Two-generation Reproductive Toxicity Research Articles

A modular one-generation reproduction study as a flexible testing system for regulatory safety assessment

The European Union's Registration, Evaluation and Authorisation of Chemicals (REACH) legislation mandates testing and evaluation of approximately 30,000 existing substances within a short period of time, beginning with the most widely used "high production volume" (HPV) chemicals. REACH testing requirements for the roughly 3000 HPV chemicals specify three separate tests for reproductive toxicity: two developmental toxicity studies on different animal species (OECD Test Guideline 414) and a two-generation reproduction toxicity study (OECD TG 416). These studies are highly costly in both economic and animal welfare terms. OECD TG 416 is a fertility study intended to evaluate reproductive performance of animals in the P and F1-generations following repeated exposure to a test substance. It can also be used to detect adverse effects on structural and functional development. Thus, it has conventionally been preferred to the one-generation study (OECD TG 415). Recently, the Agricultural Chemical Safety Assessment (ACSA) Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) proposed that routine two-generation studies could in most cases be replaced with an "enhanced" one-generation study (Reuter et al. [1]). The flexible design proposed by HESI-ACSA allows for the addition of one or more specialised modules, if triggered (e.g. production of a second generation or the investigation of classical developmental toxicity or developmental neuro- or immunotoxicity). Significantly, however, the HESI-ACSA proposal was designed for use in the safety assessment of pesticidal, as opposed to industrial, chemicals. Thus for the purposes of REACH, a streamlined one-generation study that also examines structural development would be the most efficient means of addressing current information requirements for HPV chemicals. This study represents a flexible testing system that can be modified to meet regulatory needs in a variety of sectors.

Safety of a novel oxygen-coordinated niacin-bound chromium(III) complex (NBC): I. Two-generation reproduction toxicity study

The objective of this study was to evaluate the effects of a novel oxygen-coordinated niacin-bound chromium(III) complex (NBC) on the reproductive systems of male and female rats, the postnatal maturation and reproductive capacity of their offspring, and possible cumulative effects through multiple generations. Sprague-Dawley rats were maintained on feed containing NBC at dose levels of 0, 4, 15, or 60ppm for 10weeks prior to mating, during mating, and, for females through gestation and lactation, across two generations. For the parents (F(0) and F(1)) and the offspring (F(1) and F(2a)), reproductive parameters such as fertility and mating, gestation, parturition, litters, lactation, sexual maturity and development of offspring were assessed. Results from the current study indicated that dietary exposure of NBC to parental male and female rats of both (F(0) and F(1)) the generations during the premating and mating periods, for both sexes, and during gestation and lactation in case of female rats, did not cause any significant incidence of mortality or abnormal clinical signs. Compared to respective controls, NBC exposure did not affect reproductive performance as evaluated by sexual maturity, fertility and mating, gestation, parturition, litter properties, lactation and development of the offspring. Based on the findings of this study, the parental as well as the offspring no-observed-adverse-effect level for NBC was determined to be greater than 60ppm in diet or equivalent to 7.80 and 8.31mg/kg body weight/day in male and female rats, respectively.

Safety of an oxygen-coordinated niacin-bound chromium(III) complex (NBC): II. Developmental toxicity study in rats

The objective of the present study was to evaluate the teratogenic potential of a novel oxygen-coordinated niacin-bound chromium complex (NBC) in Sprague-Dawley rats. Due to its potential to affect fat synthesis and reduce food intake, processes which are often crucial in normal fetal development, this teratology study was undertaken as part of a multi-generation reproductive investigation. The animals in this study were selected randomly after weaning from each F(2b) litter of the F1 generation from the two-generation reproductive toxicity study. To start the teratology study, Sprague-Dawley rat pups ( approximately 30/sex/group) from the F(2b) generation were allowed to grow up to 10-12 weeks of age before mating. The rats in treatment group were exposed directly to NBC through feed. The dietary exposure levels were the same as those employed for the two-generation reproductive toxicity study, viz. 4, 15, or 60 ppm. Following mating at maturity, the pregnant rats were observed daily for clinical signs of adverse effects, and body weight and feed consumption were recorded. On the day 20th of the gestation, animals were subjected to a necropsy and caesarean section to examine the uterus, ovaries and fetuses for assessment of different parameters of pregnancy and embryo-fetal defects. In this study, no indications of maternal toxicity, adverse effects on the parameters evaluated for the gravid uteri, external abnormalities in the fetuses, soft tissue abnormalities in the fetuses, or skeletal abnormalities in the fetuses were noted. Based on the results of this developmental toxicity study, NBC was found to benon-teratogenic in Sprague-Dawley rat, at the dietary exposure levels of 4, 15, and 60 ppm, equivalent to the dose levels of 0.50, 2.0, or 8.0mg/kg/day, respectively.

