Assessing interaction thresholds for trichloroethylene in combination with tetrachloroethylene and 1,1,1-trichloroethane using gas uptake studies and PBPK modeling

The volatile organic solvents trichloroethylene (TCE), tetrachloroethylene (perchloroethylene, PERC), and 1,1,1-trichloroethane (methylchloroform, MC) are widely distributed environmental pollutants and common contaminants of many chemical waste sites. To investigate the mode of pharmacokinetic interactions among TCE, PERC, and MC and to calculate defined "interaction thresholds", gas-uptake experiments were performed using a closed-chamber exposure system. In each experiment, two rats (Fischer 344, male, 8-9 weeks old) were exposed to different initial concentrations of TCE, PERC, and MC, applied singly or as a mixture, and their concentration in the gas phase of the chamber was monitored over a period of 6 h. A physiologically based pharmacokinetic (PBPK) model was developed to test multiple mechanisms of inhibitory interactions, i.e., competitive, non-competitive, or uncompetitive. All mixture exposure data were accurately described by a system of equations in which a PBPK model was provided for each chemical and each was regarded as an inhibitor of the others' metabolism. Sensitivity-analysis techniques were used to investigate the impact of key parameters on model output and optimize experimental design. Model simulations indicated that, among these three chemicals, the inhibition was competitive. The PBPK model was extended to assess occupationally relevant exposures at or below the current threshold-limit values (TLVs). Based on 10% elevation in TCE blood levels as a criterion for significant interaction and assuming TCE exposure is set at TLV of 50 ppm, the calculated interaction thresholds for PERC and MC were 25 and 135 ppm, respectively. TLV exposures to binary TCE/PERC mixture were below the 10% significance level. The interaction threshold for TCE and MC co-exposure would be reached at 50 and 175 ppm, respectively. Such interactive PBPK models should be of value in risk assessment of occupational and environmental exposure to solvent mixtures.     

Toxicological studies of chemical mixtures of environmental concern at the National Toxicology Program: health effects of groundwater contaminants

In cooperation with the Agency for Toxic Substances and Disease Registry, the National Toxicology Program is participating in a Public Health Service activity related to the Comprehensive Environmental Response, Compensation and Liability Act (Superfund Act) by conducting toxicology studies on chemicals found in high-priority hazardous waste sites and for which adequate toxicological data are not available. As part of this effort, a project on the toxicology of chemical mixtures of groundwater contaminants was initiated. The first study, centered on the health effects of groundwater contaminants, is at the contractual stage. Nineteen organic and six inorganic chemicals, selected from more than 1000 known groundwater contaminants, will be given in drinking water to Fischer 344 rats and B6C3F1 mice for 3 or 6 months. Controls and five dose levels, based on average concentrations (i.e., baseline level) of individual component chemicals, or 0.1-, 10-, or 1000-fold of the baseline level, will be used. Toxicological end points include mortality, clinical signs, water and food consumption, body and organ weights, clinical pathology analytes (e.g., hematology, clinical chemistry, and urinalysis), gross and histopathology, neurobehavioral tests, sperm morphology and vaginal cytology evaluations (SMVCE), and cytogenetics. This paper summarizes the rationale behind our experimental design and the factors one must consider when designing studies of complex chemical mixtures.     

Public Health Guidance Values for Chemical Mixtures: Current Practice and Future Directions

Agency for Toxic Substances and Disease Registry (ATSDR) utilizes chemical-specific minimal risk levels (MRLs) to assist in evaluating public health risks associated with exposure to hazardous substances. The MRLs are derived based on the data compiled from current worldwide literature searches and presented in ATSDR's toxicological profiles. These documents profile not only individual chemicals, but also groups of chemically related compounds and chemical mixtures. ATSDR took several approaches when developing MRLs for chemical mixtures. In some instances, toxicity equivalency factors were used to estimate the toxicity of the whole mixture; in other instances, the most toxic chemical was assumed to drive the health assessment for the whole mixture. Another approach was to treat the mixture as one entity and develop a health guidance value for the whole mixture. In yet another approach, each chemical of the mixture was evaluated separately and several health guidance values were developed. In the future, ATSDR will evaluate priority chemical mixtures found at hazardous waste sites. A weight-of-evidence approach, physiologically based pharmacokinetic modeling and benchmark dose modeling, and quantitative structure–activity relationships will have an impact on the development of MRLs and the assessment of chemical mixtures.     

