25th anniversary of the Berlin workshop on developmental toxicology: DevTox database update, challenges in risk assessment of developmental neurotoxicity and alternative methodologies in bone development and growth.

25 years after the first Berlin Workshop on Developmental Toxicity this 10th Berlin Workshop aimed to bring together international experts from authorities, academia and industry to consider scientific, methodologic and regulatory aspects in risk assessment of developmental toxicity and to debate alternative strategies in testing developmental effects in the future. Proposals for improvement of the categorization of developmental effects were discussed as well as the update of the DevTox database as valuable tool for harmonization. The development of adverse outcome pathways relevant to developmental neurotoxicity (DNT) was debated as a fundamental improvement to guide the screening and testing for DNT using alternatives to animal methods. A further focus was the implementation of an in vitro mechanism-based battery, which can support various regulatory applications associated with the assessment of chemicals and mixtures. More interdisciplinary and translation research should be initiated to accelerate the development of new technologies to test developmental toxicity. Technologies in the pipeline are (i) high throughput imaging techniques, (ii) models for DNT screening tests, (iii) use of computer tomography for assessment of thoracolumbar supernumerary ribs in animal models, and (iv) 3D biofabrication of bone development and regeneration tissue models. In addition, increased collaboration with the medical community was suggested to improve the relevance of test results to humans and identify more clinically relevant endpoints. Finally, the participants agreed that this conference facilitated better understanding innovative approaches that can be useful for the identification of developmental health risks due to exposure to chemical substances.

Prenatal exposure to the phytoestrogen daidzein resulted in persistent changes in ovarian surface epithelial cell height, folliculogenesis, and estrus phase length in adult Sprague-Dawley rat offspring.

Daidzein (DZ), an isoflavone with the potential to interfere with estrogen signaling, is found in soy products, which have gained popularity due to purported beneficial effects on the cardiovascular and skeletal systems and potential antineoplastic properties. However, the ingestion of phytoestrogens has been associated with impaired reproductive function in many species. The aim of this study was to determine the long-term effects on the ovaries of rat offspring exposed to DZ or ethinyl estradiol (EE) during prenatal development. Gravid rats were administered either vehicle or 5 or 60 mg DZ/kg body weight/d or 0.002 mg 17-α EE /kg body weight/d on gestational days 6-21. Ovarian-related endpoints were investigated during adulthood in female offspring. The mean cell height of the ovarian surface epithelium was significantly reduced in all treated groups. Alterations in folliculogenesis included increased follicular atresia, a reduction in secondary and tertiary follicle numbers, and cyst formation. An elevated prevalence of a slightly prolonged estrus phase was also observed. The morphological changes to the ovarian surface epithelium are consistent with an antiproliferative effect, while ovarian folliculogenesis was adversely affected. The effects of the high dose DZ were similar to those observed with 17-α EE.

Biological and tumor-promoting effects of dioxin-like and non-dioxin-like polychlorinated biphenyls in mouse liver after single or combined treatment.

To assess the impact of a mixture containing dioxin-like and non-dioxin-like polychlorinated biphenyls (PCBs), male mice were initiated with N-nitroso-diethylamine and subsequently treated with PCB126, an Ah-Receptor agonist, and PCB153, acting via activation of the constitutive androstane receptor. The two congeners were given at two dose levels: the low dose was adjusted to induce ~150-fold increases in cytochrome P450 (Cyp)1a1 (PCB126) and Cyp2b10 mRNAs (PCB153), and the high dose was chosen as twice the low dose. To keep the liver PCB levels constant, mice were given initial loading doses followed by weekly maintenance doses calculated on the basis of the PCBs' half-lives. Mice were treated with the individual congeners (low and high dose) or with a mixture consisting of the low doses of the 2 PCBs. The following results were obtained: (1) the 2 PCBs produced dose-dependent increases in Cyp1a1 and Cyp2b10 mRNA, protein, and activity when given individually; (2) combined treatment caused more than additive effects on Cyp1a1 mRNA expression, protein level, and ethoxyresurofin activity; (3) changes in the levels of several proteins were detected by proteome analysis in livers of PCB-treated mice; (4) besides these biological responses, the individual PCBs caused no significant increase in the number of glucose-6-phospatase (G6Pase)-deficient neoplastic lesions in liver, whereas a moderate significant effect occurred in the combination group. These results suggest weak but significant response-additive effects of the 2 PCBs when given in combination. They also suggest that the Cyp biomarkers tend to overestimate the carcinogenic response produced by the PCBs in mouse liver.

