Alternative models in developmental toxicology

In light of various pressures, toxicologists have been searching for alternative methods for safety testing of chemicals. According to a recent policy in the European Union (Regulation, Evaluation Authorisation and Restriction of Chemicals, REACH), it has been estimated that over the next twelve to fifteen years, approximately 30,000 chemicals may need to be tested for safety, and under current guidelines such testing would require the use of approximately 7.2 million laboratory animals [ Hofer et al. 2004 ]. It has also been estimated that over 80% of all animals used for safety testing under REACH legislation would be used for examining reproductive and developmental toxicity [Hofer et al., 2004]. In addition to REACH initiatives, it has been estimated that out of 5,000 to 10,000 new drug entities that a pharmaceutical company may start with, only one is finally approved by the Food and Drug Administration at a cost of over one billion dollars [ Garg et al. 2011 ]. A large portion of this cost is due to animal testing. Therefore, both the pharmaceutical and chemical industries are interested in using alternative models and in vitro tests for safety testing. This review will examine the current state of three alternative models - whole embryo culture (WEC), the mouse embryonic stem cell test (mEST), and zebrafish. Each of these alternatives will be reviewed, and advantages and disadvantages of each model will be discussed. These models were chosen because they are the models most commonly used and would appear to have the greatest potential for future applications in developmental toxicity screening and testing.     

REACH and reproductive and developmental toxicology: still questions

Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) is a new chemicals regulation law in the European Union (EU). The law is supplemented by tens of thousands of pages of guidance documents, and the implementation of REACH is still a work in progress. Requirements for chemical testing are based on the annual volume of a chemical or 'substance' that is produced or imported into the EU. These requirements include reproductive and developmental toxicity testing in experimental animals for an annual volume of 10 metric tonnes or more. However, under REACH, the testing in vertebrate animals may not be performed without permission, and the law encourages the use of alternative methods of filling data gaps on the toxicological properties of chemicals. These alternatives might include in vitro and structure-activity relationship studies, but the REACH technical guidance indicates that these kinds of studies are not adequate to replace reproductive and developmental toxicity testing in whole animals. The most practical opportunity for the avoidance of whole animal testing may be 'read-across,' a process in which gaps are filled using data from related compounds. A method called 'weight of evidence' under REACH may also be used to avoid whole animal reproductive and developmental toxicity testing based on existing data in regulation and non-regulation studies and based on factors such as chemical structure and anticipated exposure. It is also possible that thresholds of toxicological concerns will be accepted under REACH as a method to avoid vertebrate animal testing.     

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.     

REACH: next step to a sound chemicals management

REACH is the new European Regulation for Registration, Evaluation, Authorisation and Restriction of Chemicals. It entered into force on 1st June 2007 to streamline and improve the former legislative framework on new and on existing chemical substances of the European Union. Companies which manufacture or import more than 1 tonne of a substance per year will be required to register the substance at the new EU Chemicals Agency located in Helsinki. REACH places greater responsibility on industry to manage the risks that chemicals may pose to the health and the environment and to provide safety information that will be passed down the supply chain. In principle, REACH applies to all chemicals as such, as components in preparations and as used in articles. REACH is a radical step forward in the EU chemicals management. The onus will move from the authorities to industry. In addition, REACH will allow the further evaluation of substances where there are grounds for concern, foresees an authorisation system for the use of substances of very high concern and a system of restrictions, where applicable, for substances of concern. The Authorisation system will require companies to switch progressively to safer alternatives where a suitable alternative exists. Current use restrictions will remain under REACH system.     

