Report of an independent peer review of an acrylonitrile risk assessment.

A peer review panel made up of experts in toxicology, epidemiology, cancer mode of action (MOA), cancer mechanisms, carcinogenicity, genotoxicity, dose-response, US Environmental Protection Agency (EPA) cancer and noncancer methods, pharmacokinetic modeling and acrylonitrile, met on 22-23 September 2003 in Cincinnati, OH. The purpose of the meeting was to provide an independent review of a risk assessment of acrylonitrile that had been prepared by the Acrylonitrile Group (AN Group). Toxicology Excellence for Risk Assessment (TERA) organized the peer review and selected the panel. The panel discussed the toxicity and epidemiology literature of acrylonitrile and MOA information, and reached conclusions regarding its MOA, weight of evidence (WOE) for carcinogenicity, preferred approach for dose-response assessment and risk values. This paper summarizes the discussion and conclusions of the panel regarding the acrylonitrile assessment. Subsequent to the peer review, the authors of the acrylonitrile assessment revised their report and the panel reviewed the revised report. A manuscript of the revised assessment is being published in Regulatory Toxicology and Pharmacology.

Genetic polymorphisms in assessing interindividual variability in delivered dose.

Increasing sophistication in methods used to account for human variability in susceptibility to toxicants has been one of the success stories in the continuing evolution of risk assessment science. Genetic polymorphisms have been suggested as an important contributor to overall human variability. Recently, data on polymorphisms in metabolic enzymes have been integrated with physiologically based pharmacokinetic (PBPK) modeling as an approach to determining the resulting overall variability. We present an analysis of the potential contribution of polymorphisms in enzymes modulating the disposition of four diverse compounds: methylene chloride, warfarin, parathion, and dichloroacetic acid. Through these case studies, we identify key uncertainties likely to be encountered in the use of polymorphism data and highlight potential simplifying assumptions that might be required to test the hypothesis that genetic factors are a substantive source of human variability in susceptibility to environmental toxicants. These uncertainties include (1) the relative contribution of multiple enzyme systems, (2) the extent of induction/inhibition through coexposure, (3) allelic frequencies of major ethnic groups, (4) the absence of chemical-specific data on the kinetic parameters for the different allelic forms of key enzymes, (5) large numbers of low-frequency alleles, and (6) uncertainty regarding differences between in vitro and in vivo kinetic data. Our effort sets the stage for the acquisition of critical data and further integration of polymorphism data with PBPK modeling as a means to quantitate population variability.

Applications of mechanistic data in risk assessment: the past, present, and future.

Mechanistic data, when available, have long been considered in risk assessment, such as in the development of the nitrate RfD based on effects in a sensitive group (infants). Recent advances in biology and risk assessment methods have led to a tremendous increase in the use of mechanistic data in risk assessment. Toxicokinetic data can improve extrapolation from animals to humans and characterization of human variability. This is done by the development of improved tissue dosimetry, by the use of uncertainty factors based on chemical-specific data, and in the development of physiologically based pharmacokinetic (PBPK) models. The development of the boron RfD illustrates the use of chemical-specific data in the improved choice of uncertainty factors. The draft cancer guidelines of the U.S. Environmental Protection Agency emphasize the use of mode of action data. The first choice under the guidelines is to use a chemical-specific, biologically based dose-response (BBDR) model. In the absence of a BBDR model, mode of action data are used to determine whether low-dose extrapolation is done using a linear or nonlinear (margin of exposure) approach. Considerations involved in evaluating a hypothesized mode of action are illustrated using 1,3-dichloropropene, and use of a BBDR model is illustrated using formaldehyde. Recent developments in molecular biology, including transgenic animals, microarrays, and the characterization of genetic polymorphisms, have significant potential for improving risk assessments, although further methods development is needed. Overall, use of mechanistic data has significant potential for reducing the uncertainty in assessments, while at the same time highlighting the areas of uncertainty.

Hazard identification and dose response of inhaled nickel-soluble salts.

A substantial body of occupational epidemiology data has shown that exposure to mixed soluble and insoluble nickel causes the development of lung and nasal cancer. However, due to coexposure of these populations to soluble and insoluble forms of nickel, and limitations in exposure measurements, the contribution of soluble nickel is difficult to determine. Soluble nickel was negative in an NTP inhalation bioassay, while there was some evidence for tumorigenicity in rats for less soluble nickel oxide, and there was clear evidence for tumorigenicity of insoluble nickel subsulfide in rats. Results of parenteral assays follow a similar pattern, but provide evidence of weak carcinogenicity of soluble nickel. Kinetic factors also indicate that exposure to soluble nickel alone has a low carcinogenic potential. Overall, we conclude that the carcinogenic activity of insoluble nickel compounds should not be used to predict the carcinogenic potential of water-soluble nickel salts. The overall data suggest a nonlinear dose-response relationship for carcinogenicity, but the data are insufficient to determine the doses at which such nonlinearities occur. Under the U.S. EPA's 1996 proposed "Guidelines for Carcinogen Risk Assessment," inhaled soluble nickel compounds would be classified as "cannot be determined," because the existing evidence is composed of conflicting data. A reference concentration of 2 x 10(-4) mg Ni/cu x m was calculated, based on lung fibrosis in male rats observed in the NTP study.

Hazard identification and dose response of ingested nickel-soluble salts.