Public health implications of hazardous waste sites: Findings, assessment and research

In this paper we present an overview of the chemicals released from hazardous waste sites (HWS) and some of the mixtures of substances that have been released into environmental media. We describe how this information can be used to assess the public health hazard of releases of chemical mixtures from waste sites. Wherever possible, research on chemical mixtures should use chemical mixtures actually identified in, or representative of, mixtures in environmental media. A narrative, weight-of-evidence approach to characterize the toxicity of mixtures that incorporates mechanistic insights on chemical interactions is described. The utility of this information in the context of risk analysis and public health practice is discussed.     

Chemical exposures at hazardous waste sites: Experiences from the United States and Poland

The U.S. Agency for Toxic Substances and Disease Registry (ATSDR) and the Polish Nofer Institute of Occupational Health collaborate on issues related to hazardous chemical exposure at or near hazardous waste sites. This paper outlines the scope of hazardous chemical exposure in the United States and in Poland and identifies priority chemicals and chemical mixtures. Special attention is paid to exposures to metals and to evaluation of the health risks associated with those exposures. Studies in the United States indicate that exposure to hazardous waste site chemicals may be associated with an increased risk of adverse developmental – specifically cardiovascular and neurodevelopmental – effects.     

Seeking solutions to chemical mixtures challenges in public health

The Agency for Toxic Substances and Disease Registry (ATSDR) identifies people near hazardous waste sites who are at potential health risk because of their exposure to environmental chemicals. Nearly, 2000 chemicals have been associated with such sites. Residents of U.S. communities are potentially exposed to hazardous substances through air, soil, drinking water, and food. The agency has determined that more than 73 million people live within a 4-mile radius of waste sites. More than 14 million Americans live within 1 mile of a National Priorities List site, of which 11% are 7 years of age or younger, 12% are 64 years of age or older, 24% are women of childbearing age, and 25% are minorities. The lack of adequate environmental sampling and information on human exposures often restricts ATSDR's evaluation and assessment activities. Assessing human exposure with its attendant health risks and outcomes is complex because many populations have a wide range of reported illnesses, and generally exposures are to mixtures of chemicals. This prompted ATSDR to consider mixtures issues more in depth and to establish a formal mixtures assessment and research program in 1994. In this paper, we present an overview of the agency activities, the genesis, legislative mandates, and pertinence of the mixtures program including applied research and the development of methods for evaluating the impact of multiple-chemical exposure. On the basis of 20-year experience of evaluating and researching environmental chemical mixtures at waste sites, ATSDR convened the International Conference on Chemical Mixtures (ICCM) in 2002. The conference was supported by several federal agencies and scientific organizations and attended by international and national experts. The conference addressed broad topics such as prevalence of exposures to chemical mixtures, importance of interactions at environmentally relevant levels, validity of assuming additivity (dose or response) as default for mixtures assessment, and promising avenues in the three broad areas, viz., research, assessment, and computational tools.     

Oxidative stress and DNA damage in Fischer rats following acute exposure to trichloroethylene or perchloroethylene

Oxidative DNA damage is emerging as an biomarker of effect in studies assessing the health risks of occupational chemicals. Trichloroethylene (TCE) and perchloroethylene (PERC) are used in the dry cleaning industry and their metabolism can produce reactive oxygen compounds. The present study examined the potential for TCE and PERC to induce oxidative DNA damage in rats that was detectable as increased urinary excretion of 8-hydroxydeoxyguanosine (8OHdG). Thiobarbaturic acid reactive substances (TBARS) and 8-epiprostaglandin F2alpha (8epiPGF) were also measured as biomarkers of increased oxidative stress. Male Fischer rats were administered a single i.p. injection of 0, 100, 500, or 1000 mg/kg of PERC or TCE. Control rats received only vehicle (1:4 v/v of Alkamuls/water). A positive control group received 100 mg/kg 2-nitropropane (2NP). Rats were sacrificed 24 h after dosing. In rats receiving 2NP or TCE but not PERC, TBARS and the 8OHdG/dG ratios were significantly elevated in liver. Lymphocyte 8OHdG/dG was not affected significantly by 2NP, TCE or PERC. In rats receiving 2NP, urinary excretion of 8OHdG and 8epiPGF2 were significantly increased. In rats receiving TCE or PERC, significant increases in 8epiPGF2 or 8OHdG were not evident. Results indicate that a single high dose of TCE, but not PERC, can induce an increase in oxidative DNA damage in rat liver. However, the usefulness of 8OHdG as a biomarker of TCE-induced oxidative DNA damage is questionable.     