Two-Generation Reproductive Toxicity Study of Implanted Depleted Uranium (DU) in CD Rats

Depleted uranium (DU) munitions and armor plating have been used in several conflicts over the last 17 yr, including the Persian Gulf War and the Iraq War. Because of its effectiveness and availability, DU will continue to be used in military applications into the foreseeable future. There is much controversy over the use of DU in weapons and equipment because of its potential radiological and toxic hazards, and there is concern over the chronic adverse health effects of embedded DU shrapnel in war veterans and bystanders. This study evaluated the effects of long-term implantation of DU on the reproductive success of F0 generation adults and development and survival of subsequent F1 and F2 generations in a two-generation reproductive toxicity study. F0 generation Sprague-Dawley rats, 8 wk of age, were surgically implanted with 0, 4, 8, 12, or 20 DU pellets (1 × 2 mm). Inert implant control animals were implanted with 12 or 20 tantallum (Ta) pellets. The F0 generation was then mated at 120 d post DU implantation. In the F0 generation, when measured on postimplantation d 27 and 117, uranium was present in the urine of DU‐implanted animals in a dose-dependent manner. F0 reproductive success was similar across treatment groups and the maternal retrieval test revealed no changes in maternal behavior. DU implantation exerted no effect on the survival, health, or well-being of the F0 generation. Necropsy results of F0 animals were negative with the exception of a marked inflammatory response surrounding the implanted DU pellets. For the F1 generation, measures of F1 development through postnatal day (PND) 20 were unremarkable and no gross abnormalities were observed in F1 offspring. No uranium was detected in whole-body homogenates of PND 4 or PND 20 pups. Necropsy findings of F1 PND 20 pups were negative and no instances of ribcage malformation were observed in F1 PND 20 pups. Body weight and body weight gain of F1 rats through PND 120 were similar across treatment groups. Eight of 414 F1 animals observed from PND 20 to 120 died of unknown causes; 7 were from litters of DU-implanted F0 mating pairs. F1 mating success at 10 wk of age was an overall 70% compared with 91% for F0 mating pairs. Mating success was similar between F1 animals derived from DU-implanted F0 adults and those derived from F0 implant control adults suggesting that the comparatively low mating success was not due to F1 DU exposure. The gestational index of F1 animals derived from mid-dose F0 mating pairs was found to be lower compared with F1 controls. The average gestation duration of F1 animals derived from high-dose F0 mating pairs was found to be significantly longer than F1 controls. F1 sperm motility analyses did not differ among experimental groups and no gross abnormalities were identified at necropsy among surviving F1 animals at PND 120. Histopathology of kidneys, spleen, thymus, bone marrow, ovaries, and testes of F1 high-dose animals did not differ from F1 controls. F1 high-dose females had significantly higher mean relative liver and heart weights compared with F1 controls; the biological relevance of this finding could not be determined. For the F2 generation, measures of F2 development through PND 20 were unremarkable and no gross abnormalities were observed in F2 offspring. Necropsy findings of F2 PND 20 pups were negative and no instances of ribcage malformation were observed in F2 PND 20 pups. Body weight and body weight gain of F2 rats through PND 90 were similar across treatment groups. Mean relative heart weights of males derived from high-dose F0 parents were significantly lower compared with F2 controls. Sperm motility and concentration analysis of F2 males at PND 90 were similar across F2 groups. Overall, the consistent absence of positive findings in this study seems to suggest that DU is not a significant reproductive or developmental hazard, particularly when one considers that mid- and high-dose rats were implanted with the equivalent of 0.3 and 0.5 lb of DU in a 70-kg human, respectively. However, the findings that seven of eight F1 adults that died postweaning were from DU-implanted F0 mating pairs, and that mean relative heart weights were elevated in high-dose F1 and F2 pups, suggest conservatism is warranted in characterizing the reproductive and teratogenic hazards of embedded DU until further studies are completed.

