Endocrine-mediated Effects Research Articles

Per- and poly-fluoroalkyl substances (PFAS) and female reproductive outcomes: PFAS elimination, endocrine-mediated effects, and disease

Per- and poly-fluoroalkyl substances (PFAS) are widespread environmental contaminants frequently detected in drinking water supplies worldwide that have been linked to a variety of adverse reproductive health outcomes in women. Compared to men, reproductive health effects in women are generally understudied while global trends in female reproduction rates are declining. Many factors may contribute to the observed decline in female reproduction, one of which is environmental contaminant exposure. PFAS have been used in home, food storage, personal care and industrial products for decades. Despite the phase-out of some legacy PFAS due to their environmental persistence and adverse health effects, alternative, short-chain and legacy PFAS mixtures will continue to pollute water and air and adversely influence women’s health. Studies have shown that both long- and short-chain PFAS disrupt normal reproductive function in women through altering hormone secretion, menstrual cyclicity, and fertility. Here, we summarize the role of a variety of PFAS and PFAS mixtures in female reproductive tract dysfunction and disease. Since these chemicals may affect reproductive tissues directly or indirectly through endocrine disruption, the role of PFAS in breast, thyroid, and hypothalamic-pituitary-gonadal axis function are also discussed as the interplay between these tissues may be critical in understanding the long-term reproductive health effects of PFAS in women. A major research gap is the need for mechanism of action data – the targets for PFAS in the female reproductive and endocrine systems are not evident, but the effects are many. Given the global decline in female fecundity and the ability of PFAS to negatively impact female reproductive health, further studies are needed to examine effects on endocrine target tissues involved in the onset of reproductive disorders of women.

Single and mixture toxicity evaluation of three phenolic compounds to the terrestrial ecosystem

Endocrine-disrupting chemicals (EDCs) are primarily studied regarding endocrine-mediated effects in mammals and fish. However, EDCs can cause toxicity by mechanisms outside the endocrine system, and, as they are released continuously into soils, they may pose risks to terrestrial organisms. In this work, the plant Allium cepa and the earthworm Eisenia foetida were used as test systems to evaluate the toxicity and cyto-/geno-toxicity of three environmental phenols known as EDCs (Bisphenol A – BPA, Octylphenol – OP, Nonylphenol – NP). The tested phenols were evaluated in environmentally relevant concentrations (μg/L) and in single forms and mixture. BPA, OP, and NP did not inhibit the seed germination and root development in A. cepa in their single forms and mixture. However, all single forms of the tested phenols caused cellular and DNA damages in A. cepa, and although these effects persist in the mixtures, the effects were verified at lower levels. These phenols caused acute toxicity to E. foetida after 48 h of exposure and at both conditions evaluated (single forms and mixture); however, unlike A. cepa, in earthworms, mixtures and single forms presented the same level of effects, indicating that interspecies physiological different might influence the mixture toxicity. In summary, our results suggest that BPA, OP, and NP are toxicants to earthworm and cyto-/geno-toxicants to monocotyledonous plants at low concentrations. However, interaction among these phenols reduces the magnitude of their individual effects (antagonistic effect) in the plant test system. Therefore, this study draws attention to the need to raise knowledge about the ecotoxicity of phenolic compounds to help predict their ecological risks and protect non-target terrestrial species.

The value of avian gross pathology in identifying endocrine disrupting properties

The European Chemical Agency and European Food Safety Authority recommend that gross pathology findings, from avian reproduction studies, be used to support assessment of potential endocrine disrupting properties of active pesticidal and biocidal substances. In open literature, little information is available on the utility of gross pathology data for informing endocrine evaluations. Here the gross pathology data from historical control groups of 51 northern bobwhite and 51 mallard reproduction tests is analyzed to evaluate the utility of such information. Incidence of gross morphology findings in untreated birds may aid the interpretation of some gross abnormalities, potentially indicative of an endocrine interaction (e.g. reproductive condition). Statistical analysis of the historical control data indicates that gross pathology is not likely to be useful for detecting endocrine effects as abnormalities with relatively high increases in prevalence (more than 20–30%, depending on prevalence in controls) are reliably interpreted as a treatment response. Gross pathology changes are only indicative and not diagnostic of endocrine interactions making it difficult to distinguish gross pathology abnormalities, due to endocrine-mediated effects, from systemic toxicity. This work demonstrates the utility of using historical control analyses to establish the value and properties of selected endpoints for regulatory applications.

