals. It has been incorporated into the official test guidelines published by some authorities, including the OECD. To avoid the use of radioisotopes, efforts have been made recently to develop non-radioisotopic modifications of the LLNA. A non-radioisotopic modification of the LLNA was developed previously using 5-bromo-2'-deoxyuridine (BrdU) incorporation (non-RI LLNA). However, the non-RI LLNA was found to be somewhat less sensitive than the standard assay. This study reports the advantage of using mice of the CBA/N strain in the non-RI LLNA to improve the sensitivity of this method. The non-RI LLNA was performed using CBA/JN and CBA/N mice exposed to one of four confirmed skin sensitizers, 2,4-dinitrochlorobenzene (DNCB), eugenol (EG), isoeugenol (IEG) or alpha-hexylcinnamic aldehyde (HCA), and to one non-sensitizer, propylene glycol (PG). The EC3 values for DNCB, IEG, EG, HCA and PG were calculated to be 0.1%, 9.6%, 40.6%, 45.5% and >50% in CBA/JN mice and 0.08%, 1.9%, 10.7%, 20.3% and >50% in CBA/N mice, respectively. The EC3 values for DNCB, IEG, EG, HCA and PG in the standard LLNA using CBA/Ca mice and radioisotopes were reported elsewhere as being 0.08%, 1.3%, 13.0%, 8.0% and >50%, respectively. The EC3 values derived from the CBA/N mice in the non-RI LLNA were nearly equivalent to the EC3 values obtained using the standard radioisotopic LLNA with CBA/Ca mice. These data suggest that the use of CBA/N mice may provide a realistic opportunity to develop a version of the LLNA that does not have a requirement for the use of radioisotopes, but which nevertheless has sensitivity approaching, or comparable to, the standard method.

Subacute oral toxicity study of di(2-ethylhexyl)adipate based on the draft protocol for the "Enhanced OECD Test Guideline no. 407".

We performed a 28-day repeated-dose toxicity study of di(2-ethylhexyl)adipate (DEHA) based on the draft protocol of the "Enhanced OECD Test Guideline 407" to investigate whether it has endocrine-mediated properties according to this assay. DEHA was orally administered to SD rats at doses of 0, 40, 200 and 1,000 mg/kg/day for at least 28 days, and disturbance of the estrous cycle and increased ovarian follicle atresia were detected in the 1,000 mg/kg group.

OECD validation of the Hershberger assay in Japan: Phase 3. Blind study using coded chemicals.

The Organization for Economic Co-operation and Development (OECD) has initiated the development of new guidelines for the screening and testing of potential endocrine disrupters. The Hershberger assay is one of the assays selected for validation based on the need for in vivo screening to detect androgen agonists or antagonists by measuring the response of five sex accessory organs and tissues of castrated juvenile male rats: the ventral prostate, the seminal vesicles with coagulating glands, the levator ani and bulbocavernosus muscle complex (LABC), Cowper's glands, and the glans penis. The Phase 1 feasibility demonstration stage of the Hershberger validation program has been successfully completed with a single androgen agonist and a single antagonist as reference substances. The Phase 2 validation study was performed, employing a range of additional androgen agonists and antagonists. Recently, the Phase 3 validation study was conducted and performed in several International laboratories. Three Japanese laboratories have contributed to the blind study using coded materials of Phase 3 validation. Four coded test substances in the agonistic version and seven substances in the antagonistic version were orally administered by gavage for 10 consecutive days, respectively. In the antagonist version of the assay, 0.2mg/kg/day of testosterone propionate (TP) was coadministered by subcutaneous injection. All five accessory sex reproductive organs and tissues consistently responded with statistically significant changes in weight within a narrow window in both versions. Therefore, the Japanese studies support the Hershberger assay as a reliable and reproducible screening assay for the detection of androgen agonistic and antagonistic effects.