One-generation reproduction study of esterified propoxylated glycerol (EPG) administered in the feed to CD® (Sprague-Dawley) rats.

This one-generation study assessed the potential of esterified propoxylated glycerol (EPG) to affect reproduction and offspring development in rats. Male and female Crl:CD(SD)BR rats (30/sex/group) were exposed to EPG at 0, 0.5, 1, and 2g/kg bw/day or at 5% (w/w) in the diet prior to (13 weeks), during, and after two consecutive matings. For dams, exposure continued through gestation and lactation; F1a and F1b pups were weaned to the respective diet (for up to 91 days). No consistent treatment-related effects were observed in: body weights/gains; feed consumption; clinical observations; mating indices; survival, growth and development of litters, litter sizes, body weights, sex ratios (lower % males/litter at 1 and 2g/kg bw/day), acquisition of developmental landmarks, behavioral indices, or histology of selected organs. Lower serum vitamin D, liver vitamin A, and liver vitamin E levels were seen in some EPG-treated groups. None of the reductions were judged to be biologically significant. A/G ratio was greater among males receiving 2g/kg bw/day and 5%. In the absence of any other related effects, the biological significance of this finding is doubtful.

Developmental toxicity evaluation of esterified propoxylated glycerol (EPG) administered in the diet to New Zealand white rabbits.

The safety of a "core" version of esterified propoxylated glycerols (EPGs) was assessed in a developmental toxicity study in New Zealand white rabbits, Hra:(NZW)SPF. Four groups each of 18 inseminated female rabbits received diets ad libitum containing concentrations of 0%, 2.5%, 5%, and 10% EPG (w/w) with 6% corn oil (w/w). No treatment-related effects were observed in any maternal toxicity parameter, including maternal body weight and weight gain, feed consumption, or clinical signs of toxicity. There were no statistically significant treatment-related effects in gestational parameters, including pre- and post-implantation loss, litter size, sex ratio, fetal body weight, and crown-rump length. The incidences of fetal external, visceral, and skeletal malformations or variations were also comparable across groups. A no-observable-adverse-effect level (NOAEL) of 10% EPG (approximately 4.76 g/kg bw/day) for both maternal and developmental toxicity is proposed based on the results of this study.

Developmental toxicity study of sodium molybdate dihydrate administered in the diet to Sprague Dawley rats.

Molybdenum is an essential nutrient for humans and animals and is a constituent of several important oxidase enzymes. It is normally absorbed from the diet and to a lesser extent from drinking water and the typical human intake is around 2µg/kg bodyweight per day. No developmental toxicity studies to contemporary standards have been published and regulatory decisions have been based primarily on older studies where the nature of the test material, or the actual dose levels consumed is uncertain. In the current study the developmental toxicity of sodium molybdate dihydrate as a representative of a broad class of soluble molybdenum(VI) compounds, was given in the diet to Sprague Dawley rats in accordance with OECD Test Guideline 414. Dose levels of 0, 3, 10, 20 and 40mgMo/kgbw/day were administered from GD6 to GD20. No adverse effects were observed at any dose level on the dams, or on embryofetal survival, fetal bodyweight, or development, with no increase in malformations or variations. Significant increases in serum and tissue copper levels were observed but no toxicity related to these was observed. The NOAEL observed in this study was 40mgMo/kgbw/day, the highest dose tested.

Abbreviated assessment of bisphenol A toxicology literature.

Bisphenol A (BPA), synthesized in 1891, is produced in quantities of >2 million metric tons annually for polycarbonate plastics, epoxy resins and food contact applications. BPA can be a weak estrogen mimic, and is ubiquitous in humans (in 93% US population; in 96% US pregnant women). European/US food/drug agencies conclude that current BPA levels present no risk to the general population (some include infants/children); basic endocrine disruption (ED) researchers state that entire populations are at risk from these levels. The US Food and Drug Administration banned BPA in baby bottles in 2012 'not based on safety concerns'; the US Environmental Protection Agency and its UK counterpart concurred. Basic ED researchers report reproductive/developmental effects from perinatal BPA exposure in mice at very low doses, e.g. 2 ng/g body weight (0.002 mg/kg body weight), with non-monotonic dose-response (NMDR) curves, using few animals per group and few groups; contract research organizations, in good laboratory practice- and guideline-compliant large studies in rats and mice, report no low-dose effects or NMDR curves. The argument rages!

