U.S. EPA Reference Dose/Reference Concentration Methodology: Update on a Review of the Process

The U.S. Environmental Protection Agency (USEPA) has been reviewing several approaches to testing and risk assessment related to implementation of the Food Quality Protection Act (FQPA) and the Amendments to the Safe Drinking Water Act (SDWA), both signed into law in 1996. Based on recommendations from a review of issues related to children's health protection under these laws, the USEPA established the RfD Technical Panel to evaluate in depth the current reference dose (RfD) and reference concentration (RfC) process in general, and in particular with respect to how well children and other potentially sensitive subpopulations are protected. The RfD Technical Panel also was asked to consider scientific issues that have become of greater concern in RfD and RfC derivation (e.g., neurotoxicity, immunotoxicity), and to raise issues that should be explored or developed further for application in the RfD/RfC process. This paper provides the current status of the activities of the RfD Technical Panel. The Technical Panel has recommended that acute, short- term, and intermediate reference values should be set for chemicals, where possible, and that these values should be incorporated into the USEPA's Integrated Risk Information System (IRIS) Database. A review of current testing procedures is underway, including the endpoints assessed, life stages covered by exposure and outcome evaluation, and information that can be derived from current protocols on various durations of exposure. Data gaps identified for risk assessment include the types of pharmacokinetic data that should be collected, especially for developmental toxicity studies, the impact of aging on toxic responses occurring after early exposure as well as concomitant with exposure in old age, and information available on latency to response. The implications of the RfD Technical Panel's recommendations for various uncertainty factors are also being explored.     

Setting Pesticide Reference Doses: A Retrospective Analysis Examining Key Data and Choices

Toxicity tests are widely used to set “acceptable” levels of chemical exposure. Different organizations have identified a base set of tests specifying a mix of endpoints, durations, and species to be tested. A specific test and endpoint is chosen as the basis for calculation of human health risk values like reference doses (RfDs). This study empirically evaluates the data and choices made in setting acute and chronic RfDs for 352 conventional pesticides. The results suggest that for Acute, Acute-Female Specific, and Chronic RfDs one test is used far more than others. Ninety-six percent of the 116 Acute Female-Specific RfDs relied on a developmental toxicity test and 78% of Chronic RfDs used the chronic bioassay. Tests in rats were used far more often than other species in all RfD calculations. For all types of RfDs a total uncertainty factor of 100 was most common although values as low as 1 and as high as 3000 were seen. These results provide insights not only into the science policy frameworks used, but also into ways toxicity testing and risk assessment may be streamlined and made more efficient.     

Assessment of reproductive risks

In the regulatory process, the hazards posed by potentially toxic agents to the female and male reproductive systems and to developing young are evaluated by risk assessment procedures. In this paper, toxicity testing and the regulatory process are discussed, with emphasis on risk assessment. The suggested testing protocols of the Pesticide Assessment Guidelines (U.S. EPA) are presented as an example of testing that might be done to produce toxicity data for an agent. Protocols and end points that are utilized in testing for reproductive effects are described. Included are acute, subchronic, chronic, and short-term tests. The four components of reproductive risk assessment (hazard identification, dose-response assessment, exposure assessment, and risk characterization) are examined. Effects of dibromochloropropane on rabbit testicular parameters are used to demonstrate approaches that could be taken in doing a reproductive risk assessment. Research needs for screening methods, adequate dose-response testing, toxicokinetics, end point development, and extrapolation methods are identified. Finally, this paper discusses selected areas in which changes in reproductive risk assessment are anticipated, as well as the mechanism for influencing the nature and extent of those changes.     

Selecting a more realistic uncertainty factor: Reducing compounding effects of multiple uncertainties

