Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens (2014) / Chapter Skim
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3 Independent Assessment of Styrene
3 Independent Assessment of Styrene
Pages 57-159
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The present chapter provides a brief summary of informative studies and highlights key data that informed its independent assessment of styrene. The reader is also referred to the background document (NTP 2008)
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After identifying the relevant body of literature up to November 13, 2013, the committee reviewed the primary data and applied the RoC listing criteria to human, experimental animal, and mechanistic studies. It then integrated the evidence to develop its own independent listing recommendation for styrene.
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Increased blood concentrations of styrene or styrene metabolites have been observed in experimental subjects and workers exposed to styrene. Concentrations of styrene in the blood increase rapidly after the onset of exposure and decay over the course of several hours after termination of the exposure (see the background document for styrene [NTP 2008]
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Because styrene produces lung tumors in mice, a focus of investigation has been on pulmonary metabolism in mice. Styrene is extensively metabolized in the mouse liver and lung (primarily in Clara cells)
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A similar figure can be found in the background document for styrene (NTP 2008)
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The amount of DNA adducts found in the lung vs liver in the mouse or in the rat lung vs mouse lung does not correlate with the target organ or a specific-species tumor response (Cruzan et al.
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However, specific information on capacities for detoxification of styrene metabolites (such as epoxide hydrolase and glutathione-S-transferase) in critical target tissues in humans is not available.
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The committee then reviewed and evaluated the methods and results of those informative studies and applied the RoC listing criteria to the evidence. Identification of Informative Studies The committee established a set of attributes for identifying the most informative cohort and case–control studies.
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The following factors were judged by the committee to increase the credibility of evidence on human carcinogenicity of styrene: High estimates of MRRs, IRRs, standardized mortality ratios (SMRs) , standardized incidence ratios (SIRs)
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. (Denmark, Finland, Italy, Norway, Sweden, 18,500 measurements of styrene metabolites in Cumulative exposure computed with and United Kingdom)
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and Exposure assessment at plant level, with SIRs and 95% CIs from Poisson regression years of employment (<1 vs ≥1)
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was used to Internal comparisons made -- Cox plastics facilities who were employed in estimate exposure for each plant, accounting for proportional hazards models with areas exposed to styrene for ≥6 months calendar time with 6 process categories: cumulative exposure, duration of exposure, during January 1, 1948–December 31, 1977. sex, and age included as independent 1.
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diagnosed during 1979–1986 in 19 major which the person worked, activities of surrounding Population controls -- 71% of controls hospitals; 533 population controls age- workers, and presence of gases, fumes, or dusts. who were selected to participate were stratified to cases; 533 cancer controls and interviewed.
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. Frequency and concentration coded on an ordinal Separate analyses using cancer controls, 1, 2, 3 scale and transformed to 1, 4, and 9 scores population controls, and pooled controls for estimating cumulative exposure.
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. who ever worked in the fibrous-glass or lamination Cumulative exposure assessed for two departments (TWA = 42.5 ppm/day in company A or departments at each plant and up to 1978; SMRs and 95% CI: External comparisons TWA of 71.7ppm/day in company B)
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Cumulative exposure (ppm-years)
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Cumulative exposure was categorized as 0; >0 to ≤1.5; 1.5 to ≤67.1; >67.1 ppm-years. Cocco et al.
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Cumulative exposure score, C i y i * fi 3 xi where ci = cumulative exposure score, i = study subject, y = duration of exposure, x = exposure intensity level, f = exposure frequency level; categorized into quartiles (unexposed, low, medium, and high)
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Abbreviations: CI, confidence interval; IRR, incidence rate ratio; MRR, mortality rate ratio; OR, odds ratio; SIR, standardized incidence ratio; SMR, standardized mortality ratio; TWA, time-weighted average; WHO, World Health Organization. Source: committee-generated. 75
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Given the nature of the styrene exposure assessment in many of the cohort studies, such misclassifications are likely. For example, there were no individual-based exposure data in Kolstad et al.
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Thus, the committee discussions below begin with the broadest classification of lymphohematopoietic cancers and follow with more detailed classifications. The epidemiologic data provide credible but limited evidence that styrene is a risk factor for lymphohematopoietic cancers on the basis of two European cohort studies (Kogevinas et al.