GLP-compliant evaluation and standardization of the peripubertal castrate male rat Hershberger assay for oral exposure of test agents.

Since oral exposure is more relevant than the sc route for human exposure to environmental substances, studies to evaluate and standardize this route in the Hershberger assay were conducted in 2001-2003. Interest in environmental androgen agonists is increasing, so the oral route of the Hershberger assay may be useful to quantify agonist activity of these substances. Castrated Sprague-Dawley rats were dosed (PND 60-69) with androgen receptor agonists and/or antagonists, terminated on PND 70, and body, liver, and accessory sex organs (ASOs) weighed. Methyltestosterone (MT) po, at 0.1-50mg/kg/day, resulted in dose-dependent increases in ASO weights at 5-50 mg/kg; 0.1 mg/kg/day was without statistically significant effect. Testosterone propionate (TP) (sc) at 0.1-1.6 mg/kg/day also resulted in dose-dependent increases in ASO weights, at all doses. Detection of putative androgen antagonists by the oral route was confirmed with dose-response curves of antagonism from flutamide (FLU) po at 1, 5, or 10 mg/kg/day, with MT at 5 or 10 mg/kg/day (po, 4h later). These results extend the OECD Hershberger assay evaluation and standardization to the oral route and identify and discuss challenges of the assay to detect (anti)androgen-active compounds.

Commentary on the role of maternal toxicity on developmental toxicity.

Maternal mammalian toxicity impacts prenatal development, with general systemic maternal toxicity, from reduced weight gain to morbidity, causative for reduced fetal weights/litter and increased fetal variations (especially skeletal)/litter, but not, in the author's opinion, for increased fetal malformations, reduced litter sizes or full litter losses. Increased fetal malformations are likely due to exposure to specific chemicals which alter specific maternal functions at critical point(s) in pregnancy, typically exaggerated effects from higher doses by drugs under development with known, desired pharmacological effects. Malformations can also be from genetic/epigenetic alterations, specific altered proteins, molecular pathways, etc. Full litter losses are triggered by the mother and are rare in rats. Information to inform maternal (and developmental) toxicity includes ovarian corpora lutea counts, uterine implantation profile, degree of litter reduction (if present), timing and extent of maternal toxicity relative to those of adverse embryofetal effects, etc. The view of maternal toxicity as confounding results in in vivo developmental toxicity studies, worldwide concerns about increased research animal usage, increasing time, labor, costs, and new software and hardware sophistication all drive the interest in development, validation, and performance of in vitro/in silico assays. These assays are fast, inexpensive, responsive to animal use concerns and amenable to mechanistic questions. The strength of these in vitro/in silico assays is considered by many to be the absence of the maternal organism/placenta. These assays inform mechanism and hazard, but NOT risk. The Environmental Protection Agency currently estimates that these new assays are approximately 70% accurate versus the whole animal tests.

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.

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.

Validation of the intact rat weanling uterotrophic assay with notes on the formulation and analysis of the positive control chemical in vehicle.

The intact female weanling version in the Organization for Economic Cooperation and Development (OECD) uterotrophic assay Test Guideline (TG) 440 is proposed as an alternative to the adult ovariectomized female version, because it does not involve surgical intervention (vs the ovariectomized version) and detects direct/indirect-acting estrogenic/anti-estrogenic substances (vs the ovariectomized version which detects only direct-acting estrogenic/anti-estrogenic substances binding to the estrogen receptor). This validation study followed OECD TG 440, with six female weanling rats (postnatal day 21) per dose group and six treatment groups. Females were weighed and dosed once daily by oral gavage for three consecutive days, with one of six doses of 17α-ethinyl estradiol in corn oil at 5 ml kg⁻¹ at 0 and 0.1-10 µg kg⁻¹ per day. On postnatal day 24, the juvenile females were euthanized by CO2 asphyxiation, weighed, livers weighed and uteri weighed wet and blotted. The presence or absence of vaginal patency was recorded. Absolute and relative (to terminal body weight) uterine wet and blotted weights and uterine luminal fluid weights were significantly increased at 3.0 and 10.0 (both P < 0.01) µg kg⁻¹ per day, and increased to ~140% of control values at 1.0 µg kg⁻¹ per day (not statistically significantly). In vivo body weights, weight changes, feed consumption, liver weights and terminal body weights were unaffected. Vaginal patency was not acquired in any female at any dose, although vaginal puckering was observed in one female at 10.0 µg kg⁻¹ per day. Therefore, this intact weanling uterotrophic assay is validated in our laboratory for use under US and European endocrine toxicity testing programs/legislation.