Different behavioral effect dose–response profiles in mice exposed to two-carbon chlorinated hydrocarbons: Influence of structural and physical properties

The present study aimed to clarify whether dose–response profiles of acute behavioral effects of 1,2-dichloroethane (DCE), 1,1,1-trichloroethane (TCE), trichloroethylene (TRIC), and tetrachloroethylene (PERC) differ. A test battery involving 6 behavioral endpoints was applied to evaluate the effects of DCE, TCE, TRIC, and PERC in male ICR strain mice under the same experimental conditions. The behavioral effect dose–response profiles of these compounds differed. Regression analysis was used to evaluate the relationship between the dose–response profiles and structural and physical properties of the compounds. Dose–response profile differences correlated significantly with differences in specific structural and physical properties. These results suggest that differences in specific structural and physical properties of DCE, TCE, TRIC, and PERC are responsible for differences in behavioral effects that lead to a variety of dose–response profiles.     

Lack of promotional effects of groundwater contaminant mixtures on the induction of preneoplastic foci in rat liver.

F344 rats were exposed to drinking water mixtures of seven of the most common groundwater contaminants associated with hazardous waste sites [arsenic, benzene, chloroform, chromium, lead, phenol, and trichloroethylene (TCE)] as the full mixture or submixtures of the organic and/or inorganic chemicals. The lowest concentrations (1x) of the individual chemicals were environmentally realistic and below what would be expected to induce significant short-term toxicity. This study was intended to determine if previously reported increases in localized hepatocellular proliferation in response to these chemicals might be correlated with increased risk for hepatocarcinogenesis. Rats were exposed via a drinking water solution to the full seven- chemical mixture (at 1x and 10x concentrations), submixtures of the organic or inorganic chemicals (at 10x concentrations), a mixture of TCE, lead, and chloroform (TLC submixture at 10x and 100x concentrations), or deionized water as a control. The rats were evaluated for promotion of placental glutathione-S-transferase (GST-P) positive preneoplastic liver cell foci after diethylnitrosamine (DEN) initiation and partial hepatectomy. Focus formation, cell proliferation, and apoptosis were evaluated after exposure to DEN or saline controls, the chemical mixtures or deionized water controls, or combinations of these treatments. The total number and area of GST-P positive foci in DEN-treated rats exposed to the full seven-chemical mixture was increased as compared with the DEN-water controls, but this was statistically significant only for total focus area in the 1x dose group. In DEN-treated rats, the inorganic or TLC submixtures resulted in a significant reduction in number and area of GST-P positive foci. Focus area also was decreased in the organic submixture-treated group, but not significantly. Hepatocellular proliferation was not significantly changed in the chemical mixture saline groups as compared with the mixture water controls. After DEN treatment, however, cell proliferation was significantly decreased after the 10x seven-chemical and organic mixture treatments and the 100x TLC mixture treatment. Different groups showed either increased or decreased apoptotic rates which did not correlate well with proliferation rates or focus formation. Mixtures of these seven chemicals, therefore, did not appear to act as promoters of hepatic foci at environmentally relevant concentrations, and some mixture combinations appeared to decrease promotional activity.     