我国化学品毒性鉴定技术规范与REACH标准比对研究

[目的]了解我国现有的《化学品毒性鉴定技术规范》（以下简称＂规范＂）与化学品注册、评估、授权和限制（REACH）标准方法之间的差异,以提高我国的化学品毒性鉴定的质量和水平。[方法]对规范、REACH中的毒性鉴定方法按照不同的毒性分类进行比较。[结果]（1）REACH和规范中使用的方法大部分都源于经济合作与发展组织（OECD）的化学品毒性鉴定及其健康效应评价指南。因此,两者间在方法本质和原则上没有很大的差别。（2）异同点比较：①方法基本相同,特别是原理、试验体系、试验指标和评判终点都一致;②REACH主要采用OECD标准中运用时间长、接受面广、相对成熟的方法;③我国的标准在技术内容上和国际是接轨的,由于考虑到整体的检验水平还不高,因此对一些具体的操作步骤做出了更详细的规定,使标准更具规范性和实用性。[结论]从REACH与我国现行毒理学方法的对比分析中发现,在整体结构和原则上,我国的分析方法与REACH的方法有高度的相似性,但在某些试验方法方面也存在着差异。只有尽快在我国建立国际通用标准的毒理学检测良好实验室规范（GLP）实验室,才能从根本上提高中国实验室的检测能力。     

Application of chemical toxicity distributions to ecotoxicology data requirements under REACH

The European Union's REACH regulation has further highlighted the lack of ecotoxicological data for substances in the marketplace. The mandates under REACH (registration, evaluation, authorization, and restriction of chemicals) to produce data and minimize testing on vertebrates present an impetus for advanced hazard assessment techniques using read-across. Research in our group has recently focused on probabilistic ecotoxicological hazard assessment approaches using chemical toxicity distributions (CTDs). Using available data for chemicals with similar modes of action or within a chemical class may allow for selection of a screening point value (SPV) for development of environmental safety values, based on a probabilistic distribution of toxicity values for a specific endpoint in an ecological receptor. Ecotoxicity data for acetylcholinesterase inhibitors and surfactants in Daphnia magna and Pimephales promelas were gathered from several data sources, including the U.S. Environmental Protection Agency's ECOTOX and Pesticides Ecotoxicity databases, the peer-reviewed literature, and the Human and Environmental Risk Assessment (HERA) project. Chemical toxicity distributions were subsequently developed, and the first and fifth centiles were used as SPVs for the development of screening-predicted no-effect concentrations (sPNECs). The first and fifth centiles of these distributions were divided by an assessment factor of 1,000, as recommended by REACH guidance. Use of screening values created using these techniques could support the processes of data dossier development and environmental exposure assessment, allowing for rigorous prioritization in testing and monitoring to fill data gaps.     

Children's health and the environment: public health issues and challenges for risk assessment

Infants and children are not little adults. They are uniquely vulnerable to environmental toxicants. To protect infants and children against toxicants, the National Research Council in 1993 called for development of an approach to risk assessment that considers children's unique patterns of exposure and their special vulnerabilities to pesticides. Many aspects of that call were codified into federal law in the Food Quality Protection Act (FQPA) of 1996. This report highlights the central elements needed for development of a child-protective approach to risk assessment: a) improved quantitative assessment of children's exposures at different life stages, from fetal life through adolescence, including acute and chronic exposures, exposures via multiple routes, and exposures to multiple agents; b) development of new approaches to toxicity testing of chemicals that can detect unanticipated and subtle outcomes and that evaluate experimental subjects over the entire life span from early exposure to natural death to replicate the human experience; c) development of new toxicodynamic and toxicokinetic models that account for the unique physiologic characteristics of infants and children; d) development of new approaches to assessment of outcomes, functional, organ, cellular and molecular, over the entire life span; these measures need to be incorporated into toxicity testing and into long-term prospective epidemiologic studies of children; and e) application of uncertainty and safety factors in risk assessment that specifically consider children's risks. Under FQPA, children are presumed more vulnerable to pesticides than adults unless evidence exists to the contrary. Uncertainty and safety factors that are protective of children must therefore be incorporated into risk assessment when data on developmental toxicity are lacking or when there is evidence of developmental toxicity. The adequate protection of children against toxic agents in the environment will require fundamental and far-reaching revisions of current approaches to toxicity testing and risk assessment.     

