Developmental immunotoxicity of ethanol in an extended one-generation reproductive toxicity study

The susceptibility of developing immune system to chemical disruption warrants the assessment of immune parameters in reproductive and developmental testing protocols. In this study, a wide range of immune endpoints was included in an extended one-generation reproduction toxicity study (EOGRTS) design to determine the relative sensitivity of immune and developmental parameters to ethanol (EtOH), a well-known developmental toxicant with immunomodulatory properties. Adult Wistar rats were exposed to EtOH via drinking water (0, 1.5, 4, 6.5, 9, 11.5 and 14 % (w/v EtOH)) during premating, mating, gestation and lactation and continuation of exposure of the F₁ from weaning until killed. Immune assessments were performed at postnatal days (PNDs) 21, 42 and 70. Keyhole limpet hemocyanin (KLH)-specific immune responses were evaluated following subcutaneous immunizations on PNDs 21 and 35. EtOH exposure affected innate as well as adaptive immune responses. The most sensitive immune parameters included white blood cell subpopulations, ConA-stimulated splenocyte proliferation, LPS-induced NO and TNF-α production by adherent splenocytes and KLH-specific immune responses. Most parameters showed recovery after cessation of EtOH exposure after weaning in the 14 % exposure group. However, effects on LPS-induced NO and TNF-α production by adherent splenocytes and KLH-specific parameters persisted until PND 70. The results demonstrate the relative sensitivity to EtOH of especially functional immune parameters and confirm the added value of immune parameters in the EOGRTS. Furthermore, this study identified an expanded KLH-specific parameter set and LPS-induced NO and TNF-α production by adherent splenocytes as valuable parameters that can provide additional information on functional immune effects.     

Developmental immunotoxicity in male rats after juvenile exposure to di-n-octyltin dichloride (DOTC)

To determine relevant endpoints for evaluating developmental immunotoxicity due to juvenile exposure and optimal age of the animals at assessment, a wide range of immunological parameters were assessed in a juvenile toxicity study. Rats were exposed to di-n-octyltin dichloride (DOTC) by gavage from postnatal day (PND) 10 through PND 21 and via the diet after weaning using a benchmark dose (BMD) approach. Immune assessments were performed in male rats on PNDs 21, 42, and 70 and a subset of animals was used to evaluate the T-cell dependent antibody response (TDAR) to Keyhole limpet hemocyanin. Immune effects were more pronounced on PND 21 and 42 and observed at lower doses than developmental effects. The most sensitive immune parameters affected included TDAR parameters and thymocyte subpopulations with lower confidence limits of the benchmark doses (BMDLs) below the overall no-observed-adverse-effect-level (NOAEL) for DOTC reported so far in literature. These findings illustrate the relative sensitivity of the developing immune system for DOTC, the additional value of assessing functional immune parameters, and underscore the relevance of juvenile immunotoxicity testing in view of the risk assessment of chemicals.     

Feasibility of the extended one-generation reproductive toxicity study (OECD 443)

The extended one-generation reproduction toxicity study (OECD 443, adopted 28-July-2011) produces more information with fewer animals than the two-generation study (OECD 416), by including F1 neurotoxicity and immunotoxicity assessments, and omitting an F2 generation if there are no relevant F1 findings. This saves >1000 animals per compound. Feasibility studies based on draft OECD443 were conducted in industrial GLP laboratories in Europe and USA, using vinclozolin, methimazole and lead acetate. A fourth study was conducted with 2,4-dichlorophenoxyacetic acid (2,4-D) in response to a regulatory request for reproduction and developmental neurotoxicity data. The studies effectively profiled vinclozolin as an anti-androgenic developmental toxicant, methimazole as a developmental anti-thyroid agent, and lead acetate as a systemic and developmental toxicant. The 2,4-D study demonstrated the value of toxicokinetic data in dose setting and data interpretation. These results illustrate the variety of reproductive and developmental endpoints which can be captured in this complex but manageable study design. Time constraints for triggering further (F2) testing are summarized.     