Longer Rodent Bioassay Fails to Address 2-Year Bioassay’s Flaws

In their commentary, Huff et al. (2008) proposed that exposing experimental animals to test substances in utero and for 30 months or until their natural deaths increases the sensitivity of bioassays, avoids false negative results, and strengthens the value and validity of results. Instead, longer exposure results in increased numbers of spontaneously arising tumors, as well as increased cost and animal suffering, while failing to address the bioassay’s fundamental flaws. Although it is troubling when the bioassay produces false negative results, a far more pervasive problem is that of false positives. In our analysis of > 500 National Toxicology Program (NTP) bioassays [People for the Ethical Treatment of Animals (PETA) 2006], we found that more than half of the substances evaluated (259) produced evidence of carcinogenicity in at least one group of animals, but only about one-third of these (89) were subsequently classified as known or probable human carcinogens by the NTP itself. Even fewer of the substances, 40 and 16, respectively, were classified as carcinogens by the U.S. Environmental Protection Agency (EPA) and the International Agency for Research on Cancer (PETA 2006). This high false-positive rate is thought to be largely an indirect effect of increased cell proliferation in response to cell injury and death caused by the near toxic doses of test substances used in the bioassay (Gaylor 2005). Species-specific modes of action operating in rats or mice but not in humans, such as those mediated by 2μ-globulin, peroxisomes, and thyroid-stimulating hormone, also contribute to the high rate of false positives (Cohen 2004). Huff et al. (2008) asserted that one of the “well-accepted observations” upon which the “relevance of experimental bioassays to humans” rests is that “findings from independently conducted bioassays on the same chemicals are consistent.” In fact, in a comparison of 121 bioassays from the NTP database with those in the published scientific literature, Gottmann et al. (2001) found that the studies produced consistent results only 57% of the time. Huff et al. (2008) cited questions about the safety of aspartame raised by 3-year bioassays conducted by the Ramazzini Foundation to support their conclusions. Although they noted that the European Food Safety Authority (FSA) and the U.S. Food and Drug Administration (FDA) dispute these studies’ conclusions, the FSA’s Committee on Carcinogenicity (COC 2006) observed that In view of the inadequacies in design of the [Ramazzini Foundation] study and the use of rats with a high concurrent infection rate, the COC considered that no valid conclusions could be derived from it. Further, the COC noted that groups of animals fed aspartame had lower body weights and thus lived longer, which may have compromised the results by leading to an apparent increase in spontaneously arising tumors. Considering that lower body weights are typically observed among animals in the bioassay’s experimental groups, this is likely to generally confound the interpretation of longer bioassays. We must stress that animals suffer during the bioassay: They live in the barren, stressful conditions of the laboratory—often including daily forced feeding or inhalation—and many also suffer from exposure to near toxic doses of test substances. These exposures often produce lethargy, anemia, diarrhea, weight loss, and other symptoms of sickness and distress. The proposal of Huff et al. (2008) to extend the length of the bioassay would obviously result in a proportional increase in this suffering. Further, extending the bioassay runs counter to current trends in regulatory testing. Concern for the suffering of animals has caused regulatory agencies to review the usefulness of long-term studies, resulting in elimination of the 1-year dog toxicity test (U.S. EPA 2007) and an international effort to replace the two-generation reproductive toxicity test (Cooper et al. 2006). Huff et al.’s proposal thus clearly represents a step backward for toxicological science. According to the NTP’s own estimates, each bioassay requires 5 years to plan, conduct, and evaluate; 860 animals to be killed; and $2–$4 million. As a result, the NTP has conducted an average of only 12 bioassays/year over the past several decades. Considering that humans are thought to be exposed to approximately 80,000 environmental toxicants (Ward et al. 2003), it would take more than 32 millenia, 68 million animals, and $160 billion to test them all at this rate. Once again, extending the length of the bioassay would only increase these already ridiculous numbers. The time has clearly come for antiquated animal tests such as the bioassay to be abandoned in favor of modern, human-relevant methods such as epidemiologic studies, high-throughput in vitro methods, and computational toxicology.

Study on the two-generation reproductive toxicity of paclobutrazol in SD rats

Study on the two-generation reproductive toxicity of paclobutrazol in SD rats

Safety assessment of dietary diacylglycerol oil: A two-generation reproductive toxicity study in rats

