Grouping of PFAS for human health risk assessment: Findings from an independent panel of experts.

An expert panel was convened to provide insight and guidance on per- and polyfluoroalkyl substances (PFAS) grouping for the purposes of protecting human health from drinking water exposures, and how risks to PFAS mixtures should be assessed. These questions were addressed through multiple rounds of blind, independent responses to charge questions, and review and comments on co-panelists responses. The experts agreed that the lack of consistent interpretations of human health risk for well-studied PFAS and the lack of information for the vast majority of PFAS present significant challenges for any mixtures risk assessment approach. Most experts agreed that "all PFAS" should not be grouped together, persistence alone is not sufficient for grouping PFAS for the purposes of assessing human health risk, and that the definition of appropriate subgroups can only be defined on a case-by-case manner. Most panelists agreed that it is inappropriate to assume equal toxicity/potency across the diverse class of PFAS. A tiered approach combining multiple lines of evidence was presented as a possible viable means for addressing PFAS that lack analytical and/or toxicological studies. Most PFAS risk assessments will need to employ assumptions that are more likely to overestimate risk than to underestimate risk, given the choice of assumptions regarding dose-response model, uncertainty factors, and exposure information.

Biological monitoring screening of patients provided antineoplastic drugs including adriamycin, cyclophosphamide, 5-fluorouracil, methotrexate, and vincristine.

The aim of the present study was to establish screening biomarkers of exposure to antineoplastic drugs administered to 11 patients undergoing cancer chemotherapy. Among the anticancer drugs administered were cyclophosphamide (all), Adriamycin (5 of 11), methotrexate (3 of 11), 5-fluorouracil (4 of 11), vincristine (3 of 11), megestrol acetate (1 of 11), and procarbazine (1 of 11). The noninvasive urinary parameters investigated were thioethers, D-glucaric acid, elements, and forward and reverse mutagenesis using bacterial bioassays. The data were analyzed in terms of the observed concentrations and those corrected for personal baseline. Personal baseline correction for parameters with significant nonexposure baseline levels was essential. While glucaric acid and thioethers were increased by the drug treatments, the correlations with baseline-uncorrected data showing an inverse relationship proved spurious, because saturation of the detoxification systems occurred at the high doses administered. Glucaric acid was also influenced by methotrexate and vincristine. Thioether content was affected by cyclophosphamide only. The forward mutagenesis assay was directly correlated to cyclophosphamide dose but the reverse assay was not, in the presence or absence of rat S9 fraction. The forward assay was not sensitive to the effects of smoking. Relative to controls, the elements changed by cyclophosphamide were K, S, and P. Those affected by Adriamycin were Ca, Mg, and Na; 5-fluorouracil affected Ca, Mg, Na, and C; methotrexate changed P and S. The forward mutagenesis assay and D-glucaric acid concentrations were the screening biomarkers best suited to monitoring for extent of exposure to these antineoplastic drugs.

Use of genetic toxicology data in U.S. EPA risk assessment: the mercury study report as an example.

Assessment of human health risks of environmental agents has often been limited to consideration of the potential for the agent to cause cancer or general systemic toxicity after long-term exposure. The U.S. Environmental Protection Agency (U.S. EPA) is increasingly moving toward the development of integrated assessments, which consider all potential health end points including developmental toxicity, neurotoxicity, immunotoxicity, reproductive effects, and germ cell mutagenicity. The U.S. EPA has a responsibility to assess risks to nonhuman species or ecosystems when appropriate data are available. An example of a recent integrated human health and ecological risk assessment can be found in the U.S. EPA Mercury Study Report to Congress. This report covers the following topics in separate volumes: an inventory of anthropogenic mercury emissions in the United States; an exposure assessment using measured and predicted values and including indirect dietary exposure; an evaluation of human health risks; an assessment of ecologic risk wherein water criteria are presented for several wildlife species; an overall integrated characterization of human and nonhuman risk; and a discussion of risk management considerations. In the evaluation of human health risk, genetic toxicology data were considered for three forms of mercury: elemental, inorganic (divalent), and methylmercury. These data were used in judgments of two types of potential health effects (carcinogenicity and germ cell mutagenicity). In assessment of potential carcinogenicity of inorganic and methylmercury, genetic toxicity data were key. Data for clastogenicity in the absence of mutagenicity supported the characterization of inorganic and methylmercury as materials that produce carcinogenic effects only at high, toxic doses. The evidence for clastogenicity, coupled with information on metabolism and distribution, resulted in a judgment of a moderate degree of concern (or weight of evidence) that inorganic mercury can act as a human germ cell mutagen. For methylmercury, the degree of concern for germ cell mutagenicity is high.

