Genomic and Hormonal Biomarkers of Phthalate-Induced Male Rat Reproductive Developmental Toxicity Part II: A Targeted RT-qPCR Array Approach That Defines a Unique Adverse Outcome Pathway.

Previously, we demonstrated that exposure to some diortho-phthalate esters during sexual differentiation disrupts male reproductive development by reducing fetal rat testis testosterone production (T Prod) and gene expression in a dose-related manner. The objectives of the current project were to expand the number of test compounds that might reduce fetal T Prod, including phthalates, phthalate alternatives, pesticides, and drugs, and to compare reductions in T Prod with altered testis mRNA expression. We found that PEs that disrupt T Prod also reduced expression of a unique "cluster" of mRNAs for about 35 genes related to sterol transport, testosterone and insulin-like hormone 3 hormone syntheses, and lipoprotein signaling and cholesterol synthesis. However, phthalates had little or no effect on mRNA expression of genes in peroxisome proliferator-activated receptor (PPAR) pathways in the fetal liver, whereas the 3 PPAR agonists induced the expression of mRNA for multiple fetal liver PPAR pathway genes without reducing testis T Prod. In summary, phthalates that disrupt T Prod act via a novel adverse outcome pathway including down regulation of mRNA for genes involved in fetal endocrine function and cholesterol synthesis and metabolism. This profile was not displayed by PEs that did not reduce T Prod, PPAR agonists or the other chemicals. Reductions in fetal testis gene expression and T Prod in utero can be used to establish relative potency factors that can be used quantitatively to predict the doses of individual PEs and mixtures of phthalates that produce adverse reproductive tract effects in male offspring.

Multigenerational reproductive assessment of 4-methylimidazole administered in the diet to Hsd:Sprague Dawley SD rats.

The general population, including children and adolescents, is exposed to 4-methylimidazole (4-MI) in the diet. 4-MI is a by-product of caramel color manufacturing. It has been previously classified as a possible human carcinogen and displays potential reproductive toxicity. A follow up assessment of reproductive toxicity was conducted in rats utilizing the reproductive assessment by continuous breeding paradigm, in which multiple generations were exposed to 4-MI in diet at 750, 2500, and 5000 ppm. 4-MI exposure was associated with delays in preputial separation and vaginal opening, impairment in reproductive performance, and concomitant histopathological findings in the prostate, testis, and epididymis at 2500 and 5000 ppm. The Lowest Observed Adverse Effect Level for reproductive (based on prostate atrophy) and developmental toxicity (based on delays in preputial separation and vaginal opening) was 750 ppm, equivalent to approximately 50-60 mg/kg bw/day.

In utero exposure to simvastatin reduces postnatal survival and permanently alters reproductive tract development in the Crl:CD(SD) male rat.

We showed previously that in utero exposure to the cholesterol-lowering drug simvastatin (SMV) during sex differentiation lowers fetal lipids and testicular testosterone production (T Prod) in Hsd:SD rats. Here, the effects of SMV on fetal lipids and T Prod in Crl:CD(SD) rats were correlated with postnatal alterations in F1 males. The current study was conducted in two parts: 1) a prenatal assessment to confirm and further characterize the dose response relationship among previously reported alterations of SMV on fetal T Prod and the fetal lipid profile and 2) a postnatal assessment to determine the effects of SMV exposure during the periods of major organogenesis and/or sexual differentiation on F1 offspring growth and development. We hypothesized that SMV would have adverse effects on postnatal development and sexual differentiation as a consequence of the disruptions of fetal lipid levels and testicular T Prod since fetal cholesterol is essential for normal intrauterine growth and development and steroid synthesis. In the prenatal assessment, SMV was administered orally at 0, 15.6, 31.25, 62.5, 80, 90, 100, and 110 mg SMV/kg/d from GD 14-18, the period that cover the critical window of sex differentiation in the male rat fetus. T Prod was maximally reduced by ~40% at 62.5 mg/kg/d, and higher doses induced overt maternal and toxicity. In the postnatal assessment, SMV was administered at 0, 15.6, 31.25, and 62.5 mg/kg/d from GD 8-18 to determine if it altered postnatal development. We found that exposure during this time frame to 62.5 mg SMV/kg/d reduced pup viability by 92%, decreased neonatal anogenital distance, and altered testis histology and morphology in 17% of the F1 males. In another group, SMV was administered only during the masculinizing window (GD14-18) at 62.5 mg/kg/d to determine if male rat sexual differentiation and postnatal reproductive development were altered. SMV-exposed F1 males displayed female-like areolae/nipples, delayed puberty, and reduced seminal vesicle and levator ani-bulbocavernosus weights. Together, these results demonstrate that in utero exposure to SMV reduces offspring viability and permanently disrupts reproductive tract development in the male offspring. While the effects of high dose, short term in utero exposure to SMV in the adult male are likely androgen-dependent and consistent with the 40% reduction in T Prod in the fetal testes, long-term, lower dose administration induced some effects that were likely not mediated by decreased T Prod.

