Assessing the toxicological significance of ejaculatory plugs as a clinical observation in rat toxicology studies: CNS-mediated increases in plug production vs. gustatory-mediated decreases in plug consumption.

Spontaneous ejaculation has been reported in a variety of mammalian species and may occur either as a result of pharmacological treatment or as a component of the daily behavior of normal, untreated animals. Infrequently, increased numbers of spontaneous ejaculatory plugs have been reported among the clinical signs in rat toxicology studies. This mini-review presents an overview on the presence of ejaculatory plugs in rodents and provides recommendations to consider when attempting to understand the toxicological significance of increased numbers of ejaculatory plugs in rat toxicology studies.

An oral two-generation reproductive toxicity study of S-111-S-WB in rats.

S-111-S-WB (CAS No. 72968-38-8), a mixture of perfluoro fatty acid ammonium salts, was administered daily via oral gavage to 30 Crl:CD(SD) rats/sex/group at 0.025, 0.125 and 0.6mg/(kgday) over two generations to assess potential reproductive toxicity. Reproductive performance, mean litter size, pup survival and pup weights were unaffected. Lower mean body weights were observed in 0.6mg/(kgday) group F(0) and F(1) males. Higher liver weights, correlating to hepatocellular hypertrophy in the 0.6mg/kg group, were noted for parental males in the 0.125 and 0.6mg/(kgday) groups, parental females in the 0.6mg/(kgday) group and F(1) pups in the 0.125 and 0.6mg/(kgday) groups. Higher kidney weights, correlating to renal tubule hypertrophy in the 0.6mg/kg group, were observed for parental males and females in the 0.125 and 0.6mg/(kgday) groups. Systemic exposure (measured only in females) to total S-111-S-WB was proportional to dose following 9 weeks of daily administration on the gestation day 19. Total S-111-S-WB concentration in the serum of male and female pups was 1.2-1.4-fold higher than in the dams 2h following administration to the dams on lactation day 13. A dosage level of 0.6mg/(kgday) was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive function. A dosage level of less than 0.025mg/(kgday) was considered to be the NOAEL for F(0) and F(1) parental systemic toxicity based on microscopic hepatic findings in the males of all test article groups, and a dosage level of 0.025mg/(kgday) was considered to be the NOAEL for neonatal toxicity based on higher liver weights in the F(1) and F(2) pups at 0.125mg/(kgday) and higher.

A two-generation reproductive toxicity study of octamethylcyclotetrasiloxane (D4) in rats exposed by whole-body vapor inhalation.

This study evaluated the potential toxicity of whole-body vapor inhalation of octamethylcyclotetrasiloxane (D(4)) on reproductive capabilities in exposed F(0) and F(1) parental animals and the potential effects on neonatal survival, growth, and development of the F(1) and F(2) offspring. F(0) male and female Sprague-Dawley rats (30/sex/group) were exposed to D(4) vapor at concentrations of 0, 70, 300, 500 or 700 ppm 6h per day for at least 70 consecutive days prior to mating and lasted through weaning of the pups on postnatal day (PND) 21. Female exposures were suspended from gestation day (GD) 21 through PND 4 to allow for parturition and permit continuous maternal care for the early neonates. Starting on PND 22, F(1) weanlings were exposed to D(4) as described for the F(0) generation. The F(2) pups were not directly exposed to D(4). F(0) animals were mated once to produce the F(1) generation; F(1) parental animals were mated twice to produce two F(2) litters. In addition, the F(1) males were mated with unexposed females. Prolonged estrous cycles, decreased mating and fertility indices were observed in the F(1) generation exposed to D(4) for the first and second matings. Significant reductions in the mean number of pups born and mean live litter size were observed in the 500 and 700 ppm groups for both the F(0) and F(1) generations. Implantation sites were also reduced at 700 ppm for both F(0) and F(1) generations. No adverse effects were observed at any exposure level on anogenital distance, vaginal patency and preputial separation. No adverse effects were seen on male functional reproductive parameters, spermatogenic endpoints, microscopic evaluation of male reproductive tissue, or when the D(4)-exposed F(1) males were mated with the unexposed females, demonstrating that the reproductive toxicity observed was due to D(4) exposure to the females. Based on the lack of effect on reproduction when the D(4)-exposed males were mated to näive females, the NOAEL for male reproductive toxicity was considered to be 700 ppm. Based on the statistically significant effects on fertility and litter size, NOAEL for female reproductive toxicity was considered to be 300 ppm. The findings observed in this study are consistent with suppression or delaying of LH surge as well as acceleration of the onset of female reproductive senescence in the rat. While analogous pathways control ovulation in both rats and humans, there are significant differences in the mechanism for timing and release of LH and resulting changes in the control of ovulation and mating behavior between the two species. If D(4) delays rather than causes a prolonged suppression or ablation of the LH surge, the reproductive mode of action of D(4) would not likely be relevant for humans.

