Mortality Patterns Among Industrial Workers Exposed to Chloroprene and Other Substances: Extended Follow-Up.

OBJECTIVES: To update the U.S. portion of an historical cohort mortality study of workers with potential exposure to chloroprene (CD) and vinyl chloride (VC) with focus on lung and liver cancer. METHODS: Subjects were 6864 workers from two sites with vital status determined through 2017 for 99% of subjects and cause of death for 97.2% of deaths. Historical exposures to CD and VC were estimated quantitatively. We performed external and internal mortality comparisons. RESULTS: External comparisons revealed mostly deficits in deaths; internal comparisons revealed no consistent evidence of exposure-response relationships with CD or VC. CONCLUSIONS: Our update continues to support the conclusion that the risk of death from lung or liver cancer is unrelated to exposure to CD or VC at levels experienced by workers in the two U.S. sites.

Extended Analysis and Evidence Integration of Chloroprene as a Human Carcinogen.

ß-Chloroprene is used in the production of polychloroprene, a synthetic rubber. In 2010, Environmental Protection Agency (EPA) published the Integrated Risk Information System "Toxicological Review of Chloroprene," concluding that chloroprene was "likely to be carcinogenic to humans." This was based on findings from a 1998 National Toxicology Program (NTP) study showing multiple tumors within and across animal species; results from occupational epidemiological studies; a proposed mutagenic mode of action; and structural similarities with 1,3-butadiene and vinyl chloride. Using mouse data from the NTP study and assuming a mutagenic mode of action, EPA calculated an inhalation unit risk (IUR) for chloroprene of 5 × 10-4 per µg/m3 . This is among the highest IURs for chemicals classified by IARC or EPA as known or probable human carcinogens and orders of magnitude higher than the IURs for carcinogens such as vinyl chloride, benzene, and 1,3-butadiene. Due to differences in pharmacokinetics, mice appear to be uniquely responsive to chloroprene exposure compared to other animals, including humans, which is consistent with the lack of evidence of carcinogenicity in robust occupational epidemiological studies. We evaluated and integrated all lines of evidence for chloroprene carcinogenicity to assess whether the 2010 EPA IUR could be scientifically substantiated. Due to clear interspecies differences in carcinogenic response to chloroprene, we applied a physiologically based pharmacokinetic model for chloroprene to calculate a species-specific internal dose (amount metabolized/gram of lung tissue) and derived an IUR that is over 100-fold lower than the 2010 EPA IUR. Therefore, we recommend that EPA's IUR be updated.

Incorporation of in vitro metabolism data and physiologically based pharmacokinetic modeling in a risk assessment for chloroprene.

Objective: To develop a physiologically based pharmacokinetic (PBPK) model for chloroprene in the mouse, rat and human, relying only on in vitro data to estimate tissue metabolism rates and partitioning, and to apply the model to calculate an inhalation unit risk (IUR) for chloroprene.Materials and methods: Female B6C3F1 mice were the most sensitive species/gender for lung tumors in the 2-year bioassay conducted with chloroprene. The PBPK model included tissue metabolism rate constants for chloroprene estimated from results of in vitro gas uptake studies using liver and lung microsomes. To assess the validity of the PBPK model, a 6-hr, nose-only chloroprene inhalation study was conducted with female B6C3F1 mice in which both chloroprene blood concentrations and ventilation rates were measured. The PBPK model was then used to predict dose measures - amounts of chloroprene metabolized in lungs per unit time - in mice and humans.Results: The mouse PBPK model accurately predicted in vivo pharmacokinetic data from the 6-hr, nose-only chloroprene inhalation study. The PBPK model was used to conduct a cancer risk assessment based on metabolism of chloroprene to reactive epoxides in the lung, the target tissue in mice. The IUR was over100-fold lower than the IUR from the EPA Integrated Risk Information System (IRIS), which was based on inhaled chloroprene concentration. The different result from the PBPK model risk assessment arises from use of the more relevant tissue dose metric, amount metabolized, rather than inhaled concentrationDiscussion and conclusions: The revised chloroprene PBPK model is based on the best available science, including new test animal in vivo validation, updated literature review and a Markov-Chain Monte Carlo analysis to assess parameter uncertainty. Relying on both mouse and human metabolism data also provides an important advancement in the use of quantitative in vitro to in vivo extrapolation (QIVIVE). Inclusion of the best available science is especially important when deriving a toxicity value based on species extrapolation for the potential carcinogenicity of a reactive metabolite.