Implementing the extended one-generation reproductive toxicity study (EOGRTS): important points to consider

The hallmark of the extended one-generation reproductive toxicity study (EOGRTS) is that, based on certain criteria or triggers, selected offspring are assigned at weaning to different cohorts for further investigation of sexual maturation, reproductive organ integrity and function, neuropathological and behavioral endpoints, and/or immune function. The triggers allow for a more customizable design based directly on the data, while minimizing animal usage. Compared to the two-generation reproductive toxicity study, the EOGRTS design increases the number, extent, and duration of F₁-offspring assessments resulting in more thorough and efficient utilization of the first generation while excluding the second generation of offspring unless triggered. Therefore, the EOGRTS has the potential to reduce the number of rats required by nearly 1200 animals per study. When performing the EOGRTS, the complexity of this study should not be underestimated and experienced flexible testing laboratories with sufficient resources and historical control data for all parameters are essential. The aim of this review is to discuss the important aspects of this challenging study design and to share our knowledge on the implementation of this study in our laboratories. In addition, we elaborate on the type of criteria for expansion of the study and logistical considerations. Altogether, this review can be used as guidance by other labs, study monitors, and registration officers.     

Lack of covalent binding to DNA of di-n-octyltin dichloride (DOTC) in vivo and in vitro

[¹⁴C]di-n-octyltin dichloride ([¹⁴C]DOTC) was administered by oral gavage to male and female rats. After 96 h hepatic and thymic DNA was isolated. All DNA fractions were radioactive, but analysis of DNA hydrolysates by HPLC revealed that the radioactivity was incorporated via biosynthesis and was not due to adduct formation. The limit of detection for adduct formation, expressed in units of the covalent binding index (CBI = μanol chemical bound per mol nucleotides/mmol chemical applied per kg body wt.) was approximately 0.2 for liver DNA and about 0.7 for thymus DNA. This maximum possible DNAbinding ability is about 30 000 times lower than the corresponding value for the strong carcinogen, aflatoxin b₁. In addition, [¹⁴C]DOTC did not bind covalently to calf thymus DNA in the presence or absence of rat liver S9 or to DNA of V79 Chinese hamster cells. This study therefore gives no indication for genotoxic activity of DOTC mediated by DNA binding.     

Immunological consequences of dioctyltin dichloride (DOTC)-induced thymic injury

Inbred rats fed diets containing 75 ppm dioctyltin dichloride (DOTC) for 8 or 12 weeks demonstrated marked reduction in thymic weight. This reduction was accompanied by a decrease in the number of W3/25 positive phenotypic circulating lymphocytes and an impaired ability to respond to suboptimal mitogenic stimulation. Responsiveness to alloantigenic stimulation, as measured by mixed leucocyte reactions, was depressed in DOTC-fed animals whereas no effect on natural cytotoxicity in nonimmunized treated or control rats was evident. No difference was found in the ability of treated compared to untreated animals to mount specific antibody responses against sheep red blood cells, and no evidence was found to indicate toxicity to lymphocytes in vivo. The investigation showed that DOTC administration resulted in a selective immunodeficiency.     

Guidance on the selection of cohorts for the extended one-generation reproduction toxicity study (OECD test guideline 443)

The extended one-generation reproduction toxicity study (EOGRTS; OECD test guideline 443) is a new and technically complex design to evaluate the putative effects of chemicals on fertility and development, including effects upon the developing nervous and immune systems. In addition to offering a more comprehensive assessment of developmental toxicity, the EOGRTS offers important improvements in animal welfare through reduction and refinement in a modular study design. The challenge to the practitioner is to know how the modular aspects of the study should be triggered on the basis of prior knowledge of a particular chemical, or on earlier findings in the EOGRTS itself, requirements of specific regulatory frameworks notwithstanding. The purpose of this document is to offer guidance on science-based triggers for these extended evaluations.     

