Susceptibility and risk: an overview.

This symposium on susceptibility and risk was the third in a series designed to bring together experts from diverse disciplines to discuss contemporary issues in risk assessment. The topic in 1996 was especially challenging since susceptibility is influenced by a myriad of factors including environmental, genetic, social and political elements. The delineation of the relative contribution of various `susceptibility' factors has major implications for risk management options that may be applied in a regulatory context (risk prevention and risk reduction) or by the individual (risk avoidance). Current approaches to account for susceptibility in risk assessments (e.g. application of an uncertainty factor) have frequently been challenged as to their scientific basis and thus need periodic re-examination or update to maintain a credible foundation for the assessment process. The goal of this symposium was to gain a better understanding of the dimensions of the problem and to explore the directions that the risk assessment process might follow to better quantify the contribution of susceptibility in risk calculations.

Research to strengthen the scientific basis for health risk assessment: a survey of the context and rationale for mechanistically based methods and models.

Assessment of health risks is an integral part of regulatory decision-making that occurs at the interface between science (e.g. facts) and policy (e.g. values). Because existing scientific knowledge and understanding are often inadequate to answer the most critical risk-related questions, regulatory agencies have developed sets of formalized 'science policies' to extrapolate from existing data to real-life events and situations. These science policies, as, for example, the use of default assumptions or exposure scenarios, can introduce significant uncertainties into the final risk estimate. We survey the rationale for research to reduce extrapolation-related uncertainties, focusing specifically on the need to develop mechanistically based methods and models, including test methods to identify and characterize health effects, integrated human exposure models, physiologically based pharmacokinetic (PBPK) models and biologically based dose-response (BBDR) models.

Human tissue monitoring and specimen banking: opportunities for exposure assessment, risk assessment, and epidemiologic research.

A symposium on Human Tissue Monitoring and Specimen Banking: Opportunities for Exposure Assessment, Risk Assessment, and Epidemiologic Research was held from 30 March to 1 April 1993 in Research Triangle Park, North Carolina. There were 117 registered participants from 18 states and 5 foreign countries. The first 2 days featured 21 invited speakers from the U.S. Environmental Protection Agency, the Centers for Disease Control and Prevention, the National Institute of Environmental Health Sciences, various other government agencies, and universities in the United States, Canada, Germany, and Norway. The speakers provided a state-of-the-art overview of human exposure assessment techniques (especially applications of biological markers) and their relevance to human tissue specimen banking. Issues relevant to large-scale specimen banking were discussed, including program design, sample design, data collection, tissue collection, and ethical ramifications. The final group of presentations concerned practical experiences of major specimen banking and human tissue monitoring programs in the United States and Europe. The symposium addressed the utility and research opportunities afforded by specimen banking programs for future research needs in the areas of human exposure assessment, risk assessment, and environmental epidemiology. The third day of the symposium consisted of a small workshop convened to discuss and develop recommendations to the U.S. Environmental Protection Agency regarding applications and utility of large-scale specimen banking, biological monitoring, and biological markers for risk assessment activities.

The role of specimen banking in risk assessment.

The risk assessment process is described with a focus on the hazard identification and dose-response components. Many of the scientific questions and uncertainties associated with these components are discussed, and the role for biomarkers and specimen banking in supporting these activities are assessed. Under hazard identification, the use of biomarkers in defining and predicting a) biologically adverse events; b) the progression of those events towards disease; and c) the potential for reversibility are explored. Biomarker applications to address high-to-low dose extrapolation and interindividual variability are covered under dose-response assessment. Several potential applications for specimen banking are proposed.

U.S. Environmental Protection Agency's Ozone Epidemiology Research Program: a strategy for assessing the effects of ambient ozone exposure upon morbidity in exposed populations.

The Clean Air Act Amendments of 1990 mandate a future reduction of ambient ozone levels in many areas of the country, the cost of which will be great. In order to assess the current public health burden of ambient ozone exposure and to provide information for assessment of potential health benefits of improved air quality, the Health Effects Research Laboratory of the U.S. EPA has undertaken an Ozone Epidemiology Research Program. The research strategy which will guide this scientific program is described in this paper. Criteria for selection of important research questions as well as issues which cut across all questions and study designs are discussed. In particular, this program emphasizes the study of effects which reflect morbidity in the population. The three questions identified as being of most immediate importance involve the relationship of short-term ambient ozone exposure to acute respiratory illness, the relationship of recurrent exposure to chronic respiratory disease, and the relationship of recurrent exposure to development of acute respiratory illness. Specific research approaches and initial projects to address these three questions are described.

