Multigeneration Reproduction Studies Research Articles

An evaluation of reproductive toxicity studies and data interpretation of N-methylpyrrolidone for risk assessment: An expert panel review

An expert panel was assembled to evaluate reproductive toxicology study data and their application to health risk assessment to provide input on the data quality, interpretation, and application of data from three multi-generation reproductive toxicity studies of N-methylpyrrolidone (NMP). Panelists were engaged using a double-blinded, modified Delphi format that consisted of three rounds. Key studies were scored using the U.S. Environmental Protection Agency's (EPA) questions and general considerations to guide the evaluation of experimental animal studies for systematic review. The primary conclusions of the panel are that one of the studies (Exxon, 1991) is not a high-quality study due to several design flaws that includes: (1) exceedance of the maximum tolerable dose in the high dose group; (2) failure to adjust feed concentrations of NMP during the lactation period, resulting in NMP doses that were 2- to 3-fold higher than nominal levels; and/or (3) underlying reproductive performance problems in the strain of rats used. For these reasons, the panel recommended that this study should not be considered for quantitative risk assessment of NMP. Exclusion of this study, and its corresponding data for male fertility and female fecundity, from the quantitative risk assessment results in a change in the identification of the most sensitive endpoint. Instead, changes in rat fetal/pup body weight, an endpoint previously selected by EPA, was identified as an appropriate basis for human health risk assessment based on a consideration of the best available science and weight of scientific evidence supported by the NMP toxicity database.

Machine learning models for rat multigeneration reproductive toxicity prediction.

Reproductive toxicity is one of the prominent endpoints in the risk assessment of environmental and industrial chemicals. Due to the complexity of the reproductive system, traditional reproductive toxicity testing in animals, especially guideline multigeneration reproductive toxicity studies, take a long time and are expensive. Therefore, machine learning, as a promising alternative approach, should be considered when evaluating the reproductive toxicity of chemicals. We curated rat multigeneration reproductive toxicity testing data of 275 chemicals from ToxRefDB (Toxicity Reference Database) and developed predictive models using seven machine learning algorithms (decision tree, decision forest, random forest, k-nearest neighbors, support vector machine, linear discriminant analysis, and logistic regression). A consensus model was built based on the seven individual models. An external validation set was curated from the COSMOS database and the literature. The performances of individual and consensus models were evaluated using 500 iterations of 5-fold cross-validations and the external validation data set. The balanced accuracy of the models ranged from 58% to 65% in the 5-fold cross-validations and 45%–61% in the external validations. Prediction confidence analysis was conducted to provide additional information for more appropriate applications of the developed models. The impact of our findings is in increasing confidence in machine learning models. We demonstrate the importance of using consensus models for harnessing the benefits of multiple machine learning models (i.e., using redundant systems to check validity of outcomes). While we continue to build upon the models to better characterize weak toxicants, there is current utility in saving resources by being able to screen out strong reproductive toxicants before investing in vivo testing. The modeling approach (machine learning models) is offered for assessing the rat multigeneration reproductive toxicity of chemicals. Our results suggest that machine learning may be a promising alternative approach to evaluate the potential reproductive toxicity of chemicals.

A Workflow for the Performance of the Differential Ovarian Follicle Count Using Deep Neuronal Networks.

In order to automate the counting of ovarian follicles required in multigeneration reproductive studies performed in the rat according to Organization for Economic Co-operation and Development guidelines 443 and 416, the application of deep neural networks was tested. The manual evaluation of the differential ovarian follicle count is a tedious and time-consuming task that requires highly trained personnel. In this regard, deep learning outputs provide overlay pictures for a more detailed documentation, together with an increased reproducibility of the counts. To facilitate the planned good laboratory practice (GLP) validation a workflow was set up using MLFlow to make all steps from generating of scans, training of the neural network, uploading of study images to the neural network, generation and storage of the results in a compliant manner controllable and reproducible. PyTorch was used as main framework to build the Faster region-based convolutional neural network for the training. We compared the performances of different depths of ResNet models with specific regard to the sensitivity, specificity, accuracy of the models. In this paper, we describe all steps from data labeling, training of networks, and the performance metrics chosen to evaluate different network architectures. We also make recommendation on steps, which should be taken into consideration when GLP validation is aimed for.