Developmental toxicity of dibutyltin dichloride in cynomolgus monkeys

Dibutyltin dichloride (DBTCl) has been shown to be teratogenic in rats. The present study was conducted to determine the teratogenic potential of DBTCl given to pregnant monkeys during the entire period of organogenesis. Cynomolgus monkeys were dosed once daily by nasogastric intubation with DBTCl at 0, 2.5 or 3.8 mg/kg on days 20-50 of pregnancy, the whole period of organogenesis. The pregnancy outcome was determined on day 100 of pregnancy. In both DBTCl-treated groups, a significant increase in the incidence of pregnant females with soft stool and/or diarrhea, and with yellowish stool was observed. Maternal body weight gain at 3.8 mg/kg and food consumption at 2.5 and 3.8 mg/kg were decreased during the administration period. The survival rate of fetuses at terminal cesarean sectioning was decreased in the DBTCl-treated groups and significantly decreased at 2.5 mg/kg. There were no changes in the developmental parameters of surviving fetuses, including fetal body weight, crown-rump length, tail length, sex ratio, anogenital distance and placental weight, in the DBTCl-treated groups. No external, internal or skeletal malformations were found in the fetuses in any group. Although internal and skeletal variations were found, no difference in the incidence of fetal variation was noted between the control and DBTCl-treated groups. No effect on skeletal ossification was observed in fetuses in the DBTCl-treated groups. The data demonstrate that DBTCl is embryolethal but not teratogenic in cynomolgus monkeys.     

Lack of covalent binding to DNA of di-n-octyltin dichloride (DOTC) in vivo and in vitro

[¹⁴C]di-n-octyltin dichloride ([¹⁴C]DOTC) was administered by oral gavage to male and female rats. After 96 h hepatic and thymic DNA was isolated. All DNA fractions were radioactive, but analysis of DNA hydrolysates by HPLC revealed that the radioactivity was incorporated via biosynthesis and was not due to adduct formation. The limit of detection for adduct formation, expressed in units of the covalent binding index (CBI = μanol chemical bound per mol nucleotides/mmol chemical applied per kg body wt.) was approximately 0.2 for liver DNA and about 0.7 for thymus DNA. This maximum possible DNAbinding ability is about 30 000 times lower than the corresponding value for the strong carcinogen, aflatoxin b₁. In addition, [¹⁴C]DOTC did not bind covalently to calf thymus DNA in the presence or absence of rat liver S9 or to DNA of V79 Chinese hamster cells. This study therefore gives no indication for genotoxic activity of DOTC mediated by DNA binding.     

Toxic effects of di-n-octyltin dichloride on the thymus in mice.

The effects of di-n-octyltin dichloride (DOTC) on the thymus in mice were studied in dependence on the route of administration. Single administrations of 30, 60 and 120 mg/kg of DOTC i.m. induced a dose-related decrease of the thymus weight and of the number of nucleated cells in the thymus of male mice. 4 days after intravenous injection of 8 mg/kg of DOTC the thymus weight and the number of thymocytes were reduced to 50 percent of the control. In oral route of administration higher doses of DOTC (300 mg/kg) were necessary to provoke a thymus atrophy. After pretreatment of mice with carbon tetrachloride the toxic effects of orally administered DOTC were increased. The reduction of the thymus weight and of the number of thymocytes in mice after parenteral administration was accompanied by a depression of thymus-dependent antibody formation against sheep red blood cells.     

