Dinesh Stanislaus

Effects of Food Restriction on Testis and Accessory Sex Glands in Maturing Rats

Reduced food consumption and associated lower body weights may occur in subacute toxicity studies. The short-term effects of food restriction (FR) on body and reproductive organ weights, hormones, and testis histology were assessed in Sprague-Dawley rats fed 20% to 36% less (21 g feed/day) than rats fed ad libitum (AL) starting at six, eight, ten, or twelve weeks of age for two or six weeks. Body weight and relative seminal vesicle, ventral prostate, and/or epididymis weights were reduced in rats FR for two or six weeks. Degeneration of stage VII pachytene spermatocytes was seen in rats FR for six weeks when initiated at eight, ten, and twelve weeks of age. Plasma testosterone concentrations were lower in rats FR at ages six to eight weeks, eight to ten weeks, six to twelve weeks, and eight to fourteen weeks. Luteinizing hormone was not statistically different in FR rats compared with AL counterparts. Therefore, duration of lower food intake had a greater impact on spermatogenesis, whereas a younger initial age of lower food intake was more influential on testosterone levels. These interactions are important in the interpretation of subacute toxicology studies employing FR or when test articles lower food consumption relative to AL-fed rats.

Scientific and Regulatory Policy Committee (SRPC) Paper Assessment of Circulating Hormones in Nonclinical Toxicity Studies III. Female Reproductive Hormones

Hormonally mediated effects on the female reproductive system may manifest as pathologic changes of endocrine-responsive organs and altered reproductive function. Identification of these effects requires proper assessment, which may include investigative studies to profile female reproductive hormones. Here, we briefly describe normal hormonal patterns across the estrous or menstrual cycle and provide general guidance on measuring female reproductive hormones and characterizing hormonal disturbances in nonclinical toxicity studies. Although species used in standard toxicity studies share basic features of reproductive endocrinology, there are important species differences that affect both study design and interpretation of results. Diagnosing female reproductive hormone disturbances can be complicated by many factors, including estrous/menstrual cyclicity, diurnal variation, and age- and stress-related factors. Thus, female reproductive hormonal measurements should not generally be included in first-tier toxicity studies of standard design with groups of unsynchronized intact female animals. Rather, appropriately designed and statistically powered investigative studies are recommended in order to properly identify ovarian and/or pituitary hormone changes and bridge these effects to mechanistic evaluations and safety assessments. This article is intended to provide general considerations and approaches for these types of targeted studies.

Comparing rat and rabbit embryo-fetal developmental toxicity data for 379 pharmaceuticals: on systemic dose and developmental effects.

Abstract A database of embryo-fetal developmental toxicity (EFDT) studies of 379 pharmaceutical compounds in rat and rabbit was analyzed for species differences based on toxicokinetic parameters of area under the curve (AUC) and maximum concentration (Cmax) at the developmental lowest adverse effect level (dLOAEL). For the vast majority of cases (83% based on AUC of n = 283), dLOAELs in rats and rabbits were within the same order of magnitude (less than 10-fold different) when compared based on available data on AUC and Cmax exposures. For 13.5% of the compounds the rabbit was more sensitive and for 3.5% of compounds the rat was more sensitive when compared based on AUC exposures. For 12% of the compounds the rabbit was more sensitive and for 1.3% of compounds the rat was more sensitive based on Cmax exposures. When evaluated based on human equivalent dose (HED) conversion using standard factors, the rat and rabbit were equally sensitive. The relative extent of embryo-fetal toxicity in the presence of maternal toxicity was not different between species. Overall effect severity incidences were distributed similarly in rat and rabbit studies. Individual rat and rabbit strains did not show a different general distribution of systemic exposure LOAELs as compared to all strains combined for each species. There were no apparent species differences in the occurrence of embryo-fetal variations. Based on power of detection and given differences in the nature of developmental effects between rat and rabbit study outcomes for individual compounds, EFDT studies in two species have added value over single studies.