Variability in in vivo studies: Defining the upper limit of performance for predictions of systemic effect levels

New approach methodologies (NAMs) for chemical hazard assessment are often evaluated via comparison to animal studies; however, variability in animal study data limits NAM accuracy. The US EPA Toxicity Reference Database (ToxRefDB) enables consideration of variability in effect levels, including the lowest effect level (LEL) for a treatment-related effect and the lowest observable adverse effect level (LOAEL) defined by expert review, from subacute, subchronic, chronic, multi-generation reproductive, and developmental toxicity studies. The objectives of this work were to quantify the variance within systemic LEL and LOAEL values, defined as potency values for effects in adult or parental animals only, and to estimate the upper limit of NAM prediction accuracy. Multiple linear regression (MLR) and augmented cell means (ACM) models were used to quantify the total variance, and the fraction of variance in systemic LEL and LOAEL values explained by available study descriptors (e.g., administration route, study type). The MLR approach considered each study descriptor as an independent contributor to variance, whereas the ACM approach combined categorical descriptors into cells to define replicates. Using these approaches, total variance in systemic LEL and LOAEL values (in log10-mg/kg/day units) ranged from 0.74 to 0.92. Unexplained variance in LEL and LOAEL values, approximated by the residual mean square error (MSE), ranged from 0.20 to 0.39. Considering subchronic, chronic, or developmental study designs separately resulted in similar values. Based on the relationship between MSE and R-squared for goodness-of-fit, the maximal R-squared may approach 55 to 73% for a NAM-based predictive model of systemic toxicity using these data as reference. The root mean square error (RMSE) ranged from 0.47 to 0.63 log10-mg/kg/day, depending on dataset and regression approach, suggesting that a two-sided minimum prediction interval for systemic effect levels may have a width of 58 to 284-fold. These findings suggest quantitative considerations for building scientific confidence in NAM-based systemic toxicity predictions.

Sensitivity of different generations and developmental stages in studies on reproductive toxicity

Numerous studies on reproductive toxicity are expected to be necessary under the EU program on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Therefore, it is important to analyse existing testing strategies including also the recently implemented extended one-generation reproduction toxicity study (EOGRTS, OECD guideline 443). For this purpose the responsiveness of the different generations and developmental stages in studies on reproductive toxicity is analysed and critical targets of reproductive toxicity are identified by using the Fraunhofer FeDTex database.The F1 generation is identified as most responsive generation in more than 50% of one-generation and multi-generation reproduction studies. Within the F1 generation the adult stage is mostly affected compared to the prenatal or postnatal stage. The target analysis in F1 has revealed alterations in body weight as highly sensitive for all developmental stages. Other important targets are the liver, kidney, testes, prostate, sperm parameters as well as developmental landmarks. The findings in the F2 generation have shown a higher responsiveness than F1 only in 3% of the studies. Although in 29 studies new effects are observed in F2 offspring compared to F1 irrespective of dose levels, overall no severe new effects have emerged that would change classification and labelling and justify an F1 mating.The presented data support the importance of F1 for risk assessment and demonstrate that the study design of the EOGRTS is a suitable alternative to two-generation studies. However, compared to a conventional one-generation study the EOGRTS may identify additional effects but will change risk assessment with respect to NOELs only in rare cases.