Diacylglycerol (DAG) oil is a novel edible oil with similar taste and usability characteristics as conventional edible oils. Recent studies suggest that DAG oil may be helpful in the prevention and management of obesity. The objective of the present two-generation study was to evaluate potential adverse effects of DAG oil on reproductive processes. DAG oil was administered via gavage to rats (30/sex/group) for at least 70 days prior to mating, at dose levels of 0, 1.25, 2.5 or 5.0 ml/kg/day (0, 1160, 2320 and 4630 mg/kg/day). An additional group received a triacylglycerol (TAG) oil with a similar fatty acid composition to DAG oil. The rats were treated throughout the mating, gestation and lactation periods. Administration of DAG or TAG oil did not reveal any toxicologically significant effects on reproductive performance (mating, fertility and copulation/conception indices). DAG oil did not affect mean gestation lengths, the process of parturition, spermatogenic parameters, organ weights, histopathologic findings, mean numbers of pups born, implantation sites and unaccounted sites. F1 and F2 pup viability, live litter sizes, body weights, mean age of attainment of balanopreputial separation and vaginal patency were similar to those in the control group. Based on the results of this study, a dose level of 5.0 ml/kg (4630 mg/kg/day) was considered as the no-observed-adverse-effect level for reproductive and systemic toxicity, and neonatal toxicity.

An analysis of the results from Two-Generation Reproduction Toxicity Studies to assess the value of mating animals of the second (F1) generation for the detection of adverse treatment-related effects on reproductive performance

The need to routinely perform a two-generation study to assess reproductive performance is under debate. This review has analysed the results from 22 consecutive Two-Generation Reproduction Toxicity Studies performed within our laboratories, to determine the value of mating each generation in the detection of treatment-related effects on reproductive performance. The chemicals included 13 agrochemicals, 5 industrial chemicals and 4 food additives. Four chemicals (all from the agrochemical group) were found to have clear treatment-related effects and in three studies effects were confined to the second (F1) generation. For the three studies with second (F1) generation effects, we applied the triggers, proposed by the Agricultural Chemical Safety Assessment (ASCA) Technical Committee of HESI, for extension of the one-generation study and found that mating of the second (F1) generation would have been triggered in these studies by effects on the F1 offspring growth and sexual maturation. No effects were seen in the original parental (F0) generation which would have activated the change from a one- to a two-generation study.

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.

Safety of a Novel Calcium/Potassium Saltof (-)-Hydroxycitric Acid (HCA-SX): II.Developmental Toxicity Study in Rats

ABSTRACT(-)-Hydroxycitric acid (HCA), active constituent (10%–30%) of the dried fruit rind of Garcinia cambogia, is commonly used as a dietary supplement for weight management. The objective of the present study was to evaluate the teratogenic potential of a novel calcium/potassium salt of HCA (HCA-SX) in Sprague-Dawley rats. Due to its potential to affect fat synthesis and reduce food intake, processes that are often crucial in normal fetal development, this teratology study was undertaken as part of a multigeneration reproductive investigation. The animals in this study were selected randomly after weaning from each F2b litter of the F1 generation from the two-generation reproductive toxicity study. To start the teratology study, Sprague-Dawley rat pups (∼30/sex/group) from the F2b generation were allowed to grow up to 10 to 12 weeks of age before mating. The rats in the treatment group were exposed directly to HCA-SX through feed, while prior to their weaning, they had indirect exposure to the test material during lactation. The dietary exposure levels were the same as those employed for the two-generation reproductive toxicity study, viz. 1000, 3000, or 10,000 ppm. Following mating at maturity, the pregnant rats were observed daily for clinical signs of adverse effects, and body weight and feed consumption were recorded. On day 20 of gestation, animals were subjected to a necropsy and cesarean section to examine the uterus, ovaries, and fetuses for assessment of different parameters of pregnancy and embryo-fetal defects. Despite a slight (13%) lowering of maternal body weight gain during gestation period in the group receiving 10,000 ppm HCA-SX, no evidence of maternal toxicity, adverse effects on the parameters evaluated for the gravid uteri, external abnormalities in the fetuses, soft tissue abnormalities in the fetuses, or skeletal abnormalities in the fetuses were noted. Based on the results of this developmental toxicity study, conducted in continuation of a two-generation reproductive toxicity study, HCA-SX was not found to be teratogenic in the Sprague-Dawley rat at the dietary exposure levels of 1000, 3000, and 10,000 ppm, equivalent to the dose levels of 103, 352, or 1240 mg/kg/day, respectively.