In honor of the Teratology Society's 50th anniversary: The role of Teratology Society members in the development and evolution of in vivo developmental toxicity test guidelines.

Members of the Teratology Society (established in 1960) were involved in the first governmental developmental and reproductive toxicity testing guidelines (1966) by FDA following the thalidomide epidemic, followed by other national and international governmental testing guidelines. The Segment II (developmental toxicity) study design, described in rodents and rabbits, has evolved with additional enhanced endpoints and better descriptions, mechanistic insights, range-finding studies, and toxico/pharmacokinetic ADME information (especially for pharmaceuticals). Society members were also involved in the development of the current screening assays and tests for endocrine disruptors (beginning in 1996) and are now involved with developing new testing guidelines (e.g., the extended one-generation protocol), and evaluating the current test guidelines and new initiatives under ILSI/HESI sponsorship. New initiatives include ToxCast from the U.S. EPA to screen, prioritize, and predict toxic chemicals by high throughput and high-content in vitro assays, bioinformation, and modeling to reduce (or eliminate) in vivo whole animal studies. Our Society and its journal have played vital roles in the scientific and regulatory accomplishments in birth defects research over the past 50 years and will continue to do so in the future. Happy 50th anniversary!

Basic exploratory research versus guideline-compliant studies used for hazard evaluation and risk assessment: bisphenol A as a case study.

BACKGROUND: Myers et al. [Environ Health Perspect 117:309-315 (2009)] argued that Good Laboratory Practices (GLPs) cannot be used as a criterion for selecting data for risk assessment, using bisphenol A (BPA) as a case study. They did not discuss the role(s) of guideline-compliant studies versus basic/exploratory research studies, and they criticized both GLPs and guideline-compliant studies and their roles in formal hazard evaluation and risk assessment. They also specifically criticized our published guideline-compliant dietary studies on BPA in rats and mice and 17beta-estradiol (E(2)) in mice. OBJECTIVES: As the study director/first author of the criticized E(2) and BPA studies, I discuss the uses of basic research versus guideline-compliant studies, how testing guidelines are developed and revised, how new end points are validated, and the role of GLPs. I also provide an overview of the BPA guideline-compliant and exploratory research animal studies and describe BPA pharmacokinetics in rats and humans. I present responses to specific criticisms by Myers et al. DISCUSSION AND CONCLUSIONS: Weight-of-evidence evaluations have consistently concluded that low-level BPA oral exposures do not adversely affect human developmental or reproductive health, and I encourage increased validation efforts for "new" end points for inclusion in guideline studies, as well as performance of robust long-term studies to follow early effects (observed in small exploratory studies) to any adverse consequences.

Two-generation reproductive toxicity study of dietary bisphenol A in CD-1 (Swiss) mice.

Dietary bisphenol A (BPA) was evaluated in a mouse two-generation study at 0, 0.018, 0.18, 1.8, 30, 300, or 3500 ppm (0, 0.003, 0.03, 0.3, 5, 50, or 600 mg BPA/kg/day, 28 per sex per group). A concurrent positive control group of dietary 17beta-estradiol (0.5 ppm; 28 per sex) confirmed the sensitivity of CD-1 mice to an endogenous estrogen. There were no BPA-related effects on adult mating, fertility or gestational indices, ovarian primordial follicle counts, estrous cyclicity, precoital interval, offspring sex ratios or postnatal survival, sperm parameters or reproductive organ weights or histopathology (including the testes and prostate). Adult systemic effects: at 300 ppm, only centrilobular hepatocyte hypertrophy; at 3500 ppm, reduced body weight, increased kidney and liver weights, centrilobular hepatocyte hypertrophy, and renal nephropathy in males. At 3500 ppm, BPA also reduced F1/F2 weanling body weight, reduced weanling spleen and testes weights (with seminiferous tubule hypoplasia), slightly delayed preputial separation (PPS), and apparently increased the incidence of treatment-related, undescended testes only in weanlings, which did not result in adverse effects on adult reproductive structures or functions; this last finding is considered a developmental delay in the normal process of testes descent. It is likely that these transient effects were secondary to (and caused by) systemic toxicity. Gestational length was increased by 0.3 days in F1/F2 generations; the toxicological significance, if any, of this marginal difference is unknown. At lower doses (0.018-30 ppm), there were no treatment-related effects and no evidence of nonmonotonic dose-response curves for any parameter. The systemic no observable effect level (NOEL) was 30 ppm BPA (approximately 5 mg/kg/day); the reproductive/developmental NOEL was 300 ppm (approximately 50 mg/kg/day). Therefore, BPA is not considered a selective reproductive or developmental toxicant in mice.

