Evaluation of research activities and research needs to increase the impact and applicability of alternative testing strategies in risk assessment practice

The present paper aims at identifying strategies to increase the impact and applicability of alternative testing strategies in risk assessment. To this end, a quantitative and qualitative literature evaluation was performed on (a) current research efforts in the development of in vitro methods aiming for alternatives to animal testing, (b) the possibilities and limitations of in vitro methods for regulatory purposes and (c) the potential of physiologically-based kinetic (PBK) modeling to improve the impact and applicability of in vitro methods in risk assessment practice. Overall, the evaluation showed that the focus of state-of-the-art research activities does not seem to be optimally directed at developing in vitro alternatives for those endpoints that are most animal-demanding, such as reproductive and developmental toxicity, and carcinogenicity. A key limitation in the application of in vitro alternatives to such systemic endpoints is that in vitro methods do not provide so-called points of departure, necessary for regulators to set safe exposure limits. PBK-modeling could contribute to overcoming this limitation by providing a method that allows extrapolation of in vitro concentration-response curves to in vivo dose-response curves. However, more proofs of principle are required.     

Evaluation of subchronic toxicity data using the benchmark dose approach

We used the benchmark dose (BMD) methodology devised by Crump (Fundam. Appl. Toxicol. 4, 854-871, 1984) to estimate BMDs for 90-day toxicological data and several fabricated data sets. From a toxicological perspective, dose-response modeling offers certain advantages over using a point estimate, such as the currently used no-observable-adverse-effect level (NOAEL) approach. However, there are many variables associated with the BMD that could be set to produce unreasonable BMD estimates. Some of these variables and decisions are examined in this study. BMDs were calculated for discrete and continuous endpoints using a variety of different variables (e.g., maximum likelihood estimates [MLEs], lower-confidence limits [LCLs], and different risk levels). In addition, the fabricated data sets were manipulated (i.e., dose groups eliminated) and the BMDs recalculated. This process tested how the BMD estimates varied using different forms of the data. For the 90-day toxicological studies, the BMDs were typically within an order of magnitude of the NOAEL for discrete endpoints. For the discrete endpoints, the MLEs were typically greater than the NOAEL and the LCLs were typically less than the NOAEL. The BMD was insensitive to changes in the data points one to two dose groups beyond the NOAEL/LOAEL. With the continuous data, the ratios of MLEs and LCLs to the NOAEL were highly variable, and no general trend could be determined. The BMD methodology offers potential improvements in the risk assessment process since dose-response characteristics are used to calculate the BMD. Depending upon how the BMD is defined, i.e., the form of the dose-response model, and how the BMD is used in the risk assessment process, BMD estimates may produce reference doses/concentrations that are more or less conservative than the NOAEL approach. Active involvement in discussions with regulatory agencies is needed to ensure that inappropriate models and unreasonable BMDs are not used. In addition, further discussions on how BMDs should be used in the risk assessment process are needed.     

Harmonization of cancer and noncancer risk assessment: proceedings of a consensus-building workshop.

Significant advancements have been made toward the use of all relevant scientific information in health risk assessments. This principle has been set forth in risk-assessment guidance documents of international agencies including those of the World Health Organization's International Programme on Chemical Safety, the U.S. Environmental Protection Agency, and Health Canada. Improving the scientific basis of risk assessment is a leading strategic goal of the Society of Toxicology. In recent years, there has been a plethora of mechanistic research on modes of chemical toxicity that establishes mechanistic links between noncancer responses to toxic agents and subsequent overt manifestations of toxicity such as cancer. The research suggests that differences in approaches to assessing risk of cancer and noncancer toxicity need to be resolved and a common broad paradigm for dose-response assessments developed for all toxicity endpoints. In November 1999, a workshop entitled "Harmonization of Cancer and Noncancer Risk Assessment" was held to discuss the most critical issues involved in developing a more consistent and unified approach to risk assessment for all endpoints. Invited participants from government, industry, and academia discussed focus questions in the areas of mode of action as the basis for harmonization, common levels of adverse effect across toxicities for use in dose-response assessments, and scaling and uncertainty factors. This report summarizes the results of those discussions. There was broad agreement, albeit not unanimous, that current science supports the development of a harmonized set of principles that guide risk assessments for all toxic endpoints. There was an acceptance among the participants that understanding the mode of action of a chemical is ultimately critical for nondefault risk assessments, that common modes of action for different toxicities can be defined, and that our approach to assessing toxicity should be biologically consistent.     

