Document J3MN6y51bbn8EGx0ZE6NrmZeX

R&S 020090 Thursday December 5, 1991 Part V Environmental Protection Agency Guidelines for Developmental Toxicity Risk Assessment; Notice 63798 Federal Register / Vol. 56. No. 234 / Thursday. December 5. 1991 / Notices R&S 020091 ENVIRONMENTAL PROTECTION AGENCY (FRL-4038-3J Guidelines for Developmental Toxicity Risk Assessment agency: U.S. Environmental Protection Agency (EPA). ACT) N: Final Guidelines for Developmental Toxicity Risk Assessment. . summary: The U.S. Environmental Protection Agency (EPA) is today issuing final amended guidelines for assessing the risks for developmental toxicity from exposure to environmental agents. As background information for this guidance, this notice describes the scientific basis for concern about ' exposure to agents that cause developmental toxicity, outlines the general process for assessing potential risk to humans because of environmental contaminants, summarizes the history of these guidelines, and addresses public and Science Advisory Board comments on the 19C9 "Proposed Amendments to the Guidelines for the Health Assessment of Suspect Developmental Toxicants" (54 FR 9386-9403). These guidelines, which have been renamed "Guidelines for Developmental Toxicity Risk Assessment" (hereafter "Guidelines"), outline principles and methods for evaluating data from animal and human studies, exposure data, and other information to characterize risk to human development growth, survival, and function because of exposure prior to conception, prenatally, or to infants and children. These Guidelines amend and replace EPA's 1986 "Guidelines for the Health Assessment of Suspect Developmental Toxicants" [51 FR 3402834040] by adding new guidance on the relationship between maternal and developmental toxicity, characterization of the health-related data base for developmental toxicity risk assessment, use cf the reference dose or reference concentration for developmental toxicity (RfDo- or RfCor), and use of the benchmark dose approach. In addition, the Guidelines were reorganized to combine hazard identification and doseresponse evaluation since these are usually done together in assessing risk for human health effects other than cancer. EFFECTIVE date: The Guidelines will be effective December 5.1991. FOR FURTHER INFORMATION CONTACT: Dr. Carole A. Kimmsl, Reproductive and Developmental Toxicology Branch, Human Health Assessment Group, Office of Health and Environmental Assessment (RD-6S9), U.S. Environmental Protection Agency, 401 M Street. SW., Washington, DC 20460. TEL: 202-260-7331, FAX: 202-260-3803. SUPPLEMENTARY INFORMATION! The Clean Air Act (CAA), the Toxic Substances Control Act (TSCA), the Federal Insecticide. Fungicide, and Rodenticide Act (FIFRA) and other statutes administered by the EPA authorize the Agency to protect public kealth against adverse effects from environmental pollutants. One type of adverse effect of great concern is developmental toxicity, Le,, adverse effects produced prior to conception, during pregnancy and childhood. Exposure to agents affecting development can result in any one or more of the following manifestations of developmental toxicity: Death, structural abnormality, growth alteration, and/or functional deficit These manifestations encompass a wide array of adverse developmental end points, such as spontaneous abortions, stillbirths, malformations, early postnatal mortality, reduced birth weight, mental retardation, sensory loss, and other adverse functional or physical changes that are manifested postnatally. The Role of Environmental Agents in Developmental Toxicity Several environmental agents are established as causing developmental toxicity in humans (e.g., lead, polychlorinated biphenyls, methylmercury, ionizing radiation), while many others are suspected of causing developmental toxicity in humans based on data from experimental animal studies (e.g., some pesticides, other heavy metals, glycol ethers, alcohols, and phthalates). Data for several of the agents identified as causing human developmental toxicity have been compared to the experimental animal data (Nisbet and Karch. 1983; fCimmel et al., 1984; Hemminki and Vineis, 1985; Kimmel et al,, 1990a). In these comparisons, the agents causing human developmental toxicity in almost ail cases were found to produce effects in experimental animal studies and, in at least one species tested, types of effects similar to those in humans were generaily seen. This information provides a strong basis for the use of animal data in conducting human health risk assessments. On the other hand, a number of agents found to cause developmental toxicity in experimental animal studies have not shown clear evidence of hazard in humans, but the available human data are often too limited to evaluate a cause and effect relationship. The comparison of doseresponse relationships is hampered by differences in route, timing and duration of exposure. When careful comparisons have been done taking these factors into account, the minimally effective dose for the most sensitive animal species was generally higher than that for humans, usually within 10-fold of the human effective dose, but sometimes was 100 times or more higher (e.g,, polychlorinated biphenyls [Tilscn et al., 1990]). Thus, the experimental animal data were generally predictive of adverse developmental effects in humans, but in some cases, the administered dose or exposure level required to achieve these adverse effects was much higher than the effective dose in humans. In most cases, the toxic effects of an agent on human development have not been fully studied, even though exposure of humans to that agent may have been established. At the same time, there are many developmental effects in humans with unknown causes and no clear link with exposure to environmental agents. The background incidence of human spontaneous abortion, for example, was estimated by Hertig (1967) to be approximately 50% of all conceptions, and more recently, Wilcox et al. (1985), using sensitive techniques for detecting pregnancy as early as 9 days postconception, observed that 35% of postimplantation pregnancies ended in an embryonic or fetal loss. Of those infants bom alive, approximately 7.4% are reduced in weight at birth (i.e., below 2500 g) (Selevan, 1981). approximately 3% are found to have one or more congenital malformations at birth, and by the end of the first postnatal year, about 3% more are found to have serious developmental defects (Shepard, 1986). Of those children bom with developmental defects, it has been estimated that 20% are due to genetic transmission and 10% can be attributed to known exogenous factors (including drugs, infections, ionizing radiation, and environmental agents), leaving the remaining 70% with unknown causes (Wilson, 1977). In a recent hospitalbased surveillance study (Nelson and Holmes, 1989), 50.7% of congenital malformations were estimated to be due to genetic or multifactorial causes, while 3.2% were associated with exposure to exogenous agents and 2.9% to twinning or uterine factors, leaving 43.2% to unknown causes. The proportion of the effects with unknown causes that may be attributable to environmental agents or to a combination of factors, such as environmental agents and genetic Federal Register / Vol. 56, No. 234 / Thursday, December 5, 1991 / Notices 63799 factors, nutritional deficiencies, alcohol consumption, direct or indirect exposure to tobacco smoke, use of prescribed and illicit dings. etc., is unknown. The Kial and economic impact of deveiu: cental disabilities on the popular is extremely high. Close to one-half of the children in hospital wards are there because of prenatally acquired malformations (Shepard. 1980). Acc ding to the Centers for Disease Cor congenital anomalies, sudden infs eath syndrome, and prematurity com ,ed account for more than 50% of infant mortality among all races in the United States (National Center for Health Statistics, 1988). In addition, among the leading causes of estimated years of potential life lost (YPLL) due to death before the age of 85, congenital anomalies, prematurity, and sudden infant death syndrome combined rank third (Centers for Disease Control. 1988a. b). The YPLL estimates for developmental defects may actually underestimate the public health impact because the estimates do not include prenatal deaths, they are based only on those cases that die before age 65 and do not account for limited quality of life, and pregnancies may be terminated early due to prenatal diagnosis of developmental defects. Theae data provide the basis for a long-standing interest by Federal agencies that deal with human health to protect against exposures to agents that cause developmental toxicity, and most of these regulatory agencies have provisions for considering data on developmental toxicity in protecting human health. As a step in developing procedures for interpreting toxicity data in the regulatory context, the National Academy of Sciences/National Research Council, in 1963. published a framework for the risk assessment process, which EPA uses as the basis for its risk assessment guidelines and for the assessment of risk due to environmental agents. The Risk Assessment Process and Its Application to Developmental Toxicity Risk assessment is the process by which scientific judgments are made concerning the potential for toxicity to occur in humans. The National Research Council (1983) has defined risk assessment as including some or all of the following components: Hazard identification, dose-response assessment, exposure assessment and risk characterization. In general the process of assessing the risk of human developmental toxicity may be adapted to this format. In practice, however, hazard identification for developmental toxicity and other noncanccr health effects is usually done in conjunction with an evaluation of dose-response relationships, since the determination of a hazard is often dependent on whether a dose-response relationship is pri at (Kimmel et al.. 1990b). One advan -e of this approach is that it reflects hazard within the context of dose, route, duration and timing of exposure, all of which are important in comparing the toxicity information available to potential human exposure scenarios. Secondly, this approach avoids labelling of chemicals as developmental toxicants on a purely qualitative basis. For these reasons, the Guidelines combine hazard identification and dose-response evaluation under one section (Section III), and characterize both hazard and dose information as part of the healthrelated data base for risk assessment If data are considered sufficient for risk assessment, an oral or dermal reference dose for developmental toxicity (RfDpr) or an inhalation reference concentration for developmental toxicity (RfCOT) is then derived for comparison with human' exposure estimates. A statement of the potential for human risk and the consequences of exposure can come only bom integrating the hazard identification/dose-response evaluation with the human exposure estimates in the final risk characterization. Combining hazard identification and dose-response evaluation, as well as development of the RfDmr and RfCOT, are revisions of the 1986 Guidelines. Hazard identification /dose-response evaluation involves examining all available experimental animal and human data and the associated doses, routes, timing and duration of exposures to determine if an agent causes developmental toxicity and/or maternal or paternal toxicity in that species and under what exposure conditions. The no-observed-adverse-effect-level (NOAEL) and/or the lowest-observedadverse-effect-level (LOAEL) are determined for each study and type of effect Based upon the hazard identification/dose-response evaluation and criteria provided in these Guidelines, the health-related data base can be characterized as sufficient or insufficient for use in risk assessment (Section IILC). Because of the limitations associated with the use of the NOAEL, the Agency is evaluating the use of an additional approach, Le- the benchmark dose approach (Crump. 1984), for more quantitative dose-response evaluation when sufficient data are available. The benchmark dose provides an indication of the risk associated with exposures near the NOAEL taking into account the variability in the data and the slope of the dose-response curve. For the determination of the RfDiyr or the RfCor. uncertainty factors are applied to the NOAEL (or LOAEL if a NOAEL has not been established) to account for extrapolation from experimental animals to humans and for variability within the human population. The RfDor or RfCpr is generally based on a short duration of exposure as is typically used in developmental toxicity studies in experimental animals. The use of the terms RfDtrr and RfCor distinguish them from the oral or dermal reference dose (RID) and the inhalation reference concentration (RfC) which refer primarily to chronic exposure situations (U.S. EPA, 1991). Uncertainty factors may also be applied to a benchmark dose for calculating the RfD,,r or RfCM. but the Agency has little experience with applying this approach and is currently supporting research efforts to determine the appropriate methods. As more information becomes available, guidance will be written and published as an addendum to these Guidelines. These approaches are discussed further in section IILD. The exposure assessment identifies h unan populations exposed or p initially exposed to an agent atscribes their composition and size, and presents the types, magnitudes, frequencies, and durations of exposure to the agent. The exposure assessment provides an estimate of human exposure levels for particular populations from all potential sources. In risk characterization, the hazard identification/dose-response evaluation and the exposure assessment for given populations are combined to estimate some measure of the risk for developmental toxicity. As part of risk characterization, a summary of the strengths and weaknesses in each component of the risk assessment are discussed along with major assumptions, scientific judgments, and. to the extent possible, qualitative and quantitative estimates of the uncertainties. Confidence in the healthrelated data is always presented In conjunction with information on doseresponse and the RfD* or RfC*t. If human exposure estimates are available, the exposure basis used for the risk assessment is clearly described, e.g.. highly exposed individuals, or highly sensitive or susceptible individuals. The NOAEL may be compart to the various estimates of human txposure to calculate the margin(s) of exposure (MOE). The considerations for determining adequacy of the MOE are similar to R&S 020092 63800 .Federal Register / Vol. 56. No. 234 / Thursday, December 5. 1991 / Notices R&S 020093 those used in determining the appropriate size of the uncertainly factor for calculating the RIDM or RfC^. Risk assessment is just one component of the regulatory process and defines the potential adverse health consequences of exposure to a toxic agent. The other component, risk management, combines risk assessment with statutory directives regarding socioeconomic, technical, political, and other considerations, to reach decisions about the appropriate regulation of the suspected toxic agents. Risk management is not dealt with directly in these Guidelines since the basis for decision-making goes beyond scientific consideration alone, but the use of scientific information in this process is discussed in some cases. For example, the acceptability of the MOE is a risk management decision, but the scientific bases for establishing this value are ; discussed here. History of These Guidelines In 1984, the Agency published "Proposed Guidelines for the Health Assessment of Suspect Developmental Toxicants" (49 FR 46324-46331). Following extensive scientific and public review, final guidelines were issued on September 24,1983 (51 FR 34028-34040). The 1988 Guidelines set forth principles and procedures to guide EPA scientists in the conduct of Agency risk assessments, to help promote high scientific quality and Agency-wide consistency, and to inform Agency decision makers and the public about these scientific procedures. In publishing this guidance, EPA emphasized that one purpose of its risk assessment guidelines was to "encourage research and analysis that will lead to new risk assessment methods and data," which in turn would be used to revise and improve the guidelines, and better guide Agency risk assessors. Thus, the 1988 Guidelines were developed and published with the understanding that risk assessment is an evolving science and that continued study could lead to changes. As expected. Agency experience with the 1986 Guidelines suggested that additional or alternate approaches should be considered for certain aspects of the guidance. Proposals to amend the guidelines were considered soon after their publication in September 1988, because of new reviews or reevaluations that focused on some of the issues identified for research in the guidelines. Included were several workshops and symposia cited in the Introduction to these Guidelines. In addition, much experience had been gained in using the 1986 Guidelines and in instructing others in their use. Based on this experience, amendments to the 1986 Guidelines were proposed for public comment in March 1989 (54 FR 9386-9403). Following receipt and review of the public comments, they were collated, summarized, and reviewed by scientists within the Agency. On October 27,1989, EPA's Science Advisory Board (SAB) met to review the Proposed Amendments and the summarized public comments, and to be briefed by Agency scientists concerning proposed responses. During this same period, several issues with Implications for health effects other than cancer were under discussion in the Agency and elsewhere. These issues included use of the benchmark dose (see section 1ILB), exposure descriptors (see section V.C), and risk characterization (see section V). Thus, generic discussions on risk assessment issues, along with comments from the public and the SAB, have influenced the structure and content of these Guidelines. These revised Guidelines were then reviewed by a number of Agency scientists and official panels, including the Risk Assessment Forum and the Risk Assessment Council. The revised Guidelines also were presented to the SAB on March 27,1991. for final comment. In addition, a review was conducted by the interagency Working Party on Reproductive Toxicology, Subcommittee on Risk Assessment of the Federal Coordinating Committee on Science, Engineering and Technology. Comments of these groups have been considered in the revision of these Guidelines. The full text of the final "Guidelines for Developmental Toxicity Risk Assessment" are published here. These Guidelines were developed as part of an interoffice guidelines development program under the auspices of the Risk Assessment Forum and the Office of Health and Environmental Assessment (OHEA) in the Agency's Office of Research and Development The Agency is continuing to study risk assessment issues raised in these Guidelines, and will revise them in line with new information as appropriate. Following this Preamble are two parts: Part A is the Guidelines, and part B is the Response to the Public and Science Advisory Board Comments. Part B includes a summary of the issues raised by the public and the SAB, and the Agency's responses to those comments. References, supporting documents, and comments received on the Proposed Amendments, as well as a copy of the final Guidelines, are available for inspection and copying at the Public Information Reference Unit Docket (202260-5928). EPA Headquarters Library, 401 M Street, SW,, Washington. DC, between the hours of 8 a.m. and 4:30 p.m. Dated: November 26,19S1. Wiffinm K. Relll. Administrator. Cootents Part A: Guidelines for Developmental Toxicity Risk Assessment I. Introduction IL Definitions and Terminology 1IL Hazard Identifiestion/Dose-Response Evaluation of Agents that Cause Developmental Toxicity A. Developmental Toxidty Studies: End Points and Their Interpretation 1. Laboratory Animal Studies a. End Points of Maternal Toxidty b. End Points of Developmental Toxidty: Altered Survival. Growth, and Morphological Development c. End Points of Developmental Toxidty: Functional Defidts d. Overall Evaluation of Maternal and Developmental Toxidty e. Short-Term Testing in Developmental Toxidty (1) In vivo mammalian developmental toxidty teats (2) In vitro developmental toxidty screens L Statistical Considerations 2. Human Studies a. Epidemiologic Studies (1) General design considerations (2) Selection of outcomes for study (3) Reproductive history studies (4) Community studies/surveillance programs (5) Identification of exposures important for developmental effects b. Examination of Clusters or Case Reports/Series 3. Other Considerations a. Pharmacokinetics b. Comparisons of Molecular Structure B. Dose-Response Evaluation C. Characterization of the Health-Related Data Base D. Determination of (he Reference Dose (RfDm) or Reference Concentration (RfCpr) for Developmental Toxidty E. Summary IV. Exposure Assessment V. Risk Characterization A. Overview B. Integration of the Hazard Identification/ Doss-Response Evaluation and Exposure Assessment C. Descriptors of Developmental Toxidty Risk 1. Estimation of the Number of Individuals Exposed to Levels of Concern 2. Presenting SpedSc Scenarios 3. Risk Characterization for Highly Exposed Individuals 4. Risk Characterization for Highly Sensitive or Susceptible Individuals Federal Register / Vol. 56, No. 234 / Thursday, December 5. 