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, n = 7 Unexposed: SMR = 0.91 (0.41–1.72) , n = 9 Cumulative exposure (ppm–years)
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: Open-mold processing: SMR = 1.41, n = 4 Mixing and closed-mold processing: SMR = 0.71, n = 2 Finish and assembly: SMR = 0.62, n = 4 Plant office and support: SMR = 0.65, n = 3 Maintenance and preparation: SMR = 0.93, n= 5 Supervisory and professional: SMR = 1.02, n = 2 In proportional-hazard models, cumulative exposure and duration of exposure to styrene were not significant (n = 31)
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, n = 1 Abbreviations: MRR, mortality rate ratio; n, number of cases or deaths in cohort studies or number of exposed cases in case–control studies; SMR, standardized mortality ratio; SIR, standardized incidence ratio. Source: committee-generated.
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Cumulative exposure (ppm-years) did not appear to be associated with an increase in mortality due to combined lymphohematopoietic cancers in this cohort (Kogevinas et al.
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. Additional analyses by latency, cumulative exposure (ppm–months)
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, n = 4 Unexposed: SMR = 0.99 (0.27–2.54) , n = 4 Cumulative exposure (ppm–years)
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: <10: SMR = 1.11, n = 5 10–19: SMR = 0.68, n = 4 ≥20: SMR = 0.46, n = 2 Subgroups by cumulative exposure (ppm–years) (CIs not reported)
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, n = 1 reference population = Low exposure: SMR = 0.64 (0.18–1.65) , n = 4 Washington state Abbreviations: MRR, mortality rate ratio; n, number of cases or deaths in cohort studies or number of exposed cases in case–control studies; SMR, standardized mortality ratio; SIR, standardized incidence ratio.
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. Additional analyses by latency, duration of exposure, and cumulative exposure also did not suggest an association between styrene and leukemia in these two studies.
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Average exposure (ppm) : <60 as reference: n = 3 60–99: MRR = 2.51 (0.49–12.87)
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, n = 33 Subgroups by cumulative exposure (ppm–months)
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Ever occupationally exposed to styrene: Adjusted OR = 2.0 (0.8–4.8) , number of exposed cases = 8; unadjusted OR = 2.1 Adjusted for age, family income, ethnic group, cigarette smoking, and respondent status.
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, number of exposed cases = 66; unadjusted OR = 1.6 Cumulative exposure score based on confidence, intensity of exposure, frequency of exposure: p for trend = 0.000096 Adjusted for age, sex, education, and center Abbreviations: MRR, mortality rate ratio; n, number of cases or deaths in cohort studies or number of exposed cases in case–control studies; NHL, non-Hodgkin lymphoma; OR, odds ratio; SMR, standardized mortality ratio; SIR, standardized incidence ratio. Source: committeegenerated.
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Based on a logistic regression model adjusted for age, family income, ethnic group, cigarette smoking, and respondent status, subjects who had occupational exposure to styrene appeared to have a higher odds of NHL than those who were not occupationally exposed to styrene (adjusted odds ratio [OR]
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Kidney Cancer The epidemiologic data provide credible but limited evidence that styrene is a carcinogen for the kidney (see Table 3-5) on the basis of the US cohort studies and a European case–control study.
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, n = 2 Cumulative exposure (ppm–years) : <75 reference: n=2 100–199: MRR = 4.40 (0.71–27.2)
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, n = 34 Cumulative exposure SMRs (ppm–months)
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Karami on July 29, 2013, in response to a request from the Committee to Review the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Abbreviations: MRR, mortality rate ratio; n, number of cases or deaths in cohort studies or number of exposed cases in case–control studies; OR, odds ratio; CI, confidence interval; SMR, standardized mortality ratio; SIR, standardized incidence ratio.
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for exposed persons below the median value of cumulative exposure and 6.7 (95% CI 1.8–24.3) for exposed persons above the median value of cumulative exposure; the p for trend of ORs with cumulative exposure was 0.02.
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Therefore, the evidence fulfills NTP's listing criteria for limited evidence and not sufficient evidence for an association between exposure to styrene and kidney cancer. Pancreatic Cancer The epidemiologic data on pancreatic cancer constitute credible but limited evidence that styrene exposure is associated with pancreatic cancer on the basis of four cohort studies (see Table 3-6)
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, n = 5 Cumulative exposure (ppm–years) : <75 reference: n = 9 100–199: MRR = 1.44 (0.48–4.34)
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, n = 63 ≥15 years latency: SMR= 0.90 (0.67–1.17) , n = 53 Cumulative exposure SMRs (ppm-months)
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, n = 1 Adjusted for age, family, income, ethnic group, cigarette smoking, and respondent status. Abbreviations: MRR, mortality rate ratio; n, number of cases or deaths in cohort studies or number of exposed cases in case–control studies; CI, confidence interval; SMR, standardized mortality ratio; SIR, standardized incidence ratio.