Mutagenesis assays on urines produced by patients administered adriamycin and cyclophosphamide.

The XAD-2 resin concentration/elution system for concentration of mutagens contained in urines was optimized for cancer patients who had been administered such antineoplastic agents as adriamycin (ADR; doxorubicin), cyclophosphamide (CP), methotrexate, vincristine, and 5-fluorouracil. In the reverse mutation assay, Salmonella typhimurium strains TA1535 and TA98 differentiated between CP (with S9 fraction) and ADR (without S9), respectively. No dose-response for CP was observed. There was a dose-response to ADR by TM677 in the presence of S9 using a forward mutation assay. However, while the reverse mutation assays successfully detected ADR and CP administration in the presence of each other in terms of urine mutagenicity, the forward mutation assay did not, since unidentified CP metabolites were also detected in the latter. None of these systems detected mutagenic urines from tobacco smokers, although reaction of these urines with beta-glucuronidase allowed this type of source to be detected also.

Evaluation of coal liquefaction technologies by Salmonella mutagenesis.

Coal liquefaction materials made by two processes were found to be mutagenic in the Salmonella/microsome assay. Data from this type of in vitro assay can be used in the toxicological assessment of these processes. Such evaluations of the health and environmental impacts of technologies would aid in the development of alternate energy sources.

Mutagenicity testing of chlorinated biphenyls and chlorinated dibenzofurans.

4 chlorinated biphenyl and 5 chlorinated dibenzofuran compounds have been evaluated in the reversion assay developed by B.N Ames using Salmonella typhimurium histidine auxotrophs. All these compounds (2,4,2'-4'-tetrachlorobiphenyl, 3,4,3',4'-tetrachlorobiphenyl, 4-chlorobiphenyl, 2,4,,6,2',4',6'-hexachlorobiphenyl, dibenzofuran, 2,9-dichlorodibenzofuran, 3,6-dichlorodibenzofuran, 2,3,7,8-tetrachlorodibenzofuran and octachlorodibenzofuran) were nonmutagenic for strains TA98 and TA100 when tested over a 3-log dose range. They were also not mutagenic whether or not varying concentrations of microsomal extracts (S9) from uninduced rats or from rats induced by several methods were included in the experimental protocol.

Mutagenicity of 7H-dibenzo[c,g]carbazole and metabolites in Salmonella typhimurium.

7H-Dibenzo[c,g]carbazole (DBC) is a potent carcinogen of environmental import. Reverse-mutation plate-incorporation assays for mutagenicity were undertaken in Salmonella typhimurium strains TA98 and TA100. Results were negative when no exogenous activation system was used, as well as when assays incorporated liver homogenates (S9) from rats, mice and rabbits. By contrast DBC was mutagenic in a forward mutation assay in Salmonella strain TM677 using resistance to 8-azaguanine for selection. Metabolites of DBC were generated by incubation with rat-liver microsomes and separated by HPLC. Two of these metabolites were directly mutagenic for Salmonella strain TM 677 while two others were mutagenic upon addition of S9. Synthetic phenolic derivatives of DBC were also mutagenic in this assay when further metabolized. It is likely that metabolites of DBC phenols constitute the biologically active forms.

Metabolism of mutagenic polycyclic aromatic hydrocarbons by photosynthetic algal species.