Environmental toxic Endocrine Disrupting Compounds (EDCs): Effects on environment, animal production and human

Endocrine disrupting compounds (EDCs) encompass a variety of chemical classes, including several different categories of substances. Some are natural, such as plant oestrogens and mycoestrogens, while most are chemically composed. Residues are found in water, in sewage sludge, in the atmosphere and in foodstuff of vegitative or animal origin. The most common compounds include organochlorine pesticides, alkyl phenols, pthalates, polychlorinated biphenyls (PCBs), dioxins and polybrominated diphenyl ethers (PBDEs). Their properties render some as ubiquitous and persistent in the environment, they can be transported long distances and have been found in virtually all regions of the world. Their sources can vary from direct, such as effluents from wastewater, sewage, industrial sludge processes or agricultural effluents, to indirect, such as filtering agricultural soils, transportation through rain or air from civil and industrial centers to rivers and the surrounding environment. Others are rapidly degraded in the environment or human body or may be present for only short periods of time, but at critical periods of development. Although in low concentrations, the continuous exposure of animals of many species, including humans, induces adverse effects, such as disruption of reproductive function and of the immune system, as well as carcinogenic effects. The effects are obvious in wildlife, especially fish, and laboratory experiments. Sex change (feminizing) in fish has been observed, sexual differentiation in rats, egg shelling thinning, thyroid function disruption and mental and physical function disruption in wildlife. Most important and common is the disruption in the reproductive and the immune system of animals. As far as humans are concerned, reduction in sperm count and sperm quality has been heavily linked with EDC exposure, and furthermore, suspicions arise linking several cancer incident increments, such as testicular cancer, to EDC exposure. Still, although it is clear that many environmental chemicals can interfere with normal hormonal processes, there is weak evidence that human health can be adversely and directly affected by exposure to endocrine active chemicals. However, there is sufficient evidence to conclude that adverse endocrine mediated effects have occurred in wildlife species and laboratory studies exist to support these conclusions. The routes of exposure are numerous. Food is a common route of exposure, especially in younger animals that consume food containing higher fat percentages (e.g. milk). Soil is another route of exposure. Thus grazing animals tend to have a higher risk factor, due to surface soil pollution with EDCs. A vegetative nutrition with EDC residues is another high risk factor for these animals. On the other hand, animals bred with forage tend to be at lower risk factor, given that food is tested for low levels of EDCs. Water intake is not considered a significant route of exposure. EDCs possess the ability to act as either hormone agonists or antagonists or disrupt hormone synthesis, storage or metabolism. Due to their persistence in the environment, they are concentrated in fat tissue and are released when the fat is mobilized duringpregnancy or lactation, thus exposing embryos and neonates, which are very susceptible at this stage of development, to high concentrations of EDCs. The concerns that arise regarding the exposure to these EDCs are due primarily to:- these adverse effects observed in certain wildlife, fish and ecosystems- the increased incidences of certain endocrine-related human diseases and- endocrine disruption resulting from exposure to certain environmental chemicals observed in laboratory experimental animals.

Epigenotoxicity of Endocrine-Disrupting Chemicals Makes Inroads to a Paradigm Shift in the Risk Assessment of Pesticides