Isothiocyanates are important as haptens in contact allergy to chloroprene rubber.

BACKGROUND: Contact allergy to chloroprene rubber products is well known. Thiourea compounds are considered the cause of allergy. Diethylthiourea commonly occurs in this type of product and can decompose to the sensitizer ethyl isothiocyanate. OBJECTIVES: To investigate the clinical importance of degradation products and metabolites from organic thioureas in contact allergy to chloroprene rubber with a focus on isothiocyanates and isocyanates. METHODS: Patients with contact allergy to diphenylthiourea were patch tested with phenyl isothiocyanate and phenyl isocyanate. Patients with known contact allergy to diethylthiourea were retested with diethylthiourea, while chemical analyses of their chloroprene rubber products were performed. The stability of diethylthiourea, diphenylthiourea and dibutylthiourea in patch-test preparations was investigated. Liquid chromatography/mass spectrometry and solid-phase microextraction/gas chromatography were used for determination of organic thioureas and isothiocyanates. RESULTS: All patients allergic to diphenylthiourea reacted to phenyl isothiocyanate, two of eight reacted to phenyl isocyanate and six of eight reacted to diphenylthiourea. Four patients allergic to diethylthiourea reacted at retest; diethylthiourea was detected in all chloroprene rubber samples, with levels of 2-1200 nmol cm-2 . At 35 °C, ethyl isothiocyanate was emitted from all samples. Patch-test preparations of diethylthiourea, diphenylthiourea and dibutylthiourea all emitted the corresponding isothiocyanate, with diethylthiourea showing the highest rate of isothiocyanate emission. CONCLUSIONS: Thiourea compounds are degraded to isothiocyanates, which are generally strong or extreme sensitizers, thus acting as prehaptens. This process occurs in both chloroprene rubber products and patch-test preparations. Positive reactions to phenyl isocyanate indicate cutaneous metabolism, as the only known source of exposure to phenyl isocyanate is through bioactivation of diphenylthiourea.

Inferring Alcoholism SNPs and Regulatory Chemical Compounds Based on Ensemble Bayesian Network.

AIM AND OBJECTIVE: The disturbance of consciousness is one of the most common symptoms of those have alcoholism and may cause disability and mortality. Previous studies indicated that several single nucleotide polymorphisms (SNP) increase the susceptibility of alcoholism. In this study, we utilized the Ensemble Bayesian Network (EBN) method to identify causal SNPs of alcoholism based on the verified GAW14 data. MATERIALS AND METHODS: We built a Bayesian network combining random process and greedy search by using Genetic Analysis Workshop 14 (GAW14) dataset to establish EBN of SNPs. Then we predicted the association between SNPs and alcoholism by determining Bayes' prior probability. RESULTS AND CONCLUSION: Thirteen out of eighteen SNPs directly connected with alcoholism were found concordance with potential risk regions of alcoholism in OMIM database. As many SNPs were found contributing to alteration on gene expression, known as expression quantitative trait loci (eQTLs), we further sought to identify chemical compounds acting as regulators of alcoholism genes captured by causal SNPs. Chloroprene and valproic acid were identified as the expression regulators for genes C11orf66 and SALL3 which were captured by alcoholism SNPs, respectively.

Regression analysis of current status data in the presence of a cured subgroup and dependent censoring.

This paper discusses regression analysis of current status data, a type of failure time data where each study subject is observed only once, in the presence of dependent censoring. Furthermore, there may exist a cured subgroup, meaning that a proportion of study subjects are not susceptible to the failure event of interest. For the problem, we develop a sieve maximum likelihood estimation approach with the use of latent variables and Bernstein polynomials. For the determination of the proposed estimators, an EM algorithm is developed and the asymptotic properties of the estimators are established. Extensive simulation studies are conducted and indicate that the proposed method works well for practical situations. A motivating application from a tumorigenicity experiment is also provided.

Weighted gene co-expression network analysis of pneumocytes under exposure to a carcinogenic dose of chloroprene.