Harmonization of description and classification of fetal observations: achievements and problems still unresolved: report of the 7th Workshop on the Terminology in Developmental Toxicology Berlin, 4-6 May 2011.

This article summarizes the 7th Workshop on the Terminology in Developmental Toxicology held in Berlin, May 4-6, 2011. The series of Berlin Workshops has been mainly concerned with the harmonization of terminology and classification of fetal anomalies in developmental toxicity studies. The main topics of the 7th Workshop were knowledge on the fate of anomalies after birth, use of Version 2 terminology for maternal-fetal observations and non-routinely used species, reclassification of "grey zone" anomalies and categorization of fetal observations for human health risk assessment. The paucity of data on health consequences of the postnatal permanence of fetal anomalies is relevant and further studies are needed. The Version 2 terminology is an important step forward and the terms listed in this glossary are considered also to be appropriate for most observations in non-routinely used species. Continuation of the Berlin Workshops was recommended. Topics suggested for the next Workshop were grouping of fetal observations for reporting and statistical analysis.

Sex-dependent aromatase activity in rat offspring after pre- and postnatal exposure to triphenyltin chloride.

Triphenyltin (TPT) is an organotin compound (OTC) previously widely used as an antifouling agent in paints applied in the marine environment, a fungicide, and as an agricultural pesticide. In female aquatic invertebrates, certain OTCs induce the so-called imposex, an abnormal induction of male sex characteristics. OTC-induced environmental endocrine disruption also occurs in fish and mammals and a number of in vivo and in vitro studies have argued that OTCs may act through inhibition of the aromatase enzyme. In vivo studies supporting the aromatase inhibition hypothesis in mammals are lacking. Recently, the causal relationship between inhibition of aromatase and imposex was questioned, suggesting aromatase independent mechanisms of action for this phenomenon. We conducted a comprehensive investigation to identify the most sensitive window of exposure to TPTCl and to examine the effects of pre- and postnatal exposure on postnatal development in rats. The results on brain and gonadal aromatase activity obtained from offspring of dams exposed to 2 mg TPTCl/kg bw are reported here. Female and male offspring rats were exposed to 2 mg TPTCl/kg bw/d in utero from gestation day 6 through lactation until weaning on PND 21, or from gestation day 6 until termination at adulthood. Male offspring were sacrificed from PND 58 and female offspring at first estrus after PND 58. Pre- and postnatal TPT exposure clearly affected brain and gonadal aromatase activity in a sex-dependent fashion. While brain aromatase activity was significantly increased on PND 21 and at adulthood in female offspring, male offspring exhibited a significant decrease in brain aromatase activity only at adulthood. Ovarian aromatase activity was unaffected at both time points investigated. In contrast, testicular aromatase activity was significantly increased in males on PND 21 and significantly decreased at adulthood independent from the duration of treatment. The results of the present study confirm our previously reported observations regarding sex-dependent differences in sexual development after TPT exposure with the male rat being more susceptible to disturbances through this endocrine active compound than the female. We conclude that TPT administered during the particularly vulnerable period of development can affect aromatase activity in rats.

Terminology of developmental abnormalities in common laboratory mammals (version 2).

This update (Version 2) of the Terminology of Developmental Abnormalities in Common Laboratory Mammals (Version 1) incorporates improvements and enhancements to both content and organization of the terminology to enable greater flexibility in its application, while maintaining a consistent approach to the description of findings. The revisions are the result of an international collaboration among interested organizations, advised by individual experts and the outcomes of several workshops. The terminology remains organized into tables under the broad categories of external, visceral, and skeletal observations, following the manner in which data are typically collected and recorded in developmental toxicity studies. This arrangement of the tables, as well as other information provided in appendices, is intended to facilitate the process of specimen evaluation at the laboratory bench level. Only the commonly used laboratory mammals (i.e. rats, mice, rabbits) are addressed in the current terminology tables. The inclusion of other species that are used in developmental toxicity testing, such as primates, is considered outside the scope of the present update. Similarly, categorization of findings as, for example, 'malformation' or 'variation' remains unaddressed, in accordance with the overall principle that the focus of this document is descriptive terminology and not diagnosis or interpretation. The skeletal terms have been augmented to accommodate cartilage findings.