Multigeneration Reproductive Study of Hydroxyprogesterone Caproate (HPC) in the Rat: Laboratory Results and Clinical Significance

To demonstrate reproductive safety of a new commercial product for reducing the risk of preterm birth, HPC (17α-hydroxyprogesterone caproate, Makena; manufactured by Baxter Pharmaceutical Solutions, Bloomington IN for Ther-Rx Corporation, St. Louis, MO) was administered intramuscularly in Charles River LaboratoryCD strain rats. HPC was given at intervals equal to the half-life measured in rats during three phases of embryo-fetal development: during the period of ovarian development (RP1, days 8, 14, and 20), following implantation of the embryo (TP, days 6, 12, and 18), and, corresponding to the start of the drug in week 16 or later in humans, after gonadal formation including differentiation of the testes (RP2, day 17). Dose levels up to 30× the human therapeutic doses were utilized including 0 (vehicle), 5, 25, and 150 mg/kg (volume 0.6 ml/kg). Four groups of 25 time-mated rats each were used for each phase. In addition, equal numbers of naïve (untreated) rats of opposite gender were used for F(1) breeding studies. HPC did not produce any consistent test-article-related findings in the treated F(0) dams, their developing F(1) fetuses and did not affect the ability of the latter to produce a viable F(2) generation. The F(1) offspring did not evidence any adverse effects during their behavioral, sensory, and developmental assessments, including teratogenicity. Based on the cumulative data obtained from rats treated over two generations and during development in this study, the No-observable-effect-level (NOEL) was established as 150 mg/kg. This study supports the absence of reproductive toxicity with HPC in published studies in animal models and in human clinical trials.

Economic benefits of using adaptive predictive models of reproductive toxicity in the context of a tiered testing program

A predictive model of reproductive toxicity, as observed in rat multigeneration reproductive (MGR) studies, was previously developed using high throughput screening (HTS) data from 36 in vitro assays mapped to 8 genes or gene-sets from Phase I of USEPA ToxCast research program, the proof-of-concept phase in which 309 toxicologically well characterized chemicals were testing in over 500 HTS assays. The model predicted the effects on male and female reproductive function with a balanced accuracy of 80%%. In a theoretical examination of the potential impact of the model, two case studies were derived representing different tiered testing scenarios to: 1) screen-out chemicals with low predicted probability of effect; and 2) screen-in chemicals with a high probability of causing adverse reproductive effects. We define ‘testing cost efficiency’ as the total cost divided by the number of positive chemicals expected in the definitive guideline toxicity study. This would approach $$2.11 M under the current practice. Under case study 1, 22%% of the chemicals were screened-out due to low predicted probability of adverse reproductive effect and a misclassification rate of 12%%, yielding a test cost efficiency of $$1.87 M. Under case study 2, 13%% of chemicals were screened-in yielding a testing cost efficiency of $$1.13 M per test-positive chemical. Applying the model would also double the total number of positives identified. It should be noted that the intention of the case studies is not to provide a definitive mechanism for screening-in or screening-out chemicals or account for the indirect costs of misclassification. The case studies demonstrate the customizability of the model as a tool in chemical testing decision-making. The predictive model of reproductive toxicity will continue to evolve as new assays become available to fill recognized biological gaps and will be combined with other predictive models, particularly models of developmental toxicity, to form an initial tier to an overarching integrated testing strategy.

On the impact of second generation mating and offspring in multi-generation reproductive toxicity studies on classification and labelling of substances in Europe

The possible impact on classification and labelling decisions of effects observed in second generation parental (P1) and offspring (F2) parameters in multi-generation studies was investigated. This was done for 50 substances classified as reproductive toxicants in Europe, for which a multi-generation study was available. The P1 and F2 effects were compared to parental (P0) and first generation offspring (F1) effects with regard to type of effect as well as incidence, magnitude and severity (IMS), at any dose level. For every study with unique P1/F2 effects, or differences in IMS, the influence of the P1/F2 findings on the classification decision was investigated. Unique P1/F2 generation findings did not play a crucial role in the classification decision of any of the 50 classified substances, except for fenarimol. This substance however provided abundant alerts on the basis of its endocrine activity and developmental neurotoxicity and would therefore also be expected to be identified as a developmental neurotoxicant in an Extended One Generation Reproductive Toxicity Study (EOGRTS). These findings, in addition to the increased number of parameters analysed, increased statistical power and reduced animal use, provide strong further support for replacement of the classical two-generation reproductive toxicity study by the EOGRTS in regulatory reproductive toxicity assessment.