Using the ATSDR Guidance Manual for the Assessment of Joint Toxic Action of Chemical Mixtures

The Guidance Manual for the Assessment of Joint Toxic Action of Chemical Mixtures (Mixtures Guidance Manual) is intended to assist environmental health scientists and toxicologists in determining whether exposure to chemical mixtures at hazardous waste sites may affect public health. The Agency for Toxic Substances and Disease Registry (ATSDR) approach is a semi-quantitative screening process. Step-by-step procedures for assessing noncarcinogenic and carcinogenic effects are outlined in flow charts. Exposure data and toxicological information on the mixture of concern are the preferred basis for an assessment. If suitable whole mixture studies are not available, a components-based approach is undertaken. The hazard index (HI) method is used to screen for noncancer health hazards from potential additivity of the components. Cancer risks for the components are summed to screen for health hazards from potential additivity of carcinogenic effects. A weight-of-evidence (WOE) method is used to evaluate the potential impact of interactions on noncancer and cancer health effects.     

Health effects classification and its role in the derivation of minimal risk levels: renal effects

The Agency for Toxic Substances and Disease Registry (ATSDR) derives minimal risk levels (MRLs) for priority hazardous substances. MRLs are health guidance values intended to serve as screening levels for health assessors to select contaminants of concern and to assess potential health effects at hazardous waste sites and areas affected by unplanned releases. Current MRLs are published in ATSDR toxicological profiles and are listed at the ATSDR website at . To date, ATSDR has derived 125 inhalation MRLs, 207 oral MRLs, and eight external radiation MRLs; 19 MRLs are based on renal effects. This article reports on endpoints used to derive the MRLs. It also presents the ranking of effects into less serious and serious categories as described in ATSDR's Guidance for Developing Toxicological Profiles.     

Effects of trichloroethylene and perchloroethylene on muscle contractile responses and epithelial prostaglandin release and acetylcholinesterase activity in swine trachea.

Trichloroethylene (TCE) and perchloroethylene (PERC) have been reported to induce respiratory complications such as airway hyperactivity and asthma. The present study was designed to investigate their influence on smooth muscle contraction and epithelial release of prostanoids in swine trachea. Results showed that TCE and PERC exposure did not alter the basal tone of tracheal smooth muscle. However, TCE and PERC concentration-dependently increased both ACh-induced and high K+-induced muscle contraction. In addition to potentiation of muscle contractile responses evoked by acetylcholine or histamine, pretreatment of smooth muscle with PERC at higher concentration significantly suppressed the relaxant activity of beta-adrenergic agonists. The epithelial prostaglandin (PG)E2, but not PGD2, release from tracheal epithelium was significantly increased by TCE and PERC. In addition, the acetylcholinesterse (AChE) activity of tracheal epithelia was reduced by TCE and PERC. In conclusion, our results suggest that the enhancement of spasmogen-evoked muscle contractile responses and epithelial PGE2 secretion, as well as reduction of epithelial AChE activity, may participate in airway impairment and hyperresponsiveness after TCE and PERC exposure.     

Effect of a chemical mixture on dermal penetration of arsenic and nickel in male pig in vitro

The effect of a chemical mixture on the dermal penetration of arsenic or nickel was assessed by applying arsenic-73 or nickel-63 alone or with the chemical mixture to dermatomed male pig skin samples in flow-through diffusion cells. The chemical mixture consisted of chloroform, phenanthrene, and toluene for arsenic penetration studies and phenol, toluene, and trichloroethylene (TCE) for nickel studies. These are predominant chemicals found at hazardous waste sites. Arsenic and nickel bind to skin after dermal exposure. Total penetration of arsenic and nickel in the chemical mixture were significantly increased by 33% and 20% compared to arsenic and nickel alone, respectively. While more radioactivity penetrated skin with chemical treatment than metal alone, significantly less radioactivity was loosely adsorbed to skin and could be easily washed off from the skin surface with soap and water. The results of this study indicate that the potential health risk from dermal exposure to arsenic or nickel is enhanced if other chemicals are present.     