People can ingest soluble nickel compounds as a normal constituent of food or as a contaminant in drinking water. This paper presents an assessment of the noncancer and cancer human health risks from ingestion of soluble nickel compounds. A reference dose (RfD) of 8 x 10(-3) mg Ni/kg/day in addition to the amount in food was calculated, based on albuminuria in female rats exposed to nickel sulfate in drinking water for 6 months (A. Vyskocil et al., 1994, Hum. Exp. Toxicol. 13, 689-693). This RfD is comparable to the current RfD based on decreased body weight in a chronic feeding study in rats (A. M. Ambrose et al., 1976, J. Food Sci. Technol. 13, 181-187). The potential for nickel-induced reproductive toxicity was also taken into account in the derivation of the RfD. There are a number of negative animal bioassays with soluble nickel salts, but all of them have deficiencies that preclude a definitive conclusion. According to EPA's 1996 draft cancer guidelines, the carcinogenic potential of oral exposure to soluble nickel "cannot be determined because there are inadequate data to perform an assessment."

Non-cancer risk assessment for nickel compounds: issues associated with dose-response modeling of inhalation and oral exposures.

This report presents the results of noncancer dose-response modeling for inhalation and oral exposures to nickel compounds using the NOAEL/LOAEL and benchmark dose (BMD) approaches. Several key issues associated with the implementation of the BMD approach were examined. Primary among them are difficulties associated with use of data for which the dose-response shape is poorly defined: nonuniqueness of maximum likelihood estimates and lower bounds equal to zero. In addition, several generalizable properties of the "hybrid approach" for modeling continuous endpoints were identified. A hybrid modeling approach allows one to consider "biological significance" on an individual (rather than group) basis; differences between individual- and group-based biological significance in the definition of benchmark response (BMR) levels are elucidated. In particular, it is shown that BMDs defined using group-based BMRs may be more like LOAELs than NOAELs. Application of cross-chemical and cross-endpoint comparisons suggest that, for chronic inhalation exposure, nickel sulfate appears to be as toxic or more toxic than nickel subsulfide and nickel oxide, although the high response rates for the latter two compounds at the lowest chronically administered concentration make such conclusions problematic. A nickel reference concentration could be derived based on the most sensitive benchmark concentration for chronic inhalation exposure to nickel sulfate, 1.7 x 10(-3) mg Ni/m3 for lung fibrosis in male rats. Analyses of oral studies of nickel sulfate and nickel chloride suggest that an appropriate basis for the nickel oral reference dose would be a BMD of 4-5 mg Ni/kg/day, based on increased prenatal mortality. (Uncertainty factors were not determined and neither an RfD nor an RfC was derived in this paper.) The BMD approach provides appropriate quantitative support for toxicological judgment; this paper addresses specific issues associated with the role of the BMD approach in noncancer risk assessment. Resolution of these and other issues may require the accumulation of a number of case studies such as the one presented here.

Altering the conserved nucleotide binding motif in the Salmonella typhimurium MutS mismatch repair protein affects both its ATPase and mismatch binding activities.

The Salmonella typhimurium and Escherichia coli MutS protein is one of several methyl-directed mismatch repair proteins that act together to correct replication errors. MutS is homologous to the Streptococcus pneumoniae HexA mismatch repair protein and to the Duc1 and Rep1 proteins of human and mouse. Homology between the deduced amino acid sequence of both MutS and HexA, and the type A nucleotide binding site consensus sequence, suggested that ATP binding and hydrolysis play a role in their mismatch repair functions. We found that MutS does indeed weakly hydrolyze ATP to ADP and Pi, with a Km of 6 microM and kcat of 0.26. To show that this activity is intrinsic to MutS, we made a site-directed mutation, which resulted in the invariant lysine of the nucleotide binding consensus sequence being changed to an alanine. The mutant MutS allele was unable to complement a mutS::Tn10 mutation in vivo, and was dominant over wild type when present in high copy number. The purified mutant protein had reduced ATPase activity, with the Km affected more severely than the kcat. Like the wild type MutS protein, the mutant protein is able to bind heteroduplex DNA specifically, but the mutant protein does so with a reduced affinity.

Nucleotide sequence of the Salmonella typhimurium mutS gene required for mismatch repair: homology of MutS and HexA of Streptococcus pneumoniae.

The mutS gene product of Escherichia coli and Salmonella typhimurium is one of at least four proteins required for methyl-directed mismatch repair in these organisms. A functionally similar repair system in Streptococcus pneumoniae requires the hex genes. We have sequenced the S. typhimurium mutS gene, showing that it encodes a 96-kilodalton protein. Amino-terminal amino acid sequencing of purified S. typhimurium MutS protein confirmed the initial portion of the deduced amino acid sequence. The S. typhimurium MutS protein is homologous to the S. pneumoniae HexA protein, suggesting that they arose from a common ancestor before the gram-negative and gram-positive bacteria diverged. Overall, approximately 36% of the amino acids of the two proteins are identical when the sequences are optimally aligned, including regions of stronger homology which are of particular interest. One such region is close to the amino terminus. Another, located closer to the carboxy terminus, includes homology to a consensus sequence thought to be diagnostic of nucleotide-binding sites. A third one, adjacent to the second, is homologous to the consensus sequence for the helix-turn-helix motif found in many DNA-binding proteins. We found that the S. typhimurium MutS protein can substitute for the E. coli MutS protein in vitro as it can in vivo, but we have not yet been able to demonstrate a similar in vitro complementation by the S. pneumoniae HexA protein.