Framework for use of toxicity screening tools in context-based decision-making.

One of the principal applications of toxicology data is to inform risk assessments and support risk management decisions that are protective of human health. Ideally, a risk assessor would have available all of the relevant information on (a) the toxicity profile of the agent of interest; (b) its interactions with living systems; and (c) the known or projected exposure scenarios: to whom, how much, by which route(s), and how often. In practice, however, complete information is seldom available. Nonetheless, decisions still must be made. Screening-level assays and tools can provide support for many aspects of the risk assessment process, as long as the limitations of the tools are understood and to the extent that the added uncertainty the tools introduce into the process can be characterized and managed. Use of these tools for decision-making may be an end in itself for risk assessment and decision-making or a preliminary step to more extensive data collection and evaluation before assessments are undertaken or completed and risk management decisions made. This paper describes a framework for the application of screening tools for human health decision-making, although with some modest modification, it could be made applicable to environmental settings as well. The framework consists of problem formulation, development of a screening strategy based on an assessment of critical data needs, and a data analysis phase that employs weight-of-evidence criteria and uncertainty analyses, and leads to context-based decisions. Criteria for determining the appropriate screening tool(s) have been identified. The choice and use of the tool(s) will depend on the question and the level of uncertainty that may be appropriate for the context in which the decision is being made. The framework is iterative, in that users may refine the question(s) as they proceed. Several case studies illustrate how the framework may be used effectively to address specific questions for any endpoint of toxicity.

An inhalation reproductive toxicity study of octamethylcyclotetrasiloxane (D4) in female rats using multiple and single day exposure regimens.

Octamethylcyclotetrasiloxane (D(4)) has been shown to have effects on the female rat reproductive cycle. This study evaluated the phase of the female rat reproductive cycle affected by D(4) using a study design that allowed the complete female reproductive cycle, as well as phases of the cycle, from pre-mating through gestation, to be evaluated. Rats were exposed via whole body vapor inhalation up to 700 ppm D(4) during the overall phase (28 days prior to mating through gestation day (GD) 19), the ovarian phase (31-3 days prior to mating), the fertilization phase (3 days prior to the start of mating through gestation day 3), and the implantation phase (GD 2-GD 5) of the reproductive cycle. D(4) was associated with decreases in implantation sites and litter size in the overall and fertilization phases, but not in the ovarian or implantation phases. In order to further define the sensitive period for D(4) exposure, additional groups of rats were exposed on single days. A single 6h exposure to D(4) on the day prior to mating resulted in a significant reduction in fertility. These data indicate that there is a very narrow window, around the time of ovulation and fertilization, for D(4) to exert effects on the reproductive cycle of the female rat. Subsequent research, reported elsewhere, has elucidated the mode of action and assessed its potential relevance to humans.

A two-generation reproductive toxicity study of decamethylcyclopentasiloxane (D5) in rats exposed by whole-body vapor inhalation.

This two-generation reproduction study assessed the reproductive hazard potential of decamethylcyclopentasiloxane (D(5)). Sprague-Dawley rats (30/sex/group) were exposed by whole-body vapor inhalation to a target concentration of 30, 70, or 160 ppm D(5) or filtered air for 6h/day. Exposures for the F(0) and F(1) generations started at least 70 days prior to mating and lasted through weaning of the respective pups on postnatal day (PND) 21. Female exposures were interrupted from gestation day (GD) 21 through PND 4 to allow for parturition and to permit continuous maternal care for the early neonates. F(2) pups were not directly exposed to D(5). There were no exposure-related mortalities, clinical signs of toxicity, or effects on body weight or food consumption. There were no treatment-related gross findings or organ weight effects at the F(0) and F(1) necropsies. Other than minimal alveolar histiocytosis in all exposed groups, there were no noteworthy microscopic findings. Reproductive parameters (number of days between pairing and mating, mating and fertility indices, gestation length, and parturition), spermatogenic parameters and ovarian primordial follicle counts and numbers of corpora lutea in the F(0) and F(1) parental animals were not significantly changed between treated and control groups. Mean live litter sizes, number of pups born, sex ratios, pup body weights, postnatal pup survival and general physical condition of offspring in each generation were not affected. The slight, but statistically significant, increase in the mean F(1) male pup AGD in the 160 ppm group was not considered to be related to treatment. Vaginal patency and balanopreputial separation were unchanged compared to controls. Thus, the No-Observed-Adverse-Effect-Level (NOAEL) for parental and reproductive toxicity was determined to be 160 ppm D(5).

