Matthew W. Himmelstein

Toxicology and Epidemiology of 1,3-Butadiene

Abstract1,3-Butadiene is a colorless, volatile gas that has high-volume usage in the synthesis of polybutadiene, styrene-butadiene, and other polymers. Due to its volatile nature, uptake of butadiene occurs almost exclusively by inhalation and absorption through the respiratory system. Sources of exposure include production, transport, and end-use processes in industrial settings or environmental exposures through automotive fuel, fossil fuel combustion, and cigarette smoke. Chronic inhalation studies established that butadiene is carcinogenic in B6C3F1 mice and Sprague-Dawley rats, and that mice are considerably more sensitive than rats. For the most part, epidemiologic studies for butadiene have been equivocal, although a recent retrospective follow-up study of styrene-butadiene rubber workers provides the first internally consistent evidence of a relationship between butadiene exposure and leukemia. The mechanism(s) of butadiene-induced carcinogenicity are not entirely understood but are thought to inv...

Creating context for the use of DNA adduct data in cancer risk assessment: II. Overview of methods of identification and quantitation of DNA damage

The formation of deoxyribonucleic acid (DNA) adducts can have important and adverse consequences for cellular and whole organism function. Available methods for identification of DNA damage and quantification of adducts are reviewed. Analyses can be performed on various samples including tissues, isolated cells, and intact or hydrolyzed (digested) DNA from a variety of biological samples of interest for monitoring in humans. Sensitivity and specificity are considered key factors for selecting the type of method for assessing DNA perturbation. The amount of DNA needed for analysis is dependent upon the method and ranges widely, from <1 μg to 3 mg. The methods discussed include the Comet assay, the ligation-mediated polymerase reaction, histochemical and immunologic methods, radiolabeled (14C- and 3H-) binding, 32P-postlabeling, and methods dependent on gas chromatography (GC) or high-performance liquid chromatography (HPLC) with detection by electron capture, electrochemical detection, single or tandem mass spectrometry, or accelerator mass spectrometry. Sensitivity is ranked, and ranges from ∼1 adduct in 104 to 1012 nucleotides. A brief overview of oxidatively generated DNA damage is also presented. Assay limitations are discussed along with issues that may have impact on the reliability of results, such as sample collection, processing, and storage. Although certain methodologies are mature, improving technology will continue to enhance the specificity and sensitivity of adduct analysis. Because limited guidance and recommendations exist for adduct analysis, this effort supports the HESI Committee goal of developing a framework for use of DNA adduct data in risk assessment.

The use of non-tumor data in cancer risk assessment: reflections on butadiene, vinyl chloride, and benzene.

The estimation and characterization of a cancer risk is grounded in the observation of tumors in humans and/or experimental animals. Increasingly, however, other kinds of data (non-tumor data) are finding application in cancer risk assessment. Metabolism and kinetics, adduct formation, genetic damage, mode of action, and biomarkers of exposure, susceptibility, and effects are examples. While these and other parameters have been studied for many important chemicals over the past 30-40 years, their use in risk assessments is more recent, and new insights and opportunities are continuing to unfold. To provide some perspective on this field, the ILSI Risk Science Institute asked a select working group to characterize the pertinent non-tumor data available for 1,3-butadiene, benzene, and vinyl chloride and to comment on the utility of these data in characterizing cancer risks. This paper presents the findings of that working group and concludes with 15 simple principles for the use of non-tumor data in cancer risk assessment.

The metabolism of β-chloroprene: preliminary in-vitro studies using liver microsomes

