Extended One-generation Reproductive Toxicity Research Articles

Differences in endocrine and reproductive responses to substance exposure across generations: highlighting the importance of complementary findings.

This article analyzes the results from 112 Extended One-Generation Reproductive Toxicity studies. The objective was to determine if test animals show consistent endocrine and reproductive effects within the same and across different generations and life stages. The analysis, grounded in a comprehensive Binary Matrix, included 530 observed effects and 193 unique, statistically significant associations. Associations' strength was quantified using Jaccard (J) coefficients to measure effect co-occurrence in the same study. Associated effects co-occur infrequently across the whole dataset (median J = 0.231). However, specific patterns emerged: associations of same effects across generations exhibited a higher strength (median J = 0.400) compared to associations of different effects (median J = 0.222). Notably, associations with effects observed in both the parental animals of the adult first filial generation (P1) and developing second filial generations (dF2) demonstrated J coefficients (with medians ranging from 0.300 to 0.430) that were approximately twofold higher than those of other associations. Consistently, equivalent life stage associations across generations revealed statistically significant higher association strengths for the P1 and dF2 generations (medians of 0.375 and 0.333, respectively) compared to other generations (medians of 0.200 and 0.174), possibly due to longer exposure duration and altered cross-talk between pregnant P1 dam and its conceptus. Overall, it is concluded that co-occurrence of associated effects in the same study is rather infrequent and that associations with effects in P1 and dF2 are stronger than all other associations. In general, the findings underscore the importance of independently analyzing each effect per generation due to the generally low co-occurrence rates of associated effects, challenging traditional expectations of generational continuity in toxic effects.

‘It is the Dose That Makes the Poison’ – A Critical View on Dose Level Setting in Extended-One Generation Reproductive Toxicity (EOGRT) Studies

Adequate dose level setting is essential for the evaluation of the toxicity of a substance, meaning that the dose level should not be too low to miss the potential of a substance to exert any toxic effects; it should also not be too high to induce various effects related to systemic toxicity that would hamper a proper evaluation of specific toxicity endpoints such as reproduction and fetal development. This discussion paper is meant to highlight the challenges faced by industry as a result of ECHA’s advice to select high dose levels when conducting EOGRT studies. In our view, in its concern for finding all possible hazards, ECHA is focused on testing at dose levels that may be too high for repeat dose toxicity studies. Such high dose levels may lead to an unethical increase in animal use and animal suffering, an inability to correctly interpret results and unacceptable requests for the repetition of studies leading to an even higher demand for experimental animals.

Perinatal exposure to the immune-suppressant di-n-octyltin dichloride affects brain development in rats

Disruption of the immune system during embryonic brain development by environmental chemicals was proposed as a possible cause of neurodevelopmental disorders. We previously found adverse effects of di-n-octyltin dichloride (DOTC) on maternal and developing immune systems of rats in an extended one-generation reproductive toxicity study according to the OECD 443 test guideline. We hypothesize that the DOTC-induced changes in the immune system can affect neurodevelopment. Therefore, we used in-vivo MRI and PET imaging and genomics, in addition to behavioral testing and neuropathology as proposed in OECD test guideline 443, to investigate the effect of DOTC on structural and functional brain development. Male rats were exposed to DOTC (0, 3, 10, or 30 mg/kg of diet) from 2 weeks prior to mating of the F0-generation until sacrifice of F1-animals. The brains of rats, exposed to DOTC showed a transiently enlarged volume of specific brain regions (MRI), altered specific gravity, and transient hyper-metabolism ([18F]FDG PET). The alterations in brain development concurred with hyper-responsiveness in auditory startle response and slight hyperactivity in young adult animals. Genomics identified altered transcription of key regulators involved in neurodevelopment and neural function (e.g. Nrgrn, Shank3, Igf1r, Cck, Apba2, Foxp2); and regulators involved in cell size, cell proliferation, and organ development, especially immune system development and functioning (e.g. LOC679869, Itga11, Arhgap5, Cd47, Dlg1, Gas6, Cml5, Mef2c). The results suggest the involvement of immunotoxicity in the impairment of the nervous system by DOTC and support the hypothesis of a close connection between the immune and nervous systems in brain development.