The absorption, tissue distribution and excretion of di-n-octyltin dichloride in rats

In this study the absorption, tissue distribution and excretion of 14C-labeled di-n-octyltin dichloride ([14C]DOTC) in rats were investigated after oral and intravenous (i.v.) administration. Although after i.v. administration with 1.2 mg [14C]DOTC/kg body weight the tissue radioactivity was about 3-4 times higher than after oral administration with 6.3 mg [14C]DOTC/kg body weight, the relative tissue accumulation was found to be the same after the oral and i.v. dosage. The highest amount of radioactivity was found in liver and kidney, and to a lesser degree in adrenal, pituitary and thyroid glands. The lowest activity was recovered from blood and brain. No selective accumulation was observed in thymus, although it has been reported that thymus atrophy is the most sensitive parameter of DOTC toxicity in rats. For all tissues a time dependent decrease in radioactivity was found, except for kidney. The excretion of radioactivity in feces and urine was determined after a single i.v. or oral dose of 1.2 and 2 mg [14C]DOTC, respectively. After i.v. administration most of the radioactivity was excreted in the feces which was characterized by a biphasic excretion pattern. In orally treated rats more than 80% of the radioactivity was already excreted in the feces during the first day after administration. This indicated that only a small part of the DOTC was absorbed, which was calculated to be approximately 20% of the dose. Similar half-life values of 8.3 and 8.9 days were obtained from the fecal excretion of radioactivity after the i.v. and oral administration, respectively. The urinary excretion of radioactivity appeared to be independent of the body burden, since the daily amount of radioactivity excreted in urine was nearly the same independent of the route of administration as well as the time after administration.     

Developmental toxicity study of tetramethylurea (TMU) in rats.

The developmental toxicity of tetramethylurea (TMU) was assessed in rats by inhalation exposure of the test material over days 6-20 of gestation. Groups of 25 mated female Crl:CD BR rats were exposed whole-body for 6 hours/day to concentrations of either 0, 2, 20 or 100 ppm TMU. The dams were euthanized on day 21 and the offspring were weighted, sexed, and examined for external, visceral, and skeletal alterations. Maternal toxicity was demonstrated at both 20 and 100 ppm. Maternal body weights, weight changes, and food consumption were statistically significantly reduced at these concentrations; effects were more pronounced at 100 ppm. There was evidence of developmental toxicity only at 100 ppm. The only finding was a decrease in mean fetal weight. No fetal malformations or variations occurred in fetuses derived from rats exposed to all 3 test concentrations (up to 100 ppm). The maternal no-observed-effect-level (NOEL) was 2 ppm, the fetal NOEL was 20 ppm. Thus, TMU was not considered to be uniquely toxic to the rat conceptus.     

Endocrine effects of tetrabromobisphenol-A (TBBPA) in Wistar rats as tested in a one-generation reproduction study and a subacute toxicity study

Endocrine effects of the brominated flame retardant tetrabromobisphenol-A (TBBPA) were studied in a one-generation reproduction assay in Wistar rats via repeated dietary exposure, applying eight dose groups at 0-3-10-30-100-300-1,000-3,000 mg/kg body weight/day (mkd). This design enables dose-response analysis and calculation of benchmark doses (BMDL). This reproduction study was preceded by a 28-day repeat dose subacute toxicity study, at 0-30-100-300 mkd. Major effects in the reproduction study included decreased circulating thyroxine (T4) with BMDLs of 31 (m) and 16 (f) mkd, and increased weight of testis and male pituitary (BMDLs of 0.5 and 0.6 mkd). The hypothyroxinemia correlated to a cluster of developmental parameters including delayed sexual development in females, decreased pup mortality, and effects on brainstem auditory evoked potentials [Lilienthal, H., Verwer, C.M., Van der Ven, L.T.M., Piersma, A.H., Vos, J.G., 2008. Neurobehavioral effects of tetrabromobisphenol A (TBBPA) in rats after pre- and postnatal exposure. Toxicology]. A second cluster of parameters in F1 animals was correlated to increased testis weight, and included female gonad weight, endometrium height, CYP19/aromatase activity in the ovary, and plasma testosterone levels in males. These two correlation clusters suggest a dual action of TBBPA. The only effects in the subacute study were decreased circulating T4 and increased T3 levels in males (BMDLs 48 and 124mkd), and non-significant trends for these parameters in females, suggesting that the other effects in the reproduction study were induced during development. Combined with data of human exposure to environmental TBBPA, the margin of exposure for highly exposed populations can be calculated at 2.6, and current use of TBBPA may therefore be a matter of concern for human health.     

