Science-Based Approach to Harmonize Contraception Recommendations in Clinical Trials and Pharmaceutical Labels.

This review presents a European Federation of Pharmaceutical Industries and Association/PreClinical Development Expert Group (EFPIA-PDEG) topic group consensus on a data-driven approach to harmonized contraception recommendations for clinical trial protocols and product labeling. There is no international agreement in pharmaceutical clinical trial protocols or product labeling on when/if female and/or male contraception is warranted and for how long after the last dose. This absence of consensus has resulted in different recommendations among regions. For most pharmaceuticals, contraception recommendations are generally based exclusively on nonclinical data and/or mechanism. For clinical trials, contraception is the default position and is maintained for women throughout clinical development, whereas appropriate information can justify removing male contraception. Conversely, contraception is only recommended in product labeling when warranted. A base case rationale is proposed for whether or not female and/or male contraception is/are warranted, using available genotoxicity and developmental toxicity data. Contraception is generally warranted for both male and female subjects treated with mutagenic pharmaceuticals. We propose as a starting point that contraception is not typically warranted when the margin is 10-fold or greater between clinical exposure at the maximum recommended human dose and exposure at the no observed adverse effect level (NOAEL) for purely aneugenic pharmaceuticals and for pharmaceuticals that induce fetal malformations or embryo-fetal lethality. Other factors are discussed, including contraception methods, pregnancy testing, drug clearance, options for managing the absence of a developmental toxicity NOAEL, drug-drug interactions, radiopharmaceuticals, and other drug modalities. Overall, we present a data-driven rationale that can serve as a basis for consistent contraception recommendations in clinical trials and in product labeling across regions.

Tolerability and age-dependent toxicokinetics following perinatal hydroxyurea treatment in Sprague Dawley rats.

Hydroxyurea (HU) is a valuable therapy for individuals with sickle cell anemia. With increased use of HU in children and throughout their lives, it is important to understand the potential effects of HU therapy on their development and fertility. Thus, studies were conducted to identify appropriate doses to examine long-term effects of prenatal and early postnatal HU exposure and to understand kinetics of HU at various life stages. Pregnant Sprague Dawley dams were administered HU (0-150 mg/kg/day) via oral gavage from gestation days 17 to 21 and during lactation. Pups were dosed with the same dose as their respective dam starting on postnatal day (PND) 10 and up to PND 34. There was minimal maternal toxicity, and no significant effects on littering at any dose of HU. Starting on ~PND 16, offspring displayed skin discoloration and alopecia at doses ≥75 mg/kg/day and lower body weight compared to controls at doses ≥100 mg/kg/day. Gestational transfer of HU was observed, but there was minimal evidence of lactational transfer. Our toxicokinetic studies suggest that the internal dose in offspring may be altered due to age, but not due to sex. The plasma area under the curve, a measure of systemic exposure, at doses tolerated by offspring was threefold to sevenfold lower than the internal therapeutic dose in humans. Therefore, strategies to establish clinically relevant exposures in animal studies are needed. Overall, these data are useful for the design of appropriate nonclinical studies in the future to evaluate the consequences of long-term HU treatment starting in childhood.

Butylparaben multigenerational reproductive assessment by continuous breeding in Hsd:Sprague Dawley SD rats following dietary exposure.

Butylparaben (BP) is an antimicrobial agent utilized for decades as a preservative in numerous consumer products. The safety of parabens has recently come under scrutiny based on reports of estrogenic activity and suggested adverse effects upon the reproductive system. Due to the limited availability of studies that address the potential for BP exposure to induce reproductive toxicity, and clear evidence of human exposure, the National Toxicology Program conducted a multigenerational continuous breeding study to evaluate the impact of dietary BP-exposure at 0, 5000, 15,000, or 40,000 ppm on reproductive and developmental parameters in Hsd:Sprague Dawley SD rats. BP-exposure was not associated with adverse alterations of fertility, fecundity, pubertal attainment, or reproductive parameters in F0, F1, or F2 generations. Exposure-dependent increases in liver weights, and incidences of non-neoplastic liver lesions suggest the liver is a target organ of BP toxicity. No findings were observed that would support the purported mechanism of BP-induced endocrine disruption in perinatally-exposed rodents.