A Conflicted Tale of Two Novel AR Antagonists In Vitro and In Vivo: Pyrifluquinazon Versus Bisphenol C.

Chemicals that disrupt androgen receptor (AR) function in utero induce a cascade of adverse effects in male rats including reduced anogenital distance, retained nipples, and reproductive tract malformations. The objective of this study was to compare the in vitro and in utero activities of two novel AR antagonists, bisphenol C (BPC) and pyrifluquinazon (PFQ). In vitro, BPC was as potent an AR antagonist as hydroxyflutamide. Furthermore, BPC inhibited fetal testis testosterone production and testis gene expression ex vivo. However, when BPC was administered at 100 and 200 mg/kg/d in utero, the reproductive tract of the male offspring was minimally affected. None of the males displayed reproductive malformations. For comparison, in utero administration of flutamide has been shown to induce malformations in 100% of males at 6 mg/kg/d. In vitro, PFQ was several orders of magnitude less potent than BPC, vinclozolin, or procymidone. However, in utero administration of 12.5, 25, 50, and 100 mg PFQ/kg/d on GD 14-18 induced antiandrogenic effects at all dosage levels and 91% of the males displayed reproductive malformation in the high dose group. Overall, BPC was ∼380-fold more potent than PFQ in vitro, whereas PFQ was far more potent than BPC in utero. Incorporating toxicokinetic and toxicodynamic data into in vitro to in vivo extrapolations would reduce the discordance between the in vitro and in utero effects of PFQ and BPC and combining in vitro results with a short-term Hershberger assay would reduce the uncertainty in predicting the in utero effects of antiandrogenic chemicals.

Screening for Developmental Neurotoxicity at the National Toxicology Program: The Future Is Here.

The National Toxicology Program (NTP) receives requests to evaluate chemicals with potential to cause adverse health effects, including developmental neurotoxicity (DNT). Some recent requests have included classes of chemicals such as flame retardants, polycyclic aromatic compounds, perfluoroalkyl substances, and bisphenol A analogs with approximately 20-50 compounds per class, many of which include commercial mixtures. However, all the compounds within a class cannot be tested using traditional DNT animal testing guideline studies due to resource and time limitations. Hence, a rapid and biologically relevant screening approach is required to prioritize compounds for further in vivo testing. Because neurodevelopment is a complex process involving multiple distinct cellular processes, one assay will unlikely address the complexity. Hence, the NTP sought to characterize a battery of in vitro and alternative animal assays to quantify chemical effects on a variety of neurodevelopmental processes. A culmination of this effort resulted in a NTP-hosted collaborative project with approximately 40 participants spanning across domains of academia, industry, government, and regulatory agencies; collaborators presented data on cell-based assays and alternative animal models that was generated using a targeted set of compounds provided by the NTP. The NTP analyzed the assay results using benchmark concentration (BMC) modeling to be able to compare results across the divergent assays. The results were shared with the contributing researchers on a private web application during the workshop, and are now publicly available. This article highlights the overview and goals of the project, and describes the NTP's approach in creating the chemical library, development of NTPs data analysis strategy, and the structure of the web application. Finally, we discuss key issues with emphasis on the utility of this approach, and knowledge gaps that need to be addressed for its use in regulatory decision making.