Human health risk assessment of processing-related compounds in food

Many types of food processing techniques have been employed throughout human history, mainly to ensure microbiological and chemical safety of foods and to improve palatability. Growing consumer demand for healthy, nutritious and convenient food is a key driver for improvements and new developments in food processing. New processes or newly recognized compounds, often identified due to improved analytical capabilities, require careful evaluation of potential human health impact. As examples for processing-related contaminants, the risk assessments for 3-monochloropropanediol (3-MCPD) and acrylamide are discussed, exemplifying two traditional approaches in food safety assessment. 3-MCPD is formed in a variety of industrially and domestically produced foods in the presence of fat and chloride. 3-MCPD is considered a non-genotoxic carcinogen, hence a threshold of effect is assumed and sufficient data are available to establish a safe level of intake. The recent discovery of the formation of acrylamide in certain high heat-treated foods, domestically or industrially, has raised considerable concern, since acrylamide, besides neurotoxicity and reproductive toxicity, is considered as genotoxic carcinogen. Unavoidable substances considered to be genotoxic carcinogens are unwanted in foods and are usually dealt with via the ALARA principle. However, for such unavoidable substances, a quantitative risk assessment may give more useful advice to risk managers for appropriate actions. Currently, adequate dose-response relationships and mechanistic information regarding carcinogenicity of acrylamide are lacking. Once this information becomes available, the health risk from acrylamide exposure through food can be assessed.     

Risk assessment of acrylamide in foods.

Daily mean intakes of acrylamide present in foods and coffee in a limited Norwegian exposure assessment study have been estimated to be 0.49 and 0.46 microg per kg body weight in males and females, respectively. Testicular mesotheliomas and mammary gland adenomas have consistently been found in 2-year drinking water rat cancer studies with acrylamide. Acrylamide also shows initiating activity in mouse skin after systemic administration. Since acrylamide is converted to the mutagenic metabolite glycidamide and forms adducts to hemoglobin in rodents and humans, the tumorigenic endpoints in rats were assumed to be an expression of acrylamide genotoxicity. Using the default linear extrapolation methods LED10 and T25, the lifetime cancer hazard after lifelong exposure to 1 microg acrylamide per kg body weight per day, scaled to humans, was calculated to be, on average, 1.3 x 10-3. Using this hazard level and correlating it with the exposure estimates, a lifetime cancer risk related to daily intake of acrylamide in foods for 70 years in males was calculated to be 0.6 x 10-3, implying that 6 out of 10,000 individuals may develop cancer due to acrylamide. For females, the risk values were slightly lower. It must be emphasized that this risk assessment is conservative. A number of processes may result in nonlinearity of the dose-response relationships for acrylamide carcinogenicity in the low-dose region, including detoxication reactions, cell cycle arrest, DNA repair, apoptosis, and immune surveillance. Thus, the true risk levels related to acrylamide intake may be considerably lower.     

Summary of Dose-Response Modeling for Developmental Toxicity Studies

Developmental toxicity studies are an important area in the field of toxicology. Endpoints measured on fetuses include weight and indicators of death and malformation. Binary indicator measures are typically summed over the litter and a discrete distribution is assumed to model the number of adversely affected fetuses. Additionally, there is noticeable variation in the litter responses within dose groups that should be taken into account when modeling. Finally, the dose-response pattern in these studies exhibits a threshold effect. The threshold dose-response model is the default model for non-carcinogenic risk assessment, according to the USEPA, and is encouraged by the agency for the use in the risk assessment process. Two statistical models are proposed to estimate dose-response pattern of data from the developmental toxicity study: the threshold model and the spline model. The models were applied to two data sets. The advantages and disadvantages of these models, potential other models, and future research possibilities will be summarized.     