Statistical issues in risk assessment of reproductive outcomes with chemical mixtures.

Establishing the relationship between a given chemical exposure and human reproductive health risk is complicated by exposures or other concomitant factors that may vary from pregnancy to pregnancy. Moreover, when exposures are to complex mixtures of chemicals, varying with time in number of components, doses of individual components, and constancy of exposure, the picture becomes even more complicated. A pilot study of risk of adverse reproductive outcomes among male wastewater treatment workers and their wives is described here. The wives of 231 workers were interviewed to evaluate retrospectively the outcomes of spontaneous early fetal loss and infertility. In addition, 87 workers participated in a cross-sectional evaluation of sperm/semen parameters. Due to the ever-changing nature of the exposure and the lack of quantification of specific exposures, six dichotomous variables were used for each specific job description to give a surrogate measure of exposure. Hence, no quantitative exposure-response relationships could be modeled. These six variables were independently assigned by two environmental hygienists, and their interrater reliability was assessed. Results are presented and further innovations in statistical methodology are proposed for further applications.

Correlation of sperm motion parameters with fertility in rats treated subchronically with epichlorohydrin.

We investigated the relationship between fertility and sperm motin endpoints in rats treated subchronically with the male reproductive toxicant, epichlorohydrin (ECH). Male rats were given ECH orally for 23 days at dosages of 0, 6.25, 12.5, or 25 mg/kg/day. They were mated twice (at 19 and 22 days) to estimate fertility by (1) detection of fertilized ova (presence of sperm head and tail or two pronuclei) 18 hours after mating and by (2) counting implants on day 14 of gestation. Both indices showed dose-related reductions (P less than 0.001). Motion parameters of cauda epididymal sperm were assessed using the CellSoft computer-assisted sperm motion analysis (CASA) system after the rats were asphyxiated on day 25. Curvilinear velocity, straight-line velocity, linearity, and amplitude of lateral head displacement were reduced in a dose-related manner. The fertility indices, percent fertilized ova, and percent implantation on day 14 of gestation were correlated significantly (r = 0.68; P = 0.0001). The following motion parameters were also correlated significantly with fertility (P less than 0.0003; r1 = percent fertilized ova and r2 = percent implantation): linearity (r1 = 0.42; r2 = 0.40), amplitude of lateral head displacement (r1 = 0.54; r2 = 0.48), curvilinear velocity (r1 = 0.53; r2 = 0.50), straight-line velocity (r1 = 0.55; r2 = 0.50), and percent motile sperm (r1 = 0.42; r2 = 0.32). These results suggest a relationship between toxicant-induced reductions in sperm motion and fertility.

Fertility of workers chronically exposed to chemically contaminated sewer wastes.

Few studies have investigated the reproductive effects of exposure to chemical mixtures. The purpose of this study was to assess fertility in males exposed to mixed industrial and domestic wastes. A detailed reproductive history was obtained from the wives of 231 employees in order to evaluate fertility. Daily work records were used to define exposure status. To ascertain problems of infertility, the ratios of observed live births to expected live births (generated from U.S. birth probabilities) for exposed and nonexposed groups were calculated, and the ratios of these Standardized Fertility ratios (SRFs) were compared. Other analyses considered the couples' contraceptive history and preexposure versus postexposure experience. Though multiple statistical approaches were used to examine the data, the conclusion of this study was that exposure to chemical mixtures was not associated with a decrease in the couples' fertility.

The automated analysis of rat sperm motility following subchronic epichlorohydrin administration: methodologic and statistical considerations.

The automated analysis of sperm motion endpoints is potentially useful in identifying male reproductive toxicants and ultimately in predicting fertility in humans. The present study was designed to evaluate the automated analysis of rat sperm motility characteristics following subchronic administration of epichlorohydrin. This type of validation is a prerequisite for inclusion of sperm motion measurements in the process of reproductive risk assessment. In the present studies videotapes were made of cauda epididymal spermatozoa from Long-Evans rats, both untreated and treated with epichlorohydrin. From analysis of videotapes of control epididymal spermatozoa, the relationship of various sperm motion endpoints and settings of the CellSoft computer-assisted sperm motion analysis system (Cryo Resources, Ltd., New York, NY) is described. Optimal settings of the system for analysis of rat spermatozoa are detailed. Employing data from both control and epichlorohydrin-treated animals, a statistical methodology is described that evaluates: (1) the distributions of CellSoft generated sperm motion endpoints, (2) the correlations between these endpoints, and (3) techniques for detection of dose-related effects.