Available toxicology datasets provide a unique opportunity to validate some of the currently used Uncertainty Factors in the development of acceptable exposure levels for noncancer effects. Toxicity studies from two separate sources, the FAO/WHO database on pesticides (1978–1987) and the Monsanto database (through 1988) were chosen to evaluate three of the five currently used Uncertainty Factors. Interspecies differences in NOELs between the three mammalian species evaluated are equal to or less than a factor of 10 for both the FAO/WHO data and the Monsanto data in greater than 90% of the cases evaluated. Median values for the comparison of interspecies NOELs were 3.0 or less for all comparisons except the comparison between the mouse and rat for the Monsanto dataset where the median value was 7.5. Analyses of the Monsanto toxicity database show that the reprotoxicity NOELs were always equivalent to or higher than the chronic or subchronic NOELs for the same material. Therefore, even without conduct of a specific study to address reproductive effects, reasonable protection from adverse reproductive effects can be afforded by use of either subchronic or chronic study NOELs without application of UFD. The median ratio of subchronic NOELs and chronic NOELs was 4, and for a majority of the studies the difference between the NOELs was within one order of magnitude. Our analysis aids in validating the assumption that the upperbound for individual uncertainties maybe accounted for by use of 10‐fold uncertainty factors. However, the current U.S. Environmental Protection Agency (USEPA) reference doses/concentrations may be overly conservative because upperbounds of each of the uncertainty factors are used and each of the uncertainty factors are considered to be independent variables. Because uncertainties are probably not independent variables, the influence of compounding upperbounds when multiple uncertainty factors are used is generally only considered when four or more areas of uncertainty are outstanding. When multiple uncertainties exist, we recommend upperbound estimates only be used for the first two Uncertainty Factors, and median values be used to account for the remaining uncertainties.     

Predictive Value of Species Sensitivity Distributions for Effects of Herbicides in Freshwater Ecosystems

In this article we present a review of the laboratory and field toxicity of herbicides to aquatic ecosystems. Single-species acute toxicity data and (micro)mesocosm data were collated for nine herbicides. These data were used to investigate the importance of test species selection in constructing species sensitivity distributions (SSDs), and in estimating hazardous concentrations (i.e., HC5) protective for freshwater aquatic ecosystems. A lognormal model was fitted to toxicity data (acute EC50s and chronic NOECs) and the resulting distribution used to estimate lower (95% confidence), median (50% confidence), and upper (5% confidence), HC5 values. The taxonomic composition of the species assemblage used to construct the SSD does have a significant influence on the assessment of hazard and only sensitive primary producers should be included for the risk assessment of herbicides. No systematic difference in sensitivity between standard and non-standard test species was observed. Hazardous concentrations estimated using laboratory-derived acute and chronic toxicity data for sensitive freshwater primary producers were compared to the response of herbicide-stressed freshwater ecosystems using a similar exposure regime. The lower limit of the acute HC5 and the median value of the chronic HC5 were protective of adverse effects in aquatic micro/mesocosms even under a long-term exposure regime. The median HC5 estimate based on acute data was protective of adverse ecological effects in freshwater ecosystems when a pulsed or short-term exposure regime was used in the microcosm and mesocosm experiments. There was also concordance between the predictions from the effect model PERPEST and the concentrations at which clear effects started to emerge in laboratory and field studies. However, compared to the SSD concept, the PERPEST model is able to provide more information on ecological risks when a common toxicological mode of action is evaluated as it considers both recovery and indirect effects.     

A review of toxicity testing protocols and endpoints with Artemia spp.

Artemia spp. is an historically popular biological model still requiring an official internationally based standardization. Several endpoints are currently available. Short-term acute endpoints include biomarker (acetylcholinesterase; heat stress proteins; lipid peroxidation; thiobarbituric acid reactive substances; thioredoxin reductase; glutathione-peroxidase; glutathione S-transferase; glutathione reductase; aldehyde dehydrogenase; and adenylpyrophosphatase and Fluotox), hatching (dry biomass, morphological disorders and size), behavioral (swimming speed and path length), teratogenicity (growth), and immobilization (meaning mortality after 5–30 s observation). Long-term chronic tests focus on growth, reproduction and survival or mortality after 7–28 d exposure from larval to adulthood stage. We analyzed each test looking at its endpoint, toxicant and experimental design including replicates, exposure time, number of exposed cysts or organisms and their relative life stage, exposure conditions during hatching and testing (salinity, pH, light intensity, aeration dilution media, and food supply), type of testing chambers, and quality assurance and quality control criteria. Similarities and differences between the identified approaches were highlighted. Results evidenced that hatching 24 h short-term and 14 d long-term mortality are the most promising Artemia spp. protocols that should go forward with international standardization.     