Cytotoxicity of trichloroethylene and perchloroethylene on normal human epidermal keratinocytes and protective role of vitamin E

Trichloroethylene (TCE) and perchloroethylene (PERC), the most common alkenyl halides, have been extensively used in industry, and can cause skin damage. To evaluate their cytotoxic potential on skin, the effects of these agents on the normal human epidermal keratinocytes (NHEK) were investigated. Their action on cell viability, membrane integrity and lipid peroxidation (LPO) was assessed by neutral red uptake (NRU) assay, lactate dehydrogenase (LDH) release test and measurement of malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity. In addition, the protective effect of antioxidatant vitamin E on the cytotoxicity was also studied. Incubation of NHEK with various concentrations (0.01-31.6 mM) of TCE or PERC caused a dose-dependent decrease in cell viability, with 80% reduction at 31.6 mM. NR50 values from the cytotoxicity assay was found to be 4.53 and 2.16 mM for TCE and PERC, respectively. A time- and concentration- dependent release of LDH were observed at 1, 2, 3, 4 h after cells were exposed to different doses of TCE or PERC. These agents also caused an increase of MDA, whilst an inhibition of SOD activity, in a concentration-dependent manner. Pre-treatment of the cells with vitamin E at 10-200 mM dose-dependently attenuated the cytotoxic effect of TCE or PERC. Pre-treatment with vitamin E also reversed subsequent TCE or PERC-induced release of LDH, elevation of lipid peroxidation and decline of anti-oxidant enzyme activities. These results suggest that TCE and PERC could induce cytotoxicity to NHEK associated with oxidative stress and antioxidatant vitamin E could effectively protect NHEK from TCE- or PERC-induced cytotoxicity, which may be associated to the superoxide scavenging effect and enhancement of anti-oxidant enzyme activities.     

SAR/QSAR methods in public health practice

Methods of (Quantitative) Structure-Activity Relationship ((Q)SAR) modeling play an important and active role in ATSDR programs in support of the Agency mission to protect human populations from exposure to environmental contaminants. They are used for cross-chemical extrapolation to complement the traditional toxicological approach when chemical-specific information is unavailable. SAR and QSAR methods are used to investigate adverse health effects and exposure levels, bioavailability, and pharmacokinetic properties of hazardous chemical compounds. They are applied as a part of an integrated systematic approach in the development of Health Guidance Values (HGVs), such as ATSDR Minimal Risk Levels, which are used to protect populations exposed to toxic chemicals at hazardous waste sites. (Q)SAR analyses are incorporated into ATSDR documents (such as the toxicological profiles and chemical-specific health consultations) to support environmental health assessments, prioritization of environmental chemical hazards, and to improve study design, when filling the priority data needs (PDNs) as mandated by Congress, in instances when experimental information is insufficient. These cases are illustrated by several examples, which explain how ATSDR applies (Q)SAR methods in public health practice.     

Chemical risk assessment and uncertainty associated with extrapolation across exposure duration

The Agency for Toxic Substances and Disease Registry (ATSDR) prepares toxicological profiles on priority substances in which available epidemiologic and toxicologic data are reviewed, summarized, and interpreted. When adequate data are available, ATSDR derives health guidance values called minimal risk levels (MRLs) for acute, intermediate, and chronic durations of exposure for oral and inhalation routes of exposure. The MRLs are generally derived by use of the no-observed-adverse-effect level (NOAEL) or the lowest-observed-adverse-effect level/uncertainty factor (LOAEL/UF) approach. The UF usually employed are for LOAEL-to-NOAEL extrapolation, animal to -human extrapolation, and inter-human variability. These health guidance values are intended to serve as screening tools for health assessors and other responders to identify contaminants of concern and potential health effects in the community at hazardous waste sites and during unplanned releases. When guidance values are not available for a specific exposure scenario because of a lack of chronic data, extrapolation across exposure durations may be made. For example, chronic guidance values may be derived from subchronic data by applying an additional uncertainty factor of 10 for extrapolation to chronic exposure duration. In this paper, we analyzed the ratio of chemical-specific LOAELs from acute to intermediate and from intermediate to chronic durations for oral and inhalation exposure routes. In addition, we investigated the impact of chemical structure and chemical structure activity relationship on validation of predictions across exposure durations.     