Terminology of developmental abnormalities in common laboratory mammals (version 2).

This update (version 2) of the Terminology of developmental abnormalities in common laboratory mammals (version 1) by Wise et al. [Wise LD, Beck SL, Beltrame D, Beyer BK, Chahoud I, Clark RL, Clark R, Druga AM, Fueston MH, Guittin P, Henwood SM, Kimmel CA, Lindstrom P, Palmer AK, Petrere JA, Solomon HM, Yasuda M, York RG. Terminology of developmental abnormalities in common laboratory mammals (version 1). Teratology 1997;55:249-92] incorporates improvements and enhancements to both content and organization of the terminology, to enable greater flexibility in its application, while maintaining a consistent approach to the description of findings. The revisions are the result of an international collaboration among interested organizations, advised by individual experts and the outcomes of several workshops. The terminology remains organized into tables under the broad categories of external, visceral, and skeletal observations, following the manner in which data are typically collected and recorded in developmental toxicity studies. This arrangement of the tables, as well as other information provided in appendices, is intended to facilitate the process of specimen evaluation at the laboratory bench level. Only the commonly used laboratory mammals (i.e., rats, mice, rabbits) are addressed in the current terminology tables. The inclusion of other species that are used in developmental toxicity testing, such as primates, is considered outside the scope of the present update. Similarly, categorization of findings as, for example, "malformation" or "variation" remains unaddressed, in accordance with the overall principle that the focus of this document is descriptive terminology and not diagnosis/interpretation. The skeletal terms have been augmented to accommodate cartilage findings.

Terminology of developmental abnormalities in common laboratory mammals (Version 2).

This update (Version 2) of the Terminology of Developmental Abnormalities in Common Laboratory Mammals (Version 1) by Wise et al. (1997) incorporates improvements and enhancements to both content and organization of the terminology, to enable greater flexibility in its application, while maintaining a consistent approach to the description of findings. The revisions are the result of an international collaboration among interested organizations, advised by individual experts and the outcomes of several workshops. The terminology remains organized into tables under the broad categories of external, visceral, and skeletal observations, following the manner in which data are typically collected and recorded in developmental toxicity studies. This arrangement of the tables, as well as other information provided in appendices, is intended to facilitate the process of specimen evaluation at the laboratory bench level. Only the commonly used laboratory mammals (i.e., rats, mice, rabbits) are addressed in the current terminology tables. The inclusion of other species that are used in developmental toxicity testing, such as primates, is considered outside the scope of the present update. Similarly, categorization of findings as, for example, "malformation" or "variation" remains unaddressed, in accordance with the overall principle that the focus of this document is descriptive terminology and not diagnosis/interpretation. The skeletal terms have been augmented to accommodate cartilage findings.

Sex differences in effects on sexual development in rat offspring after pre- and postnatal exposure to triphenyltin chloride.