1991 / Notices 63801 5. Other Risk Descriptors E. Communicating Results VI. Summary and Research Needs VII. References Part 6: Response to Public and Science Advisory Board Comments I. Introduction II. Intent of the Guidelines III. Basic Assumptions IV. Matemal/Developmental Toxicity V. Functional Developmental Toxicity VI. Weight-of-Evidence Scheme V1L Applicability of the RfDj*. Concept and the Benchmark Dose Approach Part A: Guidelines for Development Toxicity Risk Assessment I. Introduction These Guidelines describe the procedures that the EPA follows in evaluating potential developmental toxicity associated with human exposure to environmental agents. The Agency has sponsored or participated in several conferences that addressed issues related to such evaluations and that provide some of the scientific basis for these Guidelines (U.S. EPA 1982a: Kimmel et al.. 1982b, 1987; Hardin. 1987; Perlin and McCormack, 1988; Kimmel et al,, 1989; Kimmel and Francis. 1990; Kimmel et al,,1990a). The Agency's authority to regulate substances that have the potential to interfere with human development is derived from a number of statutes that are implemented through multiple offices within the EPA. The procedures described herein are intended to promote consistency in the assessment of developmental toxic effects across program offices within the Agency. These Guidelines provide a general format for analyzing and organizing the available data for conducting risk assessments. The Agency previously has issued testing guidelines (U.S. EPA, 1982b. 198Sa. 1989a, 1991a) that provide protocols designed to determine the potential of a test substance to induce structural and/or other adverse effects during development These risk assessment Guidelines do not change any prescribed statutory or regulatory standards for the type of data necessary for regulatory action, but rather provide guidance for the interpretation of studies that follow the testing guidelines, and in addition, provide limited information for interpretation of other studies (e.g, epidemiologic data, functional developmental toxicity studies, and short-term tests) that are not routinely required, but may be encountered when reviewing data on particular agents. Since the purpose of risk assessment is to make inferences about potential risks to human health, the most appropriate data to be used are those deriving from studies of humans. If adequate human data are not available, then it is necessary to use data obtained from other species. There are a number of unknowns in the extrapolation of data from animal studies to humans. Therefore, a number of assumptions must be made on the relevance of effects to potential human risk which are generally applied in the absence of data. These assumptions provide the inferential basis for the approaches taken to risk assessment in these Guidelines. First it is assumed that an agent that produces an adverse developmental effect in experimental animal studies will potentially pose a hazard to humans following sufficient exposure during development. This assumption is based on the comparisons of data for agents known to cause human developmental toxicity (Nisbet and Karch, 1983: Kimmel et al, 1984: Hemminki and Vineis. 1985; Kimmel et at. 1990a). which indicate that, in almost all cases, experimental animal data are predictive of a developmental effect in humans. It is assumed that all of the four manifestations of developmental toxicity (death, structural abnormalities, growth alterations, and functional deficits) are of concern. In the past, there has been a tendency to consider only malformations or malformations and death as end points of concern. From the data on agents that are known to cause human developmental toxicity (Nisbet and Karch, 1983; Kimmel et al,, 1984; Hemminki and Vineis, 1985: Kimmel et al.. 1990a). there is usually at least one experimental species that mimics the types of effects seen in humans, but in other species tested, the type of developmental perturbation may be different. Thus, a biologically significant increase in any of the four manifestations is considered indicative of an agent's potential for disrupting development and producing a developmental hazard. It is assumed that the types of developmental effects seen in animal studies are not necessarily the same as those that may be produced in humans.This assumption is made because it is impossible to determine which will be the most appropriate species in terms of predicting the specific types of effects seen in humans. The fact that every species may not react in the same way could be due to species-specific differences in critical periods, differences in timing of exposure, metabolism, developmental patterns, placentation. or mechanisms of action. The most appropriate species is used to estimate human risk when data are available (e.g.. pharmacokinetics). In the absence of such data, it is assumed that the most' sensitive species is appropriate for use. based on observations that humans are as sensitive or more so than the most sensitive animal species tested for the majority of agents known to cause human developmental toxicity (Nisbet and Karch, 1983; Kimmel et al.. 1984; Hemminki and Vineis. 1985; Kimmel et al, 1990a). In general, a threshold is assumed for the dose-response curve for agents that produce developmental toxicity. This is based on the known capacity of the developing organism to compensate for or to repair a certain amount of damage at the cellular, tissue, or organ level. In addition, because of the multipotency of cells at certain stages of development, multiple insults at the molecular or cellular level may be required to produce an effect on the whole organism. IL Definitions and Terminology The Agency recognizes that there are differences in the use of terms in the field of developmental toxicology. For the purposes pf these Guidelines the following definitions will be used. Developmental toxicology--The study of adverse effects on the developing organism that may result from exposure prior to conception (either parent), during prenatal development, or postnatally to the time of sexual maturation. Adverse developmental effects may be detected at any point in the life span of the organism. The major manifestations of developmental toxicity include: (1) Death of the developing organism, (Z) structural abnormality. (3) altered growth, and (4) functional deficiency. Alteredgrowth--An alteration in offspring organ or body weight or size. Changes in one end point may or may not be accompanied by other signs of altered growth (e.g, changes in body weight may or may not be accompanied by changes in crown-rump length and/or skeletal ossification). Altered growth can be induced at any stage of development, may be reversible, or may result in a permanent change. Functional developmental toxicology--The study of alterations or delays in the physiological and/or biochemical competence of an organism or organ system following exposure to an agent during critical periods of development pre- and/or postnatally. Structural abnormalities--Structural alterations in development that include both malformations and variations. Malformations and variations--A malformation is usually defined as a R&S 020094 63802 Federal Register / Vol. 56, No. 234 / Thursday, December 5, 1991 / Notices R&S 020095 permanent structural change that may protocols, such as the multigeneration A. Developmental Toxicity Studies: End adversely affect survival, development, study or the continuous breeding study. Points and Their Interpretation or function. The term teratogenicity is used in these Guidelines to refer only to malformations. The term variation is used to indicate a divergence beyond the usual range of structural constitution that may not adversely affect survival or health. Distinguishing between variations and malformations is difficult since there exists a continuum of responses from the normal to the extremely deviant. There is no generally accepted classification of malformations 'and variations. Other terms that are often used, but no better defined, include anomalies, deformations, and aberrations. IIL Hazard Identification/DoseResponse Evaluation of Agents That Cause Developmental Toxidty This section discusses the evaluation and interpretation of hazards for a variety of end points of developmental toxicity seen in both human and animal studies, and describes the criteria for characterizing the sufficiency of the health-related data base for conducting a developmental toxicity risk assessment. It also details the use of dose-response data for determining potential hazards, and describes the calculation of the RfD,,x or RfCnr. a dose or concentration that is assumed to be without appreciable risk of deleterious developmental effects for a given agent. Developmental toxicity is expressed as one or more of a number of possible end points that may be used for evaluating the potential of an agent to cause abnormal development Developmental toxicity generally occurs in a dose-related manner, may result from short-term exposure (including single exposure situations) or from longer-term low-level exposure, may be produced by various routes of exposure, and the types of effects may vary depending on the timing of exposure Although functional deficits seldom have been evaluated in routine testing studies in experimental animals, functional evaluations are beginning to be required in certain regulatory situations (U.S. EPA, 1986a. 1988a. 1989b. 1991a). Developmental toxicity can be considered a component of reproductive toxicity, and often it is difficult to distinguish between effects mediated through the parents versus direct interaction with developmental processes. For example, developmental toxicity may be influenced by the effects of toxic agents on the maternal system when exposure occurs during pregnancy or lactation. In addition, following parental exposure prior to conception, developmental toxicity may result in their offspring and, potentially, in subsequent generations. Therefore, it is useful to consult the "Proposed Guidelines for Assessing Male Reproductive Risk" [U.S. EFA. 1988b) and the "Proposed Guidelines for Assessing Female Reproductive Risk" (U.S. EPA, 1988c) in conjunction with these Guidelines. Mutational events that occur as a result of exposure to agents that cause developmental toxicity may be difficult to discriminate from other possible mechanisms in standard studies of developmental toxicity. When mutational events are suspected, the "Guidelines for Mutagenicity Risk Assessment" (U.S.EPA, 1986c), which specifically address the risks of heritable mutation, should be consulted. Carcinogenic effects have occurred in .humans following developmental exposures to diethylstilbestrol (Herbst et al.. 1971). Several additional agents (e.g., direct-acting alkylating agents) have been shown to cause cancer 1. Laboratory Animal Studies This section discusses the end points examined in routinely used protocols as weil as the use of other types of studies, including functional studies and short term tests. The most commonly used protocol for assessing developmental toxicity in laboratory animals involves the administration of a test substance to pregnant animals (usually mice, rats, or rabbits) during the period of major organogenesis, evaluation of maternal responses throughout pregnancy, and examination of the dam and the uterine contents just prior to term (U.S. EPA, 1982b, 1985a; Food and Drug Administration (FDA). 1966,1970; Organization for Economic Cooperation and Development (OECD). 1981). Some studies may use exposures of one to a few days to investigate periods of particular sensitivity for induction of abnormalities in specific organs or organ systems. In addition, developmental toxicity may be evaluated in studies involving exposure to one or both parents prior to conception, to the conceptus during pregnancy and over several generations, or to offspring during the prenatal and preweaning periods (U.S. EFA. 1982b, 1985a. 1986a, 1988a. 1991a; FDA, I960,1970; OECD, 1981; Lamb, 1985). These Guidelines ar intended to provide information for interpreting developmental effects related to any of these types of exposure. Appropriate study designs include a number of important factors. For example, test animal selection is generally based on considerations of species, strain, age. weight, and health status. Assignment of animals to dose groups by stratified randomization (an the basis of body weigh1.) reduces bias because of a number of critical periods following developmental exposures in and provides a basis for performing of development for various organs and experimental animals, and it appears valid statistical tests. At a minimum, a functional systems. from the data collected thus far that high dose, a low dose, and one The four major manifestations of agents capable of causing cancer in intermediate dose are included. The high developmental toxicity are death, adults may also cause transplacental or dose is selected to produce some structural abnormality, altered growth, neonatal carcinogenesis (Anderson et minimal maternal or adult toxicity (i.c,, and functional deficit. The relationship al., 1985). Currently, there is no way to a level that al the least produces among these manifestations may vary with increasing dose, and especially at higher doses, death of the conceptus may preclude expression of other manifestations. Of these, all four manifestations have been evaluated in human studies, but only the first three are traditionally measured in laboratory animals using the conventional developmental toxicity [also called teratogenicity or Segment U) testing protocol as well as in ether study predict whether the developing offspring or adult will be more sensitive to tha carcinogenic effects of an agent. At present, testing for carcinogenesis following developmental exposure is not routinely required. However, if this type of effect is reported for an agent, it is considered appropriate to use the "GuideJines for Carcinogen Risk Assessment" ('J.S. EPA. 1930b) for assessing human risk. marginal but significantly reduced body weight, reduced weight gain, or specific organ toxicity, and at the most produces no more than 10% mortality). At doses that cause excessive maternal toxicity (that is. significantly greater than the minimal toxic level), information on developmental effects may be difficult to interpret and of limiied value. The low dose is generally a NOAEI. for adult and offspring effects, although if the low (lose produces a biologically or Federal Register / Vol. 56. No. 234 / Thursday, December 5. 1991 / Notices 63803 statistically significant increase in response, it is considered a LOAEL (see section HI.A.l.f for a discussion of biological versus statistical significance). A concurrent control group treated with the vehicle used for agent administration is a critical component of a well-designed study. The route of exposure in these studies is usually oral unless the chemical or physical characteristics of the test substance or pattern of human exposure suggest a more appropriate route of administration. In the case of dermal exposure, developmental toxicity studies showing no indication of maternal or developmental toxicity are considered insufficient for risk assessment unless accompanied by absorption data (Klmmel and Francis. 1990). Dermal developmental toxicity studies in which skin irritation is too marked (moderate erythema and/or moderate edema, i.e., raised approximately 1 mm) also are considered insufficient since excessive maternal toxicity may be produced from the irritation rather than from systemic exposure to the agenL Assessment of maternal toxicity is based on signs of systemic toxicity rather than on local effects such as skin irritation. Absorption data and limited pharmacokinetic data collected in dermal developmental toxicity studies provide very useful information in the evaluation of study design and data interpretation (Khnmel and Francis. 1990). Many of these points also are pertinent to studies by other routes of exposure. The evaluation of specific end points of maternal and developmental toxicity is discussed in the next several sections. Appropriate historical control data sometimes can be very useful in the interpretation of these end points. Comparison of data from treated animals with concurrent study controls should always take precedent over comparison with historical control data. The most appropriate historical control data are those from the same laboratory in which studies were conducted. Even data from the same laboratory, however, should be used cautiously and examined for subtle changes over time that may result from genetic alterations in the strain or stock of the species used, changes in environmental conditions both in the breeding colony of the supplier and in the laboratory, and changes in personnel conducting studies and collecting data (Klmmel and Price. 1990). Study data should be compared with recent as well as cumulative historical data. Any change in laboratory procedure that might affect control data should be noted and the data accumulated separately from previous data. The next three sections (a-c) discuss individual end points Df maternal and developmental toxicity as measured in the conventional developmental toxicity study, the multigeneration study, and. when available, in postnatal studies. Other end points specifically related to reproductive toxicity are covered in the relevant risk assessment guidelines (U.S. EPA. 1988b, 1968c). The fourth section (d) deals with the integrated evaluation of all data, including the relative effects of exposure on maternal animals and their offspring, which is important in assessing the level of concern about a particular agent a. EndPoints ofMaternal Toxicity. A number of end points that may be observed as possible indicators of maternal toxicity are listed in Table 1. Maternal mortality is an obvious end point of toxicity, however, a number of other end points can be observed that may give an indication of the more subtle adverse effects of an agent For example, in well conducted studies, the mating and fertility indices provide information on the general fertility rate of the animal stock used and are important indicators of toxic effects to adults if treatment begins prior to mating or implantation. Changes in gestation length may indicate effects on the process of parturition. Table 1.--End Points of Maternal Toxicity Mortality Mating Index ((no. with seminal plugs or sperm/no. mated) X 100) Fertility Index ((no. with implants/no. of matings) X 100| Gestation Length (useful when animals arc allowed to deliver pups) Body Weight Day 0 During gestation Day of necropsy Body Weight Change Throughout gestation During treatment (including increments of time within treatment period) Post-treatment to sacrifice Corrected maternal (body weight change Table 1.--End Points of Maternal Toxicity--Continued throughout gestation minus gravid uter ine weight or litter weight at sacrifice! Organ Weights (in cases of suspected target organ toxicity and especially when sup ported by adverse histopathology findings) Abs-.>: '>e Re' - ' to body weight Re to brain weight Food -J Water Consumption (where rele vant i Clinical Evaluations Types, incidence, degree, and duration of clinical signs Enzyme mailer* Clinical chemistries Cross Necropsy and Histopathology Body weight and the change in body weight are viewed collectively as indicators of maternal toxicity for most species, although these end points may not be as useful in rabbits, because body weight changes are usually more variable (Kimmel and Price, 1990), and in some strains of rabbits, body weight is not a good indicator of pregnancy status. Body weight changes may provide more information than a daily body weight measured during treatment or during gestation. Changes in weight gain during treatment could occur that would not be reflected in the total weight change throughout gestation, because of compensatory weight gain that may occur following treatment but before sacrifice. For this reason, changes in weight gain during treatment can be examined as another indicator of maternal toxicity. Changes in maternal body weight corrected for gravid uterine weight at sacrifice may indicate whether the effect is primarily maternal or intrauterine. For example, a significant reduction in weight gain throughout gestation and in gravid uterine weight without any change in corrected maternal weight gain generally would indicate an intrauterine effect Conversely, a change in corrected weight gain and no change in gravid uterine weight generally would suggest maternal toxicity and little or no intrauterine effect An alternate estimate of maternal weight change during gestation can be obtained by subtracting the sum of the weights of the fetuses. However, this weight does not include the uterine or placental tissue, or the amniotic fluid. 63804 Federal Register / Vol. 56, No. 234 / Thursday, December 5, 1991 / Notices R&S 020097 Changes in other end points may also Table 2.