Routes and Modes of Administration of Resorcinol and Their Relationship to Potential Manifestations of Thyroid Gland Toxicity in Animals and Man

Medical case reports published in the 20th century over the course of several decades show that resorcinol caused reversible adverse effects on the human thyroid gland (TG) manifested as hypothyroidism. Affected patients had ulcerating leg varicosities and underwent prolonged treatment with ointments containing high concentrations of resorcinol. In animal studies resorcinol failed to induce TG toxicity, unless pharmacokinetic/toxicokinetic (PK/TK) conditions were manipulated (e.g., injection of resorcinol in oil or application in a slow release formulation). A recently completed two-generation reproductive toxicity study in rats did not detect any adverse effects on either reproductive or TG end points (Welsch, Nemec, and Lawrence, 2008, Int. J. Toxicol. 37, this issue). Resorcinol intake via drinking water up to the palatability limit had resulted in average daily intakes (mg/kg) of 233 in F0 and F1 males and 304 (premating/gestation) or 660 (lactation) in females. Free resorcinol in blood plasma was barely detectable in a few parental animals, indicating rapid metabolism. This short review communication offers a perspective on compromised human skin barrier function as a likely cause of drastic increases in resorcinol absorption. In conjunction with multiple daily applications over many months to hyperemic, inflamed, and lesioned human skin much higher absorption was likely responsible for the reported human TG toxicity.

Two-generation reproductive toxicity study of the rubber accelerator N, N-dicyclohexyl-2-benzothiazolesulfenamide in rats

Male and female Crl:CD(SD) rats were fed a diet containing rubber accelerator N, N-dicyclohexyl-2-benzothiazolesulfenamide (DCBS) at 0, 80, 600 or 4500 ppm throughout the study beginning at the onset of a 10-week pre-mating period and continuing through the mating, gestation, and lactation periods for two generations. At 4500 ppm, decreases in the body weight, body weight gain, and food consumption were found in F0 males and females. No changes in the estrous cyclicity, copulation index, fertility index, gestation index, delivery index, number of implantations, precoital interval, or gestation length were observed in any generation at any dose of DCBS. Delayed preputial separation at 4500 ppm as well as delayed vaginal opening and higher body weight at the age of vaginal opening at 600 and 4500 ppm were found in the F1 generation. A transient change in performance in a water-filled multiple T-maze was found at 600 and 4500 ppm in F1 females. There were no compound-related changes in number of pups delivered, sex ratio of pups, viability of pups, anogenital distance, surface righting reflex, negative geotaxis reflex, mid-air righting reflex, pinna unfolding, incisor eruption, or eye opening in the F1 and F2 generations. The body weight of F1 and F2 male and female pups was lowered at 4500 ppm. Reduced uterine weight of the weanlings was noted in the F1 generation at 4500 ppm and in the F2 generation at 600 and 4500 ppm. The data indicate that the NOAEL of DCBS for two-generation reproductive toxicity is 80 ppm (5.2 mg/kg bw per day) in rats.

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 0.6 mg/(kg day) 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 0.6 mg/(kg day) group F 0 and F 1 males. Higher liver weights, correlating to hepatocellular hypertrophy in the 0.6 mg/kg group, were noted for parental males in the 0.125 and 0.6 mg/(kg day) groups, parental females in the 0.6 mg/(kg day) group and F 1 pups in the 0.125 and 0.6 mg/(kg day) groups. Higher kidney weights, correlating to renal tubule hypertrophy in the 0.6 mg/kg group, were observed for parental males and females in the 0.125 and 0.6 mg/(kg day) 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 2 h following administration to the dams on lactation day 13. A dosage level of 0.6 mg/(kg day) was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive function. A dosage level of less than 0.025 mg/(kg day) 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 0.025 mg/(kg day) was considered to be the NOAEL for neonatal toxicity based on higher liver weights in the F 1 and F 2 pups at 0.125 mg/(kg day) and higher.

A retrospective analysis of the two-generation reproductive toxicity study

A retrospective analysis of the two-generation reproductive toxicity study

Evaluation of two-generation reproductive toxicity of a vulcanization accelerator N, N-dicyclohexyl-2-benzothiazolesulfenamide (DCBS) in rats

Evaluation of two-generation reproductive toxicity of a vulcanization accelerator N, N-dicyclohexyl-2-benzothiazolesulfenamide (DCBS) in rats

Assessment of the Long-Term and Transgenerational Consequences of Perturbing Preimplantation Embryo Development in Mice1