Dose--Response Assessment for Developmental Toxicity: III. Statistical Models

Although quantitative modeling has been central to cancer riskassessment for years, the concept of dose-response modelingfor developmental effects is relatively new. The benchmark dose(BMD) approach has been proposed for use with developmental(as well as other noncancer) endpoints for determining referencedoses and reference concentrations. Statistical models appropriatefor representing the unique features of developmental toxicitytesting have been developed and applied (K. Rai and J. Van Ryzin,1985, Biometrics 41, 1–9; L. Kupper, C. Portier, M. Hogan,and E. Yamamoto, 1986, Biometrics 42, 85–98; R. Kodell,R. Howe, J. Chen, and D. Gaylor, 1991, Risk Anal. 11, 583–590).Generalizations of those models (designated the RVR, LOG, andNCTR models, respectively) account for the correlations amongobservations in individual fetuses or implant within litters;the potential for variables other than dose, such as littersize, to affect the probability of adverse outcome; and thepossibility of a threshold dose below which background responserates are unaltered. The generalized models were applied toa database of 607 endpoints with significant dose-related increasesin response rate. It was determined that the models were generallycapable of fitting the observed dose-response patterns, withthe LOG model appearing to be superior with respect to fit.A significant contributor to the ability of the LOG model tofit the data was its flexibility with respect to the representationof the dependence of response probability on litter size, atrait not shared by the other two models. Litter size appearedto be a significant covariable for predicting response rates,even when intralitter correlation was accounted for by assuminga ß-binomial distribution for the observations amongindividual fetuses. In contrast, a threshold dose parameterdid not appear to be necessary to adequately describe the observeddose-response patterns. BMD estimates (corresponding to 5% additionalrisk) from all three models were similar to one another andto BMDs estimated from other, generic dose-response models (notspecifically designed for developmental toxicity testing) thatmodeled average proportion of fetuses affected. The BMDs atthe 5% level of risk were similar to no observed adverse effectlevels determined by statistical tests of trend. Greater emphasison and further examination of dose-response modeling for developmentaltoxicity testing are needed; biologically based approaches thatconsider the continuum of developmental effects induced in suchtests should be encouraged.     

A statistical evaluation of toxicity study designs for the estimation of the benchmark dose in continuous endpoints.

The benchmark approach is gaining attention as an alternative to the No-Observed-Adverse-Effect-Level (NOAEL) approach. However, current guidelines for the design of toxicity tests are based on assessing a NOAEL. It has been suggested that the current study design may not be optimal for assessing a Benchmark Dose (BMD). To further investigate this we performed three simulation studies in which a large number of designs were compared, focusing on continuous endpoints. Four fictitious endpoints were considered, their underlying dose-response curves having a linear, sublinear, supralinear, or sigmoidal shape. In each simulation run the BMD was derived from a model fitted to the generated data, where the selection of the model was based on that particular data set (according to a formal likelihood ratio test procedure). Thus, the model used for deriving the BMD in a single generated data set may not be the same as the one used for generating the data. In this way, model uncertainty is taken into account as well. The results show that the performance of a design is, first of all, determined by the total number of animals used. Distributing them over more dose groups does not result in a poorer performance of the study, despite the smaller number of animals per dose group. Dose placement is another crucial factor, and to minimize the risk of inadequate dose placement, the use of multiple dose studies is favorable. As a concomitant advantage, the use of multiple doses mitigates the disturbing effect of potential systematic errors in single dose groups. However, for endpoints with large residual variation (CV > or = 18%) there is a substantial probability of not detecting the overall dose-response, and this probability increases in designs with increasing number of dose groups. In such situations, six dose groups may be used as a compromise. Designs with high dose levels (i.e., associated with relatively high effects) are helpful in estimating doses with smaller effects (such as the benchmark dose), and it appears bad practice to omit higher dose groups to improve the fit at lower doses. The typical 28-day study design of four dose groups with five animals (per sex) may not be adequate to assess endpoints with large residual variation (CV > or = 18%), both in assessing a benchmark dose and in assessing a NOAEL.     