--End Points of Developmental be important. For example, changes in Toxicity relative and absolute organ weights may be signs of a maternal effect especially when an agent is suspected of causing Litters with implants No. implantation sites/dam No. corpora lutea (CL]/dam * specific organ toxicity and when such Percent preimplantation loss findings are supported by adverse (CL--implantations) x 100* histopathologic findings in those organs. Food and water consumption data are useful, especially if the agent is CL No. and percent live offspring k/litter No. and percent resorptions/litter administered in the diet or drinking No. and percent litters with resorptions water. The amount ingested (total and relative to body weight) and the dose of ..the agent (relative to body weight) can then be calculated, and changes in food and water consumption related to No. and percent late fetal deaths/litter No. and percent nonlive (late fetal deaths + resorptions) implants/litter No. and percent litters with nonlive im plants No. and percent affected (nonlive + mal treatment can be evaluated along with formed) implants/litter changes in body weight and body weight gain. Data on food and water consumption also are useful when an' f` agent is suspected of affecting appetite, water intake, or excretory function. No. and percent litters with affected im plants No. and percent litters with total resorp tions No. and percent stillbirths/litter No. and percent litters with live offspring Utters with live offspring Clinical evaluations of toxicity also No. and percent live offspring/litter may be used as indicators of maternal toxicity. Daily clinical observations may be useful in describing the profile of maternal toxicity and alterations in general homeostasis. Enzyme markers Viability of offspring * Sex ratio/litter Mean offspring body weight/litter* Mean male or female body weight/litter* No. and percent offspring with external, visceral, or skeletal malfonnations/litter and clinical chemistries may be useful No. and percent malformed offspring/litter indicators of exposure but must be No. and percent litters with malformed interpreted carefully as to whether or not a change constitutes toxicity. Cross ^necropsy and histopathology data (when ^specified in the protocol) may aid in determining toxic dose levels. The offspring No. and percent malformed males or fe males/litter No. and percent offspring with external, visceral, or skeletal variations/litter No. and percent offspring with variations/ minimum amount of information litter considered useful for evaluating maternal toxicity (as noted in the "Proceedings of the Workshop on the Evaluation of Maternal and Developmental Toxicity" (Kimmel et al., No. and percent litters having offspring with variations _ Types and incidence of individual malfor mations Types and incidence of individual vari ations 1987)]. includes: morbidity or mortality, Individual offspring and their malforma maternal body weight and body weight gain, clinical signs of tokicity, food and water consumption (especially if dosing is via food r water), and necropsy for gross evidence of organ toxicity. In a well-designed study, maternal toxicity is determined in the pregnant and/or lactating animal over an appropriate part of gestation and/or the neonatal period, and is not assumed or extrapolated from other adult toxicity studies. tions and variations (grouped according to litter and dose) Clinical signs (type, incidence, duration, and degree) Gross necropsy and histopathology `Important when treatment begins prior to im plantation. May be difficult to aaeess in mice. `Offspring refers both to fetuses observed prior to term or to pups following birth. The end points examined depend on the protocol used for each study. 'Measured at selected intervals until termina tion of the study. When treatment of females begins prior to implantation, an increase in b. EndPoints ofDevelopmental Toxicity: Altered Survival, Growth, and Morphological Development. Because the maternal animal, and not the conceptus, is the individual treated during gestation, data generally are calculated as incidence per litter or as nuumber and percent of litters with rticular end points. Table 2 indicates e ways in which offspring and litter end points may be expressed. preimplantation loss could indicate an adverse effect on gamete transport, the fertilization process, uterine toxicity, the developing blastocyst, or on the process of implantation itself. If treatment begins around the time of implantation (i.e, day 6 of gestation in the mouse, rat, or rabbit), an increase in preimplantation loss probably reflects variability that is not treatment-related in the animals being used, but the data should be examined carefully to determine if there is a dose-response relationship. If preimplantation loss is related to dose, further studies would be necessary to determine the mechanism and extent of such effects. The number and percent of live offspring per litter, based on all litters, may include Utters that have no live implants. The number and percent of resorptions and late fetal deaths give some indication of when the conceptus died, and the number and percent of nonUve implants per litter (postimpiantation loss) is a combination of these two measures. Expression of data as the number and percent of litters showing an increased incidence for these end points may be less useful than incidence per litter because, in the former case, a litter is counted whether one or all implants were resorbed, dead, or nonUve. If a significant increase in postimpiantation loss is found after exposure to an agent, the data may be compared not only with concurrent controls, but also with recent historical control data (preferably from the same laboratory), since there is considerable interlitter variability in the incidence of postimpiantation loss (Kimmel and Price, 1990). If a given study control group exhibits an unusually high or low incidence of postimpiantation loss compared to historical controls, then scientific judgment must be used to determine the adequacy of the study for risk assessment purposes. The end point "affected implants" (i.e- the combination of nonlive and malformed conceptuses) sometimes reflects a better dose-response relationship than does the incidence of nonUve or malformed offspring taken individually. This Is especially true at the high end of the dose-response curve in cases when the incidence of nonUve implants per Utter is greatly increased. In such cases, the malformation rate may appear to decrease because only unaffected offspring have survived. If the incidence of prenatal deaths or malformations is unchanged, then the incidence of affected implants will not provide any additional dose-response informaUon. In studies where maternal animals are aUowed to deliver pups nonnaUy, the number of stillbirths per Utter should also be noted. The number of live offspring per Utter, based on those Utters that have one or more live offspring, may be unchanged even though the incidence of nonUve in all Utters is increased. This could occur either because of an increase in the number of litters with no live offspring, or an increase in the number of implants per litter. A decrease in the number of Federal Register / Vol. 56, No. 234 / Thursday, December 5, 1991 / Notices 63805 live offspring per litter is generally accompanied by an increase in the incidence of nonlive implants per litter unless the implant numbers differ among dose groups. In postnatal studies, the viability of live-bom offspring should be determined at selected intervals until termination of the study. The sex ratio per litter, as well as the body weights of males and females, can be examined to determine whether or not one sex is preferentially affected by the agent However, this is an unusual occurrence. A change in offspring body weight is a sensitive indicator of developmental toxicity, in part because it is a continuous variable. In some cases, offspring weight reduction may be the only indicator of developmental toxicity. While there is always a question as to whether weight reduction is a permanent or transitory effect, little is known about the long-term consequences of short-term fetal or neonatal weight changes. Therefore, when significant weight reduction effects are noted, they are used as a basis to establish the NOAEL Several other factors should be considered in the evaluation of fetal or neonatal weight changes; for example, in polytocous animals, fetal and neonatal weights are usually inversely correlated with litter size, and the upper end of the dose-response curve may be affected by smaller litters and increased fetal or neonatal weight Additionally, the average body weight of males is greater than that of females in the more commonly used laboratory animals. Live offspring are generally examined for external. visceral and skeletal malformations and variations. If only a portion of the litter is examined for one or more end points, then random selection of those pups examined introduces less bias in the data. An increase in the incidence of malformed offspring may be indicated by a change in one or more of the following end points; the incidence of malformed offspring per litter, the number and percent of litters with malformed offspring, or the number of offspring or litters with a particular malformation that appears to increase with dose (as indicated by the incidence of individual types of malformations). Other ways of examining the data include determining the incidence of external, visceral, and skeletal malformations end variations that may indicate the organs or organ systems affected. A listing of individual offspring with their malformations and variations may give an indication of the pattern of developmental deviations. All of these methods of expressing and examining the data are valid for determining the effects of an agent on structural development. However, care must be taken to avoid counting offspring more than once in the evaluation of any single end point based on number or percent of offspring or litters. The incidence of individual types of malformations and variations may indicate significant changes that are masked if the data on all malformations and/or variations are pooled. Appropriate historical control data can be especially helpful in the interpretation of malformations and variations, particularly those that normally occur at a low incidence and may or may not be related to dose in an individual study. Although a dose-related increase in malformations is interpreted as an adverse developmental effect of exposure to an agent, the biological significance of an altered incidence of anatomical variations is more difficult to assess, and must take into account what is known about developmental stage (e.g- with skeletal ossification), background incidence of certain variations (e.g, 12 or 13 pairs of ribs in rabbits), or other strain- or speciesspecific factors. However, if variations are significantly increased in a doserelated manner, these should also be evaluated as a possible indication of developmental toxicity. In addition, although some investigators have considered certain of these effects to simply be associated with manifestations of maternal toxicity noted at similar dose levels (Khera, 1984,1985,1987). such effects are stiil toxic manifestations and as such are generally considered a reasonable basis for Agency regulation and/or risk assessment. On a somewhat similar note, the conclusion of participants in a "Workshop on Reproductive Toxicity Risk Assessment" (Kimmel et &L. 1986) was that dose-related increases in defects that may occur spontaneously are as relevant as dose-related increases in any other developmental toxicity end points. & EndPoints ofDevelopmental Toxicity: Functional Deficits, Developmental effects that are induced by exogenous agents are not limited to death, structural abnormalities, and altered growth. Rather, it has been demonstrated in a number of instances that alterations in the functional competence of an organ or a variety of organ systems may result from exposure during critical developmental periods that may occur between conception and sexual maturati n. Sometimes, these functional defer are observed at dose levels below those at which other indicators of developmental toxicity are evident (Rodier. 1978). Such effects may be transient or reversible in nature, but generally are considered adverse effects. Testing for functional developmental toxicity has not been required routinely by regulatory agencies in the United States, but studies in developmental neurotoxicity are beginning to be required by the EPA when other information indicates the potential for adverse functional developmental effects (U.S. EPA, 1988a. 1988a. 1989b, 1991a). Data from postnatal studies, when available, are considered very useful for further assessment of the relative importance and severity of findings in the fetus and neonate. Often, the long-term consequences of adverse developmental outcomes noted at birth are unknown, and further data on postnatal development and function are necessary to determine the full spectrum of potential developmental effects. Useful data can also be derived from wellconducted multigeneration studies, although the dose levels used in these studies may be much lower than in studies with shorter-term exposure. Much of the early work in functional developmental toxicology was related to behavioral evaluations, and the term "behavioral teratology*' became prominent in the mid 1970s. Recent advances in this area have been reviewed in several publications (Riley and Vorhees, 1986: Kimmel 1988; Kimmel et al.. 1990a). Several expert groups have focused on the functions that should be included in a behavioral testing battery (World Health Organization (WHO). 1984; Buelke-Sam et al,, 1985; Leukroth, 1986). These include: sensory systems, neuromotor development locomotor activity, learning and memory, reactivity and/or habituation, and reproductive behavior. No testing battery has fully addressed all of these functions, but it is important to include as many as possible, and several testing batteries have been developed and evaluated for use in testing (Buelke-Sam et al, 1985; Tanimura, 1986; Eisner et al.. 1986). The Agency recently has developed a "generic" developmental neurotoxicity test guideline that can be used for both pesticides and industrial chemicals (U.S. EPA 1991a). Because of its design, the developmental neurotoxicity testing protocol may be conducted as a separate study, concurrently with or as a follow-up to a developmental toxicity (Segment II) study, or be folded into a multigcneration study in the second generation. Testing is generally conducted in the rat. In the protocol for the separate study, the test agent is 63806 Federal Register / Vol. 56. No. 234 / Thursday. December 5, 1991 / Notices R&S 020099 administered orally (other routes may be used on a case-by-case basis) to at least three treated groups and one concurrent control group of animals on day 6 of gestation through day 10 postnatally. The highest dose level is selected to induce some overt signs of maternal toxicity, but not result in more than a 20% reduction in weight gain during gestation and lactation. This dose also is selected to avoid in utero or neonatal death or malformations sufficient to preclude a meaningful evaluation of developmental neurotoxicity. At least 20 litters are required per treatment group. For behavioral tests, one female and one male pup per litter are randomly selected and assigned to one of the following tests: motor activity, auditory startle, and learning and memory in animals at weaning and as adults. Neuropathological evaluation and determination of brain weights are conducted on selected pups at postnatal day 11 and at termination of the Study- Several criteria for selecting agents for developmental neurotoxicity testing have been suggested (Buelke-Sam et al., 1985; Levine and Butcher. 1890), including: Agents that cause central nervous system malformations, psychoactive drugs and chemicals, agents that cause adult neurotoxicity, hormonally-active agents, and chemicals that are structurally related to others that cause developmental neurotoxicity or for which wide-spread exposure and/ or release is expected. Data from developmental neurotoxicity studies should be evaluated in light of the data that may have triggered such testing as well as all other toxicity data available. Less work has been done on other developing functional systems, but the assessment of postnatal renal morphological and functional development may serve as a model for the use of postnatal evaluations in the risk assessment process. As an example, standard morphological analyses of the kidneys of fetal rodents have detected treatment-related changes in the relative growth of the renal papilla versus the renal cortex, an effect considered in some cases to be a malformation (hydronephrosis), while in other cases a variation (apparent hydronephrosis, enlarged or dilated renal pelvis). While some investigators (Woo and Hoar, 1972) have provided data suggesting that the morphological effect represents a transient developmental delay, others have shown that it can persist well into postnatal life and that physiological function is compromised in the affected individuals (Kavlock et al.. 1987a, 1988; Daston et al.. 1988; Couture, 1990). Thus, the biological interpretation of this effect on the basis of fetal examinations alone is tenuous (U.S. EPA. 1985b). In addition, the critical period for inducing renal morphological abnormalities extends into the postnatal period (Couture, 1990), and studies on perinatally-induced renal growth retardation (Kavlock et al., 1988.1987b; Slotkin et al,, 1988; Cray et al., 1989; Gray and Kavlock, 1991) have shown that renal function is generally altered in such conditions, but that manifestation of the dysfunction is not readily predictable. Thus, both morphological and functional assessment of the kidneys after birth can provide useful and complementary information on the persistence and biological significance of expressions of developmental toxicity. Although not as well-studied, data indicate that the cardiovascular, respiratory, immune, endocrine, reproductive, and digestive systems also are subject to alterations in functional competence (Kavlock and Grabowski. 1983; Fujii and Adams. 1987) following exposure during development. Currently, there are no standard testing procedures for these functional systems; however, when data are encountered on a chemical under review, they are considered in the risk assessment process. Direct extrapolation of functional developmental effects to humans is limited in the same way as for other end points of developmental toxicity. i.e.. by the lack of knowledge about underlying toxicological mechanisms and their significance. In evaluations of a limited number of agents known to cause developmental neurotoxic effects in humans, Adams (1986) concluded that these agents produce similar developmental neurotoxic effects in animals and humans. This conclusion was strongly supported by the results of a recent "Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity," sponsored by EPA and the National Institute on Drug Abuse (NIDA), at which participants critically evaluated and compared the effects of agents known to cause human developmental neurotoxicity with the effects seen in experimental animal studies (Klmmel et al., 1990a). The high degree of qualitative correlation between human and experimental animal data for the agents evaluated lends strong support for the use of experimental animals in assessing the potential risk for developmental neurotoxicity in humans. Thus, as for other end points of developmental toxicity, the assumption can be made that functional effects in animal studies indicate the potential for altered development in humans, although the types of developmental effects seen in experimental animal studies will not necessarily be the same as those that may be produced in humans. Thus, when data from functional developmental toxicity studies are encountered for particular agents, they should be considered in the risk assessment process. Some guidance is provided here concerning important general concepts of study design and evaluation for functional developmental toxicity studies. Several aspects of study design are similar to those important in standard developmental toxicity studies (e.g., a dose-response approach with the highest dose producing minimal overt maternal or perinatal toxicity, number of litters large enough for adequate statistical power, randomization of animals to dose groups and test groups, litter generally considered the statistical unit. etc.). A replicate study design provides added confidence in the interpretation of data. Use of a pharmacological/ physiological challenge may be valuable in evaluating function and "unmasking" effects not otherwise detectable, particularly in the case of organ systems that are endowed with a reasonable degree of functional reserve capacity. Use of functional.tests with a moderate degree of'background variability may be more sensitive to the effects of an agent on behavioral end points than are tests with low variability that may be impossible to disrupt without being life-threatening (Butcher et al., 1980). A battery of functional tests, in contrast to a single test, is usually needed to evaluate the full complement of organ function in an animal; tests conducted at several ages may provide more information about maturational changes and their persistence. Critical periods for the disruption of functional competence include both the prenatal and the postnatal periods to the time of sexual maturation, and the effect is likely to vary depending on the time and degree of exposure. Interpretation of data from studies in which postnatal exposure is included should take into account possible interaction of the agent with maternal behavior, milk composition, pup suckling behavior, possible direct exposure of pups via dosed feed or water, etc. Federal Register / Vol. 56, No. 234 / Thursday, December 5. 1991 / Notices . 