Polycyclic aromatic hydrocarbons (PAH) known to produce carcinogenic and mutagenic effects have been shown to contaminate waters, sediments and soils. While it is accepted that metabolites of these compounds are responsible for most of their biological effects in mammals, their metabolism, and to a large extent their bioactivity, in aquatic plants have not been explored. Cultures of photosynthetic algal species were assayed for their ability to metabolize benzo[a]pyrene (BaP), a carcinogenic PAH under conditions which either permitted (white light) or disallowed (gold light) photooxidation of the compound. Growth of Selenastrum capricornutum, a fresh-water green alga, was completely inhibited when incubated in white light with 160 micrograms BaP/l medium. By contrast concentrations at the upper limit of BaP solubility in aqueous medium had no effect on algal growth when gold light was used. BaP quinones and phenol derivatives were found to inhibit growth of Selenastrum under white light incubation. BaP phototoxicity and metabolism were observed to be species-specific. All 3 tested species of the order Chlorococcales were growth-inhibited by BaP in white light whereas neither the green alga Chlamydomonas reinhardtii nor a blue-green, a yellow-green or an euglenoid alga responded in this fashion. Assays of radiolabeled BaP metabolism in Selenastrum showed that the majority of radioactivity associated with BaP was found in media as opposed to algal cell pellets, that the extent of metabolism was BaP concentration dependent, and that the proportion of various metabolites detected was a function of the light source. After gold light incubation, BaP diols predominated while after white light treatment at equal BaP concentrations, the 3,6-quinone was found in the highest concentration. Extracted material from algal cell pellets and from media was tested for mutagenicity in a forward mutation suspension assay in Salmonella typhimurium using resistance to 8-azaguanine for selection. Direct-acting mutagens were detected in extracted media from incubation of Selenastrum with 400 micrograms BaP/l for 1 day in gold light. Extracts of media from algae incubated in gold light from 1 to 4 days with 1200 micrograms BaP/l were found to have direct-acting mutagens as well as those requiring further metabolism. Media extracts from white light incubations of BaP were mutagenic upon addition of rat liver homogenates. Activity of these materials from white light treatment are largely attributable to unmetabolized BaP.

Non-mutagenicity for Salmonella of the chlorinated hydrocarbons aroclor 1254, 1,2,4-trichlorobenzene, mirex and kepone.

A polychlorinated biphenyl mixture, Aroclor 1254, two commercial grade insecticides, mirex and kepone, and a pesticide breakdown product, 1,2,4-trichlorobenzene were evaluated for mutagenicity and hepatic enzyme induction potential in the Salmonella/microsomal assay. None was found to revert strains TA1535, TA1537, TA98 or TA100 when tested with or without metabolic activation. Liver microsomal extracts (S9) from rats induced with 1,2,4-trichlorobenzene were shown to differ from S9 of either control or Aroclor 1254-induced rats in the capacity to activate 2-aminoanthracene mutagenesis.

Nurses' and pharmacists' exposure to antineoplastic drugs: findings from industrial hygiene scans and urine mutagenicity tests.

Data from 83 nurses and pharmacists handling antineoplastic drugs and 35 nurse/pharmacist controls who participated in a national study of antineoplastic drug-handling risks were examined to investigate antineoplastic drug exposure. Measures of external exposure included self-completion drug logs and industrial hygiene scans conducted in clinical settings. Internal exposure was measured by urine mutagenicity tests on end-of-week 24-hour urine specimens. To control for potential confounders, the staff was asked to complete food and hobby diaries and to avoid identified mutagenic substances for 1 week before collection of 24-hour urine samples. On the scans of the drug handlers, 13% showed one or more spots of drug contamination on gloved and ungloved hands, gowns, or shoes. Of the 24-hour urine samples, 15% were mutagenic for Salmonella typhimurium: Rates did not differ significantly for drug handlers and controls. Among nurses who both prepared and administered antineoplastics, those with positive mutagenicity tests handled more doses of the drugs, used less skin protection, and had more skin contact with the drugs than those with negative tests. Nurses who only administered the drugs and had positive mutagenicity tests handled fewer doses of drugs than those with negative tests, but they also reported less use of protection and more skin contact. For both groups of nurses, skin contact with antineoplastics was associated with positive mutagenicity test results (p < 0.01).