Use of epidemiologic data in Integrated Risk Information System (IRIS) assessments

In human health risk assessment, information from epidemiologic studies is typically utilized in the hazard identification step of the risk assessment paradigm. However, in the assessment of many chemicals by the Integrated Risk Information System (IRIS), epidemiologic data, both observational and experimental, have also been used in the derivation of toxicological risk estimates (i.e., reference doses [RfD], reference concentrations [RfC], oral cancer slope factors [CSF] and inhalation unit risks [IUR]). Of the 545 health assessments posted on the IRIS database as of June 2007, 44 assessments derived non-cancer or cancer risk estimates based on human data. RfD and RfC calculations were based on a spectrum of endpoints from changes in enzyme activity to specific neurological or dermal effects. There are 12 assessments with IURs based on human data, two assessments that extrapolated human inhalation data to derive CSFs and one that used human data to directly derive a CSF. Lung or respiratory cancer is the most common endpoint for cancer assessments based on human data. To date, only one chemical, benzene, has utilized human data for derivation of all three quantitative risk estimates (i.e., RfC, RfD, and dose-response modeling for cancer assessment). Through examples from the IRIS database, this paper will demonstrate how epidemiologic data have been used in IRIS assessments for both adding to the body of evidence in the hazard identification process and in the quantification of risk estimates in the dose-response component of the risk assessment paradigm.     

Copper and human health: biochemistry, genetics, and strategies for modeling dose-response relationships

Copper (Cu) and its alloys are used extensively in domestic and industrial applications. Cu is also an essential element in mammalian nutrition. Since both copper deficiency and copper excess produce adverse health effects, the dose-response curve is U-shaped, although the precise form has not yet been well characterized. Many animal and human studies were conducted on copper to provide a rich database from which data suitable for modeling the dose-response relationship for copper may be extracted. Possible dose-response modeling strategies are considered in this review, including those based on the benchmark dose and categorical regression. The usefulness of biologically based dose-response modeling techniques in understanding copper toxicity was difficult to assess at this time since the mechanisms underlying copper-induced toxicity have yet to be fully elucidated. A dose-response modeling strategy for copper toxicity was proposed associated with both deficiency and excess. This modeling strategy was applied to multiple studies of copper-induced toxicity, standardized with respect to severity of adverse health outcomes and selected on the basis of criteria reflecting the quality and relevance of individual studies. The use of a comprehensive database on copper-induced toxicity is essential for dose-response modeling since there is insufficient information in any single study to adequately characterize copper dose-response relationships. The dose-response modeling strategy envisioned here is designed to determine whether the existing toxicity data for copper excess or deficiency may be effectively utilized in defining the limits of the homeostatic range in humans and other species. By considering alternative techniques for determining a point of departure and low-dose extrapolation (including categorical regression, the benchmark dose, and identification of observed no-effect levels) this strategy will identify which techniques are most suitable for this purpose. This analysis also serves to identify areas in which additional data are needed to better define the characteristics of dose-response relationships for copper-induced toxicity in relation to excess or deficiency.     

Copper and Human Health: Biochemistry,Genetics, and Strategies for Modeling Dose-response Relationships