Finding the bad actor: Challenges in identifying toxic constituents in botanical dietary supplements.

Botanical-derived dietary supplements have widespread use in the general population. The complex and variable nature of botanical ingredients and reports of adverse responses have led to concern for negative human health impacts following consumption of these products. Toxicity testing of the vast number of available products, formulations, and combinations is not feasible due to the time and resource intensive nature of comprehensive testing. Methods are needed to assess the safety of a large number of products via more efficient frameworks. Identification of toxicologically-active constituents is one approach being used, with many advantages toward product regulation. Bioassay-guided fractionation (BGF) is the leading approach used to identify biologically-active constituents. Most BGF studies with botanicals focus on identifying pharmacologically-active constituents for drug discovery or botanical efficacy research. Here, we explore BGF in a toxicological context, drawing from both efficacy and poisonous plant research. Limitations of BGF, including loss of mixture activity and bias toward abundant constituents, and recent advancements in the field (e.g., biochemometrics) are discussed from a toxicological perspective. Identification of active constituents will allow better monitoring of market products for known toxicologically-active constituents, as well as surveying human exposure, two important steps to ensuring the safety of botanical dietary supplements.

Transcript profiling in the testes and prostates of postnatal day 30 Sprague-Dawley rats exposed prenatally and lactationally to 2-hydroxy-4-methoxybenzophenone.

2-hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet light-absorbing compound that is used in sunscreens, cosmetics and plastics. HMB has been reported to have weak estrogenic activity by in vivo and in vitro studies, making it a chemical with potential reproductive concern. To explore if prenatal and lactational HMB exposure alters gene expression profiles of the developing reproductive organs, we performed microarray analysis using the prostate and testis of postnatal day (PND) 30 male Sprague-Dawley rats offspring exposed to 0, 3000, or 30,000 ppm of HMB from gestational day 6 through PND 21. Gene expression profiles of the prostate and testis were differentially affected by HMB dose with significant alterations observed at the 30,000 ppm HMB group. Tissue-specific gene expression was also identified. These genes, whose expression was altered by HMB exposure, may be considered as candidate biomarker(s) for testicular or prostatic toxicity; however, further studies are necessary to explore this potential.

Uterine Paramesonephric Cysts in Sprague-Dawley Rats from National Toxicology Program Studies.

Congenital uterine wall cysts arising from paramesonephric (Müllerian) and mesonephric (Wolffian) ducts are typically incidental findings in most species. We used immunohistochemistry to characterize and determine the origin of uterine cysts in Sprague-Dawley (SD) rats from multigeneration studies conducted by the National Toxicology Program. Subserosal uterine cysts were observed in 20 of the 2,400 SD rats evaluated in five studies, and 10 cysts were characterized for this study. Single cysts were unilocular, fluid-filled, and occurred throughout the uterus. Microscopically, all cysts had a well-developed smooth muscle wall, lined by flattened to cuboidal, sometimes ciliated, epithelium that stained intensely positive for cytokeratin 18 and paired box protein 8 (PAX8). Most cyst epithelia displayed weak to moderate positivity for progesterone receptor (PR) and/or estrogen receptor α (ER-α), as well as were negative for GATA binding protein 3 (GATA3). Cyst lumens contained basophilic flocculent material. The cysts appeared to be developmental anomalies arising from paramesonephric tissue based on positive PAX8 and ER-α and/or PR staining. Additionally, 70% of the cysts lacked GATA3 expression. Taken together, the subserosal uterine cysts observed in adult rats in these studies most likely arose from the paramesonephric duct.

Influence of Study Design on Developmental and Reproductive Toxicology Study Outcomes.