The most ferocious chronic effect a pesticide could have is disrupting the functions mediated by the endocrine system. When this happens during early development of the life cycle, it leads to profound and lasting adverse human-health and wildlife effects. These effects may not only stop at interrupting some plain physiological functions, but could also play havoc with the epigenetic machinery regulating DNA-transcription in exposed individuals. Even worse is that this havoc and its supervened epigenotoxic effects could be stably transmitted to descendants without further exposing them to the same pesticide or changing their ancestral DNA base sequences. In this manuscript the author reviews the literature with the aim of building a case around the expandable risk posed by Endocrine-Disrupting Pesticides (EDPs); a risk beyond their traditionally-assumed or regulatory-promulgated safety measures. The fact that some EDPs elicit their endocrine-mediated effects at doses far below their acceptable daily intake (ADI) destroys our confidence in the current safety thresholds of human exposure and environmental contamination proclaimed for these pesticides. It seems that the precautionary principle of ‘reasonable certainty of no harm’ for EDPs is only a lip-serviced phrase that has no practical value, especially in long terms, due to the possible transgenerational epigenotoxicity caused by these pesticides. The most important message of this review is that the effects of EDPs and their possible epigenetic inheritance significantly amplify the negative impacts and health hazards of these pesticides and require a paradigm shift from the traditional risk assessment approach to a one that includes evidence-based epigenotoxicology (EBE).

Abstract P1-12-01: CYP19A1 and ESR1 polymorphisms and selected early-onset side effects during combined endocrine therapy in the IBCSG TEXT trial for premenopausal women with hormone receptor-positive (HR+) early breast cancer

Abstract Background: Single nucleotide polymorphisms (SNPs) of the aromatase enzyme (CYP19A1) and estrogen receptor alpha (ESR1) may be associated with breast cancer susceptibility and endocrine-mediated side effects. The IBCSG Tamoxifen (Tam) and Exemestane (Exe) Trial (TEXT) includes a translational research project to assess whether selected SNPs may influence treatment efficacy and toxicity. We report on early-onset hot flashes and sweating (HF/S) and musculoskeletal symptoms (MS; myalgia, arthralgia, stiffness) with respect to CYP19A1 and ESR1 SNPs under combined endocrine therapy. Patients and Methods: 2,672 premenopausal women with HR+ early breast cancer were randomized to treatment with the GnRH-agonist triptorelin (Trip)+Tam or Trip+Exe for 5 years. Randomization was stratified according to intended use of chemotherapy (yes/no) and lymph node status (N- vs N+). Estrogen-depletion side effects (HF/S and MS) were recorded at baseline, 3-monthly during the first year and 6-monthly thereafter using the NCI CTCAE v3.0. DNA was centrally extracted from whole blood with Qiagen kits. SNPs of CYP19A1 (rs4646 and rs10046) and ESR1 (rs207764, rs2234693 and rs9340799) were analyzed by a pyrosequencing method (Diatech Pharmacogenetics S.r.l., Jesi, Italy). Control genotypes (wt/wt; wt/v; v/v) for all SNPs were processed in each run. Logistic regression was used to analyze two endpoints: presence or absence of grade (gr) 2-3 HF/S during first 6 months and gr 2-4 MS during first 12 months. Four genetic models were used to explore associations with side effects: genetic (wt/wt; wt/v; v/v), additive, dominant and recessive. Results: DNA was isolated and genotyped for 1970 (74%) consenting women. Clinical characteristics and outcomes of this cohort were consistent with the overall trial. At baseline median age was 44, median BMI 24 kg/m2 and 86% had regular menses. During follow-up, 43% reported gr 2-3 HF/S and 27% reported gr 2-4 MS. The 5 SNPs did not deviate from Hardy-Weinberg equilibrium (p>0.30) and minor allele frequency ranged from 29%-48%. The CYP19A1 SNP rs10046 (C>T) was associated with gr 2-3 HF/S. Specifically, women with variant homozygote genotype (T/T) had a reduced risk of HF/S (39% T/T vs 45%). The univariate odds ratio (OR) was 0.77 (95%CI: 0.62-0.96; p=0.02) compared with other variant groups. The multivariate model showed consistent results after adjusting for age, BMI, menstrual status, chemo use, treatment allocation (Tam vs Exe) and baseline HF/S. The other 4 SNPs were not associated with the selected side effects. Conclusions: Our analysis indicates association of HF/S with CYP19A1 rs10046 genetic variants. The lack of association between early-onset of selected estrogen-depletion side effects and the other 4 SNPs may be masked by the concurrent Trip. Ovarian reserve before treatment may also influence the impact of SNP genotypes on early-onset side effects. Further analysis with details of concurrent medications and treatment adherence will contribute to the interpretation of these results. Funded by Susan G. Komen for the Cure. Citation Format: Harriet Johansson, Kathryn P Gray, Olivia Pagani, Meredith M Regan, Giuseppe Viale, Valentina Aristarco, Debora Macis, Antonella Puccio, Susanne Roux, Rudolf Maibach, Marco Colleoni, Manuela Rabaglio-Poretti, Alan S Coates, Richard D Gelber, Aron Goldhirsch, Roswitha Kammler, Bernardo Bonanni, Barbara A Walley. CYP19A1 and ESR1 polymorphisms and selected early-onset side effects during combined endocrine therapy in the IBCSG TEXT trial for premenopausal women with hormone receptor-positive (HR+) early breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-12-01.