Effects of epichlorohydrin on male and female reproduction in Long-Evans rats.

Male and female Long-Evans rats were treated with epichlorohydrin (ECH) by oral gavage (males: 12.5, 25, and 50 mg/kg/day; females: 25, 50, and 100 mg/kg/day) for 21 and 14 days, respectively, prior to mating trials with untreated animals. Treated females were further dosed until delivery. Fertility was assayed in the high-dose males only and was found to be totally impaired. No measured parameters of female reproduction were changed relative to controls. Treated males showed normal copulatory behavior. Sperm morphology and percentage motile sperm were not statistically different from control values in both ejaculated and cauda epididymal samples from ECH-treated animals. The number of sperm in ejaculates was normal while cauda epididymal sperm count was slightly decreased in males at the 50 mg ECH/kg dose level. Mean curvilinear velocity, straight-line velocity, and amplitude of lateral head displacement of cauda epididymal sperm were significantly reduced by ECH at 12.5 mg/kg/day and above. Sperm track linearity was also reduced, but only at 50 mg/kg/day. Beat/cross frequency of sperm was significantly increased at 12.5 mg/kg/day and above. All of the above sperm motion parameters showed dose-dependent trends. These effects are consistent with the spermatozoal metabolic lesions reported for alpha-chlorohydrin, a metabolite of ECH.

Factors associated with reduced fertility and implantation rates in females mated to acrylamide-treated rats.

A series of studies was conducted to examine the role of copulatory dysfunction, spermatotoxicity, and/or impaired fertilization in the reduced rates of fertility and implantation observed in females mated to acrylamide-treated male rats. In initial experiments, males were gavaged with 0, 5, 15, 30, 45, or 60 mg/kg acrylamide (ACR) for 5 days and then mated serially to naive females. ACR treatment reduced fertility and increased pre- and post-implantation loss, primarily over the first 3 weeks post-treatment. The effects at Week 1 appeared to result from an interference in sperm transport as demonstrated by the absence of sperm in the uteri of females following a single ejaculation by ACR-treated male rats. The effect however was transient, with recovery of fertility in all but the 60 mg/kg group by Week 2. Attempts to explain the reduced rate of implantation concentrated on characterizing changes in measures of ejaculated sperm count and various motility parameters and evaluating sperm fertilizing ability. Males were again dosed acutely with ACR (p.o.). ACR produced statistically significant, but modest, alterations in sperm motility at Week 3. More prominent was the marked decrease in the number of fertilized ova recovered from females mated to ACR-treated males at Week 3. These data suggest that events critical to the fertilizing ability of the sperm appear to play a major role in the reduced reproductive competence associated with ACR treatment in male rats.

Developmental toxicity of halogenated acetonitriles: drinking water by-products of chlorine disinfection.

The developmental toxicity of acetonitrile and 5 halogenated derivatives was examined with an in vivo teratology screen adapted for use in the Long-Evans rat. The screen was extended to an evaluation of growth till postnatal Days 41-42, and weight of several organs at sacrifice. Acetonitrile was without developmental effects even at doses toxic to the dam. Of the halogenated compounds, treatment with trichloroacetonitrile (TCAN) and dichloroacetonitrile (DCAN) resulted in reduced fertility and increased early implantation failure. There was no effect on litter size in females bearing live litters, but pup birth weight was reduced in all litters exposed to halogenated compounds. Perinatal survival of the pups was adversely impacted by DCAN and TCAN. Postnatal growth till Day 4 was reduced by DCAN and bromochloroacetonitrile (BCAN) while growth till Day 42 was consistently affected only by TCAN. Some general observations were made on the usefulness of the criteria used in the screen, and TCAN, the most toxic of the halogenated compounds, was selected for further in-depth evaluation.

Reproductive toxicology of disinfection by-products.