Perturbations of the development of preimplantation embryos may have long-term consequences for the health of progeny. There are no standardized methods for assessing such risks. The OECD/OCDE 416 Guideline for Testing of Chemicals (Two-Generation Reproduction Toxicity Study) is a standardized assay for detecting potential toxic effects of chemicals. The present study assessed the utility of this guideline for identifying long-term consequences of perturbing preimplantation development. Extended culturing of mammalian zygotes commonly results in retarded preimplantation development. Mouse zygotes were cultured in vitro for 96 h until the blastocyst stage (cultured blastocysts) or blastocysts were collected from the Day-3.5 uterus (in vivo blastocysts). The resulting blastocysts were transferred to the uteri of pseudopregnant recipients (P generation). Progeny from both treatments were mated for a further two generations (F1 and F2 generations). There was no effect of treatment group on gross fertility across the generations tested. Progeny of the cultured blastocysts had lower body weights to the time of weaning compared to in vivo blastocysts in the P and F1 generations, but not in the F2 generation. At maturity, there was no effect of treatment group on body weight, although thyroid weight was higher in the in vivo blastocyst group in the P generation, while the brain, pituitary, and kidneys were larger in the progeny of the cultured blastocysts of the F1 generation. The OECD/OCDE 416 assessment may have a role as a standardized test for the assessment of the biological consequences of perturbing the growth environment of the preimplantation embryo. Embryo culture influenced the somatometric parameters of the resulting progeny, some of which were maintained across a generation.

A retrospective analysis of the added value of the rat two-generation reproductive toxicity study versus the rat subchronic toxicity study

This study aims to evaluate the added value of the two-generation reproductive toxicity study when a subchronic study (90-day repeated dose toxicity study) is available. The analysis includes a total of 47 reproductive toxic and 75 non-reproductive toxic substances, for which a two-generation study was available. For each of these compounds the outcomes of both study types were compared, in view of the question what the impact would have been both for the derived NOAEL and for classification regarding toxicity to fertility. On average, only a small difference (less than twofold) in overall NOAELs was found between the rat two-generation study and the rat subchronic study. For individual compounds the differences could be larger (up to around a factor of 10), but differences of this magnitude equally occur between NOAELs of subchronic studies (testing the same substance). The two generation study did have an impact on classification for toxicity to fertility: about one-third of the substances shown to be toxic to fertility in the two-generation study did not show any sign of that in the 90-day study. If the subchronic study did show toxicity to reproductive organs this often occurred at (much) higher doses than other toxic effects in the same study. Therefore, apart from including more fertility endpoints, a larger dose spacing (or more dose groups) in the subchronic study might increase its detection rate of fertility toxic substances. The consequences that these findings may have for risk assessment and risk management are discussed, especially in the context of REACH.

A two-generational reproductive toxicity study of zinc in rats

A two-generation reproductive toxicity study of zinc chloride (ZnCl2) was conducted in rats. Fo male and female rats were administered 0.00 (control), 7.50 (low), 15.00 (mid) and 30.00 (high) mg/kg/day of ZnCl2. Selected F1 male and female rats were exposed to the same doses received by their parents (Fo). Exposure of F0 parental rats to ZnCl2 showed significant reduction in fertility, viability (days 0 and 4), and the body weight of F1 pups from the high-dose group but caused no effects on litter size, weaning index, and sex ratio. Similarly, the continued exposure of F1 parental rats to ZnCl2 also reduced fertility, liter size, viability (day 0), and the body weight of F2 pups within the high-dose group but caused no effects on weaning index and sex ratio. Exposure of ZnCl2 to F0 and F1 parental males resulted in a significant reduction in their body weights, and the F0 and F1 parental females did not show any significant difference in their body weights compared to their control groups. However, the postpartum dam weights of both F0 and F1 female rats were significantly reduced compared to their controls. Exposure of ZnCl2 to Fo and F1 generation parental rats did not produce any significant change of their clinical signs as well as their clinical pathology parameters, except the alkaline phosphotase (ALK) level, which showed an upward trend in both sexes of both generations. Exposure of ZnCl2 to F0 rats resulted in a reduction of brain, liver, kidney, spleen and seminal vesicles weights of males and in the spleen and uterus of females. Similarly, exposure of F1 rats to ZnCl2 also resulted in reduction of brain, liver, kidney, adrenal, spleen, prostate and seminal vesicles weights of males and in spleen and uterus of females. ZnCl2 exposure resulted in grossly observed gastro-intestianla (GI) tract, lymphoreticular/hematopoietic, and reproductive tract lesions in parental rats in both generations. Reduced body fat was also recorded in F1 parental rats.

A two-generation reproductive toxicity study of octamethylcyclotetrasiloxane (D 4) 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 6 h 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.

A two-generation reproductive toxicity study of decamethylcyclopentasiloxane (D 5) 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 6 h/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.