Sexually dimorphic behavior after developmental exposure to characterize endocrine-mediated effects of different non-dioxin-like PCBs in rats

Many chemicals are known to exhibit endocrine activity and affect reproductive functions in vertebrates and invertebrates. Endocrine effects include influences on sexual differentiation of the brain during development and reproductive and non-reproductive behavior in adult offspring. We previously demonstrated that developmental exposure to a mixture of polychlorinated biphenyls (PCBs) which was reconstituted according to the congener pattern found in human breast milk caused feminization of sweet preference as a sexually dimorphic behavior in adult male rats, following decreases in aromatase activity in the brain of newborn male pups. This result may be due to dioxin-like or non-dioxin-like (NDL) PCBs and their respective effects on steroid hormones. The aim of the present experiments was to determine if exposure to highly purified NDL-PCBs (to remove Ah receptor active contaminants) also results in alteration of sweet preference. Pregnant rats were orally exposed to PCB52 (6 dose groups, total dose of 0–3000mg/kg body weight) or PCB180 (6 dose groups, total dose of 0–1000mg/kg body weight). In a further experiment rat dams were treated with equimolar doses of PCB74 or PCB95 (total dose, 760μmol/kg body weight, corresponding to 229mg/kg or 248mg/kg body weight of PCB74 and PCB95, respectively). Adult male and female offspring were given a choice between a bottle of saccharin solution (0.25%) and a bottle of tap water on five consecutive days. Control females consumed approximately twice as much sweetened solution compared with control males, thus, demonstrating sexual dimorphism of this behavior. Only non-significant reduction of sweet preference was found at the top dose level in female offspring after exposure to PCB52. Female offspring exposed to PCB180 exhibited signs of supernormal behavior as illustrated by increased saccharin consumption at intermediate dose levels. Decreased sweet preference was observed in females after developmental PCB74, whereas males were unaffected. Only PCB95 increased saccharin consumption in exposed males, leading to decreased sexual dimorphism of this behavior and behavioral feminization. The results demonstrate that different NDL-PCBs exhibit differential effects on sexually dimorphic behavior and that feminization occurs after removal of Ah receptor active contaminants. Comparison with data from the literature reveals little evidence for a relation to anti-androgenic activity of the studied NDL-PCBs.

Endocrine-Mediated Effects of Genistein on Pups Born to Dams Fed a Phytoestrogen-Enriched Diet

Endocrine-Mediated Effects of Genistein on Pups Born to Dams Fed a Phytoestrogen-Enriched Diet

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 250mg/kg/day of 1-chloro-4-(chloromethyl)benzene, and 0, 25, 150, and 1000mg/kg/day of 1,3-diethyl benzene for at least 28days, beginning at 8weeks 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 1000mg/kg group of female rats, and TSH values also increased in the 1000mg/kg groups of both sexes after 28days’ administration. Decreased T3 values were observed in the 1000mg/kg group of female rats after 28days’ administration, and hormone values increased in the 1000mg/kg groups of both sexes after the 14-day recovery period. In addition, thyroid weight increased in the 1000mg/kg groups and thyroid follicular cell hyperplasia was detected in one male rat from the 1000mg/kg group after 28days’ 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.