Consumers are exposed to organotin compounds (OTCs) via contaminated fish and seafood due to the accumulation of these compounds in marine organisms. Certain OTCs are immunotoxic and may also have endocrine disrupting properties resulting in adverse effects on the reproductive tract in mollusks and mammals. Since effects of in utero exposure to endocrine disrupting chemicals on the reproductive system are dependent on the critical window of exposure during its development, we conducted a comprehensive study with the aim to identify the most sensitive window of exposure to TPTCl and to investigate the effects of pre- and postnatal treatment on sexual development in rats. Male and female offspring rats were exposed to 2 or 6 mg TPTCl/kg b.w. and day either in utero and during lactation (gestation day 6 until weaning on PND 21) or from gestation day 6 until termination. As previously reported, offspring in the 6 mg TPTCl dose group exhibited high perinatal mortality and therefore no further evaluation was carried out at this dose level (Grote, K., Hobler, C, Andrade, A.J.M., Wichert Grande, S., Gericke, C., Talsness, C.E., Appel, K.E., Chahoud, I., 2007. Effects of in utero and lactational exposure to triphenyltin chloride on pregnancy outcome and postnatal development in rat offspring. Toxicology 238, 177-185). In the present paper, results on postnatal development obtained from surviving offspring of dams exposed to 2mg TPTCl/kg b.w. are reported. Male offspring were sacrificed on PND 64 or 65 and female offspring at first estrus after PND 58. A clear sex difference in response to treatment was observed. Male postnatal development was severely affected with decreases in body weight gain, reproductive organ weights and testosterone concentration as well as a significant delay in the age at preputial separation. In contrast, females exhibited a precocious completion of vaginal opening while all other endpoints were unaffected. Most of these effects were already present in animals that were only exposed until weaning indicating that these effects may be irreversible and continued treatment until termination had contributed less than expected to the severity of the observed effects. The results of the present study suggest that the sensitive window for the evaluated endpoints seems to be the period of prenatal development and that male offspring rats were more susceptible to treatment.

Harmonization of terminology in developmental toxicology: the quest for a more precise description and a harmonized classification of fetal observations.

Harmonization of terminology in developmental toxicology is a prerequisite to ensure a better risk assessment of chemicals. As part of an international effort of the International Programme on Chemical Safety (IPCS) to harmonize terminology in developmental toxicology, workshops have taken place in Berlin since 1995. This publication reports the main outcomes of the Fifth and Sixth Berlin Workshops held in 2005 and 2007, respectively. The objective of the Fifth workshop was to discuss a draft international proposal for updating the glossary of descriptive terms for fetal abnormalities put forward by Wise et al. [Wise LD, et al. Terminology of developmental abnormalities in common laboratory mammals (version 1). Teratology 1997;55:249-92]. The participants were asked to classify the new external, visceral and skeletal observations included within this new version 2 of Terminology of Developmental Abnormalities in common Laboratory Mammals according to the two-category scheme (malformation and variation) agreed at previous Berlin workshops. The discussions held during the Sixth Workshop were mainly focused on the causes of uncertainty and low agreement regarding classification of some fetal observations as malformations or variations. Lack of precision in descriptive terms and insufficient knowledge of the postnatal consequences of fetal observations had been identified as major causes of uncertainty and lower agreement among evaluators regarding the classification of "grey zone anomalies", i.e. abnormalities that do not fit readily into one of the two categories (malformation or variation). Imprecise anatomical terms, observation terms that are too broad, lack of information on severity and the use of different terms for the same change or different severities of the same change, were found to be the main reasons that descriptive terms are often not sufficiently precise to allow accurate classification of findings. It was agreed that provision of additional information, including sub-location within the affected structure, more detailed description of the nature of the change, in conjunction with presentation of photographs wherever possible, and a grading for severity would make descriptive terms more precise, thereby reducing misclassifications. A better knowledge of the adversity and postnatal consequences of fetal observations was considered as the key issue for achieving a substantial reduction in the number of misclassifications and grey zone anomalies. The urgent need for additional research along this line as a prerequisite for a better risk assessment was emphasized by the participants.

Epoxiconazole causes changes in testicular histology and sperm production in the Japanese quail (Coturnix coturnix japonica).

The fungicide epoxiconazole (Epox), a triazole, belongs to the group of azole compounds that are extensively used as fungicides in various fruit crops. The frequent use of agricultural lands for wintering by migrating birds can be the source of their increased dietary intake of agricultural pesticides. We investigated whether exposure to Epox causes effects on avian fertility and reproduction, using the Japanese quail (Coturnix coturnix japonica) as a model species for the assessment of reproductive effects of pesticides in wild birds. Epox was administered to adult Japanese quail for three weeks at dietary levels of 10, 50, and 500 ppm, and possible effects on reproduction were investigated. Epox administration resulted in a significantly decreased number of spermatids in the 50- and 500-ppm dose groups. Histopathology showed a reduced number of testicular canaliculi with visible germ cells and a reduction in spermatid number. However, testis weight was not affected up to the highest dose level. No impact was observed on hormone levels, fertility, and reproductive outcome, as laying rate and percentage of fertile eggs were not altered. Likewise, treatment had no influence on the egg or chick parameters evaluated. A time- and dose-related transfer of Epox into the eggs was determined in all treatment groups. We conclude that dietary treatment of Japanese quail with 50 and 500 ppm of the triazole fungicide Epox resulted in a clear impact on the testis. The evaluation of the additional endpoints spermatid count and testicular histology have proven useful and are recommended for future studies on avian reproduction.