Based on analogy with butadiene and isoprene, the metabolism of beta-chloroprene (2-chloro-1,3-butadiene, CD) to reactive intermediates is likely to be a key determinant of tumor development in laboratory rodents exposed to CD by inhalation. The purpose of this study is to identify species differences in toxic metabolite (epoxide) formation and detoxification in rodents and humans. The in-vitro metabolism of CD was studied in liver microsomes of B6C3F1 mice, Fischer/344 and Wistar rats, Syrian hamsters, and humans. Microsomal oxidation of CD in the presence of NADP(+), extraction with diethyl ether, and analysis by GC-mass selective detection (MSD) indicated that (1-chloroethenyl)oxirane (CEO) was an important metabolite of CD in the liver microsomal suspensions of all species studied. Other potential water-soluble oxidative metabolites may have been present. The oxidation of CD was inhibited by 4-methyl pyrazole, an inhibitor of CYP 2E1. CEO was sufficiently volatile at 37 degrees C for vial headspace analysis using GC-MSD single ion monitoring (m/z=39). CEO was synthesized and used to conduct partition measurements along with CD and further explore CEO metabolism in liver microsomes and cytosol. The liquid-to-air partition coefficients for CD and CEO in the microsomal suspensions were 0.7 and 58, respectively. Apparent species differences in the uptake of CEO by microsomal hydrolysis were hamster approximately human>rats>mice. Hydrolysis was inhibited by 1,1,1-trichloropropene oxide, a competitive inhibitor of epoxide hydrolase. A preliminary experiment indicated that the uptake of CEO in liver cytosol by GSH conjugation was hamster>rats approximately mice (human cytosol not yet tested). In general, the results suggest that metabolism may help explain species differences showing a greater sensitivity for CD-induced tumorigenicity in mice, for example, compared with hamsters. Additional experiments are in progress to quantify the kinetic parameters of CD oxidation and CEO metabolism by enzymatic hydrolysis and conjugation by glutathione S-transferase for in cytosol. A future goal is to use the kinetic rates to parameterize a physiologically based toxicokinetic model and relate the burden of toxic metabolite to the cancer dose-response observed in experimental animals.

In vitro genotoxicity testing of (1-chloroethenyl)oxirane, a metabolite of β-chloroprene

(1-Chloroethenyl)oxirane (CEO) is a metabolite of beta-chloroprene (2-chloro-1,3-butadiene, CD). The purpose of this study was to evaluate the in vitro mutagenic and clastogenic (chromosome breaking) potential of CEO. For comparative purposes, the study also included an evaluation of the racemic compounds, 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Mutagenicity was evaluated in a bacterial reverse mutation test (Ames), using the pre-incubation method in the presence and absence of an exogenous metabolism system (Aroclor)-induced rat liver S9). Four Salmonella typhimurium tester strains, TA97a, TA98, TA100 and TA1535 were used. The exposure concentrations in the sealed incubation vials ranged from 0 to 69 mM for CEO, 0 to 102 mM for EB, and 0 to 83 mM for DEB. All three compounds showed signs of toxicity, with DEB being substantially more toxic than either CEO or EB. Mutagenic activity was observed with all three chemicals in primarily the base pair substitution strains (S. typhimurium TA100 and TA1535), but some activity was also seen in the frameshift elimination strains (S. typhimurium TA97a and TA98). The observed mutagenic responses after exposure with CEO or EB were greater than the observed response for DEB, most likely because of the higher toxicity of DEB. Generally, the mutagenic responses were unchanged in the frameshift strains and base pair substitution strains in the presence of S9 metabolism. In vitro clastogenicity was evaluated using the cytochalasin-B blocked micronucleus test in cultured Chinese hamster V79 cells. The test was conducted without S9 metabolism because of the absence of substantial changes in the Ames test. Exposure concentrations ranged from 0 to 0.943 mM for CEO, 0 to 3.0 mM for EB, and 0 to 0.035 mM for DEB, with the upper exposure concentrations dictated by cytotoxicity. Cytotoxicity, measured as a reduction in the proportion of binucleated cells and altered cell morphology, was observed for CEO at concentrations > or =0.175 mM. Exposure to EB led to a reduced proportion of binucleated cells at concentrations > or =2.0 mM, and cell death was observed after DEB exposure at concentrations > or =0.025 mM. No clastogenicity was observed in the V79 cells when tested up to cytotoxic concentrations of CEO, whereas an elevated frequency of micronuclei was observed after exposure to either EB (> or =1.0 mM) or DEB (> or =0.0125 mM). These results suggest that CEO-induced mutagenicity, but not clastogenicity, may contribute to the observed beta-chloroprene-induced carcinogenicity in the rodent bioassay studies.

8:2 fluorotelomer alcohol: A one-day nose-only inhalation toxicokinetic study in the Sprague-Dawley rat with application to risk assessment