Is there any added value of the second generation in the Extended One-Generation Reproductive Toxicity Study (EOGRTS)? A retrospective analysis of 24 EOGRTS

The Extended One-Generation Reproductive Toxicity Study (EOGRTS) is a standard information requirement under Registration, Evaluation, Authorization and restriction of Chemicals (REACH) in Europe. Inclusion of an F2 generation is considered useful if this impacts hazard identification or when it provides additional information altering the reproductive or developmental No Observed Adverse Effect Level (NOAEL) and subsequent risk assessment. In this retrospective analysis the added value of the F2 generation in 24 EOGRTS was evaluated. Our retrospective analysis showed an added value of the second generation in 1/24 EOGRTS only, where extending with the F2 generation lowered both the reproductive and developmental NOAEL. For this substance, extension with the F2 generation led to classification for effects on fertility. Hazard identification for developmental effects was not impacted based on clear developmental toxic effects already observed in the F1 generation and in previous studies with this substance. Risk assessment may be impacted if the developmental toxic effects are the most critical effects observed for this substance. For 23/24 studies included in this retrospective analysis, hazard and risk assessment were unaffected by inclusion of the F2 generation. Moreover, for 13/13 studies with external triggers for inclusion of the F2 generation hazard and risk assessment were unaffected. By decreasing the number of EOGRTS that are extended with an F2 generation, the number of animals used in regulatory developmental and reproductive toxicity studies may be significantly reduced in the future without negatively affecting the ability to identify potentially harmful chemicals.

S03-01: The design and conduct of Extended One-Generation Reproductive Toxicity studies (EOGRTS)

S03-01: The design and conduct of Extended One-Generation Reproductive Toxicity studies (EOGRTS)

S03-02: Extended One-Generation Reproductive Toxicity study (EOGRTS) in the EU regulatory context

S03-02: Extended One-Generation Reproductive Toxicity study (EOGRTS) in the EU regulatory context

Extended one-generation reproductive toxicity study evaluating gardenia blue in Sprague Dawley rats

Gardenia blue powder was administered at 0.5%, 2.5%, or 5.0% in feed to male and female Sprague Dawley rats in an Extended One-Generation Reproductive Toxicity Study (OECD Test Guideline 443). The dosed diet began 14 days before mating and was continued at the same concentration level for the entire study for all parental animals (P0) and offspring (F1). At weaning, offspring were allocated into one of 5 cohorts for different endpoints. P0 and F1 animals had blue urine, blue or black feces, and blue discolorations in gastrointestinal organs, mesenteric lymph nodes, and kidneys. This treatment-related finding was not considered adverse as there were no histopathologic correlates. There was a dose-related increase in sperm concentration in P0 and F1 males. There were dose-related increases in heart weights of F1 postnatal day (PND) 21 males, male and female thyroid weights, and female TSH levels of PND 91 F1 offspring, with no histopathological correlate. There were no consistent treatment-related adverse effects on any other parameters evaluated for general toxicity, reproductive toxicity, developmental neurotoxicity, or developmental immunotoxicity. The highest dietary concentration (5.0%) of gardenia blue powder was the no observed adverse effect level (NOAEL) for male and female rats at all life stages evaluated.

Extended one generation reproductive toxicity study and effect on gut flora of genetically modified rice rich in β-carotene in wistar rats

To evaluate the reproductive toxicity of gene modified rice generated by introducing phytoene synthase (Psy) and bacterial phytoene desaturase (CrtI) from maize and Erwinia uredovora, Wistar rats were allocated into 3 groups and fed with Psy and CrtI gene modified rice mixture diet (GM group), non-gene modified rice mixture diet (non-GM group), and AIN-93 diet (Blank control group) from parental generation (F0) to the offsprings (F1). GM rice, Heijinmi (HJM) and Non-GM rice, Heishuai (HS), were both formulated into diets at ratios of 73.5% and 75.5% according to the AIN93 diet for rodent animals, respectively. Relative to the non-GM group, no biologically relevant differences were observed in GM group rats concerning reproductive performance such as fertility rate, gestation rate, mean duration, hormone level, and reproductive organ pathology. The developmental parameters results were not significantly different from the non-GM group such as body weight, food consumption, developmental neurotoxicity, behavior, hematology, and serum chemistry. In terms of immunotoxicity, the IgG indicators of offspring from the GM group improved in contrast with the non-GM group. Additional gut flora analysis of F0 generation rats resulted as that the treatment elicited an increased gut microflora diversity of F0 rats. And no horizontal gene transfer of Psy and CrtI genes in rats fed a GM rice HJM diet. In conclusion, we found no adverse effects related to GM rice in the extended one-generation reproductive toxicity study, indicating that GM rice is a safe alternative for its counterpart rice regarding reproductive toxicity.