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.

Preliminary investigation of changes in the sexually dimorphic nucleus of the rat medial preoptic area following prenatal exposure to fenitrothion.

In vitro, the organophosphate insecticide fenitrothion is a potent competitive androgen receptor antagonist, whereas in vivo it affects the development of the male rat reproductive system. The purpose of this pilot study was to determine whether prenatal exposure to fenitrothion affects development of the rat sexually dimorphic nucleus of the medial preoptic area (SDN-POA). Pregnant rats (n = 5-6 litters/group) were orally dosed with corn oil (vehicle) or fenitrothion (20 or 25 mg kg(-1) day(-1)) from gestation day (GD) 12-21. Offspring were euthanized after reaching sexual maturity (females 60-65 days old and males 96-105 days old) and the SDN-POA volumes determined for two rats/sex/litter. Tremors, increased lacrimation and decreased body weight gain were observed in dams from both fenitrothion exposure groups. Reproductive effects in male offspring, including reduced anogenital distance on postnatal day (PND) 1 and increased retention of areolae (PND 13) were observed following fenitrothion exposure at these dose levels. These effects did not persist into adulthood. There was a dose-related increase in the SDN-POA volume in males and a dose-related decrease in SDN-POA volume in females exposed to fenitrothion. These SDN-POA volume changes contrast with those seen with flutamide, another potent anti-androgen, and suggest that fenitrothion may have mixed endocrine effects on the developing brain.

Assessment of developmental toxicity of vorinostat, a histone deacetylase inhibitor, in Sprague-Dawley rats and Dutch Belted rabbits.

BACKGROUND: The developmental toxicity potential of vorinostat (suberoylanilide hydroxamic acid [SAHA], ZOLINZA), a potent inhibitor of histone deacetylase (HDAC), was assessed in Sprague-Dawley rats and Dutch Belted rabbits. HDAC inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation, and apoptosis of tumor cells. Range-finding studies established oral dose levels of 5, 15, or 50 mg/kg/day and 20, 50, or 150 mg/kg/day in rats and rabbits, respectively. METHODS: Animals were dosed on Gestation Days 6-20 or 7-20, respectively, with litter/fetal parameters evaluated on GD 21 and 28, respectively. Separate studies evaluated toxicokinetic parameters at the mid- and high-dose levels. RESULTS: There was no maternal toxicity observed at the highest dose levels; however, hematology and serum biochemistry changes were characterized in the range-finding studies. Vorinostat did not induce morphological malformations in either rat or rabbit fetuses. In rats, drug-related developmental toxicity was observed only in the high-dose group and consisted of markedly decreased fetal weight and increases in fetuses with a limited number of skeletal variations. In rabbits, drug-related developmental toxicity was also observed only in the high-dose group and consisted of slightly decreased fetal weight and increases in fetuses with a short 13th rib and incomplete ossification of metacarpals. Maternal exposures to vorinostat based on AUC and Cmax values were comparable at the high-dose levels of both species. Rabbits tolerated higher dosages probably due to more extensive metabolism. Maternal concentrations of vorinostat were approximately 1,000-fold above the known in vitro HDAC inhibitory concentration. CONCLUSIONS: Review of previous work with valproic acid, another HDAC inhibitor, suggest that the developmental toxicity profiles of these 2 compounds are not the result of HDAC inhibition but involve other mechanisms.

Altered gene expression during rat Wolffian duct development following di(n-butyl) phthalate exposure.