Predictive Model of Rat Reproductive Toxicity from ToxCast High Throughput Screening1

The U.S. Environmental Protection Agency's ToxCast research program uses high throughput screening (HTS) for profiling bioactivity and predicting the toxicity of large numbers of chemicals. ToxCast Phase I tested 309 well-characterized chemicals in more than 500 assays for a wide range of molecular targets and cellular responses. Of the 309 environmental chemicals in Phase I, 256 were linked to high-quality rat multigeneration reproductive toxicity studies in the relational Toxicity Reference Database. Reproductive toxicants were defined here as having achieved a reproductive lowest-observed-adverse-effect level of less than 500 mg kg(-1) day(-1). Eight-six chemicals were identified as reproductive toxicants in the rat, and 68 of those had sufficient in vitro bioactivity to model. Each assay was assessed for univariate association with the identified reproductive toxicants. Significantly associated assays were linked to gene sets and used for the subsequent predictive modeling. Using linear discriminant analysis and fivefold cross-validation, a robust and stable predictive model was produced capable of identifying rodent reproductive toxicants with 77% ± 2% and 74% ± 5% (mean ± SEM) training and test cross-validation balanced accuracies, respectively. With a 21-chemical external validation set, the model was 76% accurate, further indicating the model's potential for prioritizing the many thousands of environmental chemicals with little to no hazard information. The biological features of the model include steroidal and nonsteroidal nuclear receptors, cytochrome P450 enzyme inhibition, G protein-coupled receptors, and cell signaling pathway readouts-mechanistic information suggesting additional targeted, integrated testing strategies and potential applications of in vitro HTS to risk assessment.

Combined retrospective analysis of 498 rat multi-generation reproductive toxicity studies: On the impact of parameters related to F1 mating and F2 offspring

The multi-generation reproductive toxicity study (OECD TG 416 and USEPA 870.3800) has been extensively used internationally to assess the adverse effects of substances on reproduction. Recently the necessity of producing a second generation to assess the potential for human health risks has been questioned. The present standardized retrospective analysis of the impact of the second generation on overall study outcome combines earlier analyses and includes 498 rat multi-generation studies representing 438 different tested substances. Detailed assessment of study reports revealed no critical differences in sensitivities between the generations on the basis of a consideration of all endpoints evaluated. This analysis indicates that the second generation mating and offspring will very rarely provide critical information. These findings are consistent with the conclusions of previous retrospective analyses conducted by RIVM, USEPA and PMRA and support adoption of the proposed OECD extended one-generation reproductive toxicity study protocol in regulatory risk assessment testing strategies.

Scientific Opinion on Bisphenol A: evaluation of a study investigating its neurodevelopmental toxicity, review of recent scientific literature on its toxicity and advice on the Danish risk assessment of Bisphenol A

Bisphenol A (BPA) is used in the manufacture of plastics, to produce reusable drinking bottles, infant feeding bottles and other food storage containers. EFSA was asked to evaluate a dietary developmental neurotoxicity study in rats (Stump, 2009) and recent scientific literature (2007–2010) in terms of relevance for the risk assessment of BPA. The impact of these studies on the current Tolerable Daily Intake (TDI) of 0.05 mg BPA/kg body weight (b.w.)/day as set by EFSA in 2006 was assessed. Advice on the Danish risk assessment underlying the Danish ban of BPA in food contact materials for infants aged 0–3 years is included. Overall, based on this comprehensive evaluation of recent toxicity data, the Panel on food contact materials, enzymes, flavourings and processing aids (CEF) concluded that no new study could be identified, which would call for a revision of the current TDI. This TDI is based on the No-Observed-Adverse-Effect-Level (NOAEL) of 5 mg/kg b.w./day from a multi-generation reproductive toxicity study in rats, and the application of an uncertainty factor of 100. This factor is regarded as conservative based on all information on BPA toxicokinetics. The Panel noted that some studies conducted on developing animals have suggested other BPA-related effects of possible toxicological relevance, in particular biochemical changes in brain, immune-modulatory effects and enhanced susceptibility to breast tumours. These studies had several shortcomings. At present the relevance of these findings for human health cannot be assessed. Should any new relevant data become available in the future, the Panel will reconsider this opinion. A minority opinion is expressed by a Panel member and presented in an Annex to this opinion.