Developmental and reproductive toxicity testing of vaccines

The majority of new preventative and therapeutic vaccines are now assessed for developmental toxicity according to guidelines issued by the FDA in 2006. Despite the absence of confirmed effects in humans, vaccines are frequently suspected of having adverse side-effects on the development of children. Such suspicions are perhaps unavoidable considering the extremely widespread use of vaccines. The preclinical developmental toxicology studies are designed to assess possible influences of each component of the vaccine formulation-and the induced antibodies-on the development of the conceptus, neonate and suckling organism. Immune modulation by a vaccine or an adjuvant could, for instance, affect the outcome of pregnancy by interfering with the natural shift in immune balance of the mother during gestation. Maternal immunoglobulins are transferred from the mother to the offspring in order to confer passive immunity during early life. This maternal antibody transport is prenatal in humans and monkeys, but tends to be delayed until after birth in other species. Therefore, a suitable model species needs to be chosen for preclinical studies in order to ensure exposure of the foetus to the induced maternal antibodies following vaccination. Rabbits are the best laboratory model for prenatal immunoglobulin transfer, but rodents are more practical for the necessary postnatal investigations. Non-human primates are the only appropriate models for the testing of vaccines that are not immunogenic in lower species. It is advisable to test new adjuvants separately according to the ICH S5(R2) guidelines. Preclinical paediatric investigations are not currently required for vaccines, even though most vaccines are given to children. Other areas of regulatory concern include developmental immunotoxicity and effects on the preimplantation embryo. Because of the limitations of the available animal models for developmental toxicity testing, pharmacovigilance is essential.     

Developmental and reproductive toxicity evaluation of toluene vapor in the rat II. Developmental toxicity

The developmental toxicity of toluene was evaluated via whole body inhalation exposure, in pregnant Sprague Dawley rats exposed to toluene (99.9% pure) from gestation day (GD) 6–15 inclusive, 6 h/day, at concentrations of 0, 250, 750, 1500 and 3000 ppm (0, 938, 2812, 5625 and 11250 mg/m³). Doses were selected from a preliminary study performed over a range of concentrations from 0 to 5000 ppm, in which maternal and fetal toxicity were observed at 2000 ppm and above. This study has been cited in various regulatory documents and is presented here to allow greater accessibility to results and conclusions.

Toluene induced clinical signs in pregnant dams (ataxia, hyper-responsivity, increased water intake, decreased food consumption) at 3000 ppm, ataxia and hyper-responsivity at 1500 ppm, and reduced maternal body weight gain at 1500 during the exposure period only and at 3000 ppm from initiation of exposure to GD20. At Caesarean section on GD20, no adverse effects on implantation, number and viability of fetuses, or fetal sex distribution were observed. Litter weight and mean fetal weight was reduced at 3000 ppm and mean fetal weight was reduced at 1500 ppm. Instances of reduced or unossified skeletal elements occurred at the same dose levels. Mean fetal weight was also reduced at 250 ppm but not at 750 ppm. Extensive statistical analysis of fetal body weight data support the conclusion that there is no toxicologically significant dose-related effect on fetal body weight at or below 750 ppm. Low incidences (≤2.5%) of various malformations occurred in the 250, 1500, and 3000 ppm groups; there was no increase in the incidence of specific or total malformations with increased exposure and thus these were not attributed to toluene.

In this Toluene study, the maternal toxicity NOAEL was 750 ppm with a defined maternal and developmental toxicity LOAEL of 1500 ppm.     