Regulatory studies of developmental and reproductive toxicity (DART) studies have remained largely unchanged for decades, with exposures occurring at various phases of the reproductive cycle and toxicity evaluations at different ages/times depending on the study purpose. The National Toxicology Program has conducted studies examining the power to detect adverse effects where there is a prenatal exposure, but evaluations occur postnatally. In these studies, examination is required of only 1 male and female pup from each litter beyond weaning. This provides poor resolving power to detect rare events (e.g., reproductive tract malformations). If an adverse effect is detected, there is little confidence in the shape of the dose-response curve (and the Benchmark Dose or No Observed Adverse Effect Level [NOAEL]). We have developed a new protocol to evaluate DART, the modified one generation study, with exposure commencing with pregnant animals and retention of 4 males and females from each litter beyond weaning to improve statistical power. These animals can be allocated to specific cohorts that examine subchronic toxicity, teratology, littering, and neurobehavioral toxicity in the same study. This approach also results in a reduction in animal numbers used, compared with individual stand-alone studies, and offers increased numbers of end points evaluated compared with recent Organization for Economic Cooperation and Development proposals.

Establishing the "Biological Relevance" of Dipentyl Phthalate Reductions in Fetal Rat Testosterone Production and Plasma and Testis Testosterone Levels.

Phthalate esters (PEs) constitute a large class of compounds that are used for many consumer product applications. Many of the C2-C7 di-ortho PEs reduce fetal testicular hormone and gene expression levels in rats resulting in adverse effects seen later in life but it appears that relatively large reductions in fetal testosterone (T) levels and testis gene expression may be required to adversely affect reproductive development (Hannas, B. R., Lambright, C. S., Furr, J., Evans, N., Foster, P. M., Gray, E. L., and Wilson, V. S. (2012). Genomic biomarkers of phthalate-induced male reproductive developmental toxicity: a targeted RT-PCR array approach for defining relative potency. Toxicol. Sci. 125, 544-557). The objectives of this study were (1) to model the relationships between changes in fetal male rat plasma testosterone (PT), T levels in the testis (TT), T production (PROD), and testis gene expression with the reproductive malformation rates, and (2) to quantify the "biologically relevant reductions" (BRRs) in fetal T necessary to induce adverse effects in the offspring. In the fetal experiment, Harlan Sprague-Dawley rats were dosed with dipentyl phthalate (DPeP) at 0, 11, 33, 100, and 300 mg/kg/day from gestational days (GD) 14-18 and fetal testicular T, PT levels, and T Prod and gene expression were assessed on GD 18. In the postnatal experiment, rats were dosed with DPeP from GD 8-18 and reproductive development was monitored through adulthood. The dose-response curves for TT levels (ED(50) = 53 mg/kg) and T PROD (ED(50) = 45 mg/kg) were similar, whereas PT was reduced at ED50 = 19 mg/kg. When the reductions in TPROD and Insl3 mRNA were compared with the postnatal effects of in utero DPeP, dose-related reproductive alterations were noted when T PROD and Insl3 mRNA were reduced by >45% and 42%, respectively. The determination of BRR levels may enable risk assessors to utilize fetal endocrine data to help establish points of departure for quantitative risk assessments.

Effects of maternal and lactational exposure to 2-hydroxy-4-methoxybenzone on development and reproductive organs in male and female rat offspring.

BACKGROUND: 2-Hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet (UV) absorbing compound used in many cosmetic products as a UV-protecting agent and in plastics for preventing UV-induced photodecomposition. HMB has been detected in over 95% of randomly collected human urine samples from adults and from premature infants, and it may have estrogenic potential. METHODS: To determine the effects of maternal and lactational exposure to HMB on development and reproductive organs of offspring, time-mated female Harlan Sprague-Dawley rats were dosed with 0, 1000, 3000, 10,000, 25,000, or 50,000 ppm HMB (seven to eight per group) added to chow from gestation day 6 until weaning on postnatal day (PND) 23. RESULTS AND CONCLUSION: Exposure to HMB was associated with reduced body and organ weights in female and male offspring. No significant differences were observed in the number of implantation sites/litter, mean resorptions/litter, % litters with resorptions, number and weights of live fetuses, or sex ratios between the control and HMB dose groups. Normalized anogenital distance in male pups at PND 23 was decreased in the highest dose group. Spermatocyte development was impaired in testes of male offspring in the highest dose group. In females, follicular development was delayed in the highest dose group. However, by evaluating levels of the compound in rat serum, the doses at which adverse events occurred are much higher than usual human exposure levels. Thus, exposure to less than 10,000 ppm HMB does not appear to be associated with adverse effects on the reproductive system in rats.