AIMS: Occupational exposure to chloroprene via inhalation may lead to acute toxicity and chronic pulmonary diseases, including lung cancer. Currently, most research is focused on epidemiological studies of chloroprene production workers. The specific molecular mechanism of carcinogenesis by chloroprene in lung tissues still remains obscure, and specific candidate therapeutic targets for lung cancer are lacking. The present study identifies specific gene modules and valuable hubs associated with lung cancer. MAIN METHODS: We downloaded the dataset GSE40795 from the Gene Expression Omnibus (GEO) and divided the dataset into the non-carcinogenic dose chloroprene exposed mice group and the carcinogenic dose chloroprene exposed mice group. With a systemic biological view, we discovered significantly altered gene modules between the two groups and identified hub genes in the carcinogenic dose exposed group using weighted co-expression network analysis (WGCNA). KEY FINDINGS: A total of 2434 differentially expressed genes were identified. Twelve gene modules with multiple biological activities were related to the carcinogenesis of chloroprene in lung tissue. Seven hub genes that were critical for the carcinogenesis of chloroprene in lung tissue were ultimately identified, including Cftr, Hip1, Tbl1x, Ephx1, Cbr3, Antxr2 and Ccnd2. They were implicated in inflammatory response, cell transformation, gene transcription regulation, phase II detoxification, angiogenesis, cell adhesion, motility and the cell cycle. SIGNIFICANCE: The seven hub genes may become valuable candidates for risk assessment biomarkers and therapeutic targets in lung cancer.

A constrained maximum likelihood approach to evaluate the impact of dose metric on cancer risk assessment: application to ß-chloroprene.

ß-Chloroprene (2-chloro-1,3-butadiene, CD) is used in the manufacture of polychloroprene rubber. Chronic inhalation studies have demonstrated that CD is carcinogenic in B6C3F1 mice and Fischer 344 rats. However, epidemiological studies do not provide compelling evidence for an increased risk of mortality from total cancers of the lung. Differences between the responses observed in animals and humans may be related to differences in toxicokinetics, the metabolism and detoxification of potentially active metabolites, as well as species differences in sensitivity. The purpose of this study was to develop and apply a novel method that combines the results from available physiologically based kinetic (PBK) models for chloroprene with a statistical maximum likelihood approach to test commonality of low-dose risk across species. This method allows for the combined evaluation of human and animal cancer study results to evaluate the difference between predicted risks using both external and internal dose metrics. The method applied to mouse and human CD data supports the hypothesis that a PBK-based metric reconciles the differences in mouse and human low-dose risk estimates and further suggests that, after PBK metric exposure adjustment, humans are equally or less sensitive than mice to low levels of CD exposure.

Excretion of mercapturic acids in human urine after occupational exposure to 2-chloroprene.

A pilot study was conducted for human biomonitoring of the suspected carcinogen 2-chloroprene. For this purpose, urine samples of 14 individuals occupationally exposed to 2-chloroprene (exposed group) and of 30 individuals without occupational exposure to alkylating substances (control group) were analysed for six potential mercapturic acids of 2-chloroprene: 4-chloro-3-oxobutyl mercapturic acid (Cl-MA-I), 4-chloro-3-hydroxybutyl mercapturic acid (Cl-MA-II), 3-chloro-2-hydroxy-3-butenyl mercapturic acid (Cl-MA-III), 4-hydroxy-3-oxobutyl mercapturic acid (HOBMA), 3,4-dihydroxybutyl mercapturic acid (DHBMA) and 2-hydroxy-3-butenyl mercapturic acid (MHBMA). In direct comparison with the control group, elevated levels of the mercapturic acids Cl-MA-III, MHBMA, HOBMA and DHBMA were found in the urine samples of the exposed group. Cl-MA-I and Cl-MA-II were not detected in any of the samples, whereas HOBMA and DHBMA were found in all analysed urine samples. Thus, for the first time, it was possible to detect HOBMA and Cl-MA-III in human urine. The mercapturic acid Cl-MA-III could be confirmed as a specific metabolite of 2-chloroprene in humans providing evidence for the intermediate formation of a reactive epoxide during biotransformation. The main metabolite, however, was found to be DHBMA showing a distinct and significant correlation with the urinary Cl-MA-III levels in the exposed group. The obtained results give new scientific insight into the course of biotransformation of 2-chloroprene in humans.