Mutagenicity of benzo(a)pyrene metabolites generated on the isolated perfused lung following particulate exposure.

The isolated perfused rabbit lung (IPL) is being used to study the effects of particulate exposure on the pulmonary metabolism of benzo(a)pyrene (BaP). Pasturealla-free New Zealand white rabbits were treated intraperitoneally with BaP prior to kill. The isolated lungs were then administered either 14C-labeled BaP alone or BaP plus Fe2O3 or fly ash by intratracheal injection. Rates of appearance of BaP metabolites in the perfusing blood were determined. The extent of metabolism, distribution of metabolites, and types of metabolites produced were quantified for various lung tissue types by high-performance liquid chromatography and liquid scintillation spectrometry. Procedures were developed to apply the Salmonella/microsome test in the assay of mutagenicity of lung tissue and blood extracts as an indicator of their biologic activity. With few exceptions, blood extracts from IPL receiving BaP only were not mutagenic. Lung, trachea-bronchi, and macrophage extracts, by contrast, were mutagenic. A part of this activity could be attributed to BaP metabolites rather than to parent compound remaining in extracts. When lungs were exposed to Fe2O3 or to fly ash, only macrophage extracts were consistently mutagenic. This activity was due to significant amounts of unmetabolized BaP.

Evaluating comparative potencies: developing approaches to risk assessment of chemical mixtures.

The U.S. EPA must provide guidance as to health risk assessment of mixtures from a variety of sources such as wastewaters, hazardous waste sites and air particulates. One approach to risk assessment of mixtures is to add up risk assessments for individual components identified as part of the mixture, after considering the potential for interaction among those components. This provides an index of hazard potential but not a quantitative estimate. When data on mixture components are incomplete, but these components are isomers or congeners of a well-studied chemical, another technique--use of toxic equivalency factors--can be applied. This approach has been proposed for estimating risk associated with chlorinated dioxins and dibenzofurans. A third approach, that of relative or comparative potency, is based on the assumption that for similar but not necessarily definable complex mixtures, a measure of relative potency based on data from in vitro tests can be correlated in a constant fashion with relative potency from an in vivo bioassay. The degree of confidence in the appropriateness of a relative potency method rests upon the way potency is measured and the validity of underlying assumptions (the degree to which these assumptions can be tested). One class of assumptions involves choice of the model or procedure for deriving the quantitative risk estimates. A second set of assumptions deals with mechanism of action, and whether such considerations add bias or, in fact, refine the relative potency judgment. The choice of short-term tests appropriate for use in establishing the potency relationships is also a factor to be considered. This paper presents examples of proposed uses of relative potency in risk assessment and outlines some areas for further study.

Derivation of U.S. EPA's oral Reference Dose (RfD) for methylmercury.

Mercury (Hg) cycles in the environment through a series of complex chemical and physical transformations that occur in air, soils, and water bodies. One component of the environmental mercury cycle is the formation of methylmercury (MHg) primarily by aquatic and marine microorganisms and the accumulation of MHg in foodwebs, particularly in piscivorous species. Human consumption of piscivorous fish and other piscivorus animals is the most common pathway of exposure to MHg. For non-carcinogenic toxic endpoints, the U.S. EPA typically develops a Reference Dose (RfD). This is generally interpreted to be a concentration of a chemical which can be consumed on a daily basis over a lifetime without expectation of adverse effect. There is substantial evidence in both animal and humans that MHg is a neurotoxicant in the adult and the child as well as a developmental neurotoxicant for the fetus. Epidemics of MHg poisoning in Japan and Iraq have resulted from high-dose exposures to MHg. In these epidemics adults, children, nursing infants and fetuses were affected by MHg. The epidemics demonstrate that neurotoxicity is the health effect of greatest concern and that effects on the developing human nervous system apparently occur at lower exposures than those affecting the adult nervous system. We describe how the data from the Iraqi MHg epidemic were used to derive the current RfD of 0.1 microgram/Kgbw/day (U.S. EPA, 1995;).