Chronic vs. short-term acute O3 exposure effects on nocturnal transpiration in two Californian oaks

We tested the effect of daytime chronic moderate ozone (O3) exposure, short-term acute exposure, and both chronic and acute O3 exposure combined on nocturnal transpiration in California black oak and blue oak seedlings. Chronic O3 exposure (70 ppb for 8 h/day) was implemented in open-top chambers for either 1 month (California black oak) or 2 months (blue oak). Acute O3 exposure (approximately 1 h in duration during the day, 120-220 ppb) was implemented in a novel gas exchange system that supplied and maintained known O3 concentrations to a leaf cuvette. When exposed to chronic daytime O3 exposure, both oaks exhibited increased nocturnal transpiration (without concurrent O3 exposure) relative to unexposed control leaves (1.8x and 1.6x, black and blue oak, respectively). Short-term acute and chronic O3 exposure did not further increase nocturnal transpiration in either species. In blue oak previously unexposed to O3, short-term acute O3 exposure significantly enhanced nocturnal transpiration (2.0x) relative to leaves unexposed to O3. California black oak was unresponsive to (only) short-term acute O3 exposure. Daytime chronic and/or acute O3 exposures can increase foliar water loss at night in deciduous oak seedlings.     

Methyl Ethyl Ketone Safety Characterization for Infants and Children: Assessment in the USEPA Voluntary Children's Chemical Evaluation Program

A safety characterization specific to children was performed for methyl ethyl ketone (MEK) according to the guidelines of the Voluntary Children's Chemical Evaluation Program (VCCEP). The characterization indicates that MEK exposures are not expected to pose an acute or chronic risk to children. Hazard information, summarized as per the VCCEP Tier structure, indicated no need for additional studies. All exposure pathways potentially relevant to children were considered, including child contact with environmental media, food, drinking water, parental transfer to child (human milk or dermal contact), direct consumer product use, and presence during product use. The assessment found that exposures from anthropogenic sources that children may encounter on a daily basis are very low, and in particular well below the chronic inhalation and oral health benchmarks (RfC and RfD) derived by the U.S. Environmental Protection Agency (USEPA). Indoor uses of consumer products can result in higher acute exposures, but these are short-lived and also fall below chronic benchmarks adjusted to an acute timeframe. In addition, MEK is rapidly metabolized and excreted, thus acute exposures do not lead to an increase in body burden over time. The USEPA concluded the VCCEP submission sufficiently characterized potential risks to children, and that no additional toxicity tests were needed for MEK.     

Sensitivity of Daphnia to toxic cyanobacteria: effects of genotype and temperature

1. We studied the effects of both acute and chronic exposure of Daphnia pulex to toxic Microcystis aeruginosa . We focused on the effects of Daphnia genotype and temperature (19 and 24 °C).
2. The study revealed variation among ten Daphnia pulex clones in survivorship under acute Microcystis exposure, measured as EC₅₀. An increase in temperature caused a clear decrease in EC₅₀, although the ranking of clones according to sensitivity remained the same at both temperatures.
3. In the chronic exposure of two of the clones, toxic Microcystis reduced survival and reproduction. The two clones differed in their responses, indicating different means of coping with toxic cyanobacteria. Toxic cyanobacteria reduced slightly more at 24 °C than 19 °C.
4. The clonal difference in sensitivity to toxic cyanobacteria at acute exposure was reversed at chronic exposure. This indicates that the results from short-term toxicity cannot be used to predict life history responses under sublethal exposure.     

Sensitivity of Daphnia to toxic cyanobacteria: effects of genotype and temperature

1. We studied the effects of both acute and chronic exposure of Daphnia pulex to toxic Microcystis aeruginosa . We focused on the effects of Daphnia genotype and temperature (19 and 24 °C).
2. The study revealed variation among ten Daphnia pulex clones in survivorship under acute Microcystis exposure, measured as EC₅₀. An increase in temperature caused a clear decrease in EC₅₀, although the ranking of clones according to sensitivity remained the same at both temperatures.
3. In the chronic exposure of two of the clones, toxic Microcystis reduced survival and reproduction. The two clones differed in their responses, indicating different means of coping with toxic cyanobacteria. Toxic cyanobacteria reduced slightly more at 24 °C than 19 °C.
4. The clonal difference in sensitivity to toxic cyanobacteria at acute exposure was reversed at chronic exposure. This indicates that the results from short-term toxicity cannot be used to predict life history responses under sublethal exposure.     