8:2 fluorotelomer alcohol (8:2 FTOH) inhalation exposure was investigated to (1) compare plasma metabolites to oral data, (2) conduct a route-to-route extrapolation (oral to inhalation), (3) develop a human equivalent air concentration (HEC) from a 90-day oral sub-chronic study in rats using BMD analysis, and (4) calculate a margin of exposure (MOE) between the HEC and measured air concentrations. Male and female rats were exposed nose-only for 6h at 3 or 30mg/m(3). Blood was collected at 1, 3 and 6h during exposure and 6 and 18h post exposure. Alcohol, perfluorocarboxylic acid and polyfluorinated acid metabolites were determined in plasma by LC-MS/MS. 8:2 FTOH was <LOQ (32nM) at the low exposure and quantifiable (37-69nM) at the high exposure. The quantifiable metabolites in plasma were dose proportional and comprised mainly of 8:2 FTCA, 7:3 Acid, and PFOA. By kinetic modeling, the yields of the terminal products 7:3 Acid (1.6-2.1 and 0.9mol%) and PFOA (1.0-1.2 and 0.3mol%) of the inhaled dose were low for male and female rats, respectively. The kinetic yield of PFOA after oral dosing was similar (1.1-1.7-fold) for male rats and greater (8-9-fold) for female rats relative to inhalation exposure, an observation confirmed by non-compartmental analysis. A BMDL10% (3.7mg/kg/day) was derived for mild hepatic necrosis observed in male rats following a 90-day oral dose study with 8:2 FTOH. The corresponding HECs were 1.8 and 3.7mg/m(3), which gave MOE values ranging from 1.8×10(4) to 6.1×10(6) based on reported ambient air concentrations of 0.3-209ng/m(3). These findings demonstrate rapid 8:2 FTOH uptake and clearance by the inhalation route and a consistent metabolite profile between inhalation and oral exposures in rats. No toxicity is expected in humans from typical ambient 8:2 FTOH air exposures.

An organizational approach for the assessment of DNA adduct data in risk assessment: case studies for aflatoxin B1, tamoxifen and vinyl chloride

Abstract The framework analysis previously presented for using DNA adduct information in the risk assessment of chemical carcinogens was applied in a series of case studies which place the adduct information into context with the key events in carcinogenesis to determine whether they could be used to support a mutagenic mode of action (MOA) for the examined chemicals. Three data-rich chemicals, aflatoxin B1 (AFB1), tamoxifen (Tam) and vinyl chloride (VCl) were selected for this exercise. These chemicals were selected because they are known human carcinogens and have different characteristics: AFB1 forms a unique adduct and human exposure is through contaminated foods; Tam is a pharmaceutical given to women so that the dose and duration of exposure are known, forms unique adducts in rodents, and has both estrogenic and genotoxic properties; and VCl, to which there is industrial exposure, forms a number of adducts that are identical to endogenous adducts found in unexposed people. All three chemicals produce liver tumors in rats. AFB1 and VCl also produce liver tumors in humans, but Tam induces human uterine tumors, only. To support a mutagenic MOA, the chemical-induced adducts must be characterized, shown to be pro-mutagenic, be present in the tumor target tissue, and produce mutations of the class found in the tumor. The adducts formed by AFB1 and VCl support a mutagenic MOA for their carcinogenicity. However, the data available for Tam shows a mutagenic MOA for liver tumors in rats, but its carcinogenicity in humans is most likely via a different MOA.

Overview of the acute, subchronic, reproductive, developmental and genetic toxicology of β-chloroprene

beta-Chloroprene (CD), the 2-chloro derivative of 1,3-butadiene, is used for the manufacture of the synthetic rubber, polychloroprene. Acute inhalation studies show that CD is lethal to Crl:CD rats at >2300 p.p.m. (4 h); the primary target organ effects were pulmonary hemorrhage and edema, and hepatic necrosis. In 2- and 4-week inhalation studies in Fischer 344 (F344) and Wistar rats, early deaths occurred at 500 and > or =161 p.p.m., respectively. Organ system injury was found in the nose (degeneration/metaplasia of olfactory epithelium), liver (centrilobular necrosis), and blood (decreased red blood cell count in F344 rats only). In a 90-day inhalation study with F344 rats, degeneration/metaplasia of the olfactory epithelium and reduced nonprotein sulfhydryl content of lungs and liver were found in animals exposed to 80 p.p.m., and anemia, hepatocellular necrosis, and forestomach inflammation were observed at 200 p.p.m. In a 90-day study with B6C3F1 mice, CD caused deaths at 200 p.p.m., the highest concentration tested, and epithelial hyperplasia of the forestomach at 80 p.p.m. Other than a slight (<10%) reduction in sperm motility in male rats at 200 p.p.m., all other reproductive parameters (sperm count or morphology in males, and estrous cyclicity or cycle length in females) were unaffected in these 90-day rat/mouse studies. There were no significant indications of neurological toxicity. The study No-Observable Adverse Effect Level was 32 p.p.m. based on nasal injury in rats. Despite some early reports of reproductive system abnormalities at levels <1 p.p.m., recent studies show no embryotoxic or developmental toxicity in female Wistar or Crl:CD rats, or in New Zealand White rabbits at CD exposure concentrations up to 25 or 175 p.p.m., respectively. In a one-generation reproduction study with Wistar rats, CD produced growth retardation in the F(0) generation exposed to 100 p.p.m., and in the F(1) offspring at 33 and 100 p.p.m.; no effects on reproductive parameters or histopathology were found. CD is nonmutagenic in standard plate incorporation bacterial reverse mutation assays (Ames assays) but positive using direct gas-phase incubation methods. Bacterial mutagenicity (primarily base pair substitution) was either negative or weakly positive when freshly prepared CD was tested. Mutagenicity increased markedly with time, presumably from CD dimer formation, and also by addition of liver S9 metabolic activation system. In vivo micronucleus, chromosome aberration and sister chromatid exchange studies in mice showed no structural chromosomal damage. Overall, the pathological effects in the liver and nose dominate the subchronic toxicity of CD. The genotoxicity of CD is inconsistent and requires further study.