Perspectives on reproductive and developmental toxicity

Human reproduction and development is a cycle of interdependent events. Virtually all of its phases have been shown to be the primary target of one or more non-mutagenic exogenous agents. Such agents interfere with certain of the countless epigenetic or ontogenic events essential for normal completion of the cycle. Mutagens disrupt this cycle at some points, but the overwhelming majority of reproductive and developmental toxins are not mutagenic. As in all aspects of toxicology, the reproductive and developmental effects of chemicals are determined by the intrinsic nature of the chemical, the quantity of the chemical exposure, the duration of exposure and the stage of the cycle at which it occurs. Signs of reproductive toxicity range from reduced fertility to spontaneous abortion. Adverse effects on the conceptus are categorized as functional deficits, developmental retardation, structural abnormality and death. One or more of these is anticipated to occur as a result of excess exposure to most chemicals. Although the degree of hazard and risk potential can be calculated in each instance, chemicals differ markedly in their ability to interfere with reproduction (Amann, 1982) and/or development (Johnson, 1984). Standardized methods for reproductive and developmental toxicity safety evaluation are available for detecting adverse effects upon any aspect of reproduction and development. Data currently available establish that these state-of-the-art tests conducted in laboratory animals are often highly predictive of the type of adverse effect a particular chemical will have in humans, as well as the exposure level at which it will occur. By adding a modest safety factor to the no-observed-effect-level of well-executed animal studies, safe human exposure levels can be established. Responsibility for determining the intrinsic hazard potential and the risk estimate of exposure rests with manufacturers and major users of occupational and other environmental chemicals. As public awareness of reproductive and developmental hazards has increased in recent years, it has come to be understood that some chemicals have a predilection for causing reproductive impairment and/or disrupting development in the absence of other toxicity. Such substances must be identified to establish safe exposure levels and to determine the types of effects to be expected, should excessive human exposure occur. The setting of safe exposure levels is necessary both from the standpoints of ensuring public safety and avoiding product liability.     

Assessment of the U.S. Environmental Protection Agency methods for identification of hazards to developing organisms, Part II: The developmental toxicity testing guideline.

BACKGROUND: The effects of toxins on developing animals depend not only on the nature of the chemical but also on the timing of exposure and assessment of outcomes. This complicates the task of regulatory agencies such as the U.S. Environmental Protection Agency (EPA), which must comply with the 1996 Food Quality Protection Act to ensure that their standards and policies protect infants and children from environmental toxins. For this task, the Agency relies heavily on scientific data obtained by manufacturers of industrial chemicals and pesticides following protocols collected under EPA's Health Effects Test Guidelines. METHODS AND RESULTS: This article reviews the protocols included in the EPA guidelines to assess developmental toxicity, which are required for food-use pesticides under the core testing battery. We reviewed these protocols on the basis of their adequacy for identifying hazards to infants and children. Our analysis found limitations in the protocols that hinder their potential for identifying developmental hazards. CONCLUSIONS: Methods that the EPA currently depends upon to identify developmental toxicity of chemicals have limitations that impede obtaining complete and reliable data on which to base regulatory decisions that protect children. Other methodological approaches need to be explored as alternatives or supplements to the current protocols. Until more accurate testing protocols become available, it may well be necessary under existing laws to employ safety factors that are more protective of the health of children at all stages of development.     

SAR and QSAR methods in the study of dioxin action

Thousand chemicals are present within our environment, and for many of them, there is a little reliable information detailing their relative hazard. Added to that increasing concern over the use of animals in toxicity testing, high costs of these tests has made the search for and validation of alternative methods to predict the hazard and the relative risk of xenobiotics. QSAR (quantitative structure activity relationship) attempt to relate statistically the biological activity of compound with its physicochemical and structural properties. QSAR methods are often seen as the first step for valid toxicological prediction. Halogenated aromatic hydrocarbons typified by polychlorinated dibenzo-p-dioxins (PCDD), dibenzofurans (PCDF) and biphenyls (PCBs) have been identified as residues in almost every component of the ecosystem. Some chemicals in this class cause adverse biological effects after binding to an intracellular cytosolic protein called the Ah receptor (AhR). Because of importance of the Ah receptor in determining toxicity, there have been a number of attempts to model the relationship between receptor binding and structure of xenobiotics. QSAR have found wide use in correlating the bioactivity of dioxins and related compounds with many kinds of biological entities. This paper will briefly summarise some SAR and QSAR study for halogenated aromatics as ligand for Ah receptor and the characteristic biological and toxic responses elicit by this class of chemicals. These study strongly support the role of AhR in dioxin toxicity.     