Dietary phytoestrogen diosgenin interrupts metabolism, physiology, and reproduction of Swiss albino mice: Possible mode of action as an emerging environmental contaminant, endocrine disruptor and reproductive toxicant.

Dietary phytoestrogens are the main source of environmental contamination due to their estrogen-mimicking and endocrine-disrupting effects, posing a threat to microbial, soil, plant, and animal health. Diosgenin, a phytosteroid saponin, is used in many traditional medicines, nutraceuticals, dietary supplements, contraceptives, and hormone replacement therapies against numerous diseases and disorders. It is important to be aware of the potential risks associated with diosgenin, as well as its potential to cause reproductive and endocrine toxicity. Due to the lack of research on the safety and probable adverse side effects of diosgenin, this work evaluated the endocrine-disrupting and reproductive toxicity of diosgenin in albino mice by following acute toxicity (OECD-423), repeated dose 90-day oral toxicity (OECD-468), and F1 extended one-generation reproductive toxicity (OECD-443) studies. Diosgenin was found to be slightly toxic, with LD50 for male and female mice being 546.26 and 538.72 mg/kg, respectively. Chronic exposure of diosgenin (10, 50, 100, and 200 mg/kg) generated oxidative stress, depleted antioxidant enzymes, disturbed homeostasis of the reproductive hormones, and interrupted steroidogenesis, germ cell apoptosis, gametogenesis, sperm quality, estrous cycle, and reproductive performance in the F0 and F1 offspring. Long-term oral exposure of diosgenin to the mice disturbed the endocrine and reproductive functions and generated transgenerational reproductive toxic effects in F0 and F1 offspring. These results suggest that diosgenin should be used carefully in food products and medical applications due to its potential endocrine-disrupting and reproductive toxic effects. The findings of this study provide a better understanding of the potential adverse effects of diosgenin and the need for appropriate risk assessment and management of its use.

Safety assessment of 2-methyloxolane as a food extraction solvent.

The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) assessed the safety of 2‐methyloxolane as an extraction solvent under the intended conditions of use and the maximum residue limits (MRLs) proposed by the applicant. 2‐Methyloxolane is intended to be used in processes currently applying hexane for oil and protein extraction from plant sources or for extraction of food additives. The proposed MRLs for the following uses are: (i) 1 mg/kg in fat, oil or butter; (ii) 10 mg/kg in defatted protein products, defatted flour and other defatted solid ingredients; (iii) 1 mg/kg in food category 13 (foods intended for particular nutritional uses as defined by Directive 2009/39/EC); and (iv) 1 mg/kg for the extraction of food additives. The Panel calculated the dietary exposure with the highest potential maximum (95th percentile) for toddlers as 0.32 mg/kg body weight (bw) per day. Based on the available toxicological data, the Panel concluded that 2‐methyloxolane was rapidly metabolised with a low bioaccumulation potential and does not raise a concern for genotoxicity. The Panel identified different no observed adverse effect levels (NOAELs) in a subchronic oral toxicity study in rats, an oral developmental toxicity study and an extended one‐generation reproductive toxicity study, and a TDI of 1 mg/kg bw per day for 2‐methyloxolane was derived based on the lowest identified NOAEL (100 mg/kg bw per day) for reproductive and developmental toxicity. This TDI was not exceeded in any of the population groups at the mean and 95th percentile exposure. The Panel concluded that the extraction solvent 2‐methyloxolane does not raise a safety concern when used according to the intended conditions and at the proposed MRLs in the extracted foods or food ingredients.