63807 Although interpretation of functional data may be limited at present, it is clear that functional effects must be evaluated in light of other toxicity data, including other forms of developmental toxicity (e.g., structural abnormalities, perinatal death, and growth retardation). The level of confidence in an adverse effect may be as important as the type of change seen, and confidence may be increased by such factors as replicability of the effect either in another study of the same function or by convergence of data from tests that purport to measure similar functions. A dose-response relationship is considered an important measure of chemical effect; in the case of functional effects, both monotonic and biphasic dose-response curves are likely, depending on the function being tested. Finally, there are at least three general ways in which the data from these studies may be useful for risk assessment purposes: (1) To help elucidate the long-term consequences of fetal and neonatal effects; (2) to indicate the potential for an agent to cause functional alterations and the effective doses relative to those that produce other forms of toxicity; and (3) for existing environmental agents, to suggest organ systems to be evaluated in exposed human populations. ' d. Overall Evaluation ofMaternal and Developmental Toxicity. As discussed previously, individual end points of maternal and developmental toxicity are evaluated in developmental toxicity studies. In order to interpret the data fully, an integrated evaluation must be performed considering all maternal and developmental end points. Agents that produce developmental toxicity at a dose that is not toxic to the maternal animal are especially of concern because the developing organism is affected but toxicity is not apparent in the adult. However, the more common situation is when adverse developmental effects are produced only at doses that cause minimal maternal toxicity; in these cases', the developmental effects are still considered to represent developmental toxicity and should not be discounted as being secondary to maternal toxicity. At doses causing excessive maternal toxicity (that is. significantly greater than the minimal toxic dose), information on developmental effects may be difficult to interpret and of limited value. Current information is inadequate to assume that developmental effects at maternally toxic doses result only from maternal toxicity; rather, when the LOAEL is the same for the adult and developing organisms, it may simply indicate that both are sensitive to that dose level. Moreover, whether developmental effects are secondary to maternal toxicity or not. the maternal effects may be reversible while effects on the offspring may be permanent. These are important considerations for agents to which humans may be exposed at minimally toxic levels either voluntarily or involuntarily, since several agents are known to produce adverse developmental effects at minimally toxic doses in adult humans (e.g.. smoking, alcohol, isotretinoin). Since the final risk assessment not only takes into account the potential hazard of an agent, but also the nature of the dose-response relationship, it is important that the relationship of maternal and developmental toxicity be evaluated and described. Then, information from the exposure assessment is used to determine the likelihood of exposure to levels near the maternally toxic dose for each agent and the risk for developmental toxicity in humans. Although the evaluation of developmental toxicity is the primary objective of standard studies within this area, maternal effects seen within the context of developmental toxicity studies should be evaluated as part of the overall toxicity profile for a given chemical. Maternal toxicity may be seen in the absence of or at dose levels lower than those producing developmental toxicity. If the maternal effect level is lower than that in other evaluations of adult toxicity, this implies that the pregnant female is likely to be more sensitive than the nonpregnant female. Data from reproductive and developmental toxicity studies on the pregnant female should be used in the overall assessment of risk. Approaches for ranking agents according to their relative maternal and developmental toxicity have been proposed; Schardein (1983) has reviewed several of these. Several approaches involve the calculation of ratios relating an adult toxic dose to a developmental^ toxic dose (Johnson, 1981; Fabro et al.. 1982; Johnson and Gabel. 1983; Brown and Freeman. 1984). Such ratios may describe in a qualitative and roughly quantitative fashion the relationship of maternal (adult) and developmental toxicity. However, at the U.S. EPA-sponsored "Workshop on the Evaluation of Maternal and Developmental Toxicity" (Kimmel et al-1987). there was no agreement as to the validity or utility of these approaches in other aspects of the risk assessment process. This is due in part to uncertainty about factors that can affect the ratios. For example, the number and spacing of dose levels, differences in study design (e.g.. route and/or timing of exposure), the relative thoroughness in the assessment of maternal and developmental end points examined, species differences in response, and differences in the slope of the dose-response curves for maternal and developmental toxicity, can all influence the maternal and developmental effects observed and the resulting ratios (Kimmel et al., 1987; U.S. EPA, 1985b). Also, maternal and developmental end points used in the ratios need to be better defined to permit cross-species comparison. Until such informetion is available, the applicability of these approaches in risk assessment is not justified. e. Short-Term Testing in Developmental Toxicity. The need for short-term tests for developmental toxicity has arisen from the need to establish testing priorities for the large number of agents in or entering the environment, the interest in reducing the number of animals used for routine testing, and the expense of testing. These approaches may be useful in making preliminary evaluations of potential developmental toxicity, for evaluating structure activity relationships, and for assigning priorities for further, more extensive testing. Furthermore, as the risk assessment process begins to incorporate more pharmacokinetic and mechanistic data, short-term tests should be particularly useful. Kimmel (1990) has recently discussed the potential application of in vitro systems in risk assessment in a context that is broader than chemical screening. However, the Agency currently considers a short-term test as "insufficient" by itself to carry out a risk assessment (see Section I1I.C). Although short-term tests for developmental toxicity are not routinely required, such data are encountered in the review of chemicals. Two approaches are considered here in terms of their contribution to the overall testing process: (1) An in vivo mammalian screen, and (2) in vitro test systems. (1) In vivo*mammalian developmental toxicity tests. Hie most widely studied in vivo short-term approach is that developed by Chemoff and Kavlock (1982). This approach is based on the hypothesis that a prenatal injury, which results in altered development, well be manifested postnatally as reduced viability and/or impaired growth. When originally proposed, the test substance 63808 Federal Register / Vol. 56, No. 234 / Thursday, December 5. 1991 / Notices R&S 020101 ,* was administered lo mice over the day 4. Male animals are exposed litters do not respond independently, the period of major organogenesis at a following mating for a period statistical analyses are generally single dose level that would elicit some corresponding to that of the females. designed to analyze the relevant data degree of maternal toxicity. At the Adult animals are evaluated for general based on incidence per litter or on the N10SH "Workshop on the Evaluation of toxicity and effects or. reproductive number of litters with a particular end the Chernoff/Kavlock Test for organs. Pups are counted, weighed and point. The analytical procedures used Developmental Toxicity" (Hardin, 1387). examined for any gross physical or and the results, as well as an indication use of a second lower dose level was behavioral abnormalities at birth and on of the variance in each end point, should encouraged to potentially reduce the postnatal day 4. This protocol permits be evaluated carefully when reviewing chances of false positive results, and the evaluation of reproductive and data for risk assessment purposes. recording of implantation sites was developmental toxicity following Analysis of variance (ANOVA) recommended lo provide a more precise repeated dosing with an agent, provides techniques, with litter nested within estimate of poslimplantation loss an indication for the need to conduct dose in the modeL take the litter IKavlock et aL. 1987c). In this approach, additional studies, and provides variable into account while allowing use the pups are counted and weighed guidance in the design of further studies. of individual offspring data and an shortly after birth, and again after 3-4 Currently, this study design is evaluation of both within and between days. End points that are considered in insufficient by itself to make an estimate litter variance as well as dose effects. the evaluation include: general maternal of human risk without further studies to Nonparametric and categorical toxicity (including survival and weight confirm and extend the observations. procedures have also been widely used gain), litter size, pup viability and (2) In vitro developmental toxidty for binomial or incidence data. In weight, and gross malformations in the screens. Test systems that fall under the addition, tests for dose-response trends offspring. Several schemes have been general heading of "in vitro" can be applied. Although a single proposed for ranking the results as a developmental toxicity screens include statistical approach has not been agreed means of prioritizing agents for further any system that employs a test subject upon, a number of factors important in testing (Chemoff and Kavlock. 1982; ' other than the intact pregnant mammal. the analysis of developmental toxicity Brown, 1984; Schuler et al,, 1984). Examples of such systems include: data have been discussed (Haseman The mouse was chosen originally for isolated whole mammalian embryos in and Kuppcr, 1979; Kimmei et al, 1980). this test because of its low cost, but the culture, tissue/organ culture, cell Studies that employ a replicate procedure has been applied to the rat as culture, and developing nonmammalian experimental design (e.g., two or three well (Wickramaratne. 1987). The test organisms. These systems have long replicates with 10 litters per dose per can predict the potential for been used to assess events associated replicate rather than a single experiment developmental toxicity of an agent in with normal and abnormal development, with 20 to 30 litters'per dose group) the species used while extrapolation of but more recently they have been allow broader interpretation of study risk to other species, including humans, considered for their potential as screens results since the variability between has the same limitations as for other in testing (Wilson, 1978; Kimmei et aL, replicates can be accounted for using testing protocols. The EPA Office of 19B2b; Brown and Fabro. 1982). Many of ANOVA techniques. Replication of Toxic Substances has developed testing these systems are now being evaluated effects due to a given agent within a guidelines for this procedure (U.S* EPA, for their ability to predict the study, as well as among studies or 1985c). and the Office of Pesticide developmental toxicity of various agents laboratories, provides added strength in Programs has applied similar protocols in intact mammalian systems. This the use of data for the estimation of risk. on a case-by-case basis (U.S. EPA, validation process requires certain An important factor to consider in 19&5b). The National Toxicology considerations in study design, including evaluating data is the power of a study Program also has developed a protocol defined end points for toxicity and an (i.e, the probability that a study will that incorporates aspects of a range . understanding of the system's ability to demonstrate a true effect), which is finding study, with the intent of handle various test agents (Kimmei et limited by the sample size used in the providing information on appropriate al., 1982a; Kimmei, 1385; FDA. 1987: study, the background incidence of the exposure levels should a standard Brown, 1987). end point observed, the variability in the developmental toxicity study be While in vitro test systems can incidence of the end point, and the required (Morrissey et aL, 1989). provide significant information, they are analysis method. As an example. Nelson Although testing guidelines are considered insufficient by themselves, and Holson (1978) have shown that the available, such procedures are required for carrying out a risk assessment (see number of litters needed to detect a 5% on a case-by-case basis. Application of section 11LC). In part, this is due to or 10% change was dramatically lower this procedure in the risk assessment limitations in the application of the data for fetal weight (a continuous variable process within the Office of Toxic to the whole animal situation. But it is with low variability) than for Substances has been described (Francis also due to the lack of assays that have resorptions (a binomial response wi:h end Farland, 1987), and the experiences been fully validated, as has been noted high variability). With the current of a number of laboratories are detailed in several reviews of available in vitro recommendation in testing protocols in the proceedings of a NI05H- systems (FDA, 1987; Brown, 1987; being 20 rodents per dose group (U.S. sponsored workshop (Hardin. 1987). Faustman. 1988) and at a recent EPA, 1982b. 1985a), the minimum change Recently, the OECD developed a workshop on in vitro teratology detectable is an increased incidence of screening protocol to be used for (Morrissey et al., 1991). malformations 5 to 12 times above prioritizing existing chemicals for further f. Statistical Considerations. In the control levels, an increase 3 to 6 times testing (draft as of March 22.1990). This assessment of developmental toxicity the in utero death rate, and a decrease protocol is similar to the design of the data, statistical considerations require 0.15 to 0.25 times the fetal weight. Thus, Chernoff-Kavlock Test except that it special attention. Since the litter is even within the same study, the ability involves exposure of male end female generally considered the experimental to detect a change in fetal weight is rats 2 weeks prior to mating, throughout unit in most developmental toxicity much greater than for the other end mating and gestation, and postnatally to studies, and fetuses or pups within points measured. Consequently, for Federal Register / Vol. 56. No. 234 / Thursday, December 5. 1991 / Notices 63809 statistical reasons only, changes in fetal weight are often observed at doses below those producing other signs of developmental toxicity. Any risk assessment should present the detection sensitivity for the study design used and for the end point(s) evaluated. Although statistical analyses are important in determining the effects of a particular agent, the biological significance of data Is most relevant It is important to be aware that with the number of end points that can be observed in standard protocols for developmental toxicity studies, a few statistically significant differences may occur by chance. On the other hand, apparent trends with dose may be biologically relevant even though pair wise comparisons do not indicate a statistically significant effect This may be true especially for the incidence of malformations or in utero death because of the low power of standard study designs in which a relatively large difference is required to be statistically significant. It should be apparent from this discussion that a great deal of scientific judgment based on experience with developmental toxicity data and with principles of experimental design and statistical analysis, may be required to adequately evaluate such data. 2. Human Studies In principle, human data are preferred for risk assessment However, the complexities of obtaining sufficient human data are such that these data are not available for many potential toxicants. The following describes the methods of generation of human data, their evaluation, and the weight they should be given in risk assessments. The category of "human studies" includes both epidemiologic studies and other reports of individual Cases or clusters of events. Greatest weight should be given to carefully designed epidemiologic studies with more precise measures of exposure, since they can best evaluate exposure-response relationships (see Section IV). Epidemiologic studies in which exposure is presumed based on occupational title or residence (e.g., some case-referent and all ecologic studies) may contribute data to qualitative risk assessments, but are of limited use for quantitative risk assessments because of the generally broad categorical groupings. Reports of individual cases or clusters of events may generate hypotheses of exposureoutcome associations, but require further confirmation with well-designed epidemiologic or laboratory studies. These reports of cases or clusters may give added support to associations suggested by other human or animal data, but cannot stand by themselves in risk assessments. Risk assessors should seek the assistance of professionals trained in epidemiology when conducting a detailed analysis. a. Epidemiologic Studies. Good epidemiologic studies provide the most relevant information for assessing human risk. As there are many different designs for epidemiologic studies, simple rules for their evaluation do not exist (1) General design considerations. The factors that enhance a study and thus increase its usefulness for risk assessment have been noted in a number of publications (Selevan. 1980: Bloom, 1981: U.S. EPA. 1981; Wilcox. 1983; Sever and Hessol. 1984; Axelson, 1985; Tilley et al.. 1985; Kimmel et at., 1986). Some of the more prominent factors are as follows: (a) The power of the study: The power, or ability of a study to detect a true effect, is dependent on the size of the study group, the frequency of the outcome in the general population, and the level of excess risk to be identified. In a cohort study, common outcomes, such as recognized fetal loss, require hundreds of pregnancies in order to have a high probability of detecting a modest increase in risk (e.g., 133 in both exposed and unexposed groups to detect a doubling of background; alpha = 0.05. power = 80%). while less common outcomes, such as the total of all malformations recognized at birth, require thousands of pregnancies to have the same probability (e.g.. more than 1.200 in both exposed and unexposed groups) (Bloom. 1981; Selevan. 1981: Sever and Hessol. 1984: Selevan, 1985; Stein et al.. 1985; Kimmel et al.. 1986). In case-referent studies, study sizes are dependent on the frequency of exposure within the source population. The confidence one has in the results of a study without positive findings is related to the power of the study to detect meaningful differences in the end points studied. Power may be enhanced by combining populations from several studies using a metaanalysis (Greenland. 1987). The combined analysis would increase confidence in the absence of risk for agents with negative findings. However, care must be exercised in the combination of potentially dissimilar study groups. A posteriori determination of power of the actual study may be useful in evaluating contradictory studies in risk assessment. Absence of positive findings in a study of lew power would be given less weight than either a positive study or a null study (one with no significant differences) with high power. Positive findings from very small studies are open to question due to the instability of the risk estimates and the potential for highly selected study groups. (b) Potential bias in data collection: Sources of bias may include selection bias and information bias (Rothman. 1988). Selection bias may occur when an individual's willingness to participate varies with certain characteristics relating to the exposure status or health status of that individual In addition, selection bias may operate in the identification of subjects for study. For example, in studies of embryonir loss, use of hospital records to identuy embryonic or early fetal loss will underascertain events, because women are not always hospitalized for these outcomes. More weight might be given in a risk assessment to a study in which a more complete list of pregnancies is obtained by. for example, collecting biological data (e.g.. human chorionic gonadotropin (hCC) measurements] on pregnancy status from study members. These studies may also be affected by bias. The representativeness of these data may be affected by selection factors related to the willingness of different groups of women to continue participation over the total length of the study. Interview data result in more complete ascertainment; however, this strategy carries with it the potential for recall bias, discussed in further detail beiow. A second example of different levels of ascertainment of events is the use of hospital records to study congenital malformations. Hospital records contain more complete data on malformations than do birth certificates (Mackeprang et al. 1972). Consequently, birth defects registries that are based on searches of hospital records are more complete than those based on vital records (Selevan. 1986). Thus, a study using hospital records to identify congenital malformations would be given more emphasis in a risk assessment than one using birth certificates. Studies of working women present the potential for additional bias since some factors that influence employment status may also be associated with reproductive end points. For example, due to child-care responsibilities, women may terminate employment, as might women with a history of reproductive problems who wish to have children and are concerned about workplace exposures (Joffe. 