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Therefore, the evidence fulfills NTP's listing criteria of limited evidence, not sufficient evidence, for an association between exposure to styrene and esophageal cancer. Lung and Breast Cancers The committee does not consider there to be credible epidemiologic evidence of an association of styrene exposure and lung or breast cancer (Table 38)
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, n = 5 Other exposed jobs: no = 0 Unexposed: 0.82 (0.47–1.31) , n = 17 Cumulative exposure (ppm–years)
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Abbreviations: MRR, mortality rate ratio; n, number of cases or deaths in cohort studies or number of exposed cases in case–control studies; CI, confidence interval; SMR, standardized mortality ratio; SIR, standardized incidence ratio. Source: committee-generated.
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but reported inverse linear trends for cumulative exposure (p < 0.001)
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, n = 37 Cumulative exposure (ppm–years) : <75 reference: n = 73 100–199: MRR = 0.75 (0.47–1.19)
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: Open-mold processing: 0.90, n = 8 Mixing and closed-mold processing: 1.24, n = 10 Finish and assembly: 1.43, n = 31 Plant office support: 1.07, n = 17 Maintenance and preparation: 1.49, n = 30, p < 0.05 Supervisory and professional: 0.66, n = 5 Proportional-hazard models, cumulative exposure, duration of exposure to styrene were not significant. Nested case control from same cohort at earlier period (Wong 1990)
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, n = 501 Cumulative exposure SMRs (ppm–months)
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Abbreviations: MRR, mortality rate ratio; n, number of cases or deaths in cohort studies or number of exposed cases in case–control studies; CI, confidence interval; OR, odds ratio; SMR, standardized mortality ratio; SIR, standardized incidence ratio. Source: committee-generated.
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The lung tumors occurred late in the study, and no increases were observed in subgroups of animals terminated after 52 and 78 weeks of exposure. After oral exposure by gavage, a significant increase in alveolar and bronchiolar tumors combined was observed in male mice at the highest dose, and there was a significant dose-related trend (NCI 1979a)
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study, the committee considers the use of the historical controls to be inappropriate in that they were not well matched to the treatment conditions of the study. As discussed in Chapter 2, many factors are related to the genetic makeup of the animals and husbandry practices that can influence tumor incidences in control and treated animals.
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No significant increases in lung tumors were observed in females, and tumors at other sites were not significantly increased in male or female mice. In view of the importance of the observation of increased lung tumors in mice following oral exposure in the NCI (1979a)
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TABLE 3-11 Statistical Comparison of Mouse Lung Tumor Data from the 1979 NCI Study Using the Peto Test Tumor Comparison P-value Males Alveolar/Bronchiolar Carcinoma Low Dose vs. Control 0.296 – 0.318 High Dose vs.
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The major organs in all organ systems (respiratory, gastrointestinal, cardiovascular, urinary, reproductive, nervous, lymphohematopoietic, endocrine, musculoskeletal, and cutaneous) and grossly abnormal tissues were examined histopathologically in all groups.
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The major organs in all organ systems (respiratory, gastrointestinal, cardiovascular, urinary, reproductive, nervous, lymphohematopoietic, endocrine, musculoskeletal, and cutaneous) and grossly abnormal tissues were examined histopathologically in all groups except for some moribund animals.
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Huff (1984) analyzed data from oral exposure of Sprague-Dawley rats to styrene in the same study and found a significant increase in combined mammary tumors in high-dose females and a significant trend with dose.
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Significant increases in forestomach tumors were observed after both high and low doses in both species and sexes. Similarly, significant increases in forestomach tumors were observed in male and female SpragueDawley rats (40 per treatment group per sex)
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The mutagenic and carcinogenic effects of DNA adducts are affected by the efficiency of DNA repair (both base-excision repair, as in the case of oxidative DNA damage, and nucleotide-excision repair of adducts derived from styrene)
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, one must go beyond simply counting the numbers of studies that report statistically significant results or statistically nonsignificant results on carcinogenesis and related modes of action to reach credible conclusions about the relative strength of the evidence and the likelihood of causality. Accordingly, the committee first categorized evidence pertaining to styrene and styrene-7,8-oxide genotoxic and clastogenic mechanistic events into tables on DNA damage (Table 3-12)
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TABLE 3-12 Studies of DNA Damage Associated with Styrene or Styrene-7,8-oxide (Including Adducts and Strand Breaks) a Styrene Styrene-7,8-oxide Positive Negative Positive Negative In vitro -- -- Bastlová et al.