Adverse postnatal developmental effects in offspring from humidifier disinfectant biocide inhaled pregnant rats

Inhalation exposure to polyhexamethylene guanidine phosphate (PHMG-P), one of the primary biocides used in humidifier disinfectants, caused a fatal pulmonary disease in Korea. Pregnant women were also exposed to PHMG-P, and subsequent studies showed that PHMG-P inhalation during pregnancy adversely affects their health and embryo-fetal development. However, the postnatal developmental effects after birth on prenatally PHMG-P-exposed offspring have not yet been investigated. Therefore, in this study, we aimed to examine the postnatal development of prenatally PHMG-P-exposed offspring. Pregnant rats (22 or 24 females per group) were exposed to PHMG-P during pregnancy in a whole-body inhalation chamber at the target concentrations of 0, 0.14, 1.60, and 3.20 mg/m3. After parturition, the prenatally exposed offspring were transferred to non-exposed surrogate mothers to minimize the secondary effects of severe maternal toxicities. Postnatal development of offspring was then examined with a modified extended one-generation reproductive toxicity study design. At 3.20 mg/m3 PHMG-P, increased perinatal death rates and decreased viability index (postnatal survival of offspring between birth and postnatal day 4) were observed. In addition, F1 offspring had lower body weight at birth that persisted throughout the study. PHMG-P-exposed pregnant rats also had severe systemic toxicities and increased gestation period. At 1.60 mg/m3 PHMG-P, a decreased viability index was also observed with systemic toxicities of PHMG-P-exposed pregnant rats. These results indicate that prenatal PHMG-P exposure adversely affects the offspring's future health and could be used for human risk assessment.

Safety assessment of titanium dioxide (E171) as a food additive.

The present opinion deals with an updated safety assessment of the food additive titanium dioxide (E 171) based on new relevant scientific evidence considered by the Panel to be reliable, including data obtained with TiO2 nanoparticles (NPs) and data from an extended one‐generation reproductive toxicity (EOGRT) study. Less than 50% of constituent particles by number in E 171 have a minimum external dimension < 100 nm. In addition, the Panel noted that constituent particles < 30 nm amounted to less than 1% of particles by number. The Panel therefore considered that studies with TiO2 NPs < 30 nm were of limited relevance to the safety assessment of E 171. The Panel concluded that although gastrointestinal absorption of TiO2 particles is low, they may accumulate in the body. Studies on general and organ toxicity did not indicate adverse effects with either E 171 up to a dose of 1,000 mg/kg body weight (bw) per day or with TiO2 NPs (> 30 nm) up to the highest dose tested of 100 mg/kg bw per day. No effects on reproductive and developmental toxicity were observed up to a dose of 1,000 mg E 171/kg bw per day, the highest dose tested in the EOGRT study. However, observations of potential immunotoxicity and inflammation with E 171 and potential neurotoxicity with TiO2 NPs, together with the potential induction of aberrant crypt foci with E 171, may indicate adverse effects. With respect to genotoxicity, the Panel concluded that TiO2 particles have the potential to induce DNA strand breaks and chromosomal damage, but not gene mutations. No clear correlation was observed between the physico‐chemical properties of TiO2 particles and the outcome of either in vitro or in vivo genotoxicity assays. A concern for genotoxicity of TiO2 particles that may be present in E 171 could therefore not be ruled out. Several modes of action for the genotoxicity may operate in parallel and the relative contributions of different molecular mechanisms elicited by TiO2 particles are not known. There was uncertainty as to whether a threshold mode of action could be assumed. In addition, a cut‐off value for TiO2 particle size with respect to genotoxicity could not be identified. No appropriately designed study was available to investigate the potential carcinogenic effects of TiO2 NPs. Based on all the evidence available, a concern for genotoxicity could not be ruled out, and given the many uncertainties, the Panel concluded that E 171 can no longer be considered as safe when used as a food additive.