Comparison of rat and rabbit embryo–fetal developmental toxicity data for 379 pharmaceuticals: on the nature and severity of developmental effects

Abstract Regulatory non-clinical safety testing of human pharmaceuticals typically requires embryo–fetal developmental toxicity (EFDT) testing in two species (one rodent and one non-rodent). The question has been raised whether under some conditions EFDT testing could be limited to one species, or whether the testing in a second species could be decided on a case-by-case basis. As part of a consortium initiative, we built and queried a database of 379 compounds with EFDT studies (in both rat and rabbit animal models) conducted for marketed and non-marketed pharmaceuticals for their potential for adverse developmental and maternal outcomes, including EFDT incidence and the nature and severity of adverse findings. Manifestation of EFDT in either one or both species was demonstrated for 282 compounds (74%). EFDT was detected in only one species (rat or rabbit) in almost a third (31%, 118 compounds), with 58% (68 compounds) of rat studies and 42% (50 compounds) of rabbit studies identifying an EFDT signal. For 24 compounds (6%), fetal malformations were observed in one species (rat or rabbit) in the absence of any EFDT in the second species. In general, growth retardation, fetal variations, and malformations were more prominent in the rat, whereas embryo–fetal death was observed more often in the rabbit. Discordance across species may be attributed to factors such as maternal toxicity, study design differences, pharmacokinetic differences, and pharmacologic relevance of species. The current analysis suggests that in general both species are equally sensitive on the basis of an overall EFDT LOAEL comparison, but selective EFDT toxicity in one species is not uncommon. Also, there appear to be species differences in the prevalence of various EFDT manifestations (i.e. embryo–fetal death, growth retardation, and dysmorphogenesis) between rat and rabbit, suggesting that the use of both species has a higher probability of detecting developmental toxicants than either one alone.

Society of toxicologic pathology position paper: review series: assessment of circulating hormones in nonclinical toxicity studies: general concepts and considerations.

This is an introductory paper to a series of papers intended to provide the basis for understanding the contribution of endocrine axis disruption or dysfunction to the pathogenesis of morphological findings and to aid in the interpretation of study outcomes. This is the first in this series of guidance papers prepared by the Working Group and outlines general concepts of study design and assay conduct and validation for hormone studies in general.

Comparative Aspects of Pre- and Postnatal Development of the Male Reproductive System

This review describes pre‐ and postnatal development of the male reproductive system in humans and laboratory animals, and highlights species differences in the timing and control of hormonal and morphologic events. Major differences are that the fetal testis is dependent on gonadotropins in humans, but is independent of such in rats; humans have an extended postnatal quiescent period, whereas rats exhibit no quiescence; and events such as secretion by the prostate and seminal vesicles, testicular descent, and the appearance of spermatogonia are all prenatal events in humans, but are postnatal events in rats. Major differences in the timing of the developmental sequence between rats and humans include: gonocyte transformation period (rat: postnatal day 0–9; human: includes gestational week 22 to 9 months of age); masculinization programming window (rat: gestational day 15.5–17.5; human: gestational week 9–14); and mini‐puberty (rat: 0–6 hr after birth; human: 3–6 months of age). Endocrine disruptors can cause unique lesions in the prenatal and early postnatal testis; therefore, it is important to consider the differences in the timing of the developmental sequence when designing preclinical studies as identification of windows of sensitivity for endocrine disruption or toxicants will aid in interpretation of results and provide clues to a mode of action. Birth Defects Research 110:190–227, 2018.

A Probability Analysis of Historical Pregnancy and Fetal Data from Dutch Belted and New Zealand White Rabbit Strains from Embryo-Fetal Development Studies.

Embryo-fetal development (EFD) studies, typically in pregnant rats and rabbits, are conducted prior to enrolling females of reproductive age in clinical trials. Common rabbit strains used are the New Zealand White (NZW) and Dutch Belted (DB). As fetal abnormalities can occur in all groups, including controls, Historical Control Data (HCD) is compiled using data from control groups of EFD studies, and is used along with each studys concurrent control group to help determine whether fetal abnormalities are caused by the test article or are part of background incidences. A probability analysis was conducted on 2014 HCD collected at Charles River Inc., Horsham PA on Covance NZW, Covance DB, and Charles River (CR) NZW rabbits. The analysis was designed to determine the probability of 2 or 3 out of a group of 22 does aborting their litter or of having a fetal abnormality by chance. Results demonstrate that pregnancy parameters and fetal observations differ not only between strains, but between sources of rabbits of the same strain. As a result the probability of these observations occurring by chance in two or three litters was drastically different. Although no one single strain is perfect, this analysis highlights the need to appreciate the inherent differences in pregnancy and fetal abnormalities between strains, and points out that an apparent isolated increased incidence of an observation in one strain will not necessarily be test-article related in another strain. A robust HCD is critical for interpretation of EFD rabbit studies, regardless of the rabbit strain used.

The inhibin B response to a motilin receptor agonist in male rats.