1985). Information bias may result from misclassification of characteristics of individuals or events identified for 63810 Federal Register / Vol. 56, No. 234 / Thursday, December 5, 1991 / Notices R&s 020103 study. Recall bias.'one type of ' information bias, may occur when respondents with specific exposures or outcomes recall information differently than these without the exposures or outcomes. Interview bias may result when the interviewer knows a priori the category' of exposure (for cohort studies) or outcome (for case-referent studies) in which the respondent belongs. Use of highly structured questionnaires and/or "blinding" of the interviewer will reduce the likelihood of such bias. Studies with lower likelihood of the above-listed bias should carry more weight in a risk assessment When data are collected by interview or questionnaire, the appropriate respondent depends on the type of data r study. For example, a comparison of husband-wife interviews on reproduction found the wives' responses to questions on pregnancy-related events to be considerably more complete and valid than those of the - husbands (Selevan, 1980). A more recent study (Schnatter, 1990) found small, nonsignificant improvements In reporting of birth weights by mothers compared to fathers, and that males who provide early fetal loss data with the aid of their wives give better data (borderline significance). Studies based on interview data from the appropriate ^^respondent(s) would carry more weight ^Bhan those from proxy respondents (e.g., ^^the specific individual when examining exposure history and the woman or both partners when examining pregnancy history). Data from any source may be prone to errors or bias. All types of bias are difficult to assess: however, validation with an independent data source (e.g., vital or hospital records), or use of biomarkers of exposure r outcome, where possible, may indicate the degree of bias present and increase confidence in the results of the study. Those studies with a low probability of biased data should cany more weight (Axelson, 1985: Stein and Hatch, 1987). Differential misclassification, i.e., when certain subgroups are more likely to have misclassifted data than others, may either raise or lower the risk estimate. Nondifferential misclassification will bias the results toward a finding of "no effect" (Rothman. 1986). (c) Collection of data on other risk factors, effect modifiers, and confounders: Risk factors for reproductive and developmental toxicity include such characteristics as age, smoking, alcohol consumption, drug use, nd past reproductive history, ^duitinnally. occupational and Environmental exposures are potential risk factors for reproductive and developmental effects. Known and potential risk factors should be examined to identify those that may be effect modifiers or confounders. An effect modifier is a factor that produces different exposure-response relationships at different levels of that factor. For example, maternal age would be an effect modifier if the risk associated with a given exposure increased with the mother's age. A confounder is a variable that is a risk factor for the disease under study and la associated with the exposure under study, but is not a consequence of the exposure. A confounder may distort both the magnitude and direction of the measure of association between the exposure of interest and the outcome. For example, socioeconomic status might be a confounder in a study of the association of smoking and fertility, since socioeconomic status may be associated with both. Studies that fail to account for effect modifiers and confounders should be given less weight in a risk assessment Both of these important factors heed to be controlled in the study design and/or analysis to improve the estimate of the effects of exposure (Kleinbaum et al., 1982). A more in-depth discussion may be found elsewhere (Epidemiology Workgroup, 1981; Kleinbaum et al., 1982; Rothman^ 1986). The statistical techniques used to control for these factors require careful consideration in their application and interpretation (Kleinbaum et al., 1982; Rothman, 1986). (d) Statisticalfactors: As in animal studies, pregnancies experienced by the same woman are not independent events (Kissling, 1981; Selevan. 1985). Women who have had embryo/fetal loss are reported to be more likely to have subsequent losses (Leridon. 1977). In animal studies, the litter is generally used as the unit of measure to deal with nonindependence of events. In studies of humans, pregnancies are sequential with the risk factors changing for different pregnancies, making analyses considering nonindependence of events very difficult (Epidemiology Workgroup. 1981; Kissling. 1981). If more than one pregnancy per woman is included, as is often necessary due to small study groups, the use of nonindependent observations overestimates the true size of the groups being compared, thus . artificially increasing the probability of reaching statistical significance (StirateUi et al., 1981). Biased estimates of risk might also result if family size confounds the relationship between exposure and outcome. Some approaches to deal with these issues have been suggested (Kissling. 1981: Stiraintii el al.. 1984: Selevan. 1985). At this point in time, a generally accepted solution to this problem has not been developed. (2) Selection of outcomes for study. As already discussed, a number of end points can be considered in the evaluation of adverse developmental effects. However, some of the outcomes are not easily observed in humane, such as early embryonic loss and reproductive capacity of the offspring. Currently, the most feasible end points for epidemiologic studies are reproductive history studies of some pregnancy outcomes (e.g., embryo/fetal loss, birth weight, sex ratio, congenital malformations, postnatal function, and neonatal growth and survival) and measures of fertility/infertility which would include indirect evaluations of very early embryonic loss. Postnatal outcomes for examination could include physical growth and development, organ or system function and behavioral effects of exposure. Factors requiring control in the design or analysis (such as effect modifiers and confounders] may vary depending on the specific outcomes selected for study. Hie developmental outcomes available for epidemiologic examination are limited by a number of factors, including the relative magnitude of the exposure since differing spectra of outcomes may occur at different exposure levels, the size and demographic characteristics of the population, and the ability to observe the developmental outcome in humans. Improved methods for identifying some outcomes such as very early embryonic loss using new bCG assays may change the spectrum of outcomes available for study (Wilcox et ah, 1985; Sweeney et al, 1988). Demographic characteristics of the population, such as marital status, age distribution, education, socioeconomic status (SES) and prior reproductive history are associated with the probability of whether couples will attempt to have children. Differences in the use of birth control would also affect the number of outcomes available for study. In addition, women with live births are more likely to terminate employment than are those with other outcomes, such as infertility or early embryonic loss. Thus, retrospective studies of female exposure that do not include terminated women workers may be of limited use in risk assessment because the level of risk for these outcomes is likely to be overestimated (Lernastcrs and Finney, 1 %i`). In addition to the above-mentioned factors, developmental end points may Federal Register / VoL. 56* No. 234 / Thursday, December 5, 1991 / Notices 63811 be envisioned as effects recognized at various po in a continuum, starting 31 concep .rough death of the offspring a malformed stillbirth would no. eluded in a study of defects obt ed at live birth, even though the t uology could be identical [Stein et al,, 1975; Bloom, 1961). A shift in the patterns of outcomes could result from differences in timing or in level of exposure (Selevan and LeMasters, 1987). (3) Reproductive history studies, (a) Measures of fertility; Normally, studies cf sub-.or infertility would not be included in an evaluation of developmental effects. However, in humans it is difficult to identify very early embryonic loss, and distinguish it from sub- or infertility. Thus, studies that examine sub- or infertility indirectly L-xamine loss very early in the gestational period. Infertility or subfertility may be thought of as a nonevent: A couple is unable to have children within a specific time frame. Therefore, the epidemiologic measurement of reduced fertility is typically indirect, and is accomplished by comparing birth rates or time intervals between births or pregnancies. In these evaluations, the couple's joint ability to procreate is estimated. One method, the Standardized Birth Ratio (SBR; also referred to as the Standardized Fertility Ratio), compares the number of births observed to those expected based on the person-years of observation stratified by factors such as time period, age, race, marital status, parity, contraceptive use, etc. (Wong et at.. 1979; Levine et al, 1989.1981; Levine. 1983; Starr et al, 1986). The SBR is analogous to the Standardized Mortality Ratio (SMR). a measure frequently used in studies of occupational cohorts, and has similar limitations in interpretation (Gaffey, 1976; McMichael, 1976; Tsai and Wen, 1586). Analysis of the time period between recognized pregnancies or live births has been suggested as another indirect measure of fertility (Dobbins et al,, 1978; Baird et al.. 1986; Weinberg and Gladen, 1986). Because the time interval between births increases with increasing parity (Leridon. 1977), comparisons within birth order (parity) are more appropriate. A statistical method (Cox regression) can stratify by birth or pregnancy order to help control for nr ' .independence of these events in the r woman. iity may also be affected by a. Jtions in sexual behavior. However, limited data are available linking toxic exposures to these alterations in humans. Moreover, such data are not easily obtained in epidemiology studies. More information on this subject is available in the proposed male end female reproductive risk assessment guidelines (U.S. EPA. 1933b, 1988c). (b) Pregnancy outcomes: Pregnancy outcomes examined in human studies of parental exposures may include embryo/fetal loss, congenital malformations, birth weight, sex ratio at birth, and postnatal effects (e.g., physical growth and development, organ or system function, and behavioral effects of exposure). Postnatal effects are discussed in more detail in the next section. As mentioned previously, epidemiologic studies that focus on only one type of pregnancy outcome may miss a true effect of exposure due to the continuum of outcomes. Examination of individual outcomes could mask a true effect due to reduced power resulting from fewer events for study. Studies that examine multiple end points could yield more information, but the results may be difficult to interpret Evidence of a dose-response relationship is usually an important criterion in the assessment of a toxic exposure. However, traditional doseresponse relationships may not always be observed for some end points. For example, with increasing dose, a pregnancy might end in a fetal loss rather than a live birth with malformations. A shift in the patterns of outcomes could result from differences either in level of exposure or in timing (Wilson, 1973; Selevan and Lemasters. 1987) (for a more detailed description, see Section IILA2.a.5). Therefore, a risk assessment should, wheirpossible, attempt to look at the interrelationship of different reproductive end points and patterns of exposure. (c) Postnatal developmental effects: These effects may include changes in growth, behavior, organ or system function, or cancer. Studies of neurological and reproductive function are discussed here as examples. Postnatal behavioral and functional effects in humans have been examined for a small number of environmental and occupational agents (e.g, lead, PCBs, methyl mercury, alcohol). For some agents (e.g, lead and PCBs), subtle changes have been observed in groups of children at lower exposures than for other developmental effecta (e.g, Bellinger et al, 1987; Needleman, 1988; Davis et al, 1990; TUson et al, 1990). This may not be true for all toxic agents. These subtle differences would be difficult to identify in individuals, but could result iri an overall shifting of mean values when comparing groups of exposed and unexposed children. Some postnatal studies have examined infants cr young children using standard developmental scales (e.g, Brazelton Neonatal Behavioral Assessment Scale, Bayley Scales of Infant Development, Stanford Binet IV, and Wechsler Scales) and some biologic measure of exposure (c.g, blood lead levels). These tests are designed to examine certain end points and have been developed to cover certain age ranges. Certain tests examine specific aspects of development. For example, the Bayley Scales look at motor and language development, but do not examine sensory function. Batteries of tests are important for a proper evaluation due to the possibility of interrelated effects, e.g, hearing deficits and language development Thus, batteries of tests will give a clearer indication of direct effects of exposure resulting in postnatal developmental deficits. Factors that may influence the examination of these effects include parental education, SES, obstetrical history, and health characteristics independent of exposure that may affect functional measurement (e.g, injuries and infections). Many social and lifestyle factors may also affect scoring on these scales fe.g, neonatal-maternal interactions, SES, home environment). Studies of premature infants cany special problems. For proper comparisons, tests keyed to age in very young children (less than 2.5 years of age) need to "correct" the age for premature infants to the age they would have been had they been born at term. In addition, premature infants or those with low birth weight for their gestational age may have problems resulting from the birth process not directly related to exposure (e.g, intraventricular hemorrhage in the brain which can then cause developmental problems). Thus, the developmental effects resulting from exposure may have their own sequelae. Other studies may examine effects occurring at a later age (e.g, in utero exposure and cancer in young women). This long time interval typically carries with it the need for retrospective studies, with the inherent limitations in accurate determination of exposure, effect modifiers, and confounders. Risk assessment methods for cancer are described in the "Guidelines for Carcinogen Risk Assessment" (U.S. EPA, 1986b). Reproductive effects may result from developmental exposures. For example, environmental exposures may result in oocyte toxicity, in which a loss of primordial oocytes irreversibly affects a woman's fertility. The exposures of importance may occur during both the 3io4 63812 Federal Register / Vol. 5& No. 234 / Thursday. December 5, 1991 / Notices prenatal period and after birth. Oocyte depletion is difficult to examine directly in women due to the invasiveness of the tests required; however, it can be studied indirectly through evaluation of the age at reproductive senescence (menopause) (Everson et ah, 1986). Risk assessment methods for female reproductive effects are described in the "Proposed Guidelines for Assessing Female Reproductive Risk" (U.S. EPA. 1988c). Developmental exposures to males could affect their reproductive function (e.g- deplete stem or Sertoli cells potentially affecting sperm production) (Zenick and Clegg, 1989). If stem cell death occurs with exposure at any age. recovery is possible as long as some stem cells survive. The same is true fop. Sertoli cells, except that they cease multiplication before puberty. Thus, cell replication cannot compensate for Sertoli cell death after puberty. Human studies of stem and Sertoli cells would be difficult due to the invasiveness of the measure. Less direct measures, e.g., sperm count morphology, and motility, could be evaluated but this would not indicate what cells or stage of spermatogenesis had been affected. Risk assessment methods for male reproductive effects are described in the "Proposed Guidelines for Assessing Male Reproductive Risk" (U.S. EPA, 1988b). In addition to the above effects, genetic damage to germ cells may result from developmental exposures. Outcomes resulting from germ-cell mutations could include reduced probability of conception as well as increased probability of embryo/fetal loss and ther developmental effects. These end points could be studied using the approaches described above. However, a human germ-cell mutagen has not yet been demonstrated (U.S. EPA, 1986c). Based on animal studies, critical exposures are to germ cells or early zygotes. Germcell mutagenicity could also be expressed as genetic diseases in future generations. Unfortunately, these studies would be very difficult to conduct in human populations due to the long time lag between exposure and outcome. For more information, refer to the "Guidelines for Mutagenicity Risk Assessment" (U.S. EPA 1986c). (4) Community studies/surveillance programs. Epidemiologic studies may also be based on broad populations such as a community, a nationwide probability sample, or surveillance programs (such as birth defects registries). Other studies have examined environmental exposures, such as toxic agents in the water system, and adverse pregnancy outcome (Swan et al., 1989; Deane et al., 1989). Unfortunately, in these studies maternally-mediated effects may be difficult to distinguish from paternally-mediated effects. In addition, the presumably lower exposure levels (compared to industrial settings) may require very large groups for study. A number of case-referent studies have examined the relationship between broad classes of parental occupation in certain communities or countries, and embryo/fetal loss (Silverman et al., 1985). birth defects (Hemminki et al- 1980; Kwa and Fine, 1980; Papier. 1985), and childhood cancer (Kwa and Fine, 1980; Zack et al., 1980; Hemminki et aL, 1981; Peters et aL 1981). In these reports, jobs are typically classified into broad categories based on the probability of exposure to certain classes or levels of exposure (e.g.. Kwa and Fine. 1980). Such studies are most helpful in the identification of topics for additional study. However, because of the broad groupings of types or levels of exposure, such studies are not typically useful for risk assessment of a particular agent Surveillance programs may also exist in occupational settings. In this case, reproductive histories and/or clinical evaluations could be followed to monitor for reproductive effects of exposures. Both could yield very useful data for risk assessment; however, a clinical evaluation program would be costly to maintain, and there are numerous impediments to the collection of reliable and valid information in the workplace. These might include similar concerns to those previously discussed plus potentially low participation rates due to employee sensitivities and confidentiality concerns. (5) Identification of exposures important for developmental effects. For all examinations of the relationship between developmental effects and potentially toxic exposures, the identification of the appropriate exposure is crucial. Preconceptional exposures to either parent and in utero exposures have been associated with the more commonly examined outcomes (e.g.. fetal loss, malformations, birth weight, and measures of infertility). These exposures, plus postnatal exposure from breast milk, food, and the general environment, may be associated with postnatal developmental effects (e.g., changes in behavioral and cognitive function, or growth). The magnitude of exposure may affect the spectrum of outcomes observed. This issue is discussed in more detail in sections HI.A.l.b and II1.B. Infants and young children may receive disproportionate levels of exposure due to their tendency to "put everything" in their mouths (pica) and the greater time they spend on the floor Carpets may serve as a reservoir for toxic agents (e.g., pesticides and lead dust), and the air nearer the floor may have greater levels of certain airborne toxicants (e.g~ mercury from latex paints). Exposures in environmental settings are frequently lower than in industrial and agricultural settings. However, this relationship may change as exposures are reduced in workplaces, and as more is learned about environmental exposures (e.g,, indoor air exposures, pesticides usage). Larger populations are necessary in settings with lower exposures (Lemasters and Selevan, 1984). Other factors affect the identification of reproductive or developmental events with various levels of exposure. Exposed individuals may move in and out of areas with differing levels and types of exposures, affecting the number of exposed and comparison events for study. Thus, exposures can be short-term or chronic. Data on exposure from human studies are frequently qualitative, such as employment or residence histories. More quantitative data may be difficult to obtain due to the nature of certain study designs (e.g., retrospective studies) and historical limitations in exposure measurements. Many developmental outcomes result from exposures during certain critical times. The appropriate exposure classification depends on the outcome(s) studied, the biologic mechanism affected by exposure, and the biologic half-life of the agent. The biologic half-life, in combination with the patterns of exposure (e.g., continuous or intermittent) affect the individual's body burden and consequently the "true" dose during the critical period. The probability of misclassification of exposure status may affect the ability to recognize a true effect in a study [Selevan. 