Inhalation and oral toxicokinetics of 6:2 FTOH and its metabolites in mammals.

The toxicokinetics of 6:2 fluorotelomer alcohol (6:2 FTOH) and its terminal perfluorinated and polyfluorinated metabolites (PFBA, PFHxA, PFHpA and 5:3 Acid) have been calculated from laboratory studies of rats and from a biomonitoring study of humans. In vitro studies with mouse, rat and human hepatocytes indicate qualitatively similar metabolic pathways of 6:2 FTOH. In a one-day inhalation study of 6:2 FTOH in rats, PFBA, PFHxA, PFHpA and 5:3 Acid were determined to be the major metabolites in plasma with calculated elimination half-lives of 1.3-15.4h and metabolic yields up to 2.7 mol%. In five-day and 23-day inhalation studies and a 90-day oral study of 6:2 FTOH, the plasma or serum concentration profile of 5:3 Acid was several-fold higher than concentrations observed in the single day study, resulting in an estimated elimination half-life of 20-30 d. In contrast, the concentrations of PFBA, PFHxA and PFHpA showed little or no concentration increase with repeated exposure. Elimination half-lives of PFHxA, PFHpA and 5:3 Acid in humans were estimated from a study of professional ski wax technicians who were occupationally exposed to aerosolized and volatilized components of fluorinated glide wax. The resulting human elimination half-life values of PFHxA, PFHpA and 5:3 Acid were 32, 70 and 43 d, respectively. Based on a one compartment toxicokinetic model, current environmental air concentrations of 6:2 FTOH are estimated to result in plasma concentrations of PFHxA, PFHpA and 5:3 Acid that are less than or equal to typical LOQ values, in agreement with extant biomonitoring results.

Development of a preliminary physiologically based toxicokinetic (PBTK) model for 1,3-butadiene risk assessment

Potential health effects of human exposure to 1,3-butadiene (BD) are of concern due to the use of BD in industry and its low-level presence throughout the environment. Physiologically based toxicokinetic (PBTK) models of BD in rodents have been developed by multiple research groups in an effort to explain species differences in toxicity (especially carcinogenic potency) through toxicokinetics. PBTK modeling of dose metrics related to a non-cancer endpoint, ovotoxicity in experimental animals, was conducted. The cumulative area under the blood concentration vs. time curve (AUC) for the metabolite diepoxybutane (butadiene diepoxide, DEB) was found to be consistent with ovotoxicity in mice and rats exposed to BD by inhalation or epoxybutene (butadiene monoepoxide, EB) or DEB by intraperitoneal injection. This suggests that cumulative DEB AUC may also be an appropriate metric for possible human risk. A preliminary human PBTK model was assembled for the eventual assessment of reproductive risk to humans and for prioritizing the determination of model parameters. The preliminary model accurately predicted published data on exhaled breath BD concentrations in a human volunteer exposed to BD by inhalation. The fit was relatively insensitive to the rate constant for BD epoxidation. Sensitivity analyses were conducted on this human PBTK model. Using a range of published rate constants, human blood DEB was found to be sensitive to rates of epoxidation of EB to DEB and hydrolysis of EB and DEB, but not BD epoxidation. Because of the large ranges of rates measured in vitro for these reactions, different combinations of in-vitro rates produce varying predictions of blood DEB concentration. Thus, validation of a human PBTK model with human biomonitoring data will be essential to produce a PBTK model that can be applied to risk assessment.