BACKGROUND In a repeat oral dose toxicity study, all of 16 male rats given 100 mg/kg/day GSK1322888 sustained testicular injury after 4 weeks of treatment; the findings were not reversible after 12 weeks off-dose. The current study was conducted to further characterize testicular toxicity and to explore the possible relationship between onset of lesions, and changes in circulating hormone levels. METHODS Male Sprague Dawley rats were orally administered 30 or 100 mg/kg/day GSK1322888 for 2 weeks with a 4-week off-dose period. Blood was collected via tail vein twice during the treatment period (days 4 and 11) and three times during the off-dose period (days 28, 36, and 42) for measurement of serum testosterone, dihydrotestosterone, and Inhibin B, luteinizing hormone, and follicle stimulating hormone concentrations. A histopathologic examination of testes was performed at the end of the treatment and off-dose periods. RESULTS At 100 mg/kg/day, microscopic findings of the testis (degeneration of the germinal epithelium) were evident for 9 of 10 male rats on day 14 and all 10 rats at the end of the 4-week recovery period. There was no testicular toxicity observed at 30 mg/kg/day. During all stages of evaluation, there was no apparent difference among control and treated animals in hormone concentrations. CONCLUSION There was poor correlation between changes in serum levels of Inhibin B and testis histopathology. Based on these observations, the utility of Inhibin B as a hormonal marker for germ cell toxicity is limited.

The Potential Role for Corticosterone in the Induction of Cleft Palate in Mice After Treatment With a Selective NK‐1 Receptor Antagonist, Casopitant (GW679769B)

BACKGROUND Casopitant is a potent and selective NK-1 receptor antagonist that has shown clinical efficacy in the prevention of chemotherapy-induced and postoperative-induced nausea and vomiting. METHODS In an embryo-fetal development study, pregnant mice were given vehicle (sterile water) or doses of 30, 100, or 300 mg/kg/day casopitant on Gestation Day (GD) 6 to 15. Fetuses were evaluated for external, visceral, and skeletal abnormalities on GD 18. In a follow-on study to investigate casopitant-induced hormonal changes during the developmental period for palate formation, pregnant mice were given vehicle (sterile water) or 300 mg/kg/day casopitant once daily on GD 6 to 13. Blood was collected on GD 13 at various time-points for measurement of plasma adrenocorticotropic hormone and corticosterone (CRT) concentrations. RESULTS There was no evidence of developmental toxicity in mice at 30 or 100 mg/kg/day but 9% of fetuses at 300 mg/kg/day had cleft palate. Mice are sensitive to glucocorticoid-induced cleft palates, and NK-1 antagonists are known to modulate the hypothalamic-pituitary-adrenal axis leading to increases in corticosterone. On GD 13, mean plasma adrenocorticotropic hormone levels at 300 mg/kg/day were elevated by approximately twofold from vehicle-treated levels at 1 hr post-dose and mean plasma CRT levels were elevated by 3, 5, and 10-fold at 0.5, 1, and 2 hr post-dose, respectively. CONCLUSIONS The increased level of CRT was in the range previously shown in the literature to cause cleft palates in mice and was likely the underlying mechanism behind casopitant-induced cleft palate in mice.

Rethinking developmental toxicity testing: Evolution or revolution?

BACKGROUND Current developmental toxicity testing adheres largely to protocols suggested in 1966 involving the administration of test compound to pregnant laboratory animals. After more than 50 years of embryo-fetal development testing, are we ready to consider a different approach to human developmental toxicity testing? METHODS A workshop was held under the auspices of the Developmental and Reproductive Toxicology Technical Committee of the ILSI Health and Environmental Sciences Institute to consider how we might design developmental toxicity testing if we started over with 21st century knowledge and techniques (revolution). We first consider what changes to the current protocols might be recommended to make them more predictive for human risk (evolution). RESULTS The evolutionary approach includes modifications of existing protocols and can include humanized models, disease models, more accurate assessment and testing of metabolites, and informed approaches to dose selection. The revolution could start with hypothesis-driven testing where we take what we know about a compound or close analog and answer specific questions using targeted experimental techniques rather than a one-protocol-fits-all approach. Central to the idea of hypothesis-driven testing is the concept that testing can be done at the level of mode of action. It might be feasible to identify a small number of key events at a molecular or cellular level that predict an adverse outcome and for which testing could be performed in vitro or in silico or, rarely, using limited in vivo models. Techniques for evaluating these key events exist today or are in development. DISCUSSION Opportunities exist for refining and then replacing current developmental toxicity testing protocols using techniques that have already been developed or are within reach.