In utero and lactational exposures to low doses of polybrominated diphenyl ether-47 alter the reproductive system and thyroid gland of female rat offspring.

BACKGROUND: Polybrominated diphenyl ethers (PBDEs) are capable of disrupting thyroid hormone homeostasis. PBDE-47 (2,2',4,4'-tetrabromodiphenyl ether) is one of the most abundant congeners found in human breast adipose tissue and maternal milk samples. OBJECTIVES: We evaluated the effects of developmental exposure to low doses of PBDE-47 on the female reproductive system. METHODS: Pregnant Wistar rats were administered vehicle (peanut oil) or PBDE-47 [140 or 700 microg/kg body weight (bw)] on gestation day (GD) 6, or 5 mg 6-n-propyl-2-thiouracil (PTU)/L in the drinking water from GD7 through postnatal day (PND) 21. RESULTS: In female offspring sacrificed on PND38, there was a significant decrease in ovarian weight after exposure to PTU or 140 microg/kg PBDE-47. Alterations in folliculogenesis were apparent: we observed a decrease in tertiary follicles and serum estradiol concentrations in the offspring exposed to either PTU or 700 microg/kg PBDE-47. PTU exposure also resulted in a decrease in primordial follicles. On PND100, persistent effects on the thyroid glands included histologic and morphometric changes after exposure to either PTU or PBDE-47. No relevant changes in reproductive indices were observed after mating the exposed F1 females with nontreated males. CONCLUSIONS: Administration of PBDE-47 at doses relevant to human exposure led to changes in the rat female reproductive system and thyroid gland.

Effects of in utero and lactational exposure to triphenyltin chloride on pregnancy outcome and postnatal development in rat offspring.

The organotin compound (OTC) triphenyltin (TPT) is used extensively as a herbicide, pesticide and fungicide in agriculture as well as, together with tributyltin (TBT), in marine antifouling paints. We studied the effects of in utero exposure to 2 or 6 mg triphenyltinchloride (TPTCl)/kgb.w. on pregnancy outcome and postnatal development in rat offspring. Gravid Wistar rats were treated per gavage from gestational day 6 until the end of lactation. In the 6 mg TPTCl dose group gestational mortality in dams as well as an increased incidence of anticipated and delayed parturition was observed. Furthermore, treatment resulted in a significant increase in perinatal mortality, a decrease in lactational body weight gain as well as in delayed physical maturation of offspring. Similarily, exposure to 2mg TPTCl/kgb.w. resulted in a significant increase in perinatal mortality and in delayed eye opening. Lactational body weight gain and other landmarks of physical maturation were unaffected in the low dose group. We conclude, that in utero exposure to TPTCl at the described dose levels severely affected pregnancy outcome and perinatal survival of offspring. These results were unexpected, as in two earlier studies with pubertal rats TPTCl at the same dose levels no signs of general toxicity were observed.

A dose-response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP): reproductive effects on adult female offspring rats.

Di(2-ethylhexyl) phthalate (DEHP) is used in numerous consumer products, mainly imparting flexibility and durability to polyvinyl chloride (PVC) based plastics. It is a known reproductive and developmental toxicant in male rodents. However, data regarding effects of DEHP on female reproductive health are particularly sparse. We performed an extensive dose-response study following developmental exposure to DEHP and evaluated the effects on adult female reproductive function. Two wide ranges of doses, low and high, were tested. Female Wistar rats were treated daily with DEHP and peanut oil (vehicle control) by gavage from gestation day 6 to lactation day 21. The low doses were: 0.015, 0.045, 0.135, 0.405 and 1.215mgDEHP/kg/bw/day and the high doses were: 5, 15, 45, 135 and 405mg DEHP/kg/bw/day. At the doses tested, no effects on organ (liver, kidney, spleen, thymus, thyroid, ovary and uterus) or body weights were detected. Female offspring presented a normal pattern of estrous cyclicity with no hormonal alterations (serum estradiol and progesterone). A statistically significant increase in tertiary atretic follicles was observed at the highest dose (405mgDEHP/kg/day). Morphometric analysis indicated that uterus and vagina luminal epithelial cell height were unaffected by treatment. An increase in the number of ovarian atretic tertiary follicles was the only effect observed in adult female offspring exposed in utero and during lactation to DEHP.