Cross-species transcriptomic analysis of mouse and rat lung exposed to chloroprene.

ß-Chloroprene (2-chloro-1,3-butadiene), a monomer used in the production of neoprene elastomers, is of regulatory interest due to the production of multiorgan tumors in mouse and rat cancer bioassays. A significant increase in female mouse lung tumors was observed at the lowest exposure concentration of 12.8 ppm, whereas a small, but not statistically significant increase was observed in female rats only at the highest exposure concentration of 80 ppm. The metabolism of chloroprene results in the generation of reactive epoxides, and the rate of overall chloroprene metabolism is highly species dependent. To identify potential key events in the mode of action of chloroprene lung tumorigenesis, dose-response and time-course gene expression microarray measurements were made in the lungs of female mice and female rats. The gene expression changes were analyzed using both a traditional ANOVA approach followed by pathway enrichment analysis and a pathway-based benchmark dose (BMD) analysis approach. Pathways related to glutathione biosynthesis and metabolism were the primary pathways consistent with cross-species differences in tumor incidence. Transcriptional BMD values for the pathway were more similar to differences in tumor response than were estimated target tissue dose surrogates based on the total amount of chloroprene metabolized per unit mass of lung tissue per day. The closer correspondence of the transcriptional changes with the tumor response is likely due to their reflection of the overall balance between metabolic activation and detoxication reactions, whereas the current tissue dose surrogate reflects only oxidative metabolism.

Kinetic modeling of ß-chloroprene metabolism: Probabilistic in vitro-in vivo extrapolation of metabolism in the lung, liver and kidneys of mice, rats and humans.

ß-Chloroprene (chloroprene) is carcinogenic in inhalation bioassays with B6C3F1 mice and Fischer rats, but the potential effects in humans have not been adequately characterized. In order to provide a better basis for evaluating chloroprene exposures and potential effects in humans, we have explored species and tissue differences in chloroprene metabolism. This study implemented an in vitro-in vivo extrapolation (IVIVE) approach to parameterize a physiologically based pharmacokinetic (PBPK) model for chloroprene and evaluate the influence of species and gender differences in metabolism on target tissue dosimetry. Chloroprene metabolism was determined in vitro using liver, lung and kidney microsomes from male or female mice, rats, and humans. A two compartment PK model was used to estimate metabolism parameters for chloroprene in an in vitro closed vial system, which were then extrapolated to the whole body PBPK model. Two different strategies were used to estimate parameters for the oxidative metabolism of chloroprene: a deterministic point-estimation using the Nelder-Mead nonlinear optimization algorithm and probabilistic Bayesian analysis using the Markov Chain Monte Carlo technique. Target tissue dosimetry (average amount of chloroprene metabolized in lung per day) was simulated with the PBPK model using the in vitro-based metabolism parameters. The model-predicted target tissue dosimetry, as a surrogate for a risk estimate, was similar between the two approaches; however, the latter approach provided a measure of uncertainty in the metabolism parameters and the opportunity to evaluate the impact of that uncertainty on predicted risk estimates.

A method for the simultaneous determination of mercapturic acids as biomarkers of exposure to 2-chloroprene and epichlorohydrin in human urine.

We developed and validated an analytical method for the simultaneous determination of several chlorine and non-chlorine containing mercapturic acids in urine as specific metabolites of the hazardous chemicals 2-chloroprene and epichlorohydrin. The method involves an online column switching arrangement for online solid phase extraction of the analytes with subsequent analytical separation and detection using LC-MS/MS. The developed method enables for the first time the determination of Cl-MA-I (4-chloro-3-oxobutyl mercapturic acid), Cl-MA-II (4-chloro-3-hydroxybutyl mercapturic acid), Cl-MA-III (3-chloro-2-hydroxy-3-butenyl mercapturic acid) and HOBMA (4-hydroxy-3-oxobutyl mercapturic acid) as potential biomarkers of 2-chloroprene in urine. Additionally, CHPMA (3-chloro-2-hydroxypropyl mercapturic acid) as a specific metabolite of epichlorohydrin in urine and DHBMA (3,4-dihydroxybutyl mercapturic acid) can be determined. The analytical method proved to be both sensitive and reliable with detection limits ranging from 1.4 µg/L (for Cl-MA-III) to 4.2 µg/L (for HOBMA). Intra- and interday imprecision was determined to range from 4.7 to 11.8%. Due to the good accuracy and precision and the low limits of detection the developed method is well suited for application in biomonitoring studies in order to determine occupational exposure to 2-chloroprene and epichlorohydrin.