Answering the endocrine test questions.

Evidence suggesting that certain chemicals may bind to endogenous hormone receptors and disturb normal endocrine functioning, thereby increasing the risk of reproductive problems and cancer in humans, has led to international efforts to screen chemicals for endocrine activity and potential health effects. The U.S. Environmental Protection Agency (EPA) has recommended that some 87,000 commercial chemicals for which there currently are inadequate toxicity data be evaluated. In December 1998, the EPA's Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommended the use of a tiered system of screening and testing assays to sequentially eliminate chemicals for which further testing is deemed unnecessary. The first step toward implementing such a system-validation of the tests to be used-is presenting some challenges, however, with stakeholders disagreeing over which tests to validate, how extensively to validate them, and how much it will cost.     

An overview of structure-activity relationships as an alternative to testing in animals for carcinogenicity, mutagenicity, dermal and eye irritation, and acute oral toxicity

The use of structure-activity relationships (SAR) has proven practical for the development of equations which can be used to estimate the above-listed endpoints for a large variety of chemicals. The SAR models predict these endpoints correctly in 85 to 97% of the cases and often surpass in their predictive ability the results obtainable from the equivalent biological assays. These SAR models are being used at several levels: drug, or more generally, chemical discovery; prioritization for testing; regulatory affairs; investigation of detoxification mechanisms; and risk estimation. In the new compound (discovery) use, potential toxic effects of a set of related compounds are investigated before synthesis to select those chemicals with the lesser probabilities of producing toxic effects for further investigation, at considerable savings in research expenditure since fewer compounds need to be synthesized, and the avoidance of blind alleys. Prioritization for testing is used in numerous instances, such as selecting those chemicals in an environment which are most likely to have toxic effects for priority attention. SAR models are used by regulatory agencies to determine the possible toxic effects of chemicals for which data insufficient to render decisions have been submitted, and to gain insight into possible toxicity problems. SAR models are also used to investigate possible metabolites, and toxicity mechanisms due to the ability of making computer-based structural modifications and observing the effects on the modelled toxic endpoints. Risk analysis is a natural outgrowth of several of the above applications, and is particularly useful for SAR models of carcinogenicity. SAR models as alternatives to animal bioassays should be used in the context of other information for the chemicals of concern. Just as bioassays and in vitro methods have their limitations, so do SAR models. These include the sometimes limited data base on which to base an SAR model, the temptation to extrapolate beyond the confines of the model, and the noise inherent in the bioassays on which the models are based. Within these constraints SAR models have a considerable potential in reducing the number of animals used in toxicity testing.     

Generic exposure scenarios: their development, application, and interpretation under REACH

The European Union Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) Regulation (EC 1907/2006) places significant new obligations on the manufacturers or importers (M/Is) of chemicals in Europe. It also places new responsibilities on downstream users (DUs) of these chemicals i.e. those that purchase and use chemical products. In particular, for registered classified substances, the M/I is expected to communicate how any substance can be safely used without risk to man or the environment. This communication is in the form of an exposure scenario (ES), which is included in an Annex to the REACH extended safety data sheet. DUs then have certain obligations relating to adopting the control conditions described in the ES. The REACH Technical Guidance Documents lay down the expectations for the process of risk assessment that M/Is should adopt when developing ESs. But with many thousands of chemicals in daily commerce, it is also necessary to ensure that what is communicated to DUs not only meets the requirements of REACH but is also understandable to these groups, as well as being consistent across different chemical suppliers and supply chains. In cooperation with relevant DU groups, the European solvents industry has developed generic approaches for describing how solvents are commonly used, in order that these can subsequently be used as the basis for REACH registrations and related safety data sheet communications on health risk control. The utility of these approaches (termed 'generic exposure scenarios') is acknowledged under REACH and they are now publicly available for use both by M/Is and DUs.     