Copper (Cu) and its alloys are used extensively in domestic and industrial applications. Cu is also an essential element in mammalian nutrition. Since both copper deficiency and copper excess produce adverse health effects, the dose-response curve is U-shaped, although the precise form has not yet been well characterized. Many animal and human studies were conducted on copper to provide a rich database from which data suitable for modeling the dose-response relationship for copper may be extracted. Possible dose-response modeling strategies are considered in this review, including those based on the benchmark dose and categorical regression. The usefulness of biologically based dose-response modeling techniques in understanding copper toxicity was difficult to assess at this time since the mechanisms underlying copper-induced toxicity have yet to be fully elucidated. A dose-response modeling strategy for copper toxicity was proposed associated with both deficiency and excess. This modeling strategy was applied to multiple studies of copper-induced toxicity, standardized with respect to severity of adverse health outcomes and selected on the basis of criteria reflecting the quality and relevance of individual studies. The use of a comprehensive database on copper-induced toxicity is essential for dose-response modeling since there is insufficient information in any single study to adequately characterize copper dose-response relationships. The dose-response modeling strategy envisioned here is designed to determine whether the existing toxicity data for copper excess or deficiency may be effectively utilized in defining the limits of the homeostatic range in humans and other species. By considering alternative techniques for determining a point of departure and low-dose extrapolation (including categorical regression, the benchmark dose, and identification of observed no-effect levels) this strategy will identify which techniques are most suitable for this purpose. This analysis also serves to identify areas in which additional data are needed to better define the characteristics of dose-response relationships for copper-induced toxicity in relation to excess or deficiency.     

Dose-Response Relationships and Threshold Levels in Skin and Respiratory Allergy

A literature study was performed to evaluate dose-response relationships and no-effect levels for sensitization and elicitation in skin- and respiratory allergy. With respect to the skin, dose-response relationships and no-effect levels were found for both intradermal and topical induction, as well as for intradermal and topical elicitation of allergenic responses in epidemiological, clinical, and animal studies. Skin damage or irritation may result in a significant reduction of the no-effect level for a specific compound. With respect to the respiratory tract, dose-response relationships and no-effect levels for induction were found in several human as well as animal studies. Although dose-response relationships for elicitation were found in some epidemiological studies, concentration-response relationships were present only in a limited number of animal studies. Reported results suggest that especially relatively high peak concentrations can induce sensitization, and that prevention of such concentrations will prevent workers from developing respiratory allergy. Moreover, induction of skin sensitization may result in subsequent heightened respiratory responsiveness following inhalation exposure. The threshold concentration for the elicitation of allergic airway reactions in sensitized subjects is generally lower than the threshold to induce sensitization. Therefore, it is important to consider the low threshold levels for elicitation for recommendation of health-based occupational exposure limits, and to avoid high peak concentrations. Notwithstanding the observation of dose-response relationships and no-effect levels, due to a number of uncertainties, no definite conclusions can be drawn about absolute threshold values for allergens with respect to sensitization of and elicitation reactions in the skin and respiratory tract. Most predictive tests are generally meant to detect the potential of a chemical to induce skin and/or respiratory allergy at relatively high doses. Consequently, these tests do not provide information of dose-response relationships at lower doses such as found in, for example, occupational situations. In addition, the observed dose-response relationships and threshold values have been obtained by a wide variety of test methods using different techniques, such as intradermal exposure versus topical or inhalation exposure at the workplace, or using different endpoints, which all appear important for the outcome of the test. Therefore, especially with regard to respiratory allergy, standardized and validated dose-response test methods are urgently required in order to be able to recommend safe exposure levels for allergens at the workplace.     