Development of Factors for Estimating Swimmers' Exposures to Chemicals in Swimming Pools

The U.S. Environmental Protection Agency (USEPA) has developed a model to estimate chemical-specific exposures to swimmers following pool treatments. This model is called SWIMODEL. The model was first introduced in 1996, and the USEPA has made refinements to the model over the last decade. One of the most significant parameters in this model is the duration of exposure. Unfortunately, there was no reliable source of data for these exposure durations, so highly conservative estimates were used in the earlier version of SWIMODEL. In order to address this data deficiency, a telephone survey of competitive swimming coaches was conducted to provide data for the refinement of the SWIMODEL. The survey collected data on practice durations and other factors that are potentially relevant to swimmer exposures. Two different surveys were developed. The first survey was designed for coaches of amateur swim club teams, and the second survey was for collegiate coaches. The survey found that the average practice durations for youth, adult Masters, and collegiate teams were 9.1, 6.4, and 17.0 hours/week, respectively. The use of these data to estimate acute, chronic, and lifetime exposures is discussed. The data developed in this study were used to update the exposure duration and frequency estimates in SWIMODEL for competitive swimmers based on survey responses. The survey was also used to determine the percentage of competitive swimmers that wear eye goggles and other protective gear, and to supplement other measurements of the incidental water ingestion of competitive swimmers.     

Noncancer Risk Assessment: A Probabilistic Alternative to Current Practice

Based on imperfect data and theory, agencies such as the United States Environmental Protection Agency (USEPA) currently derive “reference doses” (RfDs) to guide risk managers charged with ensuring that human exposures to chemicals are below population thresholds. The RfD for a chemical is typically reported as a single number, even though it is widely acknowledged that there are significant uncertainties inherent in the derivation of this number.

In this article, the authors propose a probabilistic alternative to the EPA's method that expresses the human population threshold as a probability distribution of values (rather than a single RfD value), taking into account the major sources of scientific uncertainty in such estimates. The approach is illustrated using much of the same data that USEPA uses to justify their current RfD procedure.

Like the EPA's approach, our approach recognizes the four key extrapolations that are necessary to define the human population threshold based on animal data: animal to human, human heterogeneity, LOAEL to NOAEL, and subchronic to chronic. Rather than using available data to define point estimates of “uncertainty factors” for these extrapolations, the proposed approach uses available data to define a probability distribution of adjustment factors. These initial characterizations of uncertainty can then be refined when more robust or specific data become available for a particular chemical or class of chemicals.

Quantitative characterization of uncertainty in noncancer risk assessment will be useful to risk managers who face complex trade-offs between control costs and protection of public health. The new approach can help decision-makers understand how much extra control cost must be expended to achieve a specified increase in confidence that the human population threshold is not being exceeded.     

Effects of manufactured nanomaterials on fishes: a target organ and body systems physiology approach

Manufactured nanomaterials (NM) are already used in consumer products and exposure modelling predicts releases of ng to low μg l(-1) levels of NMs into surface waters. The exposure of aquatic ecosystems, and therefore fishes, to manufactured NMs is inevitable. This review uses a physiological approach to describe the known effects of NMs on the body systems of fishes and to identify the internal target organs, as well as outline aspects of colloid chemistry relevant to fish biology. The acute toxicity data, suggest that the lethal concentration for many NMs is in the mg l(-1) range, and a number of sublethal effects have been reported at concentrations from c. 100 μg to 1 mg l(-1). Exposure to NMs in the water column can cause respiratory toxicity involving altered ventilation, mucus secretion and gill pathology. This may not lead, however, to overt haematological disturbances in the short term. The internal target organs include the liver, spleen and haematopoietic system, kidney, gut and brain; with toxic effects involving oxidative stress, ionoregulatory disturbances and organ pathologies. Some pathology appears to be novel for NMs, such as vascular injury in the brain of rainbow trout Oncorhynchus mykiss with carbon nanotubes. A lack of analytical methods, however, has prevented the reporting of NM concentrations in fish tissues, and the precise uptake mechanisms across the gill or gut are yet to be elucidated. The few dietary exposure studies conducted show no effects on growth or food intake at 10-100 mg kg(-1) inclusions of NMs in the diet of O. mykiss, but there are biochemical disturbances. Early life stages are sensitive to NMs with reports of lethal toxicity and developmental defects. There are many data gaps, however, including how water quality alters physiological responses, effects on immunity and chronic exposure data at environmentally relevant concentrations. Overall, the data so far suggest that the manufactured NMs are not as toxic as some traditional chemicals (e.g. some dissolved metals) and the innovative, responsible, development of nanotechnology should continue, with potential benefits for aquaculture, fisheries and fish health diagnostics.     