Development of QSARs for the toxicity of chlorobenzenes to the soil dwelling springtail Folsomia candida

To meet the goals of Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) as formulated by the European Commission, fast and resource-effective tools are needed to predict the toxicity of compounds in the environment. We developed quantitative structure-activity relationships (QSARs) for the toxicity of nine chlorinated benzenes to the soil-dwelling collembolan Folsomia candida in natural LUFA2.2 (Landwirtschaftliche Untersuchungs und Forschungsanstalt [LUFA]) standard soil and in Organisation for Economic Co-operation and Development artificial soil. Toxicity endpoints used were the effect concentrations causing 10% (EC10) and 50% (EC50) reduction in the reproduction of the test organism over 28?d, while lethal effects on survival (LC50) were used for comparisons with earlier studies. Chlorobenzene toxicity was based on concentrations in interstitial water as estimated using nominal concentrations in soil and literature soil-water partition coefficients. Additionally, for LUFA2.2 soil the estimated concentrations in interstitial water were experimentally determined by solid-phase microextraction measurements. Measured and estimated concentrations showed the same general trend, but significant differences were observed. With the exception of hexachlorobenzene, estimated EC10 and EC50 values were all negatively correlated with their logK(ow) and QSARs were developed. However, no correlation for the LC50 could be derived and 1,2,4,5-tetrachlorobenzene and hexachlorobenzene had no effect on adult survival at all. The derived QSARs may contribute to the development of better ecotoxicity-based models serving the REACH program.     

The Future Use of in Vitro Data in Risk Assessment to Set Human Exposure Standards: Challenging Problems and Familiar Solutions

Background

The vision of a National Research Council (NRC) committee (the Committee on Toxicity Testing and Assessment of Environmental Agents) for future toxicity testing involves the testing of human cells in in vitro assays for “toxicity pathways”—normal signaling pathways that when perturbed can lead to adverse effects. Risk assessments would eventually be conducted using mathematical models of toxicity pathways (TP models) to estimate exposures that will not cause biologically significant perturbations in these pathways.

Objectives

In this commentary we present our vision of how risk assessment to support exposure standards will be developed once a suitable suite of in vitro assays becomes available.

Discussion

Issues to be faced basing risk assessments on in vitro data are more complex than, but conceptually similar to, those faced currently when applying in vivo data. Absent some unforeseen technical breakthrough, in vitro data will be used in ways similar to current practices that involve applying uncertainty or safety factors to no observed adverse effect levels or benchmark doses. TP models are unlikely to contribute quantitatively to risk assessments for several reasons, including that the statistical variability inherent in such complex models severely limits their usefulness in estimating small changes in response, and that such models will likely continue to involve empirical modeling of dose responses.

Conclusion

The vision of the committee predicts that chemicals will be tested more quickly and cheaply and that animal testing will be reduced or eliminated. Progress toward achieving these goals will be expedited if the issues raised herein are given careful consideration.     

Innovative non-animal testing strategies for reproductive toxicology: the contribution of Italian partners within the EU project ReProTect

Reproductive toxicity, with its many targets and mechanisms, is a complex area of toxicology; thus, the screening and identification of reproductive toxicants is a main scientific challenge for the safety assessment of chemicals, including the European Regulation on Chemicals (REACH). Regulatory agencies recommend the implementation of the 3Rs principle (refinement, reduction, replacement) as well as of intelligent testing strategies, through the development of in vitro methods and the use of mechanistic information in the hazard identification and characterization steps of the risk assessment process. The EU Integrated Project ReProTect (6th Framework Programme) implemented an array of in vitro tests to study different building blocks of the mammalian reproductive cycle: methodological developments and results on male and female germ cells, prostate and placenta are presented.     

Human Neurospheres as Three-Dimensional Cellular Systems for Developmental Neurotoxicity Testing

Background

Developmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the “3 Rs” (reduction, replacement, and refinement) of animal testing and the European regulation of chemicals [Registration, Evaluation, and Authorisation of Chemicals (REACH)], alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening.