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Studies were categorized as negative if there was an absence of a particular effect (that is, no statistically significant change from the appropriate control group) ; bRoute of administration is inhalation unless noted otherwise; cIdentified through committee's literature search; dDenotes chemical administration through intraperitoneal injection; e Denotes chemical administration through oral gavage.
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TABLE 3-13 Studies of Sister-Chromatid Exchanges Associated with Styrene or Styrene-7,8-oxidea Styrene Styrene-7,8-oxide Positive Negative Positive Negative Norppa et al. 1980a -- Norppa et al.
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; eIdentified through committee's literature search; f Denotes chemical administration through intraperitoneal injection.
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Studies were categorized as negative if there was an absence of a particular effect (that is, no statistically significant change from the appropriate control group) ; bRoute of administration is inhalation unless noted otherwise; cIdentified from Scott and Preston (1994a)
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TABLE 3-15 Studies of Chromosomal Aberrations Associated with Styrene or Styrene-7,8-oxidea 124 Styrene Styrene-7,8-oxide Positive Negative Positive Negative Linnainmaa et al. 1978a -- Linnainmaa et al.
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; fIdentified through committee's literature search; gDenotes chemical administration through intraperitoneal injection; hDenotes chemical administration through oral gavage.
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Genotoxic and Clastogenic Effects of Styrene and Styrene-7,8-Oxide on in Vitro Human and Rodent Cells The evidence available on all forms of DNA and genetic damage shows clearly that DNA damage (Table 3-12) and clastogenic effects (Tables 3-13 through 3-15)
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In rodent studies of styrene and styrene-7,8-oxide, evidence was less strong but mostly positive for DNA damage, sister-chromatid exchanges, and micronuclei; however, the total number of rodent studies was less than the number of studies of exposed humans. Effects of styrene or styrene-7,8-oxide were well documented, and this helps to establish that exposure to styrene or styrene7,8-oxide was associated with the positive and negative observations.
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DNA damage, reflected by the presence of styrene-7,8-oxide-derived DNA adducts in human tissues, is highly likely to generate mutations, some of which may occur in genes that lead to cancer in susceptible people. The presence of DNA adducts -- occurring predominantly at the N7, N2, and O6 positions of guanine -- has been amply demonstrated in cell culture, experimental animals, and, most important, lymphocytes of workers occupationally exposed to styrene (Table 3-12)
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The evidence reviewed by the committee also indicates that styrene-7,8-oxide, a major reactive metabolite of styrene that is produced in exposed humans, reacts with DNA to form covalent adducts and other premutagenic forms of DNA damage, which result in genotoxic effects. The committee recognizes that styrene-7,8-oxide may not be the only genotoxic metabolite of styrene.
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. In contrast, allergic responses 2 A differential blood count gives the relative percentage of white blood cell types, such as neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
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1990 could not be reached Abbreviation: BFU-E, burst-forming unit-erythroid; CFU-C, colony-forming unit in culture; CFU-E, colony-forming unit-erythrocyte; CFU-S, colony-forming unit-spleen; NK, natural killer cell; RBC, red blood cell; WBC, white blood cell.
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2000; Jahnova et al. 2002 Abbreviation: CBC, complete blood count; CD4, cluster of differentiation 4; NK, natural killer cell; RBC, red blood cell; WBC, white blood cell.
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Monocytes, macrophages, neutrophils, and NK cells are the main effector cells in innate immunity, and T and B lymphocytes are part of adaptive immunity. In the studies reviewed by the committee, styrene generally had more suppressive effects than stimulatory effects on innate immunity.
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Among the studies that investigated effects of styrene on adaptive immunity, inconsistent results were observed between the number of lymphocytes in workers exposed to styrene and the number in controls. For example, Khristeva (1986)
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In animals, inhibitory effects were observed mainly on the innate immune system, including decreases in lymphocyte counts and weights in the spleen, suppressed monocyte and macrophage activity, and suppressed NK-cell activity. In adaptive immunity, stimulatory effects were observed in cellular immunity, including increased type IV hypersensitivity and increased production of cytokines, interferon-gamma, and interleukins.
