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b. Male-Soecific Endooints Male-specific endpoints are summarized in Table 2 and are described below. o Male Gamete Number The most frequently used clinical tool for evaluation of male reproductive function is quantitation of the number of motile sperm per ejaculate. Thus both sperm number and sperm motility are assessed, and significant decreases in both (i.e., motile sperm) are considered to be sufficient evidence to list a substance as a reproductive toxicant. Although this is presumed to be related to male fecundity (17), graded fertility has not been demonstrated in a human population. The association of decreasing fertility with decreasing numbers of sperm ejaculated has been demonstrated in experimental animals (1). Unfortunately, the threshold number of sperm required to assure fertility is not known and probably varies among couples. The Expert Committee believes that substances that significantly decrease the percentage of progressively motile sperm in the ejaculate are reproductive toxicants and should be listed as such. o Snerm Moroholoav Morphological changes (e.g., a statistically significant reduction in morphologically normal sperm) would provide evidence sufficient to list; however, less severe changes would not be judged to be sufficient to list as reproductive toxicants. o Male Gamete Function There are several assays which measure functional characteristics of the male gamete. This area is also under active investigation and changing rapidly. Those presently available include human sperm/hamster egg

strengthen the conclusion that no adverse effects are associated with the measured exposures to the substance. 2. Animal Studies In the interpretation of data from animal reproductive toxicology studies, the quality of the study, design, conduct, and statistical analyses must be taken into consideration. Studies must be of high quality and designed so that the animals are exposed to the test compound by an appropriate route of administration (i.e., relevant to the human route of exposure). Other routes may be relied upon by taking into consideration pharmacokinetic information. Also, exposures should be at the proper time, and for the proper duration so as to maximize detection of an effect. Details of study design are beyond the scope of this report but can be found elsewhere (4, 23, 24). The study design must include identification of reproductive endpoints suitable for defining an adverse effect (see the following section for a discussion of endpoints). An important consideration is whether the substance is exerting a selective adverse effect on reproductive function. For a substance to be identified as a reproductive hazard, adverse reproductive effects should occur at doses that do not cause other types of toxicity that could interfere with mating ability or frequency, especially other significant systemic toxicity. When reproductive effects are seen in the presence of systemic toxicity, scientific judgment concerning the probability of reproductive toxicity in the absence of other toxicities (and at lower doses) is needed to determine whether an adverse reproductive effect has occurred. Another important consideration in evaluating animal data is a determination of the power of the study, which is the probability that the study will demonstrate a true effect. It is dependent on the sample size, as well as the background incidence and variability of the endpoint(s) examined. The apparent lack of an effect may be due to a true lack of activity or the inability of the study to identify an effect because of small sample size. Conversely, some statistically significant effects may

of results from experimental dosage levels to the normally lower levels of human exposure. An important step in characterizing the dose-response relationship in these studies is to determine the "no-observed-effect level," i.e., the highest exposure level at which no morphological, physiological, or functional modification is detectable under the test conditions. Another widely used concept in toxicology is the "no-observed-adverse-effect level" (NOAEL), i.e., the highest dose level at which no biologically adverse effects occur. In many cases, the NOEL and NOAEL both refer to the same exposure level. The Expert Committee suggests that, when the NOEL and NOAEL differ, the NOAEL, rather than NOEL, provide the basis for establishing permissible levels for human exposure, since it is possible for a substance to have a non-adverse effect at a low dose level and an adverse effect at a higher dose. Depending upon the sensitivity of the endpoint monitored and the test species utilized, different NOAELs can be derived for the same chemical. The Expert Committee recommends that the same endpoint used as a basis for listing the substance be evaluated to determine the NOAEL. If multiple endpoints provide the basis for listing, then the most sensitive one (i.e., the effect occurring at the lowest exposure) should be used. In the selection of the appropriate NOAEL, the study selected should use an exposure route that is relevant to the human exposure whenever possible. However, data from other routes should also be evaluated and taken into consideration, especially if supported by pharmacokinetic information. If sufficient data do not exist to determine the NOAEL for an endpoint, then the lowest-observed-adverse-effect level (LOAEL) and an additional safety factor should be used. If good quantitative human dose-response data exist, the NOAEL should be determined from the human data. It must be emphasized that human studies can be of widely varying quality in both design and analysis. The Expert Committee believes that if it is determined that a compound is a reproductive toxicant based on sufficient human evidence, the NOAEL should be determined from the study that resulted in the lowest NOAEL, if that study was adequately designed, conducted, and analyzed. -6-