Mutagenicity of algal metabolites of benzo(a)pyrene for Salmonella typhimurium.

The metabolism and growth effects of benzo(a)pyrene (BaP) were studied using a freshwater green alga, Selenastrum capricornutum. Algal cultures were incubated under gold light with BaP added at concentrations of 40, 160, 400, and 1,200 micrograms/liter for the periods of 1-4 days. The metabolites and BaP were identified and quantified from ethyl acetate extracts of both algal cells and incubation medium. The ethyl acetate extracts were evaluated for genotoxicity using a micro-volume Salmonella typhimurium forward mutation assay with resistance to 8-azaguanine for selection. This assay detected the presence of small quantities of BaP and was particularly sensitive to the mutagenicity of BaP diols. Of those extracts prepared from algae and medium from cultures exposed to 400 micrograms BaP/liter (10 micrograms/25 ml culture), only algal cell extracts from one day's growth were mutagenic. In cultures exposed to 1,200 micrograms BaP/liter (30 micrograms/25 ml culture), mutagenic materials were produced or persisted in both algae and media throughout the 4-day incubation. The observed mutagenic response can be attributed in part to the presence of unmetabolized BaP or to BaP diols.

Biological considerations for combining carcinogenicity data for quantitative risk assessment.

Quantitative risk assessments for carcinogens conducted by the U.S. EPA have most frequently been based on results of a bioassay from a single sex/strain/species of animal. In some cases, more than one data set derived from different sexes, strains, or species of animals is suitable for quantitative risk assessment. When there is no apparent difference among the data sets in sensitivity to the carcinogen, use of more of the available data should result in a higher level of confidence in the risk estimate. Several biological factors must be considered before combining data from different animal sexes, strains, species, or tumor sites. The relevance of the animal models, study design and execution, dose selection, and route of administration are factors which influence whether data from separate studies should be combined. The decision to combine data sets is also based on what is known of the mechanism of action of the agent, its pharmacokinetics, any species/sex specificity of the effect, and considerations regarding tumor site specificity. Statistical analysis also indicates whether the data sets may be described by the same multistage model. The evaluation of these factors in the decision to combine or not combine data sets is discussed.

A statistical test of compatibility of data sets to a common dose-response model.

Quantitative estimates of cancer risk generally involve low-dose extrapolation based on an exponential dose-response model for dichotomous response data. Frequently more than one data set is available. If a careful analysis of the biological issues indicates that more than one of the available data sets could be used in the quantitative estimate of cancer risk, it is reasonable to think of combining the data. Before combining data, however, it would be prudent to test whether the data sets are compatible with a common dose-response model. If they are not, it could be concluded that an underlying biological factor is responsible. If they are statistically compatible, the decision to combine data sets based on biological issues would be reinforced. A statistical test based on the generalized likelihood ratio method is proposed for evaluating the compatibility of different data sets with a common dose-response model. This method of constructing a statistical test and the associated asymptotic theory is consistent with the approach used by GLOBAL86 (R. B. Howe, K. S. Crump, and C. Van Landingham, GLOBAL86: A Computer Program to Extrapolate Quantal Animal Toxicity Data to Low Doses, K. S. Crump & Co., Ruston, LA, 1986) for estimating the confidence limits that are used as a basis for quantitative estimates.

Mutagenicity of products from coal gasification and liquefaction in the Salmonella/microsome assay.

As a first step in the assessment of their possible bio-effects, coal-related materials were tested for mutagenicity in the Salmonella/microsome assay. Of three coal gasification by-products tested, only a tar was mutagenic for any of four Salmonella strains. The following liquefaction materials were mutagenic for strains TA1538, TA98, and/or TA100: A liquefaction vehicle oil and coal hydrogenation filtered liquid, separated bottoms, vacuum overhead, and vacuum bottoms. Neither powdered coal nor water produced as a by-product of the hydrogenation process was positive in the Salmonella test. No coal-related material was mutagenic for the missense mutant TA1535 or for any strain in the absence of metabolic activation provided by rat hepatic homogenates (S9). In all but one instance Aroclor 1254-induced S9 provided the maximum activation for mutagenesis. Fractionation of all samples was undertaken by serial extraction with organic solvents of increasing polarity (hexane, toluene, methylene chloride, acetonitrile). Highly mutagenic materials were found in fractions of the hydrogenation filtered liquid, vacuum overhead, and vacuum bottoms. Thus far non-mutagenic samples have not yielded mutagenic components upon fractionation.