A dose response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP): reproductive effects on adult male offspring rats.

The reproductive effects of in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP) in adult male offspring rats were investigated. The selected endpoints included reproductive organ weights, testicular function, hormonal status, sexual behaviour and fertility. Two wide ranges of doses, low and high, were tested. Female Wistar rats were treated daily with DEHP and peanut oil (vehicle control) by gavage from gestation day 6 to lactation day 21. The low-doses were 0.015, 0.045, 0.135, 0.405 and 1.215 mg DEHP/kg body weight (bw)/day, and the high-doses were 5, 15, 45, 135 and 405 mg DEHP/kg bw/day. A reduction in daily sperm production of 19-25% in relation to control was observed in animals exposed to 15, 45, 135 and 405 mg/kg/day. Quantitation of specific cell types shows that the observed effects in daily sperm production are not related to changes in the number of Sertoli cells or their capability to support early stages spermatocytes. A low incidence of cryptorchidism was observed in DEHP exposed groups with a lowest observed adverse effect level of 5mg/kg/day. Serum testosterone concentration was similar to control at most doses but was significantly increased at 0.045, 0.405 and 405 mg DEHP/kg/day. In spite of this effect, the weight of seminal vesicle with coagulating glands was significantly reduced at 405 mg/kg/day. Testis, epididymis and prostate weights were similar among groups. Fertility and sexual behaviour were not affected by DEHP treatment at any dose. Overall, our results show that in utero and lactational DEHP exposure reduces daily sperm production and has the potential to induce reproductive tract abnormalities (of which cryptorchidism seems to be the most sensitive in our rat strain) in male offspring rats. The lowest observed adverse effect levels (LOAELs) for these effects were 15 and 5 mg/kg/day, respectively. Therefore, the no observed adverse effect level (NOAEL) for this study can be set at 1.215 mg/kg/day.

A dose-response study following in utero and lactational exposure to di-(2-ethylhexyl)-phthalate (DEHP): non-monotonic dose-response and low dose effects on rat brain aromatase activity.

Di-(2-ethylhexyl)-phthalate (DEHP) is a commonly used plasticizer which can act as an endocrine disruptor. It has been suggested that in addition to its antiandrogenic effects, DEHP may interfere with estrogen metabolism through suppression of aromatase enzyme activity. This enzyme catalyzes the conversion of testosterone to estradiol and plays a critical role in brain sexual differentiation. We investigated the effects of two wide ranges of DEHP doses on brain aromatase activity of male and female rat offspring. Wistar rat dams were treated daily with DEHP and peanut oil (control) by gavage from gestation day 6 to lactation day 21 at doses of 0.015, 0.045, 0.135, 0.405 and 1.215mgDEHP/kgbodyweight(bw)/day (low doses) and at 5, 15, 45, 135 and 405mgDEHP/kgbw/day (high doses). Aromatase activity was determined in hypothalamic/preoptic area (HPOA) brain sections from male and female pups on postnatal days (PNDs) 1 and 22. In males on PND 1, aromatase activity was inhibited at low doses and increased at high doses resulting in a non-monotonic dose-response profile which resembled a J-shaped curve. Inhibition was statistically significant at 0.135 and 0.405mgDEHP/kg/day, while increased activity was observed at 15, 45 and 405mg/kg/day. In contrast to findings on PND 1, aromatase activity at weaning (PND 22) was more affected in females than in males. An increase in aromatase activity was observed at only one dose in males (0.405mg/kg/day) while an increase in activity was observed at all doses in the females except for 0.045 and 5mgDEHP/kg/day. Overall, these results indicate that males and females respond differently to DEHP not only in regard to the age at which effects are manifested, but also in the shape of the dose-response curve. To our knowledge, this is the first study to report biological effects of DEHP at doses that overlap with the estimated exposure of the general human population.