arise by chance, especially if a large number of endpoints are analyzed; the use of appropriate historical control data may prevent a false assumption of biological relevance in such cases. The Expert Committee encourages appropriate statistical evaluation of studies and inclusion of biological meaningfulness and relevance in the final interpretation of study results. Negative findings from animal reproductive toxicology studies deserve special scrutiny regarding study design and conduct. Such studies must include sufficient numbers of animals to detect an adverse effect, appropriate dose levels and exposure routes must be used, and the data must be evaluated using appropriate statistical methods. Negative studies should also indicate the power to define an adverse effect or the confidence interval on the null hypothesis. C. Evaluation of Endpoints If positive studies are identified that satisfy all of the above criteria concerning the adequacy of study design, execution, and analysis, the Expert Committee recommends that the endpoints observed be evaluated in terms of their relevance to adverse human reproductive outcome. Only those endpoints derived from adequate studies that are judged to be directly relevant to human reproductive success are considered by the Expert Committee to be sufficient to identify a substance as a reproductive toxicant. Endpoints considered by the Expert Committee to be only suggestive of a reproductive hazard should be interpreted as signs of potential reproductive toxicity, but deemed insufficient to list a substance as a reproductive toxicant. Significant adverse effects elicited by a test agent in the latter endpoints should lead to additional investigations into the potential for this agent to induce adverse reproductive effects. In this section, the Expert Committee has categorized endpoints measured in humans and in laboratory animals according to their relevance to adverse reproductive outcomes. The Committee recognizes that such an evaluation of endpoints has not been undertaken heretofore and emphasizes that the following discussion is based on the collective judgment

of the Committee and reflects the Committee's understanding of the scientific knowledge in this area at this time. As new information is developed, the Committee's analysis should be reviewed and updated. 1. Human Endpoints Specific reproductive endpoints from studies in humans that the Expert Committee considered relevant to the assessment of the potential reproductive hazard of an agent are highlighted in this section. All endpoints have been evaluated in terms of their relation to any of several adverse involuntary reproductive outcomes. Only those endpoints associated with adverse reproductive outcomes are considered to provide a sufficient basis for identifying (and listing) a substance as a reproductive toxicant under Proposition 65. The Expert Committee notes that even when data are available which show that alterations have occurred in two or more endpoints that are only suggestive of reproductive hazard, sufficient evidence does not exist to cause the listing of the substance. The Expert Committee has developed a listing of human endpoints based on couple-specific measures (Table 1), male-specific measures (Table 2), and female-specific measures (Table 3). a. Couole-Snecific Endooints Couple-specific endpoints are summarized in Table 1 and are described below. o Infertility As an endpoint, "infertility" is defined in a test population as the complete absence of reproductive capability. This outcome can be achieved by meeting either of two criteria. The first requires demonstration of an absence of fertility throughout the complete reproductive life span. The second calls for evidence, prior to the completion of the reproductive life

The Expert Committee acknowledges that although the "correctness" of the flexible uncertainty factor approach cannot be objectively assessed, this approach has been adopted by all regulatory and advisory groups that have addressed reproductive toxicity risk assessment, including the U.S. Environmental Protection Agency (EPA), U.S. Food and Drug Administration, the World Health Organization, and the National Academy of Sciences. The appropriate magnitude of an uncertainty factor is influenced by many of the factors described above, all of which should be considered. In this regard, the Expert Committee believes that an approach such as that taken by the EPA in its guidelines for male reproductive toxicity risk assessment (24) is preferable to an across-the-board imposition of a "standard fixed factor" of any magnitude. As described in the EPA document Currently, uncertainty factors are applied to a NOAEL or LOAEL to estimate an exposure level for humans at or below which there should be no adverse reproductive effects (i.e., the reference dose). The total uncertainty factor usually ranges from 10 to 1,000 depending on the number of adjustments needed. Uncertainty factors are used for (1) situations in which the LOAEL must be used because a NOAEL was not established, (2) interspecies extrapolation, and (3) intraspecies adjustment for variable sensitivity among individuals. In addition, adjustments may be appropriate for length of exposure (acute to subchronic) and/or to correct for inadequacy of the NOAEL or LOAEL (including consideration of background variability in the measurements, insensitivity of the endpoint, and protection against effects of more prolonged exposure). III. SCIENTIFIC BASIS FOR THE EXPERT COMMITTEE'S RECOMMENDED CRITERIA Development of adequate criteria requires 1) definition of successful reproduction and other commonly used terms, 2) understanding of adequate test methodologies to detect reproductive toxicity, 3) interpretation and assessment of the importance of the various -8-