Di(n-butyl) phthalate (DBP) is a common plasticizer and solvent that disrupts androgen-dependent male reproductive development in rats. In utero exposure to 500 mg/kg/day DBP on gestation days (GD) 12 to 21 decreases androgen biosynthetic enzymes, resulting in decreased fetal testicular testosterone levels. One consequence of prenatal DBP exposure is malformed epididymides in adult rats. Reduced fetal testosterone levels may be responsible for the malformation, since testosterone is required for Wolffian duct stabilization and their development into epididymides. Currently, little is understood about the molecular mechanisms of Wolffian duct differentiation. The objective of this study was to identify changes in gene expression associated with altered morphology of the proximal Wolffian duct following in utero exposure to DBP. Pregnant Crl:CD(R) (SD) rats were gavaged with corn oil vehicle or 500 mg/kg/day DBP from GD 12 to GD 19 or 21. There were only small morphological differences between control and DBP-exposed Wolffian ducts on GD 19. On GD 21, 89% of male fetuses in the DBP dose group showed marked underdevelopment of Wolffian ducts, characterized by decreased coiling. RNA was isolated from Wolffian ducts on GD 19 and 21. Together with empirical information, cDNA microarrays were used to help identify candidate genes that could be associated with the morphological changes observed on GD 21. These candidate genes were analyzed by real-time RT-PCR. Changes in mRNA expression were observed in genes within the insulin-like growth factor (IGF) pathway, the matrix metalloproteinase (MMP) family, the extracellular matrix, and in other developmentally conserved signaling pathways. On GD 19, immunolocalization of IGF-1 receptor protein demonstrated an increase in cytoplasmic expression in the mesenchymal and epithelial cells. There was also a variable decrease in androgen receptor protein in ductal epithelial cells on GD 19. This study provides insight into the effects of antiandrogens on the molecular mechanisms involved in Wolffian duct development. The altered morphology and changes in gene expression following DBP exposure are suggestive of altered paracrine interactions between ductal epithelial cells and the surrounding mesenchyme during Wolffian duct differentiation due to lowered testosterone production.

Inhibin B as a potential biomarker of testicular toxicity.

Inhibin B is a glycoprotein produced predominantly by Sertoli cells which regulates pituitary FSH release by a negative feedback loop. The regulation of inhibin B is complex with changes in the pattern of secretion occurring during development, and many factors such as FSH, testosterone, Sertoli cell proliferation and germ cell complement likely to contribute to overall production. Systemic inhibin B concentrations seem to reflect the extreme ends of spermatogenic status with high levels of inhibin B observed in normal, fertile individuals and lower levels of inhibin B in individuals with severe damage to the testis as a result of germ cell depletion. Inhibin B has proved valuable in epidemiological studies exploring male infertility with data showing that inhibin B combined with FSH measurements has a higher positive predictive value for detecting male infertility than either alone. Inhibin B is proposed as a potential biomarker of testicular toxicity in rodent toxicity studies to compliment traditional endpoints. In pharmaceutical development, inhibin B might allow better linkage between animal study results and subsequent monitoring of testicular function in clinical trials. An international, intercompany project has been initiated to evaluate the overall suitability and limitations of inhibin B as a biomarker of testicular toxicity.

Altered gene expression during rat Wolffian duct development in response to in utero exposure to the antiandrogen linuron.

Linuron is an herbicide with weak androgen receptor (AR) antagonist activity. Exposure to linuron from gestation days (GD) 12 to 21 perturbs androgen-dependent male reproductive development. In utero exposure to 50-mg/kg/day linuron induces malformations of the epididymis and the vas deferens. The objective of this study was to identify alterations in gene expression within the testis and epididymis associated with abnormal Wolffian duct development and to correlate changes in gene expression with the gross morphology of the affected epididymides. Pregnant Sprague-Dawley rats were administered either corn oil vehicle or linuron (50 mg/kg/day) by gavage from GD 12 to 21 (n = 3-6 controls, n = 5-10 linuron-treated dams per time point). Changes in gene expression were evaluated in testes on GD 21 and in epididymides on GD 21 and postnatal day (PND) 7, using cDNA microarrays and confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) analyses. RNA was isolated from intact epididymides with reduced or no ductal coiling from the linuron groups, and epididymides with noncontiguous ducts were excluded. In the fetal testis, exposure to linuron did not result in reduced mRNA expression of the AR or that of several steroidogenic enzymes, supporting the hypothesis that linuron does not reduce fetal testosterone production. Linuron induced a significant decrease in AR mRNA expression in GD 21 epididymides. Significant changes in mRNA expression in GD 21 and PND 7 epididymides were also identified in the epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Notch signaling pathways. These pathways are involved in tissue morphogenesis. Changes in the expression of AR and IGF-1 receptors were detected by immunostaining in malformed epididymides from linuron-exposed rats. Linuron induced changes in epididymal gene expression suggestive of altered paracrine interactions between the mesenchyme and epithelial cells during development. The EGF, Notch, IGF-1, BMP4, and FGF signaling pathways may be involved in normal testosterone-mediated development of the Wolffian duct.