Dose-response relationships and threshold levels in skin and respiratory allergy

A literature study was performed to evaluate dose-response relationships and no-effect levels for sensitization and elicitation in skin- and respiratory allergy. With respect to the skin, dose-response relationships and no-effect levels were found for both intradermal and topical induction, as well as for intradermal and topical elicitation of allergenic responses in epidemiological, clinical, and animal studies. Skin damage or irritation may result in a significant reduction of the no-effect level for a specific compound. With respect to the respiratory tract, dose-response relationships and no-effect levels for induction were found in several human as well as animal studies. Although dose-response relationships for elicitation were found in some epidemiological studies, concentration-response relationships were present only in a limited number of animal studies. Reported results suggest that especially relatively high peak concentrations can induce sensitization, and that prevention of such concentrations will prevent workers from developing respiratory allergy. Moreover, induction of skin sensitization may result in subsequent heightened respiratory responsiveness following inhalation exposure. The threshold concentration for the elicitation of allergic airway reactions in sensitized subjects is generally lower than the threshold to induce sensitization. Therefore, it is important to consider the low threshold levels for elicitation for recommendation of health-based occupational exposure limits, and to avoid high peak concentrations. Notwithstanding the observation of dose-response relationships and no-effect levels, due to a number of uncertainties, no definite conclusions can be drawn about absolute threshold values for allergens with respect to sensitization of and elicitation reactions in the skin and respiratory tract. Most predictive tests are generally meant to detect the potential of a chemical to induce skin and/or respiratory allergy at relatively high doses. Consequently, these tests do not provide information of dose-response relationships at lower doses such as found in, for example, occupational situations. In addition, the observed dose-response relationships and threshold values have been obtained by a wide variety of test methods using different techniques, such as intradermal exposure versus topical or inhalation exposure at the workplace, or using different endpoints, which all appear important for the outcome of the test. Therefore, especially with regard to respiratory allergy, standardized and validated dose-response test methods are urgently required in order to be able to recommend safe exposure levels for allergens at the workplace.     

Can the concept of hormesis Be generalized to carcinogenesis?

The concept of hormesis (i.e., low-dose stimulation/high-dose inhibition) has been shown to be widely generalizable with respect to chemical class, animal model, gender, and biological end point. The public health implication of this lack of linearity in the low-dose area of the dose-response curve raises the question of whether low doses of carcinogens will reduce cancer risk. Articles relating to the process of carcinogenesis (i.e., initiation, promotion, tumor development, and progression) were obtained from a recently developed chemical hormesis database and evaluated for their evidence of hormesis. Numerous examples in well-designed studies indicate that U- or J-shaped dose-response relationships exist with respect to various biomarkers of carcinogenesis in different animal models of both sexes. Examples of such J-shaped dose-response relationships in each stage of the process of carcinogenesis were selected for detailed toxicological examination. These results have important implications for both the hazard assessment of carcinogens and cancer risk assessment procedures.     

Practical Risk Assessment and Management Issues Arising were we to Adopt Low-Dose Linearity for all Endpoints

The 2009 National Research Council report, Science and Decisions, proposes harmonizing dose-response approaches for cancer and non-cancer endpoints, and for non-cancer quantitative risk assessment, this would usually take the form of a low-dose linear no-threshold dose-response curve. The soundness of this recommendation has been questioned, but I focus on its consequences if adopted, many of them apparently unintended. If most endpoints for most agents are assumed to have non-zero low-dose risks, then the critical-effect concept, choosing the one endpoint on which to calculate acceptable doses, loses its basis. All regulatory decisions, since they entail substituting some exposures (and their attendant risks) for others, become risk-risk trade-off decisions, and equity questions are raised since risk transfer is inevitably involved. A valid basis for estimating low-dose linear components is not evident, and upper-bound approaches fail to be reliably public health-protective owing to the risk trade-off decisions that need to be faced.     

Fallback tests in dose-response clinical trials

This article introduces a general testing procedure for performing dose-control comparisons in dose-response trials with one or more endpoints. The procedure (termed multi-stage fallback procedure) is an extension of the fallback test proposed by Wiens (2003). The multi-stage fallback procedure features a simple stepwise form and improves the power of dose-control tests at higher doses by taking into account the ordering of the doses. It also serves as an efficient tool for handling multiplicity caused by multiple endpoints. It is shown in this article that the multi-stage fallback procedure can be formulated as a closed testing procedure and thus controls the Type I error rate with respect to multiple dose-control comparisons as well as multiple endpoints. The proposed testing method is illustrated using examples from dose-response clinical trials with single and multiple endpoints.     