Simvastatin and dipentyl phthalate lower ex vivo testicular testosterone production and exhibit additive effects on testicular testosterone and gene expression via distinct mechanistic pathways in the fetal rat.

Sex differentiation of the male reproductive tract in mammals is driven, in part, by fetal androgen production. In utero, some phthalate esters (PEs) alter fetal Leydig cell differentiation, reducing the expression of several genes associated with steroid synthesis/transport, and consequently, lowering fetal androgen and Insl3 hormone levels. Simvastatin (SMV) is a cholesterol-lowering drug that directly inhibits HMG-CoA reductase. SMV may also disrupt steroid biosynthesis, but through a different mode of action (MOA) than the PEs. As cholesterol is a precursor of steroid hormone biosynthesis, we hypothesized that in utero exposure to SMV during the critical period of sex differentiation would lower fetal testicular testosterone (T) production without affecting genes involved in cholesterol and androgen synthesis and transport. Secondly, we hypothesized that a mixture of SMV and a PE, which may have different MOAs, would reduce testosterone levels in an additive manner. Pregnant Sprague Dawley rats were dosed orally with SMV, dipentyl phthalate (DPeP), or SMV plus DPeP from gestational days 14-18, and fetuses were evaluated on GD18. On GD18, SMV lowered fetal T production and serum triglycerides, low density lipoprotein, high density lipoprotein, and total cholesterol levels, and downregulated two genes in the fetal testis that were different from those altered by PEs. When SMV and DPeP were administered as a mixture, fetal T production was significantly reduced in an additive manner, thus demonstrating that a mixture of chemicals can induce additive effects on fetal T production even though they display different MOAs.

A short-term in vivo screen using fetal testosterone production, a key event in the phthalate adverse outcome pathway, to predict disruption of sexual differentiation.

This study was designed to develop and validate a short-term in vivo protocol termed the Fetal Phthalate Screen (FPS) to detect phthalate esters (PEs) and other chemicals that disrupt fetal testosterone synthesis and testis gene expression in rats. We propose that the FPS can be used to screen chemicals that produce adverse developmental outcomes via disruption of the androgen synthesis pathway more rapidly and efficiently, and with fewer animals than a postnatal one-generation study. Pregnant rats were dosed from gestational day (GD) 14 to 18 at one dose level with one of 27 chemicals including PEs, PE alternatives, pesticides known to inhibit steroidogenesis, an estrogen and a potent PPARα agonist and ex vivo testis testosterone production (T Prod) was measured on GD 18. We also included some chemicals with "unknown" activity including DMEP, DHeP, DHEH, DPHCH, DAP, TOTM, tetrabromo-diethyl hexyl phthalate (BrDEHP), and a relatively potent environmental estrogen BPAF. Dose-response studies also were conducted with this protocol with 11 of the above chemicals to determine their relative potencies. CD-1 mice also were exposed to varying dose levels of DPeP from GD 13 to 17 to determine if DPeP reduced T Prod in this species since there is a discrepancy among the results of in utero studies of PEs in mice. Compared to the known male reproductive effects of the PEs in rats the FPS correctly identified all known "positives" and "negatives" tested. Seven of eight "unknowns" tested were "negatives", they did not reduce T Prod, whereas DAP produced an "equivocal" response. Finally, a dose-response study with DPeP in CD-1 mice revealed that fetal T Prod can be inhibited by exposure to a PE in utero in this species, but at a higher dose level than required in rats.Key words. Phthalate Syndrome, Fetal endocrine biomarkers, Phthalate adverse outcome pathway, testosterone production, fetal rat testis.

Regulatory Forum opinion piece: New testing paradigms for reproductive and developmental toxicity--the NTP modified one generation study and OECD 443.