Extended One-Generation Reproductive Toxicity (EOGRT) study of benzoic acid in Sprague Dawley rats

Benzoic acid (BA) was administered in the diet to male and female Sprague Dawley Crl:CD(SD) rats in an OECD Test Guideline 443 Extended One-Generation Reproductive Toxicity (EOGRT) study to test for effects that may occur as a result of pre- and postnatal exposure. The study included cohorts of F1 offspring to evaluate potential effects of benzoic acid on reproduction, the developing immune system, and the developing neurological system with the inclusion of learning and memory assessments. Benzoic acid was incorporated in the diet at concentrations of 0, 7,500, 11,500, and 15,000 mg/kg diet (ppm). These concentrations were selected based on the results of preliminary studies, and, based on average food consumption, were intended to supply BA doses of approximately 0, 500, 750, and 1000 mg/kg bw/day. To avoid exceeding these target dose levels, the dietary concentrations were adjusted (based on historical control body weight and food consumption data) to maintain the target mg/kg bw/day dose levels during those life periods when food intake per unit of body weight was increased to support milk production by females (gestation and lactation) and rapid pup growth (post-weaning).In the parental (F0) generation, survival, clinical observations, organ weights, pathology, hematology, serum chemistry, urinalysis, and bile acids were unaffected by BA administration. Reproductive parameters were also unaffected by BA administration.In the F1 generation, survival, growth and developmental landmarks, organ weights, pathology, immunotoxicity assessment, and neurotoxicity and neurobehavioral parameters such as auditory startle response, locomotor activity, learning and memory assessments were unaffected by BA administration, as were clinical pathology (hematology, serum chemistry, urinalysis, bile acids and thyroid hormones) and reproductive performance. Similarly, no adverse effects or systemic toxicity were observed in the F2 generation.Overall, the highest dietary concentration (15,000 ppm), providing a dosage of approximately 1000 mg/kg bw/day, was the NOAEL for benzoic acid in this EOGRT study.

Effects of endocrine disruptor furan on reproductive physiology of Sprague Dawley rats: An F1 Extended One-Generation Reproductive Toxicity Study (EOGRTS).

The present study investigated the reproductive toxicity of furan in an Extended One-Generation Reproductive Toxicity Study in rats. Sprague Dawley F0 weaning rats (30 per sex per group) were exposed to furan orally at 0, 1, 2.5, 5, and 10 mg kg-1 for 10 weeks (males) and 2 weeks (females) and then mated. Results of F0 indicated that in the furan-treated groups (5 mg kg-1 and 10 mg kg-1), body weight (bw) gain decreased during prebreed and gestational period while increased during lactation periods. F0 animals prebreeding exposure resulted in head tilt and foot splay at 10 mg kg-1. Number of live pups at birth were decreased (p < 0.001) at 10 mg kg-1. At postnatal day (PND) 70, a significant (p = 0.03) decrease in testosterone levels of male rats and estrogen levels of female rats (p = 0.05) was observed in 10 mg kg-1 furan-treated group in F1 generation. Luteinizing hormone, follicle-stimulating hormone, and progesterone levels were also reduced, but their reduction was not statistically significant in all groups. In higher dose furan group (10 mg kg-1), testicular and ovarian weights were reduced in F1 generation at PND 70, with decreased daily sperm production (p = 0.01) and disturbed estrous cyclicity (p < 0.01). Some histopathological changes were also observed in testis and ovaries in groups whose parents were previously exposed to 10 mg kg-1 bw of furan group. Based on the above results, it is suggested that exposure to food-based contaminant furan induced remarkable changes in the F0 (parental stage) and F1 (offspring, pubertal, and adult stage) generations of Sprague Dawley rats.