The chronic exposure of large segments of the population to disinfected drinking water has necessitated an evaluation of the health effects of the by-products of the chlorination process. This paper reviews the available information concerning the reproductive consequences associated with exposure to disinfection by-products. Four groups of compounds are discussed: the trihalomethanes, in particular chloroform; the chlorinated phenols; chlorinated humic substances; and the haloacetonitriles. In the pregnant female, chloroform and the 2- and 2,4-chlorophenols produced low levels of embryo- and fetotoxicity. Chloroform induced terata when administered by inhalation. The chlorinated humic substances and 2,4,6-trichlorophenol were without significant reproductive effects. The haloacetonitriles showed in utero toxicity, becoming more severe with increasing halogen substitution.

The effect of trichloroethylene on male sexual behavior: possible opioid role.

Trichloroethylene (TCE) is a chlorinated hydrocarbon solvent which is widely used as an industrial degreasing agent. Workers exposed to TCE often exhibit symptoms similar to those symptoms produced by narcotics. The present studies evaluated the effects of TCE exposure on measures of male sexual behavior in rats. The data indicated that TCE (1000 mg/kg, PO) 4 hours before testing produced an increased ejaculation latency effect on male copulatory behavior. Naltrexone (2.0 mg/kg, IP) given 15 minutes before testing blocked this TCE-induced effect. Animals given chronic TCE administration showed tolerance to TCE's effect by the end of two weeks. Cross-tolerance to morphine was also demonstrated at this time. Quaternary naloxone failed to block any of the TCE-induced effects. These data suggest that many of TCE's effects may be mediated via the endogenous opioid system at a CNS level.

Decreasing epididymal sperm reserves enhances the detection of ethoxyethanol-induced spermatotoxicity.

Current test strategies for assessing male reproductive toxicity may be inadequate for estimating risk in humans. High levels of sperm production and existence of large epididymal sperm reserves in most test species may impede the detection of spermatotoxicity at low doses. The current report reflects initial efforts to address these issues. An active schedule of copulation was employed to reduce cauda epididymal reserves in the rat. The detection of spermatotoxicity in this animal relative to its nonmated counterpart was then compared following exposure to ethoxyethanol (EE). Adult, male Long-Evans hooded rats were assigned to a "mate" or "nonmate" condition, with the former mated every other day (3-hr sessions) for 2 weeks prior to and then throughout the experiment. After 2 weeks, males from each group were randomly assigned to receive either 0, 150, or 300 mg/kg (po) of EE, 5 days/week for 6 weeks. Males were then sacrificed and organ weights, testicular spermatid counts, and cauda epididymal sperm count and sperm morphology were obtained. EE produced a significant reduction in testicular weight and spermatid counts in mated and nonmated animals receiving 300 mg/kg. Significant decreases were also noted in epididymal sperm count and percentage normal morphology. However, these effects were seen in the nonmated animals only at 300 mg/kg, whereas significant reductions in both parameters were also obtained at 150 mg/kg in the males mated bidaily. The data from this study suggest that bidaily matings, by reducing epididymal sperm reserves, can enhance the detection of spermatotoxicity.

In vivo and in vitro evaluations of spermatotoxicity induced by 2-ethoxyethanol treatment.

2-Ethoxyethanol (EE), a member of the glycol ethers, has been shown to produce testicular atrophy in laboratory animals. The present study further identified the spectrum of effects on the male reproductive system in vivo and initiated in vitro studies on possible mechanisms of action. Adult, male rats were treated (po) with 0 or 936 mg EE/kg, 5 days/week for 6 weeks. Semen samples were collected on a weekly basis during the exposure period from ovariectomized, hormonally primed females immediately following mating and analyzed for sperm count, sperm morphology, and sperm motility. Sperm count and percent normal morphology were decreased at Weeks 5 and 6, and sperm motility was decreased at Week 6. These data, along with other studies, indicated that the pachytene spermatocyte was the most sensitive target for EE. In vitro studies monitored O2 consumption and ATP concentrations in isolated pachytene spermatocytes which were treated with 10 mM EE or 1 or 10 mM ethoxyacetic acid (EAA), the reported active metabolite of EE. An increase in respiratory ratio for the lactate rate/endogenous rate and a decrease in the 2,4-dinitrophenol rate/lactate rate were observed only for cells treated with 10 mM EAA. Additionally, a decrease in ATP concentration was seen only with 10 mM EAA. These results indicated that EAA interfered with energy metabolism in the pachytene spermatocyte. This effect may, in part, explain the testicular toxicity produced by this compound.