Scientific Opinion on the safety and efficacy of Koffogran (nicarbazin) as a feed additive for chickens for fattening

Koffogran (containing 25 % nicarbazin) is intended to be used as a coccidiostat for chickens for fattening up to an age of 28 days. Feeding 125 mg nicarbazin/kg feed was safe for chickens for fattening (margin of safety about 1.5) and effective in controlling coccidiosis. Ingested nicarbazin is rapidly split into its components 2-hydroxy-4, 6-dimethylpyrimidine (HDP) and 4,4’-dinitrocarbanilide (DNC). DNC is the marker residue, liver the target tissue. HDP-related residues are much lower than those derived from DNC. Nicarbazin is not genotoxic. In sub-chronic rat studies, no NOAEL could be derived for nicarbazin, but a NOAEL of 709 mg/kg bw/day was identified for DNC. Those differences may be related to the higher systemic exposure of rats to DNC when administered as nicarbazin instead of DNC alone. No major concern appeared in multigeneration reproduction studies in rats at 300 mg DNC + 100 mg HDP/kg bw/day and in developmental studies with rats and rabbits at 70 and 60 mg nicarbazin/kg bw, respectively. As the consumer is exposed to DNC rather than nicarbazin, an ADI was set for DNC with 0.77 mg/kg bw/day, derived from the NOAEL for elevated serum ALT in a two-year dog study (154 mg DNC/kg bw/day) applying a safety factor of 200. MRLs of 15, 6, 4 and 4 mg DNC/kg liver, kidney, muscle and skin/fat, respectively, were proposed. Applying those MRLs consumer exposure would not exceed 24 % of the ADI. A one-day withdrawal period was considered as adequate. Nicarbazin was not an irritant or sensitiser to skin, but a slight irritant to eyes. Inhalatory exposure of users to nicarbazin was negligibly small. No safety concern for the soil compartment, groundwater or by secondary poisoning was identified, the risk for surface water could however not be assessed.

Profiling the Reproductive Toxicity of Chemicals from Multigeneration Studies in the Toxicity Reference Database

Multigeneration reproduction studies are used to characterize parental and offspring systemic toxicity, as well as reproductive toxicity of pesticides, industrial chemicals and pharmaceuticals. Results from 329 multigeneration studies on 316 chemicals have been digitized into standardized and structured toxicity data within the Toxicity Reference Database (ToxRefDB). An initial assessment of data quality and consistency was performed prior to profiling these environmental chemicals based on reproductive toxicity and associated toxicity endpoints. The pattern of toxicity across 75 effects for all 316 chemicals provided sets of chemicals with similar in vivo toxicity for future predictive modeling. Comparative analysis across the 329 studies identified chemicals with sensitive reproductive effects, based on comparisons to chronic and subchronic toxicity studies, as did the cross-generational comparisons within the multigeneration study. The general pattern of toxicity across all chemicals and the more focused comparative analyses identified 19 parental, offspring and reproductive effects with a high enough incidence to serve as targets for predictive modeling that will eventually serve as a chemical prioritization tool spanning reproductive toxicities. These toxicity endpoints included specific reproductive performance indices, male and female reproductive organ pathologies, offspring viability, growth and maturation, and parental systemic toxicities. Capturing this reproductive toxicity data in ToxRefDB supports ongoing retrospective analyses, test guideline revisions, and computational toxicology research.