Is PM more toxic than the sum of its parts? Risk-assessment toxicity factors vs. PM-mortality "effect functions"

Epidemiology studies of populations living in areas with good air quality report correlations between levels of ambient particulate matter (PM) and mortality rates. These associations occur at low PM concentrations that are below current air quality standards. Can such concentrations cause mortality, given the toxicity of PM chemical constituents? We examined chemical-specific, dose-response data typically used in U.S. EPA human health risk assessments. These assessments rely on established, no-effect thresholds for noncancer health endpoints. We found that chemicals identified as constituents of ambient PM are present at concentrations considerably below the regulatory thresholds used in risk assessment (i.e., below the RfCs and RfDs that identify levels for which no adverse health effects are anticipated). From the perspective of risk assessment, exposure to the concentrations of chemicals in ambient PM (e.g., sulfate, nitrate, and elemental carbon) cannot be expected to cause death. Hence, the health effects attributed to ambient PM in "regulatory impact analyses" appear to be at odds with what would be predicted from a standard U.S. EPA health-risk assessment for PM chemicals. Four possible resolutions of this paradox are that (1) the mixtures of chemicals present in ambient PM are vastly more toxic than the sum of individual components, (2) small portions of the general population are vastly more sensitive to certain ambient PM chemicals than reflected in U.S. EPA toxicity factors, (3) the toxicity of ambient PM is unrelated to its chemical constituents, or (4) PM mass concentration is not the causal factor in the reported associations. The associations may arise because ambient PM concentrations (1) are a surrogate for unmeasured copollutants (e.g., HAPs), (2) covary with confounding factors that cannot be fully controlled (e.g., weather, demographics), or (3) covary with unmeasured (e.g., societal, behavioral, or stress) factors.     

Suppression of neoplastic transformation in vitro by low doses of low LET radiation

A major concern of exposure to low doses of radiation is the risk of cancer induction. Epidemiologic data are rarely powerful enough to accurately discriminate this risk at doses <10 cGy. In order to gain insight into events at these low doses, laboratory-based studies of relevant endpoints are required. One such endpoint is radiation-induced neoplastic transformation in vitro. Such studies can provide quantitative dose-response data, as well as insights into underlying cellular and molecular mechanisms. Data are presented that indicate that low doses of low LET radiation can suppress neoplastic transformation in vitro to levels below those seen spontaneously. Mechanisms involved include both the death of a subpopulation of cells prone to spontaneous neoplastic transformation and the induction of DNA repair. The relative contributions of these mechanisms is dose-dependent. The relevance of these observations to radiation risk estimation is discussed.     

The development of a protocol to assess reproductive effects of toxicants in the rat

The determination that a chemical poses a reproductive risk to man typically relies upon fertility studies using rodents. However, fertility in rodents is often difficult to disrupt and more sensitive indicators of reproductive function should be included in the risk assessment process. The present discussion compares the sensitivity of fertility to other endpoints following exposure to known reproductive toxicants. In our studies rats were dosed from weaning through puberty, gestation, and lactation. The reproductive function of the male, the female, and the offspring was assessed. The effects of methoxychlor, carbendazim (MBC), dibutyl phthalate (DBP), and lindane are discussed and compared to fertility. For each compound a ratio (SR = sensitivity ratio) of the lowest effect level (LEL) for infertility or reduced fecundity to the LEL for the most sensitive physiologic endpoint was calculated. The SR should be large when a compound produces effects over a wide range of doses, but should equal unity when the dose-response curve is very steep. For methoxychlor, which blocked implantation, pubertal landmarks and estrous cyclicity provided rapid and sensitive indicators of the subsequent reproductive failure. The SR = 8 (100/12) for methoxychlor using data from females. In contrast, DBP and MBC directed altered testicular function, and for these compounds, sperm and testicular measures provided sensitive indicators of toxicity. The SR for MBC was 2 (100/50), while DBP had a SR of 1 (500/500). In the lindane study, fertility was not reduced but most of the pups (F1) died shortly after birth. The SR for lindane is equal to 0.5 (10/20). At 20 mg/kg the treated females were larger and their estrous cycles were erratic. The data from these studies indicate that no single endpoint will consistently be the most sensitive indicator of reproductive toxicity. Studies must include a number of well validated endpoints that provide a comprehensive assessment of the entire reproductive system of the male and female.