Developmental exposure to lead and late life abnormalities of nervous system

Role of developmental exposure to environmental agents in altering the disease process is well known. Exposure to chemical agents at critical periods of development may cause some permanent changes in the functioning of various vital systems including the nervous system in the organisms. It is not surprising to see an extensive response due to exposure to chemical agents early in life as the organ systems are more vulnerable to chemical insults during developmental stages. In some cases the response to low level environmental insults may not be obvious until adult or old age. Results from several studies have shown such latency in response to the nervous system leading to neurodegeneration in old age. Studies conducted in murine and primate models provided ample evidence for the association of developmental exposure to low levels of heavy metal lead (Pb) and Alzheimer's disease-like pathology during senescence. It is not clear about the reasons behind such response; however, the contribution of epigenetic mechanisms could explain the role of early events in life in inducing the late life abnormalities of nervous system. It is possible that environmental agents epigenetically modulate the gene regulation to persist the response silent for a long period of time and to result pathological outcomes significantly later in life. This article will summarize the association of early life exposure to environmental agents and late-life abnormalities with an emphasis on developmental exposure to Pb and neurodegeneration in old age.     

Ecologically relevant measures of the physiological tolerance of light brown apple moth, Epiphyas postvittana, to high temperature extremes

Invasive ectothermic species are limited in their geographic range expansion primarily by their capacity to withstand temperature extremes. Epiphyas postvittana is a highly polyphagous invasive leafroller that was discovered in California in 2006. To predict its potential range and future response to climate change, high temperature tolerance of this species was determined for all life stages and larval instars. Using the static method to estimate high temperature tolerance with response to probing as an endpoint, the mean time leading to 50% mortality (LT(50)) ranged from 45 to 187h at 32.3°C, 34 to 68h at 36°C, 11 to 21h at 38°C, and 1.2 to 5.6h at 40.4°C. There was no clear pattern in the relative tolerance of the life stages across the range of temperatures tested. For pupae and adults, gender did not influence the LT(50) values at any of the temperatures tested. For the larval instars, LT(50) values increased with increasing larval instar at the highest three temperatures while this trend was reversed for the lowest temperature (32.3°C). An analysis of LT(50) values obtained from acute responses to probing compared to subsequent survival to adult emergence, showed that chronic mortality severely affected all larval instars at three out of the four constant temperatures and resulted in 64-85% reduction in LT(50) values. No difference in acute and chronic mortality was found for exposure of the egg stage to high temperatures. These findings have important implications for predicting thermal limits and range expansions of insect species, since upper thermal tolerance could readily be overestimated from the use of ad hoc rather than ecologically relevant endpoint measurements such as survival to adult emergence.     

Response of the phiX174 am3, cs70 transgene to acute and chronic ENU exposure: implications for protocol design

Studies of other transgenic assays have shown that time after treatment is a very important variable in the analysis of mutation frequencies but that eventually a plateau frequency is reached, indicating that the mutations are neutral. This neutrality is very important for the design of both experiments and testing protocols. Here we show that the phiX174 am3, cs70 transgene gives qualitatively similar results to the other transgenes studied after exposure of the mice to N-ethyl-N-nitrosourea. In the small intestine, the mutant frequency induced by an acute dose did not change significantly from 10 to 70 days post-treatment, indicating that the mutations induced are, indeed, neutral. Likewise, the mutant frequency increased linearly with duration of exposure to ENU at a constant rate. Mutant frequencies obtained were 10 times higher from the chronic exposure than produced by a nearly lethal acute dose. As in previous comparisons of a transgene and the endogenous Dlb-1 locus in the small intestine, the chronic exposure was much more effective at increasing the sensitivity of the transgene than of the endogenous gene. The Dlb-1 locus shows more complex kinetics in this strain, as in others, with mutations initially accumulating at a slower rate, indicating a differential repair of genetic damage.     