Urinary biological monitoring markers of anticancer drug exposure in oncology nurses.

People handling anticancer drugs or their wastes may absorb these potent genotoxic agents. The aim of this study was to determine the utility of some general urinary markers among 24 female oncology nurses handling these drugs in comparison with 25 "unexposed" nurses. The markers were the Salmonella typhimurium reverse and forward mutation assays, total thioethers, and D-glucaric acid. The reverse mutation assay was the most specific and sensitive marker for anti-cancer drug exposure. Use of the marker battery was no great advantage as a screening tool relative to use of the reverse mutation assay alone. Better recording of work practices in nurse work logs would have improved interpretation of results.

Mutagenic studies of folic acid antagonists.

Compounds that compete with folic acid (folic acid antagonists [FAAs]) become limited in their usefulness in the treatment of leukemia, malaria, and bacterial infections by the rapid development of resistance. Assays of the plasma levels of certain of these FAAs led to the observation, in about 25% of the determinations, that a higher density of growth of Streptococcus faecium var. durans (ATCC 8043) was obtained at an FAA concentration just below the completely inhibitory level than at one-half this concentration. This and other considerations suggested that FAAs may act not only as selective agents for resistant organisms but also as mutagens. Seven FAAs including amethopterin, pyrimethamine, trimethoprim, chlorguanide triazine, an experimental quinazoline, WR-158,122, and two experimental triazines, WR-99,210 and WR-38,839, were tested for mutagenicity in the Salmonella reversion assay developed by Ames et al. (1975). All were found to be negative for strains TA1535, TA1537, TA1538, TA98, and TA100, both with and without microsomal activation. These compounds were then tested as mutagens for three traits in the folic acid-requiring S. faecium. FAAs were shown to cause mutations to folic acid independence, rifampin resistance, and FAA resistance. It is postulated that the FAAs induce mutations by causing thymine deprivation in the folic acid-requiring host.

Residue organic mixtures from drinking water show in vitro mutagenic and transforming activity.

Indications of possible health effects of residue organics in drinking water have been sought using short-term tests of mutagenic and transforming activity. Ten percent or less of the total organic material in drinking water has been identified; the remainder is believed to include thousands of unknown nonvolatile compounds. Residual organics were concentrated from drinking water from representative U.S. cities by reverse osmosis followed by liquid-liquid extraction [yielding the reverse osmosis concentrate-organic extract (ROC-OE) fraction] and sorption-desorption on XAD-2 resin. Samples of these residue organics were provided by the Environmental Protection Agency for bioassay. They were examined for mutagenic activity by using Salmonella tester strains (primarily TA98 and TA100) and for transforming activity by using mouse fibroblasts (BALB/3T3 clone 1-13). City-specific patterns of dose-dependent bacterial mutagenesis and of bacterial toxicity were observed for these samples and for subfractions generated by sequential extractions with hexane, ethyl ether, and acetone. Mutagenic effects were essentially independent of a microsome activation system prepared from liver of Aroclor 1254-induced rats. On the basis of strain-specific effects in mutagenesis and differential distributions of mutagenic activity during liquid-liquid extraction, at least some of the active compounds are thought to be acidic, frameshift mutagens. The ROC-OE fraction of a New Orleans sample transformed BALB/3T3 cells in replicate experiments. By comparison with the bacterial mutagenesis data, cell transformation is a relatively sensitive method for detecting possible mutagenic and carcinogenic activity in this sample. The appropriateness of these systems for the assay of complex mixtures and the degree to which reverse osmosis concentrates contain the unaltered organic compounds in the original samples are discussed.