1981: Hogue, 1984; Lemasters and Selevan, 1984: Sever and Hessol. 1984; Kimmel et al.. 1986). As more prospective studies are done, better estimates of exposure will be developed. b. Examination of Clusters or Case Reports/Series. The identification of cases or clusters of adverse pregnancy outcomes is generally limited to those identified by the women involved, or clinically by their physicians. Examples of outcomes more easily identified include mid to late fetal loss or congenital malformations. Identification of other effects, such os very early R&S 020105 Federal Register / Vol. 56. No. 234 / Thursday. December 5, 1991 / Notices 63813 embryonic loss may be difficult to separate from the study of sub- or infertility. Such "nonevents" (e.g., lack of pregnancies or children) are much harder to recognize than are developmental effects such as malformations resulting from in utero exposure. While case reports have been important in the recognition of some agents that cause developmental toxicity, they may be of greatest use in suggesting topics for further investigation (Hogue. 1985). Reports of clusters and case reports/series are best used in risk assessment in conjunction with strong laboratory data to suggest that effects observed in animals also occur in humans. Previous discussion of the use of human data should be taken into account wherever possible. 3. Other Considerations Several other types of information may be considered in the evaluation and interpretation of human and animal data. Information on pharmacokinetics and structure-activity relationships may be very useful, but is often lacking for developmental toxicity risk assessments. a. Pharmacokinetics. Extrapolation of toxicity data between species can be aided considerably by the availability of data on the pharmacokinetics of a particular agent in the species tested and, when available, in humans. Information on absorption, half-life, steady-state and/or peak plasma concentrations, placental metabolism and transfer, excretion in breast milk, comparative metabolism, and concentrations of the parent compound and metabolites may be useful in pr '.ting risk for developmental tc ly. Such data may also be helpful in ..^fining the dose-response curve, developing a more accurate comparison of species sensitivity (Wilson et al,, 1975,1977), determining dosimetry at target sites, and comparing pharmacokinetic profiles for various dosing regimens or routes of exposure. Pharmacokinetic studies in developmental toxicology are most useful if conducted in animals at the stage when developmental insults occur. The correlation of pharmacokinetic parameters and developmental toxicity data may be useful in determining the contribution of specific pharmacokinetic parameters to the effects observed (Kimmel and Young, 1983). While human pharmacokinetic data are often lacking, absorption data in laboratory animals for studies conducted by any relevant route of exposure may assist in the interpretation of the developmental toxicity studies in the animal models for the purposes of risk assessment. Specific guidance regarding both the development and application of pharmacokinetic data was agreed upon by the participants at the "Workshop on the Acceptability and Interpretation of Dermal Developmental Toxicity Studies" (Kimmel and Francis, 1990). It was concluded that absorption data are needed both when a dermal developmental toxicity study shows no developmental effects, as well as when developmental effects are seen. The results of a dermal developmental toxicity study showing no adverse developmental effects and without blood level data (as evidence of dermal absorption) are potentially misleading and would be insufficient for risk assessment especially if interpreted as a "negative" study. In studies where developmental toxicity is detected, regardless of the route of exposure, absorption data can be used to establish the internal dose in maternal animals for risk extrapolation purposes. b. Comparisons ofMolecular Structure. Comparisons of the chemical or physical properties of an agent with those known to cause developmental toxicity may indicate a potential for developmental toxicity. Such information may be helpful in setting priorities for testing of agents or for evaluation of potential toxicity when only minimal data are available. Structure-activity relationships have not been well studied in developmental toxicology, although data are available that suggest structure-activity relationships for certain classes of chemicals (e.g., glycol ethers, steroids, retinoids). Under certain circumstances (e.g.. in the case of new chemicals), this is one of several procedures used to evaluate the potential for toxicity when little or no data are available. B. Dose-Response Evaluation The evaluation of dose-response relationships for developmental toxicity includes the evaluation of data from both human and animal studies. When quantitative dose-response data are available in humans and with sufficient range of exposure, dose-response relationships may be examined. Since data on human dose-response relationships have been available infrequently, the dose-response evaluation is usually based on the assessment of data from tests performed in laboratory animals. Evidence for a dose-response relationship is an important criterion in the assessment of developmental toxicity, which is usually based on limited data from standard studies using three dose groups and a control group. Most agents causing developmental toxicity in humans alter development at doses within a narrow range near the lowest maternally toxic dose (Kimmel et al.. 1984). Therefore, fcr most agents, the exposure situations of concern will be those that are potentially near the maternally toxic dose range. For those few agents that produce developmental effects at much lower levels than maternal effects, the potential for exposing the conceptus to damaging doses is much greater than when the maternal and developmental toxic doses are similar. As mentioned previously (Section IlLA.l.b), however, traditional dose-response relationships may not always be observed for some end points. For example, as exposure increases, embryolethal levels may be reached, resulting in an observed decrease in malformations with increasing dose (Wilson, 1973; Selevan and LeMasters, 1987). The potential for this response pattern indicates that dose-response relationships of individual end points as well as combinations of end points (e.g., dead and malformed combined) must be carefully examined and interpreted. The evaluation of dose-response relationships includes the identification of effective dose levels as well as doses that are associated with no increased incidence of adverse effects when compared with controls. Much of the focus is on the identification of the critical effect(s) (i.e.. the adverse effect(s) observed at the lowest dose level) and the LOAEL and NOAEL associated with that developmental effect, which may be any of the four manifestations of developmental toxicity. The NOAEL is defined as the highest dose at which there is no statistically or biologically significant increase in the frequency of an adverse effect in any of the possible manifestations of developmental toxicity when compared with the appropriate control group in a data base characterized as having sufficient evidence for use in a risk assessment (see Section III.C). The LOAEL is the lowest dose at which there is a statistically or biologically significant increase in the frequency of adverse developmental effects when compared with the appropriate control group in a data base characterized as having sufficient evidence. Although a threshold is assumed for developmental effects, the existence of a NOAEL in an animal study does not prove or disprove the existence or level of a biological threshold; it only defines the highest level of exposure under the conditions of 0*0 loe 63814 Fed ral Register / Vol. 56. No. 234 / Thursday. December 5, 1991 / Notices the study that is hot associated with a significant increase in adverse effects. Several limitations in the use of the NOAEL have been described (Gaylor. 15B3; Cramp, 1984; Kimmel and Gaylor. 1988; Gaylor. 1989: Brown and Erdreich. 1989. Kimmel. 1990): (1) Use of the NO.'VEL focuses only on the dose that is the NOAEL. and does not incorporate information on the slope of the doseresponse curve or the variability in the data. (2) Since data variability is not taken into account (i.e.. confidence limits are not used), the NOAEL will likely be higher with decreasing sample size or poor study conduct either of which Is usually associated with increasing variability in the data. (3) Toe NOAEL is limited to one of the experimental doses. (4) The number and spacing of doses in a study influence th dose chosen for the NOAF.L (5) Since the NOAEL is defined as a dose that does not produce an observed increase in adverse responses from control levels and is dependent on the power of the study, theoretically, the risk associated with it may fall anywhere between zero and an incidence just below that detectable from control levels (usually in the range of 7% to 10% for quantal data). Crump (1984) end Gaylor (1989) have estimated the upper confidence limit on risk at the NOAEL to be 2% to 6% for specific developmental end points from several data sets. Because of the limitations associated with the use of the NOAEL (Kimmel and Gaylor. 1988; Gaylor. 1989; Kimmel, 1990), the Agency is evaluating the use of an additional approach for more quantitative dose-response evaluation i*hcn sufficient data are available, i.e.. the benchmark dose (Crump. 1904). The benchmark dose is based on a modelderived estimate of a particular incidence level, such as 10% incidence. More specifically, the benchmark dose (BD) is derived by modeling the data in the observed range, selecting an incidence level within or near the observed range (e.g- the effective dose to produce a 10% increased incidence of response, the EDi). and determining the upper confidence limit on the modeL The upper confidence value corresponding to. for example, a 10% excess in response is used to derive the BD which is the lower confidence limit on dose for that level of excess response, in this case, the LEDio (see Figure 1). siluno cooe ssco-s-m R&S 020107 Federal Register / Vol. 56, No. 234 / Thursday. December 5.1991 / Notices 801020 S3H Figure 1. This graphical illustration of the benchmark dose approach is based on Crump (1984) and Kimmel and Gaylor (1988), The benchmark dose (BD) is derived by modeling the data in the observed range, selecting an incidence level within or near the observed range (e.g., the effective dose to produce a 10% increased incidence of response, the EDI0), and determining the upper confidence limit on the model. The upper confidence value corresponding to, for example, a 10% excess In response is used to derive the BD which is the lower confidence limit on dose for that level of excess response, in this case, the LED^. The RfD^ or RKV estimated by applying uncertainly factors (UF) to the BD would be greater than or equal to the BD/UF, BIU.INQ CODE 4M0-50-C 63816 Federal Register / VoL 56, No. 234 / Thursday. December S. 1991 / Notices Various mathematical approaches have been proposed for deriving the benchmark dose for developmental toxicity data (e.g.. Crump. 1964; Rai and Van Ryzin. 19S5; Kimmel and Gaylor, 1968: Faustman et al.. 1989: Chen and Kodell, 1969; Kodell et al.. 1991). Such models may be used to calculate the benchmark dose, and the particular model used may be less critical since estimation of the benchmark dose is limited to the observed dose range. Since the model is only used to fit the observed data, the assumptions about the existence or nonexistence of a threshold are not as pertinent Thus, models that fit the empirical data well may provide a reasonable estimate of the benchmark dose, although biological factors known to influence data should ff be incorporated into the model (e.g,,' intralitter correlations, correlations among end points (Ryan et al., 1991)). The Agency is currently conducting , studies to evaluate the application-of several models to actual data sets for calculating the benchmark dose, to determine the minimum data required for modeling, and to develop methods for application to continuous data. In addition, information from these studies will be used to develop guidance for application of the benchmark dose approach to the calculation of the RfD* or the RfCur. since the Agency has limited experience with this approach (see Section 1I1.D for a discussion of the RIDdj and RfC^r). Using the benchmark dose approach, an LEDio can be calculated for each effect of an agent for which there is a data base with sufficient evidence to conduct a risk assessment In some cases, the data may be sufficient to aiso estimate the ED.* or EDTM which should be closer to a true no effect dose. A level between the EDTM and the EDi* usually corresponds to the lowest level cf risk that can be estimated for binomial end points from standard developmental toxicity studies. Certain principles are especially applicable for determining the NOAEL LOAEL and benchmark dose for developmental toxicity studies. First, the NOAEL. LOAEL or benchmark dose are identified for both developmental and maternal or adult toxicity, based on the information available from studies in which developmental toxicity has been evaluated. The NOAEL. LOAEL or benchmark dose for maternal or adult toxicity should be compared with the corresponding values from other adult toxicity data to determine if the pregnant or lactating female or the paternal animal (if exposure is prior to mating) may lie more sensitive to an agent than adult males or nonpregnant females in other toxicity studies that generally involve longer exposure times. Second, for developmental toxic effects, a primary assumption is that a single exposure at a critical time in development may produce an adverse developmental effect, i.e.. repeated exposure is not a necessary prerequisite for developmental toxicity to be manifested. In most cases, however, the data available for developmental toxicity risk assessment are from studies using exposures over several days of development, and the NOAEL IJ3AEL and/or benchmark dose Is most often based on a daily dose. e.g.. mg/kg/day. Usually, the daily dose is not adjusted for duration of exposure because appropriate pharmacokinetic data are not available. In cases where such data are available, adjustments may be made to provide an estimate of equal average . concentration at the site of action for the human exposure scenario of concern. For example, inhalation studies often use 6 hr/day exposures during development. If the human exposure scenario is continuous and pharmacokinetic data indicate an accumulation with continuous exposure, appropriate adjustments can be made. If. on the other hand, the human exposure scenario of concern is very brief or Intermittent pharmacokinetic data indicating a long half-life may also require adjustment of dose. When quantitative absorption data by any route of exposure are available, the NOAEL may be adjusted accordingly; e.g.. absorption of 50% of administered dose could result in a 50% reduction in the NOAEL If absorption in the experimental species has been determined, but human absorption is not known, human absorption is generally assumed to be the same as that for the species with the greatest degree of absorption. NOAELs from inhalation exposure studies are adjusted to derive a human equivalent concentration (HEC) by taking into account known anatomical and physiological species differences (e.g., minute volume, respiratory rate, etc.) (U.S. EPA. 1991b). In summary, the dose-response evaluation identities the NOAEL LOAEL or benchmark dose, defines the range of doses for a given agent that are effective in producing developmental and maternal toxicity, the route, timing and duration of exposure, species specificity of effects, and any pharmacokinetic or other considerations that might influence the comparison with human exposure scenarios.This information should always accompany the characterization of the health- related data base (discussed in the next section). C. Characterization of the HeaitkRelated Data Base This section describes the process for evaluating the health-related data base as a whole on a particular agent and provides criteria for characterizing the evidence for judging a potential developmental hazard in humans within the context of expected exposure or dose. This determination provides the basis for judging whether or not there are sufficient data for proceeding further in the risk assessment process. This section does not address the nature and magnitude of human health risks which are discussed as part of the final characterization of risk along with estimates of potential human exposure and the relevancy of available data for estimating human risk. Characterization of hazard potential within the context of exposure or dose should assist the risk assessor in clarifying the strengths and uncertainties associated with a particular data base. Because a complex interrelationship exists among study design, statistical analysis, and biological significance of the data, a great deal of scientific judgment based on experience with developmental toxicity data and with the principles of study design and statistical analysis, may be required to adequately evaluate the data base. Scientific judgment is always necessary, and in many cases, interaction with scientists in specific disciplines (e.g.. developmental toxicology, epidemiology, statistics) is recommended. A categorization scheme for characterizing the evidence for developmental toxicity is presented in Table 3. The categorization scheme contains two broad categories, sufficient evidence and insufficient evidence, which are deCned in the table. Data from all available studies, whether indicative of potential hazard or not. must be evaluated and factored into a judgment as to the strength of evidence available to support a complete risk assessment for developmental toxicity. The primary considerations are the human data, if available, and the experimental animal data. The judgment or whether the data are sufficient or insufficient should consider quality of the data, power of the studies, number and types of end points examined, replication of effects, relevance of the test species to humans, relevance of route and timing of exposure for both human and animal studies, appropriateness of the dose selection in animal studies, and number of species R&S 020109 Federal Register / VoL 50. N . 234 / Thursday. December 5, 1991 / Notices 63817 ixamined. In addition, pharmacokinetic lata and structure-activity considerations, data from other toxicity studies, as well as other factors that may affect the strength of the evidence, should be taken into account Table 3.