Environmental factors and puberty timing: expert panel research needs.

Serono Symposia International convened an expert panel to review the impact of environmental influences on the regulation of pubertal onset and progression while identifying critical data gaps and future research priorities. An expert panel reviewed the literature on endocrine-disrupting chemicals, body size, and puberty. The panel concluded that available experimental animal and human data support a possible role of endocrine-disrupting chemicals and body size in relation to alterations in pubertal onset and progression in boys and girls. Critical data gaps prioritized for future research initiatives include (1) etiologic research that focus on environmentally relevant levels of endocrine-disrupting chemicals and body size in relation to normal puberty as well as its variants, (2) exposure assessment of relevant endocrine-disrupting chemicals during critical windows of human development, and (3) basic research to identify the primary signal(s) for the onset of gonadotropin-releasing hormone-dependent/central puberty and gonadotropin-releasing hormone-independent/peripheral puberty. Prospective studies of couples who are planning pregnancies or pregnant women are needed to capture the continuum of exposures at critical windows while assessing a spectrum of pubertal markers as outcomes. Coupled with comparative species studies, such research may provide insight regarding the causal ordering of events that underlie pubertal onset and progression and their role in the pathway of adult-onset disease.

One-generation reproductive toxicity study of dietary 17beta-estradiol (E2; CAS No. 50-28-2) in CD-1 (Swiss) mice.

There is no information on reproductive/developmental effects in mice from dietary estrogen. Therefore, 10 adult CD-1 mice/sex/group were administered dietary 17beta-estradiol (E2) at 0, 0.005, 0.05, 0.5, 2.5, 5, 10, and 50 ppm for 2-week prebreed, mating, gestation, lactation. F1 weanlings (3/sex/litter) were necropsied and 2/sex/litter were retained, with exposure, until vaginal patency (VP) or preputial separation (PPS) and then necropsied. Results included complete infertility at 2.5-50 ppm with normal mating indices. At 0.5 ppm (and above), F0 adult female uterus plus cervix plus vagina weights (UCVW) were increased. At 0.5 ppm: prolonged gestational length; increased F1 stillbirth index; reduced live birth index and litter size; decreased testes and epididymides weights at weaning; unaffected AGD on pnd 0 and 21; delayed PPS; increased undescended testes; unaffected prostate weight; accelerated VP; enlarged vaginas; fluid-filled uteri. At 0.05 ppm: no F0 reproductive effects, increased F1 weanling UCVW; delayed PPS. The NOEL was 0.005 ppm ( approximately 1 microg/kg/day).

Two-generation reproductive toxicity evaluation of dietary 17beta-estradiol (E2; CAS No. 50-28-2) in CD-1 (Swiss) mice.

No information exists on reproductive/developmental effects in mice exposed to dietary 17beta-estradiol (E2) over multiple generations. Therefore, under OECD Test Guideline 416 with enhancements, CD-1 mice (F0 generation, 25 mice/sex/group) were exposed to dietary E2 at 0, 0.001, 0.005, 0.05, 0.15, or 0.5 ppm ( approximately 0, 0.2, 1, 10, 30, or 100 mug E2/kg body weight/day) for 8 weeks prebreed, 2 weeks mating, approximately 3 weeks gestation, and 3 weeks lactation. At weaning, selected F1 offspring (F1 parents; 25/sex/group) and extra retained F1 males (one per litter) were exposed to the same dietary concentrations and durations as the F0 generation; study termination occurred at F2 weaning; F1/F2 weanlings (up to three per sex per litter) were necropsied with organs weighed. At 0.5 ppm, effects were increased F1/F2 perinatal loss, prolonged F0/F1 gestational length, reduced numbers of F2 (but not F1) litters/group, reduced F1/F2 litter sizes, accelerated vaginal patency (VP) and delayed preputial separation (PPS), increased uterus + cervix + vagina weights (UCVW) in F0/F1 adults and F1/F2 weanlings, and decreased testes and epididymides weights (TEW) in F1/F2 weanlings. At 0.15 ppm, effects were increased UCVW in F0/F1 adults and F1/F2 weanlings, accelerated VP, delayed PPS, and reduced TEW in F1/F2 weanlings. At 0.05 ppm, UCVW were increased in F1/F2 weanlings, and PPS was delayed only in extra retained F1 males. There were no biologically significant or treatment-related effects on F0/F1 parental body weights, feed consumption, or clinical observations, or on F0/F1 estrous cyclicity, F0/F1 andrology, or F1/F2 anogenital distance at any dose. The no observable effect level was 0.005 ppm E2 ( approximately 1 mug/kg/day). Therefore, the mouse model is sensitive to E2 by oral administration, with effects on reproductive development at doses of 10- 100 mug/kg/day.