Developmental toxicity but no immunotoxicity in the rat after prenatal exposure to diethylstilbestrol

Studies with dioxins and PCB's have shown that the developing immune system may be especially vulnerable to xenobiotics during the perinatal period. However, current guidelines for reproductive toxicity testing do not include immune parameters. In the present study, we have explored the usefulness of including immune parameters within the prenatal developmental toxicity study in rats, using the treatment protocol as described in the OECD 414 developmental toxicity test guideline. In addition, the experimental protocol was enhanced by including ten dose groups to facilitate dose-response analysis. Diethylstilbestrol (DES) was used as the model compound, as it is known to be toxic both for embryofetal development and for the immune system. The results show developmental toxicity in terms of decreased fetal survival and decreased pup body weight in the presence of reduced maternal food consumption and reduced body weight gain. However, immune parameters, including histopathology, hematology, and antibody responses to sheep red blood cells (SRBC) in pups at 4 weeks of age were uncompromised. It is speculated that rather than the prenatal exposure protocol used here, the generation study design with both pre- and postnatal exposure may be preferable as a general screen to detect developmental immunotoxic injury after xenobiotic exposure.     

Developmental immunotoxicity of atrazine in rodents

There is a substantial literature reporting that the developing immune system is more sensitive to toxic insult and that the measurable phenotype resulting from prenatal/neonatal exposure often differs from that seen in adult exposure models (reviewed in Holladay and Steven, and Smialowicz et al.). Atrazine is a common herbicidal contaminant of groundwater in agricultural areas in the USA. The potential immunotoxicity of atrazine has been extensively studied using adult-exposure models; however, few studies have explored its immunotoxicity in a prenatal and/or lactational exposure model. Prenatal/lactational atrazine exposure affects the function of young adult rodent immune systems in both sex- and age-dependant manners. In our studies, the humoural and cell-mediated immune responses of offspring from atrazine-exposed dams were assessed at two ages, 3 and 6 months of age to test the hypothesis that prenatal/lactational atrazine exposure would cause greater health complications as the mice aged. Male offspring showed a significant immunopotentiation at three moa that was not apparent at 6 months. Three-month-old female offspring showed no significant difference in immune response from controls. However, at 6 months, female litter mates showed a significant depression in their immune function. These results indicate a decreasing trend in immune capacity. Rooney et al. showed a significant depression of the immune function of young male rat exposure prenatally and lactationally to atrazine. These results demonstrate a sex- and age-dependant effect of prenatal exposure to atrazine on the immune system of the adult offspring using two rodent strains.     

Subchronic immunotoxicity and screening of reproductive toxicity and developmental immunotoxicity following single instillation of HIPCO-single-walled carbon nanotubes: purity-based comparison

Impurity has been suggested as an important factor determining toxicity following exposure to single-walled carbon nanotubes (SWCNTs). In this study, we first compared immunotoxicity based on iron content on day 90 after a single intratracheal instillation of SWCNTs in male and female mice. The inflammatory responses were generally stronger in mice exposed to acid-purified (P)-SWCNTs compared to raw (R)-SWCNTs. In addition, both R- and P-SWCNTs induced Th1-polarized immune responses with apoptotic death of BAL cells and systemically impaired the function of antigen-presenting cells (APC). We also screened reproductive and developmental toxicity by cohabitating male and female mice on day 14 after instillation. Interestingly, the pregnancy rate rapidly decreased following exposure to both types of SWCNTs, especially R-SWCNTs. In addition, we investigated developmental immunotoxicity of the offspring on day 28 after exposure to both types of SWCNTs. Their hematological changes were clearer relative to those of the parents and a significant decrease in the alkaline phosphatase and potassium levels was observed in mice of both sexes exposed to the higher dose of R- and P-SWCNTs. In conclusion, we suggest that SWCNTs may induce Th1-polarized immune responses accompanied by suppression of APC function on day 90 after a single instillation without significant iron content dependance. In addition, the consecutive exposure of SWCNTs to the subsequent generation may exacerbate metabolic and hematological disturbance. Furthermore, our results underscore the need to clarify the reproductive and developmental health effects of SWCNTs.