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In Press: Reoroductive Toxicolostv CRITERIA FOR IDENTIFYING AND LISTING SUBSTANCES KNOWN TO CAUSE REPRODUCTIVE TOXICITY UNDER CALIFORNIA'S PROPOSITION 65* 1 2 3 4 5 6 7 Donald R. Mattisonl, Peter K. Working2, William F. Blazak3, Claude L. Hughes, Jr.4, Joanne M. Killinger5, David L. Olive6 and K.S. RaoT Division of Reproductive Pharmacology and Toxicology, Department of Obstetrics and Gynecology, Slot 518, University of Arkansas for Medical Sciences, 4301 Markham Street, Little Rock, AR 72205 and Division of Human Risk Assessment, National Center for Toxicological Research, Jefferson, AR, 72079. Department of Pharmacological Sciences, Genentech, Inc., 460 Pt. San Bruno, South San Francisco, CA 94080. Sterling Winthrop Research Institute, 81 Columbia Turnpike, Rensselaer, NY 12144-3493. Reproductive Hormone Lab, Box 3418, Duke University, Durham, NC 27710. Battelle Columbus Laboratories, 505 King Avenue, Room 6214A, Columbus, OH 43201. Department of Gynecology, University of Texas Health Sciences Center, San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284. Toxicology Research Laboratory, The Dow Chemical Company, 1803 Building, Midland, MI 48674. * The authors are members of The Expert Committee on Criteria for Listing Reproductive Toxicants convened by the International Life Sciences Institute-Nutrition Foundation. The Expert Committee wishes to extend its appreciation to the ILSI-NF Proposition 65 Task Force, Dr. Catherine St. Hilaire, ENVIRON Corporation and the ILSI Risk Science Institute staff for their assistance in the preparation of this document, in particular, Dr. Carol J. Henry, Dr. R.J. Dutton, Ms. Gretchen Bretsch, Ms. Stephanie D. Carter, and Ms. Elizabeth Jean-Paul. Correspondence and reprint requests should be directed to Dr. Mattison, at the University of Arkansas for Medical Sciences.

For example, substances identified as reproductive toxicants may produce effects that are reversible upon discontinuation of exposure, or that are pertinent only to specifically designated age groups. Provision of this type of information would be very beneficial to exposed individuals who must determine an appropriate response to a warning. Based on the above considerations, the Expert Committee suggests that the state Health and Welfare Agency actively develop education plans for the public and for health care practitioners that will precede and continue throughout the institution of warning requirements. The state should consider developing guidelines for management strategies to assist health care professionals in dealing with these issues. The Expert Committee further recommends that, when listing compounds, the SAP explicitly identify the type or types of toxicity caused. It is also recommended that the SAP, where possible, should attempt to identify the gender affected, the presumed magnitude of the effect (dose-response), the age susceptibility, the window of vulnerability, and reversibility of the effect. The Expert Committee suggests that the SAP request that this information be included in the official list. B. Determinina Acceptable Exposure Levels for Renroductive Toxicants After a substance has been identified as a reproductive toxicant and listed as such, an exposure level that complies with the requirements of the law must be developed. There are two components to the development of the exposure level: (1) identification of the no-observed-effect level, or the no-observed-adverse-effect level (NOEL or NOAEL) and (2) extrapolation of the NOEL (or NOAEL) to an acceptable human exposure level. 1. Selection of a NOEL or NOAEL It is customary to conduct animal experiments at dosage levels exceeding estimated levels of human exposure to increase the likelihood that a weak reproductive toxicant will produce a detectable effect and to compensate for the relatively small numbers of animals used in the test. This results in the necessity for extrapolation

suggestive of such. Confirmation requires a study design (preferably cohort in type) constructed around a prior hypothesis, adjusting meticulously for confounding variables and resulting in a statistical validation of the study hypothesis. Epidemiological studies or clinical fertility trials relating exposure to toxicants or indirect endpoints to direct measures of reproductive function pose unique dilemmas in study design and analysis. Well-constructed randomized clinical trials are preferable, but rare in this context. Prospective cohort trials are preferable also, but careful correction for bias should be carried out. The value of retrospective case control and cohort studies is limited by the choice of the control population. Care must be taken when using concurrent, nonrandomized controls or historical controls to adjust for all known confounding variables. The choice of statistical procedures to be applied to particular study designs can sometimes influence interpretation, especially if the procedures are based on inappropriate biologic models or theories. Simple statements of point estimates of relative risk or odds ratios are meaningless without concomitant calculations of confidence intervals or significance levels. Furthermore, calculations of statistical significance must always be considered in light of the number of comparisons made and the biological plausibility of associations noted. Regarding analysis of fertility trials, it is important to correct for time-dependent variation in follow-up of the population at risk for pregnancy. This may be done with either life table methodology or appropriate modeling techniques. As length of follow-up is never uniform in such studies, simple pregnancy rates are inappropriate for statistical comparisons. Negative studies should always be evaluated in light of the study's power to detect true associations with sufficient statistical confidence. A well-constructed and well-analyzed study of sufficient power demonstrating no association should take evidentiary precedence over a poorly designed study implicating a chemical as a reproductive toxicant. Multiple negative studies of sufficient design and power