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For the liver, serum has been analyzed for sorbitol dehydrogenase. Although those nonspecific approaches appear to provide reliable screening tools and reflect the overall responses of the organs, they lack sufficient specificity to define the response in the presumed target cells for styrene, the Clara cells.
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. Metabolism of styrene to the R- and Sstyrene oxide enantiomers was identical in the liver of wild-type and knockout mice but markedly reduced in the lungs of knockout mice (Carlson 2012)
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However, a clear understanding of the roles of CYP450s in the cytotoxicity of styrene will require further studies with a more comprehensive approach, including comparisons of not only liver and lung but other organs. Cellular Oxidative Stress Response Styrene and its principle metabolites, R- and S-styrene oxide and 4vinylphenol, have been used to define markers of oxidative stress and the cellular stress response only in Clara cells in mice.
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To define mechanisms by which the metabolites of styrene react with potential target cells more clearly, future studies will need to address oxidative stress in other cell populations that have different levels of susceptibility in the lungs and in other organs, such as the liver. Cellular Antioxidants Extracellular pools of the antioxidant glutathione were markedly altered in mice by exposure to a single intraperitoneal dose of styrene or R-styrene oxide (Carlson 2010a)
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Depletion of glutathione by styrene was increased in the liver of deficient mice but not in the lung. There was a difference between the toxic responses to R- and S-styrene oxide in microsomal epoxide hydrolase–deficient mice (Carlson 2011a)
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SUMMARY OF EVIDENCE AND CONCLUSIONS The statement of task (Appendix B) directed the committee to "integrate the level-of-evidence conclusions, and considering all relevant information in accordance with the RoC listing criteria, make an independent listing recommendation for styrene and provide scientific justification for its recommendation." As discussed throughout this report, a substance can be categorized as reasonably anticipated to be a human carcinogen on the basis of sufficient evidence in animals or limited evidence in humans and a substance can be catego
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The listing criteria state that a substance should be classified as known to be a human carcinogen if "there is sufficient evidence of carcinogenicity from studies in humans". The footnote associated with that sentence states that "this evidence can include data derived from the study of tissues or cells from humans exposed to [styrene]
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1995. Styrene oxide-induced HPRT mutations, DNA adducts and DNA strand breaks in cultured human lymphocytes.
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1983. Chromosomal aberrations and sister-chromatid exchanges in workers exposed to styrene.
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1997. Influence of duration of exposure to styrene oxide on sister chromatid exchanges and cell-cycle kinetics in cultured human blood lymphocytes in vitro.
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1978. Induction of sister chromatid exchanges by styrene and its presumed metabolite styrene oxide in the presence of rat liver homogenate.
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2009. DNA single and double-strand breaks by alkaline- and immune-comet assay in lymphocytes of workers exposed to styrene.
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1997. Recombinant expression of human microsomal epoxide hydrolase protects V79 Chinese hamster cells from styrene oxide- but not from ethylene oxide-induced DNA strand breaks.
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1979. Increased frequency of chromosome aberrations in workers exposed to sty rene.
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Karami from the Committee to Review the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Received on July 29, 2013.
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2001b. Genotoxic effects of styrene-7,8-oxide in human white blood cells: Comet assay in relation to the induction of sister chromatid exchanges and micronuclei.
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1991. Single-strand breaks, chromosome aberrations, sister-chromatid exchanges, and micronuclei in blood lymphocytes of workers exposed to styrene during the production of rein forced plastics.
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1979. Ef fects of styrene oxide on chromosome aberrations, sister chromatid exchange and hepatic drug biotransformation in Chinese hamsters in vivo.
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1998. Influence of GSTT1 genotype on sister chromatid exchange induction by styrene-7,8-oxide in cultured human lym phocytes.
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1984. Cytogenetic analysis of human peripheral blood lymphocytes in culture exposed in vitro to styrene and styrene oxide.
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2012. Insensitivity of the in vitro cytokinesis-block miconucleaus assay with human lymphocutes for the detection of DNA damage present at the start of the cell culture.
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1998. Detection of styrene and styrene oxide-induced DNA damage in various organs of mice using the comet assay.
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2004c. Markers of individual susceptibility and DNA repair rate in workers exposed to xenobiotics in a tire plant.
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2011. Low level occupational exposure to styrene: Its effects on DNA damage and DNA repair.
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