Identification and interpretation of developmental neurotoxicity effects: a report from the ILSI Research Foundation/Risk Science Institute expert working group on neurodevelopmental endpoints.

The reliable detection, measurement, and interpretation of treatment-related developmental neurotoxicity (DNT) effects depend on appropriate study design and execution, using scientifically established methodologies, with appropriate controls to minimize confounding factors. Appropriate statistical approaches should be optimized for the specific endpoints in advance, analyzing effects across time and functional domains as far as possible. If available, biomarkers of exposure are useful to assess the bioavailability of toxicants to the dam and offspring in utero and after birth. Finally, "weight of evidence" principles are used to aid assessment of the biological significance of differences from concurrent controls. These effects should be interpreted in light of available information from historical controls, positive controls, maternal and offspring systemic toxicity, and other relevant toxicological data. This review provides a framework for the integration of all these types of information in the interpretation of DNT studies.

Altered axial skeletal development.

The axial skeleton is routinely examined in standard developmental toxicity bioassays and has proven to be sensitive to a wide variety of chemical agents. Dysmorphogenesis in the skull, vertebral column and ribs has been described in both human populations and in laboratory animals used to assess potential adverse developmental effects. This article emphasizes vertebrae and rib anomalies both spontaneous and agent induced. Topics discussed include the morphology of the more common effects; incidences in both human and experimental animal populations; the types of anomalies induced in the axial skeleton by methanol, boric acid, valproic acid and others; the postnatal persistence of common skeletal anomalies; and the genetic control of the development of the axial skeleton. Tables of the spontaneous incidence of axial anomalies in both humans and animals are provided.

General background on the hypothalamic-pituitary-thyroid (HPT) axis.

This article reviews the thyroid system, mainly from a mammalian standpoint. However, the thyroid system is highly conserved among vertebrate species, so the general information on thyroid hormone production and feedback through the hypothalamic-pituitary-thyroid (HPT) axis should be considered for all vertebrates, while species-specific differences are highlighted in the individual articles. This background article begins by outlining the HPT axis with its components and functions. For example, it describes the thyroid gland, its structure and development, how thyroid hormones are synthesized and regulated, the role of iodine in thyroid hormone synthesis, and finally how the thyroid hormones are released from the thyroid gland. It then progresses to detail areas within the thyroid system where disruption could occur or is already known to occur. It describes how thyroid hormone is transported in the serum and into the tissues on a cellular level, and how thyroid hormone is metabolized. There is an in-depth description of the alpha and beta thyroid hormone receptors and their functions, including how they are regulated, and what has been learned from the receptor knockout mouse models. The nongenomic actions of thyroid hormone are also described, such as in glucose uptake, mitochondrial effects, and its role in actin polymerization and vesicular recycling. The article discusses the concept of compensation within the HPT axis and how this fits into the paradigms that exist in thyroid toxicology/endocrinology. There is a section on thyroid hormone and its role in mammalian development: specifically, how it affects brain development when there is disruption to the maternal, the fetal, the newborn (congenital), or the infant thyroid system. Thyroid function during pregnancy is critical to normal development of the fetus, and several spontaneous mutant mouse lines are described that provide research tools to understand the mechanisms of thyroid hormone during mammalian brain development. Overall this article provides a basic understanding of the thyroid system and its components. The complexity of the thyroid system is clearly demonstrated, as are new areas of research on thyroid hormone physiology and thyroid hormone action developing within the field of thyroid endocrinology. This review provides the background necessary to review the current assays and endpoints described in the following articles for rodents, fishes, amphibians, and birds.