The National Toxicology Program (NTP) has developed a new flexible study design, termed the modified one generation (MOG) reproduction study. The MOG study will encompass measurements of developmental and reproductive toxicity parameters as well as enable the setting of appropriate dose levels for a cancer bioassay through evaluation of target organ toxicity that is based on test article exposure that starts during gestation. This study design is compared and contrasted with the new Organization for Economic Co-operation and Development (OECD) 443 test guideline, the extended one generation reproduction study. The MOG study has a number of advantages, with a focus on F 1 animals, the generation of adequately powered, robust data sets that include both pre and postnatal developmental toxicity information, and the measurement of effects on reproductive structure and function in the same animals. This new study design does not employ the use of internal triggers in the design structure for the use of animals already on test and is also consistent with the principles of the 3R's.

In vitro testicular toxicity models: opportunities for advancement via biomedical engineering techniques.

To address the pressing need for better in vitro testicular toxicity models, a workshop sponsored by the International Life Sciences Institute (ILSI), the Health and Environmental Science Institute (HESI), and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT), was held at the Mt. Washington Conference Center in Baltimore, MD, USA on October 26-27, 2011. At this workshop, experts in testis physiology, toxicology, and tissue engineering discussed approaches for creating improved in vitro environments that would be more conducive to maintaining spermatogenesis and steroidogenesis and could provide more predictive models for testicular toxicity testing. This workshop report is intended to provide scientists with a broad overview of relevant testicular toxicity literature and to suggest opportunities where bioengineering principles and techniques could be used to build improved in vitro testicular models for safety evaluation. Tissue engineering techniques could, conceivably, be immediately implemented to improve existing models. However, it is likely that in vitro testis models that use single or multiple cell types will be needed to address such endpoints as accurate prediction of chemically induced testicular toxicity in humans, elucidation of mechanisms of toxicity, and identification of possible biomarkers of testicular toxicity.

An ethanolic extract of black cohosh causes hematological changes but not estrogenic effects in female rodents.

Black cohosh rhizome (Actaea racemosa) is used as a remedy for pain and gynecological ailments; modern preparations are commonly sold as ethanolic extracts available as dietary supplements. Black cohosh was nominated to the National Toxicology Program (NTP) for toxicity testing due to its widespread use and lack of safety data. Several commercially available black cohosh extracts (BCE) were characterized by the NTP, and one with chemical composition closest to formulations available to consumers was used for all studies. Female B6C3F1/N mice and Wistar Han rats were given 0, 15 (rats only), 62.5 (mice only), 125, 250, 500, or 1000 mg/kg/day BCE by gavage for 90 days starting at weaning. BCE induced dose-dependent hematological changes consistent with a non-regenerative macrocytic anemia and increased frequencies of peripheral micronucleated red blood cells (RBC) in both species. Effects were more severe in mice, which had decreased RBC counts in all treatment groups and increased micronucleated RBC at doses above 125 mg/kg. Dose-dependent thymus and liver toxicity was observed in rats but not mice. No biologically significant effects were observed in other organs. Puberty was delayed 2.9 days at the highest treatment dose in rats; a similar magnitude delay in mice occurred in the 125 and 250 mg/kg groups but not at the higher doses. An additional uterotrophic assay conducted in mice exposed for 3 days to 0.001, 0.01, 0.1, 1, 10, 100 and 500 mg/kg found no estrogenic or anti-estrogenic activity. These are the first studies to observe adverse effects of BCE in rodents.

Genomic biomarkers of phthalate-induced male reproductive developmental toxicity: a targeted RT-PCR array approach for defining relative potency.