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. Epidemiologic 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

If adequate human data are not available, animal studies must be used. If data from several species/strains are available, the most appropriate species should be used in determining the NOAEL. The Expert Committee recommends that the most sensitive species be used in determining the NOAEL, unless there is evidence that data from that species are not relevant to the human. In that case, a more relevant species should be selected. A determination of relevance is based on the effect measured and comparable anatomical, physiological, pharmacological, pharmacokinetic, metabolic, and pharmacodynamic processes for the effect in the test animal and in humans. In summary, the Expert Committee recommends that the NOAEL be used to develop the acceptable human exposure level. The NOAEL should be derived from dose-response data from the most sensitive relevant study, human whenever possible, of adequate design and execution that demonstrates the endpoint(s) that forms the basis for listing. 2. Development of an Accentable Human Exposure Level Proposition 65 mandates the use of a 1,000-fold safety or uncertainty factor to develop human exposure levels for listed substances. The Expert Committee did not consider the impact of the 1,000-fold mandatory "uncertainty factor" for establishing acceptable human exposure levels in its development of listing criteria. The Expert Committee, however, felt that it must comment on the lack of a scientific basis for this fixed approach to establishing acceptable exposure levels for reproductive toxicants. Use of a single, inflexible uncertainty factor as specified in Proposition 65 is not scientifically defensible. No single uncertainty factor is appropriate for all situations. The data available to determine that a substance is "known to cause reproductive toxicity" will vary tremendously in quantity, quality, and type. In addition, the kinds of effects caused, the sensitivity of the species studied, the nature of the dose-response relationship, and the site and mechanism of action should influence the determination of the levels that are acceptable for human exposure. -7-

Endpoints of human reproductive toxicity, which would be adequate to justify listing of a chemical as a reproductive toxicant under Proposition 65, include exposures that reduce the cumulative number of cycles that could result in conception. This could result from exposures that cause a) delay of puberty/disturbance of the pubertal sequence, b) premature menopause, and/or c) increases in cycle length. Exposures causing precocious puberty should certainly be considered an adverse effect due to the psychological, sociological, and general growth compromises that such children suffer. The Committee has not included this endpoint as sufficient to list as a reproductive toxicant because it is not necessarily related to decreased reproductive success. However, the Committee notes that it is an adverse endpoint and should be considered a developmental effect as defined in an earlier ILSI-NF document (16). On the other extreme of the reproductive lifespan, earlier age of menopause within the climacteric interval may not have any impact on lifelong fertility potential; hence the Committee has classified this endpoint as insufficient to list a reproductive toxicant. o Disturbances of Ovulation/Ovarian Cvclicitv Subfertility could result from a chemical exposure if ovulation were adversely affected. Such adverse effects could include anovulation per se, dysfunctional ovarian cycles (with abnormal levels of reproductive hormones) (2, 6, 9, 11, 12, 14, 22), or diminished functional capacity of the released oocyte (to be fertilized, initiate early development, nidate, grow, or differentiate). Strict assessment of ovulation requires detection of conception or observation of an egg outside of the ovary. Since these are not practical screening tests in assessment of ovarian function in humans,

penetration, zona attachment and penetration, and other measures of sperm function. Although it is biologically plausible that alterations in any of these parameters may be associated with decreased fertility in human populations, there is little evidence demonstrating a clear relationship between alterations in these functional parameters and decreased fertility. They are, nevertheless, used frequently as clinical tests. The Expert Committee believes that alterations in these functions are not sufficient to list a substance as a reproductive toxicant at the present time, but that a positive response in any one of them should prompt further investigation. o Sexual Behavior The assessment of sexual behavior among humans (libido or coital frequency) is difficult. Data demonstrating that subtle alterations in libido or coital frequency are associated with alterations in human fertility are not available. Therefore, alterations in these functions are not sufficient to list a substance as a reproductive toxicant. However, they may be considered indicators of the need for further studies of reproductive effects. c. Female-Soecific Endwints Female-specific endpoints of reproductive toxicity which can be reasonably considered to reflect exposure-dependent subfertility include 1) alterations of the reproductive lifespan, 2) disturbances of ovulation/ovarian cyclicity, and 3) compromise of female genital tract function. Female endpoints are summarized in Table 3 and described below. o Alterations of the Reproductive Lifesnan If an exposure shortens the reproductive lifespan of women, then logically the lifetime potential for successful reproduction will be decreased. -18-