--Categorization of the Health- Related Data Base for Hazard Identifica tion/Dose-Response Evaluation Sufficient Evidence The sufficient evidence category includes data that collectively provide enough infor mation to judge whether or not a human developmental hazard could exist within the context of dose, duration, timing and route of exposure. This category includes both human and experimental animal evidence. Sufficient Human Evidence: This category includes deta from epidemiologic studies (e.g,, case control and cohort) that provide convincing evidence for the scientific com munity to judge that e causal relationship is or is not supported. A case series in conjunc tion with strong supporting evidence may also be used. Supporting animal data may or may not be available. Sufficient Experimental Animal Evidencef Limited Human Data: This category includes data from experimental animal studies and/ or limited human data that provide convinc ing evidence for the scientific community to judge if the potential for developmental tox icity exists. The minimum evidence neces sary to judge that a potential hazard exists generally would be data demonstrating an advene developmental effect in a single, ap propriate, well-conducted study in a single experimental animal species. The minimum evidence needed to judge that a potential hazard does not exist would include date from appropriate, well-conducted laboratory animal studies in several species (at least two) which evaluated a variety of the poten tial manifestations of developmental toxicity, and showed no developmental effects at doses that were minimally toxic to the adult. Insufficient Evidence- This category includes situations for which there is less than the minimum sufficient evidence necessary for assessing the poten tial for developmental toxicity, such as when no data are available on developmental tox icity, as well at for data bases from studies in animals or humans that have a limited study design (e.g.. small numbers, inappro priate dose selection/exposure information, other uncontrolled factors), or data from a single species reported to have no adverse developmental effects, or data bases limited to reformation on structure/activity relation ships, short-term tests, pharmacokinetics, or metabolic precursors. In general the categorization is based on criteria that define the minimum evidence necessary to conduct a hazard identification/dose-response evaluation. Establishing the minimum sufficient human evidence necessary to do a hazard identification/dose-response evaluation is difficult, since there are often considerable variations in study designs and study group selection. The body of human data should contain convincing evidence as described in the "Sufficient Human Evidence" category. Because the human data necessary to judge whether or not a causal relationship exists are generally limited, there are currently few agents that can be classified in this category. In the case cf animal data, agents that have been tested adequately in laboratory animals according to current test guidelines generally would be included in the "Sufficient Experimental Animal Evidence/Limited Human Data" category. The strength of evidence for a data base increases with replication of the findings and with additional animal apecies tested. Information on pharmacokinetics or mechanisms, or on more than one route of exposure may reduce uncertainties in extrapolation to the human. More evidence is necessary to judge that an agent is unlikely to pose a hazard for developmental toxicity than that required to tudge a potential hazard- This is because it is more difficult, both biologically and statistically, to support a finding of no apparent adverse effect than a finding of an adverse effect. For example, to judge that a hazard for developmental toxicity could exist for e given agent, the minimum evidence necessary would be data from a single, appropriate, wellexecuted study in a single experimental animal species that demonstrate developmental toxicity, and/or suggestive evidence from adequately conducted clinical/epidemiologic studies- On the other hand, to judge that an agent is unlikely to pose a hazard for developmental toxicity, the minimum evidence would include data from appropriate, well-executed laboratory animal studies in several species (at least two) which evaluated a variety of the potential manifestations of developmental toxicity and showed no adverse developmental effects at doses that were minimally toxic to the adult animal. In addition, there may be human data from appropriate studies supportive of no adverse developmental effects. If a data base on a particular agent includes less than the minimum sufficient evidence (as defined in the "Insufficient Evidence" category) necessary for a risk assessment, but some data are available, this information could be used to determine the need for additional testing. In the event that a substantial data base exists for a given chemical, but no single study meets current test guidelines, the risk assessor should use scientific judgment to determine whether the composite data base may be viewed as meeting the "Sufficient Evidence" criteria. In some cases, a data base may contain conflicting data. In these instances, the risk assessor must consider each study's strengths and weaknesses wiihin the context of the overall data base in an attempt to define the strength of evidence of the data base for assessing the potential for developmental toxicity. judging that the health-related data base is sufficient to indicate a potential developmental hazard does not mean that the agent will be a hazard at every exposure level (because of the assumption of a threshold) or in every situation (e.g., hazard may vary significantly depending on route and timing of exposure). In the final risk characterization, the characterization of the health-related data base should always be presented with information on the dose-response evaluation (e.gLOAEL. NOAEL. and/or benchmark dose), exposure route, timing and duration of exposure, and with the human exposure estimate. D. Determination of the Reference Dose (RfDvr) or Reference Concentration (RfCtn)for Developmental Toxicity The RfDixr or RfCor is an estimate of a daily exposure to the human population that is assumed to be without appreciable risk of deleterious developmental effects. The use of the subscript DT is intended to distinguish these terms from the reference dose (RfD) for oral or dermal exposure or the reference concentration (RfC) for inhalation exposure, terms that refer primarily to chronic exposure situations (U.S. EPA. 1991b). The RfDor or RfCor is derived by applying uncertainty factors to the NOAEL (or the LOAEL, if a NOAEL is not available), or the benchmark dose. To date, the Agency has applied uncertainty factors only to the NOAEL or LOAEL to derive an RfDur or RfCOT. The Agency is planning eventually to use the benchmark dose approach as the basis for derivation of the RfDor or RfCOT and will develop guidance as information is acquired and analyzed from ongoing Agency studies. The most sensitive developmental effect (i.e., the critical effect) from the most appropriate and/or sensitive mammalian species is used for determining the NOAEL, LOAEL, or the benchmark dose in deriving the RfDor or RfCor (Section IIIJB). Uncertainty factors (UFs) for developmental and maternal toxicity applied to the NOAEL generally include a 10-fold factor for interspecies variation and a 10-fold factor for 63818 Federal Register / VoL 58, No. 234 / Thursday, December 5. 1991 / Notices R& s 020111 intraspecies variation. In general, an uncertainty factor is not applied to account for duration of exposure. Additional factors may be applied to account for other uncertainties or additional information that may exist in the data base. For example, the standard study design for a developmental toxicity study calls for a low dose that demonstrates a NOAEL. but in some cases, the lowest dose administered may cause significant adverse effect(s) and. thus, be identified as the LOAEL. In circumstances where only a LOAEL is available, the use of an additional uncertainty factor of up to 10 may be required, depending on the sensitivity of the end points evaluated, adequacy of dose levels tested, or general confidence in the LOAEL. In f addition, if a benchmark dose has been calculated, it may be used to help interpret how close the LOAEL is to a level that would not be detectable from controls (equivalent to the NOAELJ. and thus the size of the uncertainty factor to be applied. Other modifying factors (MFs) may be used depending on the characterization of the data base (Section UI.C), data on pharmacokinetics, or other considerations that may alter the level of confidence in the data (U.S. EPA. 1991b). The total size of the uncertainty factor will vary from agent to agent and will require the exercise of scientific judgment, taking into account interspecies differences, variability within specleB, the slope of the doseresponse curve, the background incidence of the effects, the route of administration, and pharmacokinetic data. As stated above, there is little experience with the application of uncertainty factors to the benchmark dose approach for calculating the RfDpr or RfCtrr. and there are several issues that must be addressed prior to its use for this purpose. For example, which benchmark dose (e.g.. LEDsi. 1ETV. LEDto) should be used for calculating the RfDor or RfCm* and what are theappropriate uncertainty factors that should be applied to the benchmark dose for deriving the RfDn or RfC^? That is, should the uncertainty factor applied to an LED)* be similar to that applied to a LOAEL. or should the uncertainty factor applied to an LEDsi be equal to or less than that applied to a NOAEL? These and other questions are being addressed in ongoing Agency studies on the calculation of the RfDpr or RfCw using the benchmark dose approach. As results become available, and as further guidance is developed. this information will be published as a supplement to these Guidelines. The total uncertainty factor selected is divided into the NOAEL or LOAEL (or the benchmark dose) for the critical effect in the most appropriate and/or sensitive mammalian species to determine the RfDpr or RfCOT. If the NOAEL, LOAEL. or benchmark dose for maternal toxicity is lower than that for developmental toxicity, this should be noted in the risk characterization, and this value compared with data from other studies in which adult animals are exposed. The modeling approaches that have been proposed for developmental toxicity are. for the most part statistical probability models that do not take into account underlying biological processes or mechanisms (e.g.. Crump, 1984; Rai and Van Ryzin, 1985; Kimmel and Gaylor, 1988; Faustman et al., 1989; Chen and Kodell. 1989; Kodell et al,, 1991). These models can be applied to derive dose-response curves for data in the observed dose range, but may or may not accurately predict risk at low levels of exposure. It has generally been assumed that there is a biological threshold for developmental toxicity; however, a threshold for a population of individuals may or may not exist because of other endogenous or exogenous factors that may increase the sensitivity of some individuals in the population. Thus, the addition of a toxicant may result in an increased risk for the population, but not necessarily for all individuals in the population. Models that are more biologically based should provide a more accurate estimation of low-dose risk to humans. The development of biologically based dose-response models in developmental toxicology has been limited by a number of factors, including a lack of understanding of the biological mechanisms underlying developmental toxicity, mtra/interspecies differences in the types of developmental events, appropriate pharmacokinetic data, and the influence of maternal effects on the dose-response curve. The Agency is currently supporting several major research efforts to develop biologically based dose-response models for developmental toxicity risk assessment that include the consideration of threshold under its Research to Improve Health Risk Assessment program. Summary In summary, the hazard identification/dose-response evaluation of developmental toxicity data is used as part of the final characterization of risk along with information on estimates of human exposure. This analysis depends on scientific judgment as to the accuracy and sufficiency of the healthrelated data, biological relevance of significant effects, the conditions of human exposure, and other considerations important in the extrapolation of data from animals to humans. Scientific judgment is always necessary, and in many cases, interaction with scientists in specific disciplines (e.g., developmental toxicology, epidemiology, statistics) is recommended. IV. Exposure Assessment In order to obtain quantitative estimates of risk for human populations, estimates of human exposure are required. This discussion is not intended to provide definitive guidance on exposure assessment: the "Guidelines for Estimating Exposures" have been published separately (U.S. EPA. 1988d) and will not be discussed in detail here. Rather, the issues important to developmental toxicity risk assessment are addressed. In general, the exposure assessment describes the magnitude, duration, frequency, and route(s) of exposure. This information is usually developed from monitoring data and from estimates based on various scenarios of environmental exposures. There are several exposure considerations that are unique for developmental toxicity. For example, exposure to developing individuals is often secondary via placental transfer or through breast milk. Thus, exposure to the embryo/fetus or child may not be the same as for the pregnant or lactating mother, and measurements of an agent in maternal or cord blood and in breast milk may provide a better estimate of developmental exposure. Direct exposure of neonates and children may also occur via environmental media such as water, air and soil, and thus may require estimates of exposure from multiple sources. Duration and period of exposure also must be related to stage of development, if possible (e.g., first, second, or third trimester of pregnancy, infancy, early, middle, and late childhood, adolescence, etc.). These stages of development may have different sensitivities to agents, and exposure estimates should be derived for as many as possible. In addition, exposure to either parent prior to conception must be considered in relation to adverse developmental effects. There is also a possibility that a single exposure may be sufficient to produce adverse developmental effects (i.e.. repeated exposure is not a necessary prerequisite for developmental toxicity Federal Register / Vol. 56, No. 234 / Thursday, December 5, 1991 / Notices 638X0 to be manifested, although it should be considered in cases where there is evidence of cumulative exposure or where the half-life of the agent is sufficiently long to produce an increasing body burden over time). Therefore, it is assumed that, in most cases, a single exposure at any of several developmental stages may be sufficient to produce an adverse developmental effect Most of the data available for risk assessment involve exposures over several days of development- Thus, human exposure estimates used to calculate margins of exposure (MOE, see following section) or to compare with the RfDor or RfCpr are usually based on a daily dose that is not adjusted for duration or pattern of exposure. For example, it would be inappropriate in developmental toxicity risk assessments to use time-weighted averages or adjustment of exposure over a different time frame than that actually encountered (such as the adjustment of a 6-hour inhalation exposure to account for a 24-hour exposure scenario), unless pharmacokinetic data were available to indicate an accumulation with continuous exposure. In the case of intermittent exposures, examination of the peak exposure(s), as well as the average exposure over the time period of exposure, would be important. It should be recognized that, based on the definition used in these Guidelines for developmental toxicity, exposure of almost any segment of the human population may lead to risk to the developing organism. This would include fertile men and women, the developing embryo and fetus, and children up to the age of sexual maturation. Although some effects of developmental exposures may be manifested while the exposure is occurring (e.g.. spontaneous abortion, structural abnormality present at birth, childhood mental retardation), some effects may not be detectable until later in life, long after exposure has ceased (e.g., perinatally induced carcinogenesis, impaired reproductive function, shortened lifespan). V. Risk Characterization o. Overview Risk characterization is the culmination of the risk assessment process. In this final step, risk characterization involves integration of the toxicity information from the hazard identiflcation/dose-response evaluation with the human exposure estimates and provides an evaluation of the overall quality of the assessment, describes risk in terms of the nature and extent of harm, and communicates the results of the risk assessment to a risk manager. The risk manager can then use the risk assessment, along with other risk management elements, to make public health decisions. The following sections describe these throe aspects of the risk characterization in more detail, but do not attempt to provide a full discussion of risk characterization. Rather these Guidelines point out issues that are important to risk characterization for developmental toxicity. B. Integration of the Hazard Identification/Dose-Response Evaluation and Exposure Assessment In developing the hazard identiflcation/dose-response and exposure portions of the risk assessment the risk assessor makes many judgments concerning human relevance of the toxicity data, including the appropriateness of the various animal models for which data are available, the route, timing, and duration of exposure relative to expected human exposure, etc. These judgments should be summarized at each stage of the risk assessment process (e.g.. the biological relevance of anatomical variations may be made in the hazard identification process, or species differences in metabolic patterns in the dose-response evaluation). When data are not available to make such judgments, as is often the case, the background information and assumptions discussed in the introduction (Section I) provide a default position. The risk assessor must determine if some of these judgments have implications for other portions of the assessment, and whether the various components of the assessment are compatible. The description of the relevant data should convey the major strengths and weaknesses of the assessment that arise from availability of data and the current limits of understanding of the mechanisms of toxicity. Confidence in the results of a risk assessment is a function of confidence in the results of the analysis of these elements. Each of these elements should have its own characterization as a part of it Interpretation of data should be explained, and the risk manager should be given a clear picture of consensus or lack of consensus that exists about significant aspects of the assessment Whenever more than one view is supported by the data and choosing between them is difficult both views should be presented. If one has been selected over another, the rationale should be given: if not. then both should be presented as plausible alternative results. The risk characterization should not only examine the judgments, but also explain the constraints of available data and the state of knowledge about the phenomena studied in making them, including: The qualitative conclusions about the likelihood that the agent may pose a specific hazard to human health, the nature of the observed effects, under what conditions (route, dose levels, time, and duration) of exposure these effects occur, and whether the healthrelated data are sufficient to use in a risk assessment; A discussion of the dose-response patterns for the critical effect(s), data such as the shapes and slopes of the dose-response curves for the various end points, the rationale behind the determination of the NOAEL, LOAEL, and/or calculation of the benchmark dose, end the assumptions underlying the estimation of the RfDOT or RfCm; and The estimates of the magnitude of human exposure, the route, duration, and pattern of the exposure, relevant pharmacokinetics, and the size and characteristics of the populations exposed. The risk characterization of an agent should be based on data from the most appropriate species, or, if such information is not available, on the most sensitive species tested. It should also be based on the most sensitive indicator of toxicity, whether maternal, paternal, or developmental, when such data are available, and should be considered in relationship to other forms of toxicity. . If data used in characterizing risk are from a route of exposure other than the expected human exposure, ihen pharmacokinetic data should be used, if available, to extrapolate across routes of exposure. If such data are not available, the Agency makes certain assumptions concerning the amount of absorption likely or the applicability of the data from one route to another (U.S. EPA. 1934.1985b). The level of confidence in the hazard identification/dose-response evaluation should be stated to the extent possible, including determination of the appropriate category regarding sufficiency of the health-related dale. A comprehensive risk assessment ideally includes information on a variety of end points that provide insight into the full spectrum of developmental responses. A profile that integrates both human and test species data and incorporates a broad range of developmental effects provides more confidence in a risk assessment for a given agent. 63820 Federal Register / VoL 56. No. 234 / Thursday. December 5. 1991 / Notices R&S 020113 Tie ability to describe the nature Dr The objective of looking at the upper human exposure is important for end of the exposure distribution is to prediction of specific outcomes and the derive a realistic estima'e of a relatively likelihood of permanence or reversibility highly exposed individuai(s). for ! of the effect. An important part of this example by identifying a specified upper effort is a description of the nature of percentile of exposure in the population the exposed populations. For example, and/or by estimating the exposure of the the consequences of exposure to the most highly exposed individual(s). developing individual versus the adult Whenever possible, it is important to can differ markedly and again can express the number of individuals who influence whether the effects are comprise the highly exposed group and transient or permanent. Other discuss the potential for exposure at still considerations relative to human higher levels. .exposures might include potential If population data are absent, it will synergistic effects, increased often be possible to describe a scenario susceptibility resulting from concurrent representing high end exposures using for exposures to other agents, upper percentile or judgment-based concurrent disease, and nutritional values for exposure variables. In these status. instances, caution should be taken not C. Descriptors ofDevelopmental Toxicity Risk There are a number of ways to describe risks. These include: h to overestimate the high end values if a "reasonable" exposure estimate is to be achieved. 