Mode of action: yolk sac poisoning and impeded histiotrophic nutrition--HBOC-related congenital malformations.

Rodents form an early inverted yolk sac placenta (invYSP) by apposing the yolk sac to the uterine wall. The invYSP supplies nutrients via histiotrophic nutrition involving pinocytosis of materials from uterine gland secretions, lysosomal degradation, and transfer of the products to the embryo. Interference with histiotrophic trafficking through the invYSP by high-molecular-weight molecules (such as trypan blue) causes malformations and resorptions. Later in gestation, rodents form a definitive chorioallantoic placenta (CAP). By contrast, humans and dogs never develop an invYSP, relying exclusively on the CAP. Given their large size (approximately 250 kD), hemoglobin-based oxygen carriers (HBOC), being developed as blood substitutes, could be expected to interfere with histiotrophic trafficking through the invYSP. During initial toxicity testing, intravenous infusions of HBOC caused pronounced developmental toxicity in rats exposed during the pre-CAP period. Assuming that HBOC interfered with invYSP function, we hypothesized that these findings would not apply to humans or dogs, which lack an invYSP. Subsequent extensive developmental toxicity studies in dogs produced no developmental toxicity after intravenous infusion at the maximum tolerated dose. In view of the existing species-specific placental differences and HBOC's demonstrated, exclusive interference with invYSP histiotrophic nutrition, HBOC is not expected to cause abnormal development in humans or other mammals that do not develop an invYSP.

Absence of embryo-fetal toxicity in rats or rabbits following oral dosing with nelfinavir.

The potential for nelfinavir mesylate (VIRACEPT) to induce maternal and embryo-fetal toxicity was evaluated in rats and rabbits following oral administration. The drug was administered by gavage to rats at doses of 200, 500, or 1000mg/kg/day on days 6-17 of gestation and to rabbits at doses of 200, 400, or 1000mg/kg/day on days 7-20 of gestation. Dams and does were euthanized on GD20 and 29, respectively, and the offspring were weighed and examined for external, visceral, and skeletal alterations. Maximum plasma nelfinavir concentrations (C(max)) in rats were comparable to C(max) values in humans and were 3- to 6-fold higher than the reported human trough levels, while plasma nelfinavir levels in rabbits were approximately 0.13-0.17x the human C(max) and 0.25-0.5x the human trough. In rats, no treatment-related maternal or embryo-fetal toxicity was observed at any dose level and the NOAEL for both maternal and fetal toxicity was considered to be 1000mg/kg/day. Two rabbits in the 400mg/kg/day group died prior to scheduled termination. Because no deaths occurred in the high dose group and there were no other treatment-related signs of clinical toxicity in any dose group, these deaths were considered unrelated to nelfinavir. Group mean body weight loss in rabbits was observed at 1000mg/kg/day on gestation days 7-10. Food consumption was also reduced in this treatment group throughout the dosing period. There were no treatment-related findings in other maternal or fetal parameters. Thus, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity in the rabbit was considered to be 400mg/kg/day (based on maternal body weight loss in the high dose group), while the NOAEL for embryo-fetal toxicity in the rabbit was considered to be 1000mg/kg/day. Thus, under the conditions of this study, nelfinavir was not considered to be toxic to the rat or rabbit conceptus.

Absence of reproductive and developmental toxicity in rats following oral dosing with nelfinavir.

The potential for nelfinavir mesylate (VIRACEPT) to produce reproductive toxicity was evaluated in rats administered oral doses of 200, 500, or 1000mg/kg/day. In the fertility and early embryonic development to implantation study, male rats were treated beginning 28 days prior to mating until necropsy and females for 2 weeks prior to mating and through gestation day (GD) 7. In the pre- and postnatal development study, pregnant rats were treated from GD 6 through lactation day (LD) 20. Selected F(1) pups from this study were evaluated in sensory and behavioral tests and were subsequently mated. Pregnant F(1) females were euthanized on GD 20 and their F(2) fetuses were examined. F(1) animals were not directly dosed with the drug. No treatment-related effects were observed on any male reproductive parameters. Administration of nelfinavir did not produce adverse effects on fertility, pregnancy, embryo-fetal development, parturition, or lactation in the F(0) generation. Similarly, no adverse effects of nelfinavir treatment were observed on pre- and postnatal growth, development, reproductive performance and embryo-fetal development in the F(1) offspring. Based on the results of this study, the no-observed-adverse-effect-level (NOAEL) for developmental and reproductive toxicity in rats was considered to be 1000mg/kg/day, the highest dose tested.