A dose-response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP): effects on androgenic status, developmental landmarks and testicular histology in male offspring rats.

An extensive dose-response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP) was conducted. A wide range of low and high DEHP doses were tested. Reproductive effects were evaluated on male offspring rats. Female Wistar rats were treated daily with DEHP and peanut oil by gavage from gestation day 6 to lactation day 21 at doses of 0.015, 0.045, 0.135, 0.405 and 1.215 mg DEHP/kg body weight (bw)/day (low doses) and at 5, 15, 45, 135 and 405 mg DEHP/kg bw/day (high doses). Nipple retention and reduced anogenital distance, both sensitive markers of anti-androgenic effects during development, were only seen in males exposed to the highest dose (405 mg/kg/day). Delayed preputial separation was observed in animals exposed to 15 mg DEHP/kg/day and higher doses. Histopathological examination of the testis on postnatal days (PNDs) 1 and 22 revealed changes at 135 and 405 mg DEHP/kg/day. The most prominent finding on PND 1 was the presence of bi- and multinucleated gonocytes. On PND 22 signs of reduced germ cell differentiation in seminiferous tubules of exposed animals were observed. Testis weight on PND 22 was significantly increased at 5, 15, 45 and 135 mg/kg/day, an effect that qualitatively differs from exposure to higher doses. The current results show that DEHP acts as an anti-androgen at a high dose exposure (405 mg/kg/day). However, these results also indicate that other subtle developmental effects occur at lower DEHP doses.

A dose-response study following in utero and lactational exposure to di(2-ethylhexyl)phthalate: effects on female rat reproductive development.

Phthalates, a class of chemicals used as plasticizers, are economically important due to several industrial applications. Di(2-ethylhexyl)phthalate (DEHP) is the most commonly used phthalate plasticizer, and it has been described as a potent antiandrogen in males. We performed an extensive dose-response study following developmental exposure to DEHP and evaluated the effects on female reproductive development. Two wide ranges of doses that included dose levels relevant for human exposure as well as high doses typically used in toxicological studies were tested. Female Wistar rats were treated daily with DEHP and peanut oil (vehicle control) by gavage from gestation day 6 to lactation day 22. The low doses were 0.015, 0.045, 0.135, 0.405, and 1.215 mg DEHP/kg body weight (bw)/day, and the high doses were 5, 15, 45, 135, and 405 mg DEHP/kg bw/day. At the dose levels tested, no signs of maternal toxicity were observed. A significant delay in the age at vaginal opening (approximately 2 days) at 15 mg DEHP/kg bw/day and above, as well as a trend for a delay in the age at first estrus at 135 and 405 mg DEHP/kg bw/day (approximately 2 days), was observed. Liver enlargement (characteristic of phthalate exposure in rats) was limited to the 135- and 405-mg DEHP/kg bw/day doses. Anogenital distance and nipple development were unaffected. Based on the results of delayed pubertal onset, the no observed adverse effect level for female reproductive development may be set at 5 mg DEHP/kg bw/day.

Effects of peripubertal exposure to triphenyltin on female sexual development of the rat.

Triphenyltin (TPT) belongs to the group of organotin compounds which have been shown to affect reproduction in mammals. It is used as a fungicide and antifouling agent and the main source of human exposure is via food. We studied the effects of 2 or 6 mg TPT/kg bw on female sexual development using a modification of the Rodent 20-Day Thyroid/Pubertal Female Assay. Moreover, the effect of TPT before the onset of puberty was investigated. Beginning at postnatal day (PND) 23 female Wistar rats were treated per gavage until either PND 33 or the first estrus after PND 53. A delay in the completion of vaginal opening (VO) was observed in the 6 mg TPT group, while the 2mg TPT group showed advanced VO. Significantly increased ovarian weights were observed in both treatment groups. Steroid hormone levels and ovarian aromatase activity were affected after exposure to 6 mg TPT/kg bw, while treatment with 2mg TPT/kg bw resulted in minor changes of these endpoints. We conclude that peripubertal exposure to 6 mg TPT/kg bw, and to a lesser extent to 2mg TPT/kg bw, affects female sexual development.