Integrated Disinfection By-Products Research: Assessing Reproductive and Developmental Risks Posed by Complex Disinfection By-Product Mixtures

This article presents a toxicologically-based risk assessment strategy for identifying the individual components or fractions of a complex mixture that are associated with its toxicity. The strategy relies on conventional component-based mixtures risk approaches such as dose addition, response addition, and analyses of interactions. Developmental toxicity data from two drinking-water concentrates containing disinfection by-products (DBP) mixtures were used to illustrate the strategy. The results of this study showed that future studies of DBP concentrates using the Chernoff–Kavlock bioassay need to consider evaluating DBP that are concentrated more than 130-fold and using a rat strain that is more sensitive to chemically-induced pregnancy loss than Sprague-Dawley rats. The results support the planned experimental design of a multigeneration reproductive and developmental study of DBP concentrates. Finally, this article discusses the need for a systematic evaluation of DBP concentrates obtained from multiple source waters and treatment types. The development of such a database could be useful in evaluating whether a specific DBP concentrate is sufficiently similar to tested combinations of source waters and treatment alternatives so that health risks for the former may be estimated using data on the latter.

Toxicological properties of metaflumizone

Metaflumizone is a new insecticide developed for crop protection and urban pest control by BASF. Its mammalian toxicological profile was assessed by conducting multiple toxicity studies in the rat, mouse, and dog, covering all relevant endpoints. Metaflumizone is characterized by very low acute toxicity, is not irritating to the eye or the skin and does not possess a potential to induce skin sensitization. The substance also shows relatively low toxicity following subchronic oral or dermal exposure to mammals. In addition, metaflumizone demonstrates low toxicological potential following chronic oral exposure to rats, mice, and dogs. Overall, the lowest no observed adverse effect level (NOAEL) is 12 mg/(kg day) from the 1-year chronic dog study. In a battery of in vitro and in vivo mutagenicity assays, the weight-of-the-evidence indicates a lack of potential genotoxicity for metaflumizone. Furthermore, the compound demonstrated a lack of potential oncogenicity in long-term toxicity studies in rats and mice. Results from the rat multi-generation reproductive toxicity study as well as the rat and rabbit developmental toxicity studies indicate that metaflumizone is not selectively toxic to the offspring or fetus, as compared to the parents. Also, metaflumizone is not teratogenic in the rat or rabbit. Lastly, no neurotoxicity could be detected in acute and subchronic neurotoxicity studies in rats.

Multigeneration Reproductive and Developmental Toxicity Study of bar Gene Inserted into Genetically Modified Potato on Rats

Each specific protein has an individual gene encoding it, and a foreign gene introduced to a plant can be used to synthesize a new protein. The identification of potential reproductive and developmental toxicity from novel proteins produced by genetically modified (GM) crops is a difficult task. A science-based risk assessment is needed in order to use GM crops as a conventional foodstuff. In this study, the specific characteristics of GM food and low-level chronic exposure were examined using a five-generation animal study. In each generation, rats were fed a solid pellet containing 5% GM potato and non-GM potato for 10 wk prior to mating in order to assess the potential reproductive and developmental toxic effects. In the multigeneration animal study, there were no GM potato-related changes in body weight, food consumption, reproductive performance, and organ weight. Polymerase chain reaction (PCR) was carried out using extracted genomic DNA to examine the possibility of gene persistence in the organ tissues after a long-term exposure to low levels of GM feed. In each generation, the gene responsible for bar was not found in any of the reproductive organs of the GM potato-treated male and female rats, and the litter-related indexes did not show any genetically modified organism (GMO)-related changes. The results suggest that genetically modified crops have no adverse effects on the multigeneration reproductive–developmental ability.