4. Risk Characterization for Highly Sensitive or Susceptible Individuals 1. Estimation of the Number of Individuals Exposed to Levels of Concern The purpose of this measure is to quantify exposure to identified sensitive or susceptible populations to the effect The RfDOT or RfCpr is assumed to be a of concern. Sensitive or susceptible level at or below which no significant individuals are those within the exposed risk occurs. Therefore, information on population at increased risk of the populations at or below the RfDpr or expressing the adverse effect All stages RfCor ("not likely to be at risk") and of development might be considered above the RfDor or RfCor ("may be at highly sensitive or susceptible, but risk") may be useful information for risk certain subpopulations can sometimes managers. be identified because of critical periods This method is particularly useful to a for exposure: for example, pregnant or risk manager considering possible lactating women, infants, children, actions to ameliorate risk for a adolescents. population. If the number of persons in In general, not enough is understood the "at risk" category can be estimated about the mechanisms of toxicity to then the number of persons potentially identify sensitive subgroups for all removed from the "at risk" category agents, although factors such as after a contemplated action is taken can nutrition, personal habits (e.g., smoking, be used as an indication of the efficacy alcohol consumption, illicit drug abuse), of that action. or pre-existing disease (e.g.. diabetes) 2. Presenting Specific Scenarios may predispose some individuals to be more sensitive to the developmental Presenting specific scenarios in the form of "what if?" questions is particularly useful to give perspective to effects of various agents. 5. Other Risk Descriptors the risk manager, especially where In risk characterization, dose- criteria, tolerance limits, or media response information and the human quality limits are being set. The question exposure estimates may be combined being asked in these cases is. "At this either by comparing the RfDpr or RfCpr proposed limit, what would be the and the human exposure estimate or by resulting risk for developmental toxicity calculating the margin of exposure above the RfDOT?" (MOE). The MOE is the ratio of the 3. Risk Characterization for Highly Exposed Individuals This measure and the next are NOAEL from the most appropriate or sensitive species to the estimated human exposure level from all potential sources (U.S. EPA. 1985b). If a NOAEL is examples of specific scenarios. The purpose of this measure is to describe the upper end of the exposure distribution. This allows risk managers to evaluate whether certain individuals are at disproportionately high or unacceptably high risk. not available, a LOAEL may be used in the calculation of the MOE. but considerations for the acceptability would be different than when a NOAEL is used. Consideretions for the acceptability of the MOE are similar to that for the uncertainty factor applied to the LOAEL. NOAEL or the benchmark dose. The MOE is presented along with the characterization of the data base, including the strengths and weaknesses of the toxicity and exposure data, the number of species affected, and the dose-response, route, timing, and duration information. The RfDpr or RfCor comparison with the human exposure estimate and the calculation of the MOE are conceptually similar but are used in different regulatory situations. If the MOE is equal to or more than the uncertainty factor used as a basis for an RfDOT or RfCor. then the need for regulatory concern is likely to be reduced. The choice of approach is dependent upon several factors, including the statute involved, the situation being addressed, the data base used, end the needs of the decision maker. While these methods of describing risk do not actually estimate risks per se. they give the risk manager some sense of how close the exposures are to levels of concern. The RfDpr. RfCor, and/or the MOE are considered along with other risk assessment and risk management issues in making risk management decisions, and the scientific issues that must be taken into account in establishing them have been addressed here. K Communicating Results Once the risk characterization is completed, the focus turns to communicating results to the risk manager. The risk manager uses the results of the risk characterization, other technologic factors, and nontechnoiogical social and economic considerations in reaching a regulatory decision. Because of the way in which these risk management factors may impact different cases, consistent but not necessarily Identical risk management decisions must be made on a case-by-case basis. Consequently, it is entirely possible and appropriate that an agent with a specific risk characterization may be regulated differently under different statutes. These Guidelines are not intended to give guidance on the nonscientific aspects of risk management decisions. VI. Summary and Research Needs These Guidelines summarize the procedures that the U.S. Environmental Protection Agency uses in evaluating the potential for agents to cause developmental toxicity. While these are the first amendments to the developmental toxicity guidelines issued in'1966, further revisions and updates will be made as advances occur in the Federal Register / Vol. 56, No. 234 / Thursday, December 5, 1991 / Notices 63821 field. These Guidelines discuss the assumptions that should be made in risk assessment for developmental toxicity because of gaps in our knowledge about underlying biological processes and how these compare across species. Research to improve the risk assessment process is needed in a number of areas. For example, research is needed to delineate the mechanisms of developmental toxicity and pathogenesis, provide comparative pharmacokinetic data, examine the validity of short-term in vivo and in vitro tests, elucidate possible functional alterations and their critical periods of exposure to toxic agents, develop improved animal models to examine the developmental effects of exposure during the premating and early postmating periods and in neonates, further evaluate the relationship between maternal and developmental toxicity, provide insight into the concept uf threshold, develop approaches for improved mathematical modeling of adverse developmental effects, and improve animal models for examining the effects of agents given by various routes of exposure. Epidemiologic studies with quantitative measures of exposure are also strongly encouraged. 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Washington, DC EPA 540/ 09-91-123. Available from; NTIS, Springfield, VA. PB91-154817. U.S. Environmental Protection Agency. (1991b) Integrated Risk Information System (IRIS). Online. Office of Health and Environmental Assessment. Washington. DC Weinberg. C.R.: Gladen, B.C (1938) The betageometric distribution applied to comparative fecundability studies. Biometrics 42:547-560. Wickramsratne. GA. da S. (1987) The Chemoff-Kaviock assay: Us validation and application in rats. Teratogenesis Carcinog. Mutagen. 7:73-83. Wilcox. A.J. (1983) Surveillance of pregnancy loss in human populations. Am. J. Ind. Med. 4:285-291. Wilcox, A.J.: Weinberg. CRj Wehmann. R.E.; Armstrong. E.G.; Canfield, RJL; Nisula, B.C (1985) Measuring early pregnancy loss: laboratory and Geld methods. Fertil. Steril. 44:360-374. Wilson. J.G. (1973) Environment and birth defects. New York, NY: Academic Press, pp. 30-32. Wilson, J.G. (1977) Embryotoxidty of drugs in man. In: Wilson. J.G.: Fraser. F.C, eds. Handbook of teratology. New York, NY: Plenum Press, pp. 309-355. 51:17883-17894. Wilson. J.G. (1978) Survey of in vitro systems: their potential use in teratogenicity screening. In: Wilson. J.G4 Fraser, F.C.. eds. Handbook of teratology, vol. 4. New York. NY: Plenum Press, pp. 135-153. Wilson, ).G.: Scott. W.J.; Ritter. E.).; Fradkin. R. (1975) Comparative distribution ami embryotoxidty of hydroxyurea in pregnant rats and rhesus monkeys. Teratology 11:169-171 Wilson, J.G4 Ritter, E.J4 Scott, W.J.; Fradkin, R. (1977) Comparative distribution and embryotoxidty of acetylsalicylic add in pregnant rats and rhesus monkeys. Toxicol. Appl PharmacoL 41:67-78. Wong, O4 Utidjian. HAU3; Karten. VS. (1979) Retrospective evaluation of reproductive performance of workers exposed to ethylene dibromide. J. Occun. Med. 2138-102. Woo, D.C; Hoar, R-M. (1972) "Apparent hydronephrosis" as a normal aspect of renal development in late gestation of rats: the effect of methyl salicylate. Teratology 6:191-196. World Health Organization. (1984) Prindples for evaluating health risks to progeny assodated with exposure to chemicals during pregnancy. In: Environmental Health Criteria, vol. 30. Geneva: World Health Organization. Zack. M.; Cannon. Lloyd, D.; Heath, GW., Jr- Falletta, JM4 Jones. B4 Housworth, U Crowley, S. (1980) Cancer in children ol parents exposed to hydrocarbon-related industries and occupations. Am. J. Epidemiol 3329-338. Zenick. H.: Clegg, ED. (1989) Assessment of male reproductive toxicity: a risk assessment approach. In; Hayes, A.W.. ed. Prindples and methods of toxicology Second ed. New York. NY: Raven Press, pp. 279-309. PART B: RESPONSE TO PUBLIC AND SCIENCE ADVISORY BOARD COMMENTS I. Introduction This section summarizes the major issues raised in the public and Science Advisory Board (SAB) comments on the Proposed Amendments to the Guidelines for the Health Assessment of Suspect Developmental Toxicants published March 6,1989 (54 FR 9386-9403). Comments were received from 25 individuals or organizations. The Agency's initial summary of the public comments and proposed responses were presented to the Environmental Health Committee of the SAB on October 27, 1989. The report of the SAB Committee was provided to the Agency on April 23, 199a The SAB and public comments were diverse and addressed issues from a variety of perspectives. The majority of the comments were favorable and in support of the Proposed Amendments to the Guidelines. Many praised the Agency's efforts as being timely and well-justified. Most commentors also gave specific comments or criticisms for R&S 020118 Federal Register / Vol. 58. No. 234 / Thursday. December 5. 1991 / Notices 63825 farther consideration. clarification, or re-evaluation. For example, there was concern expressed about the Guidelines impose further testing requirements, parti i isriy functional testing, and many commentors felt that the Proposed Amendments discounted the role of maternal toxicity in developmental toxicity. In addition, there was concern that the proposed weight-of-evidence scheme would promote labeling of agents as causing developmental toxicity before the entire risk assessment process was completed. " The SAB Committee also indicated that the proposed revisions were adequately founded In developmental toxicology and represented a step forward for the Agency. They suggested that the Agency revisit the weight-ofevidence scheme to avoid confusion with more commonly applied uses of such classifications, and to develop a more powerful conceptual approach. Further, the SAB Committee urged that the Agency begin to move away from the current use of the no-observedadverse-effect level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) basis for calculating the reference dose for developmental toxicity to a benchmark dose and confidence limit approach tied to empirical models of dose-response relationships. In response to the comments, the Agency has modified or clarified many sections of the Guidelines. For the purposes of this discussion, the major issues reflected by the pubiic and SAB comments are discussed. Several minor recommendations, which are not discussed specifically here, also were considered by the Agency in the revision of these Guidelines. 11. Intent of the Guidelines Many of the public comments ir cated some misunderstanding of the ir -d of the Guidelines, apparently assuming that the risk assessment guidelines impose testing requirements. In particular, seme commentors suggested that because the Agency was providing guidance on the interpretation of tests not required in the EPA testing guidelines, the Agency was suggesting that these tests be required in the future. The 1986 Guidelines and the 1989 Proposed Amendments clearly state that these guidelines are not Agency testing guidelines, but rather are intended to ensure uniform interpretation of a*1 existing, relevant data. However. io avoid any confusion, the discussion of study designs has been changed to avoid the impression that these Guidelines set testing requirements. In the evaluation of data on nn agent for risk assessment, relevant data arc often encountered that have been generated from nontraditionai tests. In such cases, it is imperative that the Agency provide guidance so that ali data considered to be relevant are included in the risk assessment and are interpreted uniformly. III. Basic Assumptions In the 1986 Guidelines, several assumptions were implicit in tire approach to risk assessment, but were not explicitly stated. These assumptions were detailed in the 1989 Proposed Amendments. Comments received from the public and the SAB favored presentation of these assumptions and generally agreed with the wording, except for the fourth assumption which concerns the use of the most relevant or most sensitive species. The 1989 Proposed Amendments stated that "it is assumed that the most sensitive species should be used to estimate human risk. When data are available (e.g.. pharmacokinetic, metabolic) to suggest the most appropriate species, that species will be used for extrapolation." The SAB recommended that, for this assumption, the basic position of tbe Agency should be to use data from the most relevant species, and that use of data from the most sensitive species should be the default position. In addition, the SAB recommended that the threshold assumption be considered carefully in the dose-response assessment of any agent, and that the Agency develop more comprehensive approaches to risk assessment as discussed further in the following sections. Changes have been made in the statement of the basic assumptions in line with the SAB and pubiic comments that clarify, but do not alter, the intent of the assumptions. IV. Maternal/Developmental Toxicity The 1989 Proposed Amendments stated that "when adverse developmental effects are produced only at maternally toxic doses, they are still considered to represent developmental toxicity and should not be discounted as being secondary to maternal toxicity." This statement and others concerning the interpretation of developmental toxicity in the presence of maternal toxicity were the subject of a considerable number of public comments and were also addressed by the SAB. In general, commentors were divided in their opinions on whether they supported the Agency's statements or felt that they discounted the role of maternal toxicity in developmental toxicity, but in general, the recommended changes did not significantly alter the intern of the statements. The SAS endorsed the o:('.posed revision, and suggested that the Agency retain the statement that was made in the Proposed Amendments. in these Guidelines, the position is further clarified by indicating that when maternal toxicity is significantly greater than the minima! maternally toxic dose, developmental effects at that dose may be difficult to interpret. This statement is added to clarify, but not to change, the intent or meaning of the statements regarding the relationship between maternal and developmental toxicity. From a risk assessment point of view, whether a developmental effect is or is not secondary to maternal toxicity, docs not impact on the selection of the NOAEL or other dose-response methodology. V. Functional Developmental Toxicity The 1989 Proposed Amendments provided information on the state-of-theart in the evaluation of functional effects resulting from developmental exposures. Several commentors voiced strong objection to this section because they perceived it as indicating an imminent requirement for testing. Several indicated there are no standard methods for functional testing, some felt that functional end points should not be used to establish the NOAEL. and others voiced concern about the problems with using postnatal exposures in animal studies. The final Guidelines further update this section to include a discussion of the latest changes in the requirements for functional developmental toxicity testing by the Agency, and reflect the current approach to interpretation of such data, with incorporation of information from the EPA/NIDAsponsored "Workshop on the Qualitative and Quantitative Comparability of Human and Animal Developmental Neurotoxicity" (1990). The intent of these Guidelines as stated above, is not to change testing requirements but to give guidance when these types of data are encountered in the risk assessment process. The Guidelines also indicate that functional developmental toxicity end points will be used for establishing the NOAEL when they are found to be the adverse effect occurring at the lowest dose in appropriate, well-conducted studies. Interpretation of postnatal exposure data is a concern, and must take into consideration effects on the mother, her offspring, and possible interactions: a statement to this effect has been added. Further interpretation of data will be i 63828 Federal Register / Vol. 56, No. 234 / Thursday, December 5. 1991 / Notices discussed in the guidance being developed by the Agency on neurotoxicity risk assessment. VI. Weight-of-Evidence Scheme The 1989 Proposed Amendments described important considerations in determining the relative weight of various kinds of data in estimating the risk of developmental toxicity in humans. The intent of the proposed weight-of-evidence (WOE) scheme was that it not be used in isolation, but be used as the first step in the risk assessment process, to be integrated with dose-response information and the exposure assessment. The WOE scheme was the subject of a considerable number of public comments, and was one of the major concerns of the SAB. The concern of public commentors was that the reference to human developmental toxicity in this scheme suggested that a chemical could be prematurely designated, and perhaps labeled, as causing developmental toxicity in humans prior to the completion of the risk assessment process. The SAB suggested that the intended use of this scheme was not consistent with the use of the term "weight of evidence" in other contexts, since WOE is usually thought of as an evaluation of the total composite of information available to make a judgment about risk. In addition, the SAB Committee proposed that the Agency consider development of a more conceptual approach using decision analytical techniques to predict the relationships among various outcomes. In the final Guidelines, the terminology used in the WOE scheme has been completely changed and retitled "Characterization of the HealthRelated Data Base." The-intended purpose of the scheme is to provide a framework and criteria for making a decision cn whether or not sufficient data are available tc conduct a risk assessment. This decision is based on the available data, whether animal or human, and does not necessarily imply human hazard. This decision process is part of. but not the complete. WOE evaluation, which also takes into account the RfDpr or RfCpr and the human exposure information, culminating in risk characterization. The final Guidelines also place strong emphasis on the integration of the doseresponse evaluation with hazard information in characterizing the sufficiency of the health-related data base. In line with this approach, the Guidelines have been reorganized to combine hazard identification and doseresponse evaluation. Finally, the SAB comments on developing a conceptual matrix provide an interesting challenge, but current data indicate that the relationships among end points of developmental toxicity are not consistent across chemicals or species. The Agency is currently supporting modeling efforts to further explore the relationship among various development toxicity end points and the development of biologically based dose-response models that consider multiple effects. VII. Applicability of the RfD^t Concept and the Benchmark Dose Approach The 1989 Proposed Amendments introduced the term "reference dose for developmental toxicity--RfDpr," based on short-term exposure, to distinguish it from the reference dose (RfD), which is used for chronic exposure situations. The public comments received generally supported the RfDpr approach. The SAB also agreed with the concept of the RfDor for developmental toxicity risk assessment, based on short-term exposure. In addition, the SAB urged the Agency to consider strengthening the RfD approach by moving to more quantitative alternatives to the NOAEL In particular, the use of a benchmark dose approach to replace the NOAEL was strongly suggested. The final Guidelines have incorporated many of the SAB Committee's suggestions concerning the development of more quantitative approaches to the RfD, and state that the Agency is beginning to use the benchmark dose approach for comparison with and interpretation of the NOAEL. That is. benchmark dose calculations may allow better interpretation of dose-response data and. in particular, what level of risk may be associated with the NOAEL The Agency also has developed the concept of an inhalation reference concentration (RfC) and the RfCOT is being calculated for inhalation concentrations based on developmental toxicity. Guidance for use of the benchmark dose in the calculation of the RfDpr or RfCpr is not included in the final Guidelines, because of the limited experience of the Agency with this approach. There are several issues that must be addressed prior to its use for this purpose; for example, which benchmark dose (e.g.. LEDoi, LEDo, LEDTM) should be used for calculating the RfDpr or RfCpr, and what are the appropriate uncertainty factors that should be applied to the benchmark dose for deriving the RfDpr or RfCpr? Should the uncertainty factor applied to an LEDTM be similar to that applied to a LOAEL or should the uncertainty factor applied to an LEDoi be equal to or less than that applied to a NOAEL? These and other questions are being addressed in ongoing Agency studies on the calculation of the RfDpr or RfC0T using the benchmark dose approach. As results become available, and as further guidance is developed, this information will be published as a supplement to these Guidelines. (FR Doc. 91-29178 Filed 12-4-91: 8:45 am) aiuwo CODE SMO-CO-M R&S 020119