Effects of in utero exposure to finasteride on androgen-dependent reproductive development in the male rat.

Finasteride is a specific inhibitor of type II 5alpha-reductase, the enzyme that converts testosterone (T) to the more potent androgen receptor agonist dihydrotestosterone (DHT). In utero exposure to androgen receptor antagonists and T biosynthesis inhibitors have induced permanent effects on androgen-sensitive end points such as anogenital distance (AGD), nipple retention, and malformations of the male rat reproductive tract. The objectives of this study were to (1) characterize the dose response of finasteride-mediated alterations in androgen-dependent developmental end points, (2) determine whether prenatal exposure to finasteride permanently decreases AGD or results in nipple retention, and (3) evaluate whether AGD or nipple retention is predictive of adverse alterations in the male reproductive tract. Pregnant Crl:CD(SD)BR rats (n=5-6/group) were gavaged with either vehicle or finasteride at 0.01, 0.1, 1.0, 10, or 100 mg/kg/day on gestation days 12 to 21. All male offspring were monitored individually until necropsy on postnatal day (PND) 90. The present study design has been used previously for other antiandrogens and is sensitive to perturbations of the male rat reproductive tract. Decreases in AGD on PND 1 and increases in areolae-nipple retention on PND 13 were significantly different from controls in all finasteride-exposed male rats. Finasteride-induced changes in AGD and nipple retention were permanent in male rats exposed to finasteride at and above 0.1 mg/kg/day. On PND 90, dorsolateral and ventral prostate lobes were absent in 21 to 24% of rats exposed to 100 mg/kg/day finasteride and weighed significantly less at and above 10 mg/kg/day. In the highest dose group, 73% of animals had ectopic testes, much higher than previously reported. The most sensitive malformation other than decreased AGD and nipple retention was the dose-dependent increase in hypospadias. The lowest observed adverse effect level (LOAEL) for finasteride-induced permanent effects in this study was 0.1 mg/kg/day based on permanent changes in AGD and nipple retention. Finasteride-induced changes in AGD and retention of nipples were highly predictive of hypospadias, ectopic testes, and prostate malformations even though some animals with retained nipples or decreased AGD may not have had other reproductive tract malformations. In summary, prenatal exposure to finasteride specifically inhibited DHT-mediated development with little to no change in T-mediated development.

Effects of in utero exposure to the organophosphate insecticide fenitrothion on androgen-dependent reproductive development in the Crl:CD(SD)BR rat.

Fenitrothion [0,0-dimethyl-O-(4-nitro-m-tolyl) phosphorothioate] is an organophosphate insecticide that has been shown to have antiandrogenic activity using in vitro and in vivo screening assays. Studies were performed to evaluate the ability of fenitrothion to disrupt androgen-dependent sexual differentiation in the male rat. Pregnant Crl:CD(SD)BR rats were administered fenitrothion by gavage at 0, 5, 10, 15, 20, or 25 mg/kg/day ( n = 6-11/group) from gestation day (GD) 12 to 21. Maternal toxicity was observed in the dams treated with 20 and 25 mg fenitrothion/kg/day based on muscle tremors and decreases in body weight gain from GD 12 to 21. Fetal death was increased in the 20 and 25 mg/kg/day exposure groups, as evidenced by a decrease in the proportion of pups born alive. Androgen-mediated development of the reproductive tract was altered in male offspring exposed in utero to maternally toxic levels of fenitrothion (25 mg/kg/day), as evidenced by reduction in anogenital distance on postnatal day (PND) 1 and retention of areolae on PND 13. However, these effects were only transient, and there were no indications of abnormal phenotypes or development of androgen-dependent tissues on PND 100. At the dose levels evaluated in this study, fenitrothion was only weakly antiandrogenic in vivo compared with other androgen receptor antagonists such as flutamide, linuron, and vinclozolin. Based on observed fetotoxicity at 20 mg/kg/day, the lowest observed adverse effect level (LOAEL) for developmental effects can be lowered from 25 to 20 mg/kg/day.