Using Human Data to Develop Risk Values

One of the criticisms of industry-sponsored human subject testing of toxicants is based on the perception that it is often motivated by an attempt to raise the acceptable exposure limit for the chemical. When Reference Doses (RfDs) or Reference Concentrations (RfCs) are based upon no-effect levels from human rather than animal data, an animal-to-human uncertainty factor (usually 10) is not required, which could conceivably result in a higher safe exposure limit. There has been little in the way of study of the effect of using human vs. animal data on the development of RfDs and RfCs to lend empirical support to this argument. We have recently completed an analysis comparing RfDs and RfCs derived from human data with toxicity values for the same chemicals based on animal data. The results, published in detail elsewhere, are summarized here. We found that the use of human data did not always result in higher RfDs or RfCs. In 36% of the comparisons, human-based RfDs or RfCs were lower than the corresponding animal-based toxicity values, and were more than 3-fold lower in 23% of the comparisons. In 10 out of 43 possible comparisons (23%), insufficient experimental animal data are readily available or data are inappropriate to estimate either RfDs or RfCs. Although there are practical limitations in conducting this type of analysis, it nonetheless suggests that the use of human data does not routinely lead to higher toxicity values. Given the inherent ability of human data to reduce uncertainty regarding risks from human exposures, its use in conjunction with data gathered from experimental animals is a public health protective policy that should be encouraged.     

Evaluation of the INS-1 832/13 Cell Line as a Beta-Cell Based Screening System to Assess Pollutant Effects on Beta-Cell Function

Environmental pollutants have recently emerged as potential risk factors for metabolic diseases, urging systematic investigation of pollutant effects on metabolic disease processes. To enable risk assessment of these so-called metabolic disruptors the use of stable, robust and well-defined cell based screening systems has recently been encouraged. Since beta-cell (dys)functionality is central in diabetes pathophysiology, the need to develop beta-cell based pollutant screening systems is evident. In this context, the present research evaluated the strengths and weaknesses of the INS-1 832/13 pancreatic beta-cell line as diabetogenic pollutant screening system with a focus on beta-cell function. After optimization of exposure conditions, positive (exendin-4, glibenclamide) and negative (diazoxide) control compounds for acute insulin secretion responses were tested and those with the most profound effects were selected to allow potency estimations and ranking of pollutants. This was followed by a first explorative screening of acute bisphenol A and bis(2-ethylhexyl)phthalate effects. The same approach was applied for chronic exposures, focusing primarily on evaluation of acknowledged chronic stimulators (diazoxide, T0901317, exendin-4) or inhibitors (glibenclamide) of insulin secretion responses to select the most responsive ones for use as control compounds in a chronic pollutant testing framework. Our results showed that INS-1 832/13 cells responded conform previous observations regarding acute effects of control compounds on insulin secretion, while bisphenol A and bis(2-ethylhexyl)phthalate had limited acute effects. Furthermore, chronic exposure to known beta-cell reactive compounds resulted in deviating insulin secretion and insulin content profiles compared to previous reports. In conclusion, this INS-1 subclone appears to lack certain characteristics needed to respond appropriately to acute pollutant exposure or long term exposure to known beta-cell reactive compounds and thus seems to be, in our setting, inadequate as a diabetogenic pollutant screening system.     

Sportsfish Consumption Surveys: A Risk Assessment Practitioner's Wish List

In the United States, risk assessments that include sportsfish consumption often rely on default values derived by the USEPA. However, local, regional, and cultural differences can result in substantial differences in consumption rates, species of fish caught, parts of fish consumed, and fish preparation methods—all of which contribute to the extent of contaminant exposure. Because of these differences, site-specific data are important for the reliable prediction of exposure. This article reviews the types of information important to a risk assessor to enhance the applicability of the exposure assessment to the population being evaluated. It provides a method for setting priorities based on the sensitivity of the exposure assessment to each type of information. Consumption rate and parts of fish consumed are the most important information to obtain. This information should be obtained for each fish species consumed, and should be obtained for both children and adults. Body weight and exposure duration have the next level of priority, and cooking method has the lowest level of priority. Results from surveys should be presented in a manner that allows the reader to understand the variability observed in the data and the survey questions that were asked. Correlations between variables should be evaluated and presented.