Review of the Potential Environmental and Human Health–Related Hazards and Risks from Long-Term Exposure to p-tert-Octylphenol

ABSTRACT Para-tert-octylphenol (OP) is an important chemical intermediate mainly used for production of phenolic resins and lacquers. OP has weak estrogen-like activity in vitro, with induction of vitellogenin in fish hepatocytes and competitive estrogen receptor binding and estrogen-responsive gene expression in mammalian systems. Induction of vitellogenin in fish plasma and liver, in vivo, has also been measured and weak estrogen-like activity in rodent screening assays occurs but at much lower potency than predicted by the in vitro activity. Conventional aquatic wildlife toxicity testing shows effects on survival, growth and development, and reproduction occurring at concentrations lower than the estrogen-like responses predicted from screening assays. Studies in rodents have provided evidence of metabolic saturation at high doses of OP, and a multigeneration reproductive study confirms the lack of estrogen-like activity below this metabolic saturation as well as the lack of reproductive toxicity. This article reviews the weight of evidence for the aquatic and mammalian data and compares the results of the definitive assays to those of the screening tests. The results show that the estrogen-like activity in screening tests is not predictive of results from definitive testing, and that risk assessment decisions should be made using conventional toxicity endpoints.

Genetic damage detected in CD-1 mouse pups exposed perinatally to 3'-azido-3'-deoxythymidine and dideoxyinosine via maternal dosing, nursing, and direct gavage.

Human immunodeficiency virus (HIV)-infected pregnant women are administered nucleoside-analogue antiretrovirals to reduce maternal-infant viral transmission. The current protocol recommends treating newborns for 6 additional weeks postpartum. The treatment is effective, but the risk of drug-induced chromosomal damage in neonates remains undefined. We used a mouse model to investigate this concern. In a multigeneration reproductive toxicity study, female CD-1 mice received 3'-azido-3'-deoxythymidine (AZT) and dideoxyinosine (ddI) (50/250, 75/375, 150/750 mg/kg/day AZT/ddI) by gavage twice daily in equal fractions beginning prior to mating and continuing throughout gestation and lactation. Direct pup dosing (same regimen) began on postnatal day (PND) 4. Peripheral blood erythrocytes of male pups were screened for micronuclei, markers of chromosomal damage, on PNDs 1, 4, 8, and 21. Extraordinary increases in micronucleated cells were noted in pups for each treatment group at each sampling time; treated dams exhibited smaller yet significant increases in micronucleated erythrocytes. The frequencies of micronucleated cells in untreated pups were higher than in the untreated dams, and all pups had markedly elevated levels of circulating reticulocytes compared to dams. These observations suggest that fetal and neonatal mouse hematopoietic precursor cells have heightened sensitivity to genotoxic agents, perhaps due to rapid cell proliferation during the perinatal period of development. The amount of genetic damage observed in treated pups raises concern for the potential of similar damage in humans. Investigations of chromosomal integrity in exposed newborns and children are recommended.

Endocrine active agents: implications of adverse and non-adverse changes.

The US Environmental Protection Agency (EPA) is currently in the process of developing screening and testing methodologies for the assessment of agents that may possess endocrine-like activity--the so-called endocrine disruptors. Moreover, the EPA has signaled its intention of placing information arising from such studies on the worldwide web. This has created significant interest in how such information may be used in risk assessment and by policymakers and the public in the potential regulation or deselection of specific chemical agents. The construction of lists of endocrine disruptors, although fulfilling the requirements of some parties, is really of little use when the nature of the response, the dose level employed, and the lifestage of the test species used are not given. Thus, we have already seen positive in vitro information available on the interaction with a receptor being used as a key indicator when the results of large, high quality in vivo studies showing no adverse changes have been ignored. Clearly a number of in vitro systems are available to ascertain chemical interaction with specific (mainly steroid) hormone receptors including a number of reporter gene assays. These assays only provide indicators of potential problems and should not be, in isolation, indicators of toxicity. Likewise, short-term in vivo screens such as the uterotrophic and Hershberger studies are frequently conducted in castrated animals and thus indicate the potential for a pharmacological response in vivo rather than an adverse effect. A number of new end points have been added to standard rodent testing protocols in the belief of providing more sensitivity to detect endocrine related changes. These include the measurement of anogenital distance (AGD), developmental landmarks [vaginal opening (VO), preputial separation (PPS)], and in some studies the counting of nipples and areolae on males. AGD, VO, and PPS are all affected by the size of the pup in which they are measured and should always be compared using bodyweight as a covariate. The historical control database for such changes is gradually growing, albeit that if pups are not individually identified it becomes problematic to associate any change with a specific malformation or to assess whether a delay or advance in, for example, developmental landmarks is biologically significant. Agents that significantly reduce AGD in males (it is an androgen-dependent variable) frequently have other more adverse changes associated with this end point (eg, reproductive tract malformations), but a 2 to 3% change in AGD although measurable is unlikely to be biologically of importance and in isolation would not necessarily be considered adverse. Retention of thoracic nipples in male rat pups is also an indicator of impaired androgen status. Recent studies have also shown that this retention for some endocrine active chemicals is permanent. Thus, the presence of a permanent structural change that is rarely found in adult control animals could be considered a malformation and therefore a developmental adverse effect on which risk assessment decisions could be made. The advent of multigeneration reproduction studies as the definitive studies for the assessment of the dose-response relationships and risk assessment for endocrine disruptors has shown that current testing protocols may be inadequate to reliably detect the adverse effects of concern as only 1 adult/sex/litter is examined. A number of the effects on reproductive development although, due to an in utero exposure, will not be manifest until after puberty or at adulthood. The use of only a limited number of animals to examine such changes, particularly for weaker acting materials indicates that some agents may have been examined in well-conducted, modern protocols but have insufficient power to detect low incidence phenomena (eg, a 5% incidence of malformations).

Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans.

Reviews on the safety of glyphosate and Roundup herbicide that have been conducted by several regulatory agencies and scientific institutions worldwide have concluded that there is no indication of any human health concern. Nevertheless, questions regarding their safety are periodically raised. This review was undertaken to produce a current and comprehensive safety evaluation and risk assessment for humans. It includes assessments of glyphosate, its major breakdown product [aminomethylphosphonic acid (AMPA)], its Roundup formulations, and the predominant surfactant [polyethoxylated tallow amine (POEA)] used in Roundup formulations worldwide. The studies evaluated in this review included those performed for regulatory purposes as well as published research reports. The oral absorption of glyphosate and AMPA is low, and both materials are eliminated essentially unmetabolized. Dermal penetration studies with Roundup showed very low absorption. Experimental evidence has shown that neither glyphosate nor AMPA bioaccumulates in any animal tissue. No significant toxicity occurred in acute, subchronic, and chronic studies. Direct ocular exposure to the concentrated Roundup formulation can result in transient irritation, while normal spray dilutions cause, at most, only minimal effects. The genotoxicity data for glyphosate and Roundup were assessed using a weight-of-evidence approach and standard evaluation criteria. There was no convincing evidence for direct DNA damage in vitro or in vivo, and it was concluded that Roundup and its components do not pose a risk for the production of heritable/somatic mutations in humans. Multiple lifetime feeding studies have failed to demonstrate any tumorigenic potential for glyphosate. Accordingly, it was concluded that glyphosate is noncarcinogenic. Glyphosate, AMPA, and POEA were not teratogenic or developmentally toxic. There were no effects on fertility or reproductive parameters in two multigeneration reproduction studies with glyphosate. Likewise there were no adverse effects in reproductive tissues from animals treated with glyphosate, AMPA, or POEA in chronic and/or subchronic studies. Results from standard studies with these materials also failed to show any effects indicative of endocrine modulation. Therefore, it is concluded that the use of Roundup herbicide does not result in adverse effects on development, reproduction, or endocrine systems in humans and other mammals. For purposes of risk assessment, no-observed-adverse-effect levels (NOAELs) were identified for all subchronic, chronic, developmental, and reproduction studies with glyphosate, AMPA, and POEA. Margins-of-exposure for chronic risk were calculated for each compound by dividing the lowest applicable NOAEL by worst-case estimates of chronic exposure. Acute risks were assessed by comparison of oral LD50 values to estimated maximum acute human exposure. It was concluded that, under present and expected conditions of use, Roundup herbicide does not pose a health risk to humans.