Dominant lethal effects of subchronic acrylamide administration in the male Long-Evans rat.

Acrylamide, a widely used vinyl monomer, is well known as a neurotoxin but inactive as a mutagen in bacterial test systems. The experiments reported demonstrate that after subchronic oral dosing in the male rat, acrylamide induced significant elevations in both pre- and post-implantation loss following dominant lethal testing. These effects were seen at doses which failed to produce clinical or pathological evidence of neurotoxicity. In an accompanying cytogenetic study, no increase in chromosome aberrations was observed in spermatogonia or spermatocytes of treated animals. When spermatocytes from treated spermatogonial stem cells were analyzed, reciprocal translocations (4) were observed in the treated animals and not in the control, but the significance of this result still needs to be established.

Evaluation of the reproductive toxicology of 2,4,6-trichlorophenol in male and female rats.

The toxicity of chlorinated organic compounds which may be generated as a by-product of drinking water chlorination has been an issue of increasing concern. Relatively few data are available concerning their reproductive toxicity. The present study was designed to evaluate the reproductive effects of one of these compounds, 2,4,6-trichlorophenol (TCP), in male and female rats. Adult males were treated with either 0, 100, 500, or 1000 mg/kg of TCP (po) for 10 weeks, at which time semen evaluations were conducted on ejaculates recovered from the genital tract of receptive females. Fertility was assessed in the 0- and 1000-mg/kg groups. Females were treated with identical doses for 2 weeks prior to pregnancy then throughout gestation. Dams were allowed to litter and pup development was monitored until Day 42 postpartum. TCP had no effect on any sperm parameter or male fertility. Treatment of females with 1000 mg/kg of TCP produced gross maternal toxicity as reflected in increased lethality and decreased weight gains in the dams. However, no treatment-related differences were seen in litter sizes or pup survival. Male and female birth weights were significantly depressed in the 500- and 1000-mg/kg groups; these differences disappeared by Day 4 postpartum, suggesting that they were a reflection of maternal toxicity. To this extent, the reproductive processes of male and female rats do not appear to be a primary target for the effects of TCP.

Reproductive toxicity associated with acrylamide treatment in male and female rats.

The present study was designed to evaluate the influence of acrylamide (ACR) on male and female reproductive function. Male rats received ACR in drinking water (50, 100, or 200 ppm) for up to 10 wk. Copulatory behavior, semen, and (for controls and 100 ppm only) fertility and fetal outcomes were evaluated. Females received ACR (25, 50, 100 ppm) for 2 wk prior to initiation of breeding and then throughout gestation and lactation. Hindlimb splaying was apparent in the 200-ppm males by wk 4; less severe splaying appeared in the 100-ppm group at wk 8. Disruptions in copulatory behavior preceded the appearance of this ataxia. These disruptions in mating performance interfered with ejaculatory processes and subsequent transport of sperm, since semen was found in the uterus of only 1 of the 15 females mated with the 100-ppm males at wk 9. Moreover, only 33% of the females mated (wk 10) to the 100-ppm males were pregnant. Postimplantation loss was also significantly increased in this group. Hindlimb splaying appeared in the females receiving 100 ppm ACR during wk 1-2 of pregnancy. Body weight and fluid intake were also depressed. Dams in the 50-ppm group showed depression in these parameters during the last 2 wk of lactation. ACR did not significantly affect mating performance of the females, pregnancy rates, litter size, or survival. However, ACR did significantly depress pup body weight at birth (100-ppm group) and weight gain during lactation through post-weaning, d 42 (50- and 100-ppm groups). Vaginal patency was delayed in the 100-ppm group only.

Influence of the uterine environment on rat sperm motility and swimming speed.

The present study compared several rat sperm parameters in semen samples recovered from a natural uterine environment (i.e., intact estrous female) to those recovered from an artificially induced uterine environment (i.e., ovariectomized hormonally primed female). The sperm parameters measured were percent motile, percent exhibiting forward progressive motility, actual swimming speed, and linear swimming speed. The comparisons were conducted at four postcopulatory time points (0.25, 1.5, 3, and 6 hours) in order to detect differences as a function of residence time within the uterus. No significant differences (P less than 0.05) in the parameters were seen between the two types of uterine environments. Residence time within the reproductive tract had no significant effect on the parameters with the exception of percent motile, which was significantly increased (P less than 0.01) at the 1.5-hour postcopulatory time point.