Endocrine-Mediated Effects of a Benzophenone Related Chemical, 2,3,4,4'-Tetrahydroxybenzophenone, Based on Uterotrophic Assay, Hershberger Assay, and Subacute Oral Toxicity Study

The purpose of this study was to investigate the endocrine-mediated effects of a benzophenone-relative compound, 2,3,4,4’-tetrahydroxybenzophenone, based on the OECD 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 300 and 1,000 mg/kg doses of the test chemical increased. In the Hershberger assay, the test chemical was orally administered at doses of 0, 60, 200, and 600 mg/kg/day to castrated male SD rats for 10 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, 60, 200, and 600 mg/kg/day for at least 28 days, serum T4 values decreased in male rats in the 600 mg/kg group. This suggests a mechanism of action related to the inhibition of thyroid peroxidase. The uterotrophic assay used in this study showed that the chemical has estrogen-agonist properties, however, estrogenic effects were not observed in a 28-day repeated-dose toxicity study. On the other hand, endocrine-mediated effects such as thyroid hormone dysfunction were detected in growing rats based on the results of the OECD test guideline No. 407.

Interlaboratory Study Comparison of the 15‐Day Intact Adult Male Rat Screening Assay: Evaluation of an Antithyroid Chemical and a Negative Control Chemical

Validation of the 15-day intact adult male rat screening assay (IAMRSA), an endocrine activity screen, was extended beyond the 28 substances evaluated to date. Two independent laboratories evaluated specificity using allyl alcohol (AA), a putative negative control, and DE-71 (technical grade pentabromodiphenyl ether) for comparison with previous pubertal assays that demonstrated thyroid effects. Male rats (15/group) were gavaged daily with AA (0, 10, 30, or 40 mg/kg/day) or DE-71 (0, 3, 30, or 60 mg/kg/day) for 15 days. Body and organ weights and serum hormone concentrations were measured, and a limited histopathological assessment was conducted. AA results were considered negative at doses that did not exceed the maximum tolerated dose (MTD); effects reported were dose-related decreases in weight gain, increased liver weights and, although the pattern varied across studies, alterations in some androgen-sensitive endpoints in the high-dose where the maximum tolerated dose was exceeded. In the DE-71 studies, dose-dependent increases in liver weights (consistent with hepatic enzyme induction), decreases in tri-iodothyronine and thyroxine, concomitant thyroid stimulating hormone increases were observed and one laboratory reported histopathological thyroid changes in mid- and high-dose groups, and the other increased thyroid weights. For DE-71, the IAMRSA was comparable in sensitivity to the pubertal assays. Overall, the specificity and sensitivity of the IAMRSA for deployment in an endocrine screening battery are supported. However, differentiating primary endocrine-mediated effects from secondary effects caused by systemic toxicity will be challenging, emphasizing the need to utilize a battery of assays and a weight of evidence approach when evaluating the potential endocrine activity of chemicals.

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.

Uterotrophic assay, Hershberger assay, and repeated 28-day oral toxicity study of flumorph based on the Organization for Economic Co-operation and Development draft protocols

To evaluate the endocrine-mediated effects of flumorph, we performed the uterotrophic assay, the Hershberger assay, and the repeated 28-day oral toxicity study based on the OECD draft protocols. In the uterotrophic assay, female ovariectomized SD rats were subcutaneously injected with flumorph at doses of 0, 50, 150, and 500 mg/kg on each of 3 days, and no changes were observed. In the Hershberger assay, castrated male SD rats were administered with flumorph by oral gavage at doses of 0, 50, 150, and 500 mg/kg/day for 10 consecutive days, and no abnormal changes were observed. However, in the repeated 28-day oral toxicity study , flumorph was orally administered at doses 0, 30, 100, and 300 mg/kg/day for at least 28 days, a significantly increase in T 4 value in male rats and TSH value in female rats were detected in the highest dosage group, respectively. Besides, the flumorph administration increase liver weights, produce hepatocellular diffuse fatty degeneration, and effect biochemical parameters related to liver function in male and/or female rats in dosed groups. Therefore, flumorph is concluded to have thyroid disruption effects and is likely a thyroid disrupter, but the further studies are needed for hazard identification.

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.

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.