Comments on article "Applying mode-of-action and pharmacokinetic considerations in contemporary cancer risk assessments: an example with trichloroethylene" by Clewell and Andersen

In their 2004 article, Clewell and Andersen provide their perspective on the application of mode-of-action (MOA) and pharmacokinetic considerations in contemporary cancer risk assessment using trichloroethylene (TCE) as a case example. TCE is a complex chemical toxicologically, with multiple metabolites, multiple sites of observed toxicity, and multiple potential MOAs. As scientists who are responsible for revising the U.S. Environmental Protection Agency's draft risk assessment of TCE, we welcome input of the quality to which the Agency is held accountable. However, in our view, Clewell and Andersen do not present a sufficiently current, complete, accurate, and transparent review of the pertinent scientific literature. In particular, their article would need to incorporate substantial recently published scientific information, better support its conclusions about MOA and choice of linear or nonlinear dose-response extrapolation, and increase its transparency as to quantitative analyses in order to make a significant contribution to the scientific discussion of TCE health risks.     

Comments on Article “Applying Mode-of-Action and Pharmacokinetic Considerations in Contemporary Cancer Risk Assessments: An Example with Trichloroethylene” by Clewell and Andersen

In their 2004 article, Clewell and Andersen provide their perspective on the application of mode-of-action (MOA) and pharmacokinetic considerations in contemporary cancer risk assessment using trichloroethylene (TCE) as a case example. TCE is a complex chemical toxicologically, with multiple metabolites, multiple sites of observed toxicity, and multiple potential MOAs. As scientists who are responsible for revising the U.S. Environmental Protection Agency's draft risk assessment of TCE, we welcome input of the quality to which the Agency is held accountable. However, in our view, Clewell and Andersen do not present a sufficiently current, complete, accurate, and transparent review of the pertinent scientific literature. In particular, their article would need to incorporate substantial recently published scientific information, better support its conclusions about MOA and choice of linear or nonlinear dose-response extrapolation, and increase its transparency as to quantitative analyses in order to make a significant contribution to the scientific discussion of TCE health risks.     

Roads and motorized transport as major sources of priority substances? A data register study

A data register study was performed in order to identify the amounts of hazardous substances in products related to motorized transport in Norway during 2012. The hazardous substances were selected from legislative investigations performed by the European Chemicals Agency (ECHA), European Union (EU), and Norwegian Environment Agency (NEA). Information regarding hazardous substances in 52 selected product categories associated with traffic-related activities was obtained from the Norwegian Product Register administrated by the NEA. Substances present on ECHA list of substances of very high concern (SVHC), NEA national priority list, and priority substances under the EU Water Framework Directive (WFD) were given most attention, with substances from ECHA community rolling action plan (CoRAP) also included. Results showed that selected products contained a diverse range of substances that were classified as hazardous to either human or environmental health. The quantities of hazardous substances in the selected products were 120 tons (SVHC), 280 tons (Norway priority list), and 2,400 tons (WFD). It proved difficult to pinpoint these quantities only to traffic-related operations since product categories included compounds used for other activities. However, data illustrate that large quantities of hazardous substances are employed concurrent with being prioritized for reduction/elimination by national and international authorities. A list of substances with annual use in 2012 >1 ton was prepared to aid a prioritization for further actions such as substitution, phasing out, or environmental monitoring. The list contains substances that are toxic to humans, especially as adverse reproductive/carcinogenic agents, and/or pose a threat to the environment.     

Lead intervention and pediatric blood lead levels at hazardous waste sites

Lead intervention at Superfund sites typically seeks to reduce pediatric blood lead levels by disrupting the surface-to-hand-to-mouth pathway. This article presents the results of a survey of the publicly available literature on the effectiveness of lead intervention on pediatric blood lead levels at hazardous waste sites. The survey includes six hazardous waste sites located in Canada, Australia, and the United States at which intervention activities were conducted and pediatric blood lead levels were sampled both pre- and postintervention. Evaluation of the effectiveness of intervention on pediatric blood lead levels is often complicated due to confounding variables and statistical limitations. Nevertheless, the outcomes of the intervention studies reviewed in this report suggest that various approaches to the intervention of the dust ingestion pathway, alone or in combination, contributed to declines in blood lead levels in children living in areas heavily contaminated with lead.