Extended one-generation reproductive toxicity of thiamethoxam in Wistar rats

Extended one-generation reproductive toxicity of thiamethoxam in Wistar rats

Suggestions for the OECD's extended one-generation reproductive toxicity study to further understand the temporality of endocrine-disrupting effects from chemical exposure: example using bisphenol A

Suggestions for the OECD's extended one-generation reproductive toxicity study to further understand the temporality of endocrine-disrupting effects from chemical exposure: example using bisphenol A

Regulatory aspects under REACH relating to the developmental immunotoxicity (DIT) cohort in the extended one-generation reproductive toxicity study (EOGRTS)

Regulatory aspects under REACH relating to the developmental immunotoxicity (DIT) cohort in the extended one-generation reproductive toxicity study (EOGRTS)

A better approach to in vivo developmental neurotoxicity assessment: Alignment of rodent testing with effects seen in children after neurotoxic exposures

High throughput screens for developmental neurotoxicity (DN) will facilitate evaluation of chemicals and can be used to prioritize those designated for follow-up. DN is evaluated under different guidelines. Those for drugs generally include peri- and postnatal studies and juvenile toxicity studies. For pesticides and commercial chemicals, when triggered, include developmental neurotoxicity studies (DNT) and extended one-generation reproductive toxicity studies. Raffaele et al. (2010) reviewed 69 pesticide DNT studies and found two of the four behavioral tests underperformed. There are now many epidemiological studies on children showing adverse neurocognitive effects, yet guideline DN studies fail to assess most of the functions affected in children; nor do DN guidelines reflect the advances in brain structure-function relationships from neuroscience. By reducing the number of test ages, removing underperforming tests and replacing them with tests that assess cognitive abilities relevant to children, the value of DN protocols can be improved. Testing for the brain networks that mediate higher cognitive functions need to include assessments of working memory, attention, long-term memory (explicit, implicit, and emotional), and executive functions such as cognitive flexibility. The current DNT focus on what can be measured should be replaced with what should be measured. With the wealth of data available from human studies and neuroscience, the recommendation is made for changes to make DN studies better focused on human-relevant functions using tests of proven validity that assess comparable functions to tests used in children. Such changes will provide regulatory authorities with more relevant data.

An extended one-generation reproductive toxicity study of plant protection product containing glyphosate on rats – Androgen- and estrogen-dependent endpoints

Roundup Transorb® (RT) is a glyphosate-based herbicide and despite its wide use around the world there are few studies comparing the effects of the active ingredient with the formulated product. In this context the purpose of this study was to compare the genotoxicity of the active ingredient glyphosate with the formulated product RT in order to clarify whether the active ingredient and the surfactant of the RT formula may exert toxic effects on the DNA molecule in juveniles of fish Prochilodus lineatus. Erythrocytes and gill cells of fish exposed to glyphosate and to RT showed DNA damage scores significantly higher than control animals. These results revealed that both glyphosate itself and RT were genotoxic to gill cells and erythrocytes of P. lineatus, suggesting that their use should be carefully monitored considering their potential impact on tropical aquatic biota.

An extended one-generation reproductive toxicity test of 1,2,4-Triazol-5-one (NTO) in rats

ABSTRACTNitrotriazolone (1,2,4-triazol-5-one; NTO), an insensitive, energetic material used in explosive formulations, induced testicular toxicity and oligospermia in repeated-dose oral toxicity tests in rats. To evaluate whether NTO produces additional reproductive and developmental effects, a modified extended one-generation reproductive toxicity test was conducted. Rats were provided ad libitum access to NTO in drinking water at 0-, 144-, 720-, or 3600-mg/L NTO. Treatment of the parental generation began 2 (females) and 4 (males) wk premating and continued until weaning of litters. Direct dosing of offspring (F1) occurred from weaning through puberty. Pups were counted and weighed on postnatal day (PND) 0/1. Anogenital distance (AGD) was measured on PND 4 and males were examined for presence of nipples on PND 13. F1 offspring were examined daily for attainment of puberty. NTO did not markedly affect measures of fertility, including mating indices, gestation index, litter size, and sex ratio. Seminiferous tubule degeneration or atrophy was observed in P1 and F1 3600-mg/L NTO males. F1 males in the 3600 mg/L group exhibited reduced reproductive organ mass (testes, epididymides, and accessory sex organs). Nipple retention was increased in NTO exposed F1 males compared to controls. Attainment of puberty was delayed by 2.6 d in the 3600-mg/L NTO-exposed males relative to controls. Comparison of the effects of NTO with those of antiandrogens suggests absence of malformations of the genital tract in NTO-exposed males. This study supports previous findings indicating that NTO is a testicular toxicant with male developmental effects that may be secondary to testicular toxicity.