Objectives

The goal of this study was to establish a three-dimensional test system for DNT screening based on human fetal brain cells.

Methods

We established assays suitable for detecting disturbances in basic processes of brain development by employing human neural progenitor cells (hNPCs), which grow as neurospheres. Furthermore, we assessed effects of mercury and oxidative stress on these cells.

Results

We found that human neurospheres imitate proliferation, differentiation, and migration in vitro. Exposure to the proapoptotic agent staurosporine further suggests that human neurospheres possess functioning apoptosis machinery. The developmental neurotoxicants methylmercury chloride and mercury chloride decreased migration distance and number of neuronal-like cells in differentiated hNPCs. Furthermore, hNPCs undergo caspase-independent apoptosis when exposed toward high amounts of oxidative stress.

Conclusions

Human neurospheres are likely to imitate basic processes of brain development, and these processes can be modulated by developmental neurotoxicants. Thus, this three-dimensional cell system is a promising approach for DNT testing.     

Chemicals in the environment and developmental toxicity to children: a public health and policy perspective

There are numerous pesticides and toxic chemicals in the environment that have yet to be evaluated for potential to cause developmental neurotoxicity. Recent legislation and testing initiatives provide an impetus to generating more information about potential hazards to children. In the United States, the 1996 Food Quality Protection Act (FQPA) required the U.S. Environmental Protection Agency (U.S. EPA) to make a finding that a pesticide food use is safe for children. In addition, the law requires U.S. EPA to incorporate an additional 10-fold factor in risk assessments for pesticide residue tolerances to take into account the special sensitivities of infants and children as well as incomplete data with respect to toxicity and exposures. The potential of chemicals in food and drinking water to cause endocrine disruption will also be examined via the Endocrine Disruptor Screening and Testing Program required by the FQPA and the 1996 Safe Drinking Water Act. In addition, a new voluntary chemical information program will provide screening-level information for the some 2,800 high-volume chemicals in commerce in the United States. These initiatives will need to be accompanied by research focused on developmental toxicity for children, including developmental disabilities. Developmental disabilities exact a large toll on children's health in the United States. Three major developmental disabilities--autism, cerebral palsy, and severe mental retardation--each affect substantial numbers of children. We know very little about the etiology of these conditions. A number of priority areas for research are suggested, including a large environmental prospective study of developmental neurotoxicity.     

高内涵筛选技术在毒理学研究中的应用进展

随着各种新、老化学物质健康风险评价的需求急剧增长,基于动物实验的测试方法难以满足当前健康风险评价的需求,迫切需要研发新型的、高通量、灵敏的毒性测试方法,整合基于体外替代模型的高通量筛选技术、计算方法和信息技术的毒性测试策略。其中,高内涵筛选（HCS）利用自动化显微镜和图像分析平台,以可视化和定量的方式对体外模型进行多参...     

Assessment of the U.S. Environmental Protection Agency methods for identification of hazards to developing organisms, Part I: The reproduction and fertility testing guidelines.

BACKGROUND: Successful reproduction depends on the coordination of many processes, particularly the normal development and subsequent maturation of the sexual organs. The Food Quality Protection Act of 1996 mandates that the U.S. Environmental Protection Agency must protect infants and children from the effects of toxins, including those that affect the reproductive system. Therefore, the Agency finds itself at a critical juncture to make sure that the methods it requires for toxicity testing, the Health Effects Test Guidelines or Series 870 Guidelines, are adequate to determine possible toxicity to children. METHODS AND RESULTS: We found that two testing protocols included in the core guidelines assess toxicological effects on developing animals. This article aims to provide a detailed analysis of the protocols included in the Reproduction and Fertility Effects Test Guideline. An accompanying article assesses the Developmental Toxicity Testing Guideline. We conducted this analysis on the basis of whether the test would yield the information needed to adequately determine risk to infants and children. CONCLUSIONS: Our analysis concludes that given the limitations inherent in testing for reproduction and fertility effects during development, it is necessary to include a safety factor during risk assessment of chemicals. This action will fulfill the mandate expressed in the FQPA to protect infants and children from environmental hazards.