Male rat fetuses exposed to certain phthalate esters (PEs) during sexual differentiation display reproductive tract malformations due to reductions in testosterone (T) production and the expression of steroidogenesis- and INSL3-related genes. In the current study, we used a 96-well real-time PCR array containing key target genes representing sexual determination and differentiation, steroidogenesis, gubernaculum development, and androgen signaling pathways to rank the relative potency of several PEs. We executed dose-response studies with diisobutyl (DIBP), dipentyl (DPeP), dihexyl (DHP), diheptyl (DHeP), diisononyl (DINP), or diisodecyl phthalate (DIDP) and serial dilutions of a mixture of nine phthalates. All phthalates, with the exception of DIDP, reduced fetal testicular T production. Several genes involved in cholesterol transport, androgen synthesis, and Insl3 also were downregulated in a dose-responsive manner by DIBP, DPeP, DHP, DHeP, DINP, and the 9-PE mixture. Despite speculation of peroxisome proliferator activated receptor (PPAR) involvement in the effects of PEs on the fetal testis, no PPAR-related genes were affected in the fetal testes by exposure to any of the tested PEs. Furthermore, the potent PPARα agonist, Wy-14,643, did not reduce fetal testicular T production following gestational day 14-18 exposure, suggesting that the antiandrogenic activity of PEs is not PPARα mediated. The overall sensitivity of the fetal endpoints (gene expression or T production) for the six phthalates from most to least was Cyp11b1 > Star = Scarb1 > Cyp17a1 = T production > Cyp11a1 = Hsd3b = Insl3 > Cyp11b2. The overall potency of the individual phthalates was DPeP > DHP > DIBP ≥ DHeP > DINP. Finally, the observed mixture interaction was adequately modeled by the dose-addition model for most of the affected genes. Together, these data advance our understanding of the collective reproductive toxicity of the PE compounds.

Critical evaluation of current developmental toxicity testing strategies: a case of babies and their bathwater.

This review is the second in a series of four papers emanating from a workshop entitled "Developmental Toxicology-New Directions," which was sponsored by the ILSI Health and Environmental Sciences Institute's (HESI) Developmental and Reproductive Toxicology Technical Committee. The present review analyzes the strengths and weaknesses of current developmental safety testing approaches in an effort to identify those strengths that should be retained in the future versus the weaknesses that should be eliminated. Workshop participants considered the following to be key strengths of current testing approaches: the integrated biology of pregnant animal models including pharmacokinetic and pharmacodynamic processes, the ability to detect low incidence malformations as well as maternally mediated toxicity, and the long history of use coupled with extensive historical data. A number of weaknesses were related to the resource-intensive nature of developmental toxicity testing (e.g., large number of animals, high costs, low throughput, the inability to keep pace with the demand for more toxicity data). Other weaknesses included the use of very high dose levels that often far exceed human exposure levels, the confounding influence of maternal toxicity, sparse understanding of basic developmental mechanisms and genetics of standard animal models relative to mouse or lower organisms, difficulties interpreting low incidence findings, and issues surrounding the interpretation of minor skeletal variations. An appreciation of these strengths and weaknesses is critical for the design of new approaches to developmental toxicity testing in the 21st century.

Toxicity and carcinogenicity of androstenedione in F344/N rats and B6C3F1 mice.

Androstenedione was marketed as a dietary supplement to increase muscle mass during training. Due to concern over long-term use, the NTP evaluated the subchronic and chronic toxicity and carcinogenicity of androstenedione in male and female F344/N rats and B6C3F1 mice. In subchronic studies, dose limiting effects were not observed. A chronic (2-year) exposure by gavage at 10, 20, or 50 mg/kg in rats and male mice, and 2, 10, or 50 mg/kg in female mice (50 mg/kg, maximum feasible dose) was conducted. Increased incidences of lung alveolar/bronchiolar adenoma and carcinoma occurred in the 20 mg/kg male rats and increases in mononuclear cell leukemia occurred in the 20 and 50 mg/kg female rats, which may have been related to androstenedione administration. In male and female mice, androstenedione was carcinogenic based upon a significant increase in hepatocellular tumors. A marginal increase in pancreatic islet cell adenomas in male (50 mg/kg) and female (2, 10, 50 mg/kg) mice was considered to be related to androstenedione administration. Interestingly, incidences of male rat Leydig cell adenomas and female rat mammary gland fibroadenomas decreased. In conclusion, androstenedione was determined to be carcinogenic in male and female mice, and may have been carcinogenic in rats.