endpoints of reproductive toxicity, and 4) consideration of the overall weight-of-evidence for reproductive toxicity. A. Definitions The primary goal of Proposition 65 is to protect the public health by restricting exposures to carcinogens and reproductive toxicants. Efforts to protect human reproductive health would focus on pre- and/or postconception exposures that alter fertility in the couple or fecundity in the male or female and/or that produce subsequent developmental toxicity in the offspring. Because postconception exposures and developmental toxicity have been addressed in an earlier document (16), the Expert Committee focused in this document on preconception exposures altering fertility and fecundity. The Expert Committee began its deliberations by developing a definition of successful human reproduction: The essential components of successful human reproduction include the ability to conceive at the appropriate time in the life cycle of the couple (this includes consideration of the optimum time for conception), the continuation of pregnancy to term, and the formation of a structurally and functionally normal offspring. Under this broad definition, substances that decrease contraceptive efficacy, as well as substances that impair reproductive ability, could be considered to be reproductive toxicants. However, in the Expert Committee's opinion, interference in contraception is beyond the scope of Proposition 65 and is not considered further in this document. Therefore, the Expert Committee has defined a reproductive toxicant as a substance that has been demonstrated to impair successful reproduction.

I. BACKGROUND The International Life Sciences Institute-Nutrition Foundation (ILSI-NF) convened a group of experts in reproductive toxicology to develop criteria for listing substances as reproductive toxicants under the provisions of California's Safe Drinking Water and Toxics Enforcement Act of 1986 ("Proposition 65"). The ILSI-NF Expert Committee on Reproductive Toxicity developed this document in coordination with the ILSI Risk Science Institute to provide additional guidance to the Governor's Scientific Advisory Panel (SAP) as it considers criteria for listing substances "known to cause reproductive toxicity." In developing this document, the Expert Committee focused its efforts on establishing criteria for reproductive toxicity endpoints. A previous document focusing on developmental toxicity was prepared in a similar manner by another expert committee convened by ILSI-NF and submitted to the SAP (16). The Expert Committee on Reproductive Toxicity began its deliberations by reviewing the draft criteria, "Identification of Chemicals as Reproductive Toxicants", proposed by the Reproductive Toxicity Subpanel of the SAP (21) and other guidelines and criteria, most notably the U.S. Environmental Protection Agency's proposed guidelines for assessing male and female reproductive risk (23, 24). Guidelines and criteria developed for making weight-of-evidence determinations of human health risks are bound by the context in which they were developed and the point in time at which they were developed. - The Expert Committee based the criteria recommended in this document on existing criteria and guidelines, making only those modifications that were deemed necessary to meet the unique requirements and objectives of Proposition 65. The Expert Committee would like to emphasize that most modifications were made in response to a fundamental difference between the listing process under Proposition 65 and other "listing" activities aimed at identifying potential toxicants for other purposes. Proposition 65 combines virtually all elements of the risk assessment and risk management process into a single step --the listing

pro,;ess. Thic iz a major difference from approaches used by federal and other state regulatory agencies, which use "listing" activities to identify substances for further evaluation before implementing risk management activities. The Expert Committee cautions that the criteria developed in this document, or any other set of criteria adopted for a particular use, must not be adopted for use in other contexts without a thorough assessment of their relevance. In addition, all criteria, including those developed herein, must be updated periodically to ensure that they reflect current scientific understanding. The criteria recommended by the Expert Committee are meant to provide guidance based on the collective experience of the Committee; they are not meant to be a rigid set of rules. II. GENERAL OBSERVATIONS AND RECOMMENDATIONS In the course of its deliberations, the Expert Committee developed observations and recommendations related to (1) the public health effects of Proposition 65 specifically related to the listing process and (2) approaches for determining acceptable levels of human exposure to potential reproductive toxicants under Proposition 65. A. Public Health Considerations Proposition 65 was adopted by California voters to protect themselves against, and to be informed about, chemicals that cause cancer, birth defects, or other reproductive harm. When the issue is viewed from a public health perspective, the state has a clear obligation to protect and promote the public health in accord with these desires. Reproductive toxicology is an extremely complex and evolving science which focuses on the effects of toxicants on adult reproductive function and development of the offspring. These effects may be produced through alterations in a wide range of processes in either the female or the male. Within each of the processes of normal reproductive function, the various events which may be altered and lead to a toxic response can represent a continuum, in the sense that observed alterations may or may not affect reproductive capacity. Therefore, it is