A fatal intoxication by chloroprene.

UNLABELLED: Objective Chloroprene, 2-chloro-1,3 butadiene, is a volatile synthetic liquid. The chloroprene monomer is extremely reactive and is used for the production of latexes and synthetic rubber such as Neoprene. Up to now an acute lethal human exposure has been described only once in the literature [19]. The intoxication is associated with nervous system depression, pulmonary edema, narcosis, and respiratory arrest. Case report A 29-year-old chemistry company worker was found unconscious in an empty vessel (depth: 3m) used for chloroprene. The man was dressed in shoes, trousers, a helmet and a respiratory mask. The upper part of the body was unclothed. In spite of reanimation, the man died three hours later in a hospital. MATERIAL AND METHODS: All analyses were performed by headspace gas chromatography (HS/GC/FID). In addition, brain, muscle and myocardial muscle were analysed by headspace GC-MS. Results and discussion Autopsy findings: The cause of death could not be determined as the macromorphological findings were unspecific. Toxicology findings The calibration curve of chloroprene in serum shows linearity from 1.0 to 200 µg/ml (r(2)=0.9999) using benzene as internal standard. The LOD is 0.28 µg/ml, the LLOQ is 0.99 µg/ml. Tissues and body fluids were stored at -20 °C till the analysis. Chloroprene was quantified after addition of benzene as the internal standard. It was found in nearly all tissues and body fluids except in the urine and lung. The highest concentrations were detected in the kidney, liver, myocardial muscle and especially in the brain. Furthermore, hexanal was found in all samples except in the urine. The amount of hexanal in some specimens is high, especially in the lung, bile, gastric content and myocardial muscle. Conclusion We assume that a significant amount of chloroprene was not only inhaled but also absorbed through the skin because the man wore a respiratory mask. Presumably the accident would not have happened if the works safety protocols had been followed. The reason why high concentrations of hexanal were found in the tissues could not be clarified.

DNA interstrand cross-linking activity of (1-Chloroethenyl)oxirane, a metabolite of beta-chloroprene.

With the goal of elucidating the molecular and cellular mechanisms of chloroprene toxicity, we examined the potential DNA cross-linking of the bifunctional chloroprene metabolite, (1-chloroethenyl)oxirane (CEO). We used denaturing polyacrylamide gel electrophoresis to monitor the possible formation of interstrand cross-links by CEO within synthetic DNA duplexes. Our data suggest interstrand cross-linking at deoxyguanosine residues within 5'-GC and 5'-GGC sites, with the rate of cross-linking depending on pH (pH 5.0 > pH 6.0 > pH 7.0). A comparison of the cross-linking efficiencies of CEO and the structurally similar cross-linkers diepoxybutane (DEB) and epichlorohydrin (ECH) revealed that DEB > CEO > or = ECH. Furthermore, we found that cytotoxicity correlates with cross-linking efficiency, supporting a role for interstrand cross-links in the genotoxicology of chloroprene.

A frailty model approach for regression analysis of multivariate current status data.

This paper discusses regression analysis of multivariate current status failure time data (The Statistical Analysis of Interval-censoring Failure Time Data. Springer: New York, 2006), which occur quite often in, for example, tumorigenicity experiments and epidemiologic investigations of the natural history of a disease. For the problem, several marginal approaches have been proposed that model each failure time of interest individually (Biometrics 2000; 56:940-943; Statist. Med. 2002; 21:3715-3726). In this paper, we present a full likelihood approach based on the proportional hazards frailty model. For estimation, an Expectation Maximization (EM) algorithm is developed and simulation studies suggest that the presented approach performs well for practical situations. The approach is applied to a set of bivariate current status data arising from a tumorigenicity experiment.