important that this continuum is recognized in establishing and assessing reproductive toxicity in order to appreciate the complexity of a particular response and better understand its applicability in assessing the risk of reproductive impairment. There are two unique issues of concern to public health that are related to adverse effects on reproduction. First is the concept that alteration of fertility is an adverse effect upon the couple rather than simply individuals. Thus, an adverse outcome is inflicted on some individuals in the absence of exposure; i.e., when one partner in a couple has been exposed to, and affected by, a toxicant, both partners are affected. Second, adverse effects can, by definition, be noted only in those couples actively attempting to conceive. Due to variation in age, marital status, or reproductive desires, the consequences of exposure may not be reflected in impaired conception. The Committee believes that any impairment of reproduction by a substance even in couples not attempting to conceive is an adverse health effect. However, it should be realized that all of the above factors are dynamic rather than static determinants. Thus, risk communication should be made available to all segments of the population regardless of perceived risk. Promotion of the public health includes addressing not only the need for accurate information on risks stemming from exposure, but also the facilitation of care and appropriate responses from both exposed persons and health care providers. From this perspective, it is insufficient to provide information on possible hazards (i.e., "warn") while failing to provide a means to manage the concerns engendered. The Expert Committee believes that providing information on possible hazards can have both beneficial and untoward public health effects. An obvious benefit is that it does provide a warning of a possible hazard of exposure about which individuals may not have been aware. Based on the information provided, individuals may have an opportunity to decide whether or not to limit or avoid exposure based on individual assessment of personal risks and benefits of the exposure. However, insufficient or ineffective communication programs which fail to adequately specify the type of toxicity and the expected consequences of such toxicity could result in unnecessary concern and inappropriate health care utilization.

span, of modification in gamete production and/or interference with the fertilization process and subsequent survival of the conceptus. o Subfertility "Subfertility" is defined as a statistically significant decrease from the fertility pattern of a control population. In comparisons of the test and control populations, adjustments must be made for known confounding variables such as age, anatomic or functional reproductive abnormalities, and exposure to other known reproductive toxicants. Two methods can be used to demonstrate such a statistical deviation: (1) Time to Preanancv (TTP) This is defined as the mean length of time required for pregnancy to occur. TTP may also be designated as its inverse value, referred to as the Cycle Fecundity Rate (CFR). This latter term is similar to a variety of indices including Cycle Fertility Rate and Monthly Fecundity Rate (19). (2) Alteration in the Cumulative Fertility Curve While measures of central tendencies, such as mean time to pregnancy, are satisfactory for continuous or similarly variant effects, more subtle alterations in reproductive ability may fail to alter these values. Analysis of cumulative pregnancy as a function of time, months, menstrual cycles, or cycles attempting conception may allow identification of more subtle alterations in reproductive performance.

exposure and some important genital tract factors (e.g., tubal occlusion and intraperitoneal adhesions). Thus, these endpoints are considered insufficient to list as reproductive toxicants. o Perturbations of Female Sexual Behavior While coital frequency and human sexuality in general are most appropriately considered in the context of the couple, female gender-specific sexual behavior changes are possible reproductive outcomes. However, current means to objectively measure alterations in female sexual behavior and current understanding of the association of behavioral changes with reproductive outcome are not sufficient at the present time to support these endpoints as being sufficient for listing as a reproductive toxicant. 2. Animal Endnoints Reproductive toxicity in animals is defined as adverse effects of chemicals on the adult or maturing organism and includes, but is not limited to, deleterious effects on gonadal structure and function, alterations in fertility (e.g., infertility or subfertility), and impaired gamete function. Although exposure occurs prior to the time of conception, reproductive toxicity may also become evident during fertilization, the embryonic or fetal periods, or even postnatally. Gestational and postnatal deficits have been addressed in a previous document on developmental toxicity (16) and are not further addressed here. Reversibility of an effect was not considered in the categorization of endpoints. A number of chemicals cause reversible reproductive effects in the adult male and female or developing offspring. This concept is important in communication of reproductive hazard warnings. Since the effects are reversible and cease after exposure stops and the chemical is cleared from the system, exposures leading to effects in this category are likely to be of lower risk to human reproduction than those that cause permanent damage. However, because

(3) Standardized Fertility Ratio The Standardized Fertility Ratio (SFR) is a measure of the deficit in birth rates (total or stratum-specific) in an exposed group versus some comparison group. o Reoroductive Loss The endpoint "reproductive loss" is defined in a test population as a statistically significant decrease in the rate of ongoing pregnancies resulting from conception when compared to the outcome of a control population. In comparisons of the test and control populations, adjustment must be made for known confounding or functional reproductive abnormalities and exposure to other known reproductive toxicants. Significant effects may be termed valid regardless of the stage of embryogenesis or fetal development at which the study is terminated, provided that the stage at which the studies ended was uniform throughout the study populations. o Other Endnoints Several couple-specific endpoints centered around sexual behavior provide indirect evidence of reproductive effects, including such endpoints as libido and coital frequency. Additionally, couple-specific functional assessments such as the postcoital test (PCT) for motile sperm provide indirect measures of reproductive function. None , of these couple-specific endpoints providing indirect evidence of reproductive effects have sufficient data available at the present time on the relationship between the endpoint and reproductive success to support a decision to list a substance under Proposition 65.