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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Page 84
Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Page 86
Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Page 93
Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Suggested Citation:"3 Independent Assessment of Styrene." National Research Council. 2014. Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens. Washington, DC: The National Academies Press. doi: 10.17226/18725.
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Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

3 Independent Assessment of Styrene The committee’s task had two parts. The first was to review the styrene substance profile as it was presented in the National Toxicology Program (NTP) 12th Report on Carcinogens (RoC) (NTP 2011a). In Chapter 2 of this report, the committee considered only literature that was available to NTP (literature pub- lished by June 10, 2011). It reviewed the primary literature, assessed NTP’s de- scription and analysis of that literature, and determined whether NTP’s argu- ments support the conclusion that styrene is “reasonably anticipated to be a human carcinogen”. To address the second part of its task, the committee carried out an inde- pendent assessment of styrene carcinogenicity, which is the focus of the present chapter. The committee used its peer review in Chapter 2 and the background document that supports the styrene profile in the 12th RoC as a starting point. The present chapter provides a brief summary of informative studies and high- lights key data that informed its independent assessment of styrene. The reader is also referred to the background document (NTP 2008) for a more detailed discussion of study methodologies, strengths, and weaknesses for literature pub- lished prior to 2011 and to the primary literature. The committee’s independent assessment of styrene carcinogenicity was based on literature that included primary data; however, the committee used published peer-reviewed review articles and reviews by other authoritative bod- ies to ensure that relevant literature was not missed and that all plausible inter- pretations of primary data were considered. The committee also considered comments and arguments that were presented during its first meeting, comments received from outside stakeholders during the study process, and independent literature searches carried out by National Research Council staff. The goal of the literature searches was to identify relevant literature that may have missed inclusion in the 12th RoC and relevant literature that was published after the release of the 12th RoC. Each search began on January 1, 2008, the year in which the background document for styrene was published (Bucher 2013). The search was first run on May 28, 2013, and it was updated on November 13, 57

58 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens 2013.1 Databases searched were PubMed, Medline (Ovid), Embase (Ovid), Sco- pus, and Web of Science. The search strategy for each database and the exclu- sion criteria are described in greater detail in Appendix D. 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. Consideration was given to all relevant information, including “dose response, route of exposure, chemi- cal structure, metabolism, pharmacokinetics, sensitive sub-populations, genetic effects, or other data relating to mechanism of action or factors that may be unique to a given substance” (NTP 2011b). In accordance with the listing criteria, expert judgment was used to inter- pret and apply the RoC listing criteria to evidence in human and animal studies. A substance can be classified in the RoC as “known to be a human carcinogen” if “there is sufficient evidence of carcinogenicity from studies in humans, which indicates a causal relationship between exposure to the agent, substance, or mix- ture, and human cancer” (NTP 2008, p. v). A substance can be classified in the RoC as “reasonably anticipated to be a human carcinogen” if at least one of the following three criteria are fulfilled (NTP 2008, p. v):  “There is limited evidence of carcinogenicity from studies in humans, which indicates that causal interpretation is credible, but that alternative explanations, such as chance, bias, or confounding factors, could not adequately be excluded.”  “There is sufficient evidence of carcinogenicity from studies in experi- mental animals, which indicates there is an increased incidence of ma- lignant and/or a combination of malignant and benign tumors (1) in multiple species or at multiple tissue sites, or (2) by multiple routes of exposure, or (3) to an unusual degree with regard to incidence, site, or type of tumor, or age at onset.”  “There is less than sufficient evidence of carcinogenicity in humans or laboratory animals; however, the agent, substance, or mixture belongs to a well-defined, structurally related class of substances whose mem- bers are listed in a previous Report on Carcinogens as either known to be a human carcinogen or reasonably anticipated to be a human carcin- ogen, or there is convincing relevant information that the agent acts through mechanisms indicating it would likely cause cancer in hu- mans.” 1 The cut-off date for the literature search was chosen to allow the committee time to review the literature within the time constraints of the project schedule.

Independent Assessment of Styrene 59 As discussed in Chapter 2, the type of information needed to meet the criterion for sufficient evidence in experimental animals is clear and transparent. The type of information needed to meet the criterion for limited or sufficient evidence in hu- mans required more interpretation and expert judgment on behalf of the commit- tee. In its evaluation of the epidemiology literature, the committee described the information it used to identify informative studies and to evaluate those studies. This chapter begins with a section on metabolism and toxicokinetics. It then reviews cancer studies in humans, cancer studies in experimental animals, and mechanistic data. The chapter ends with a section that summarizes the evi- dence and provides a final conclusion and listing recommendation for styrene that is based on the listing criteria published in the 12th RoC. METABOLISM AND TOXICOKINETICS The absorption, distribution, metabolism, and excretion of styrene have been reviewed by several organizations (Sumner and Fennell 1994; IARC 2002; Vodicka et al. 2006; NTP 2008). In brief, as expected for a lipid-soluble hydro- carbon, styrene is absorbed after inhalation, ingestion, or dermal exposure. In- creased blood concentrations of styrene or styrene metabolites have been ob- served 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 back- ground document for styrene [NTP 2008] for more information). Styrene is ex- tensively metabolized, and metabolites are excreted in urine. Humans and ro- dents differ quantitatively in whole-body metabolism and excretion, but styrene metabolic pathways are qualitatively similar in rodents and humans (IARC 2002; Vodicka et al. 2006). Several pharmacokinetic and physiologically based pharmacokinetic models of inhaled styrene absorption and metabolism have been developed (Filser et al. 2002; Sarangapani et al. 2002; Chen et al. 2008; NTP 2008; Verner et al. 2012). Metabolic activation is thought to be essential for styrene toxicity and car- cinogenicity (IARC 2002; Vodicka et al. 2006; NTP 2008, 2011a). The balance between the metabolic activation rate and the detoxification rate in a specific target tissue is critical in determining the ultimate response. This section pro- vides information on styrene phase I metabolism (metabolic activation), infor- mation on phase II (detoxification) pathways, and then a summary. Multiple target sites are relevant to the carcinogenic hazard posed by sty- rene. In humans, styrene exposure is associated with cancer of the lymphohema- topoietic system, esophagus, pancreas, and kidney (see the section “Epidemio- logic Studies” below). In mice, styrene causes lung tumors, but statistically significant increases in tumor burden were not observed for other sites. The metabolic pathways that are likely to be important in the carcinogenic response are fairly well defined (and described below), but there is no comprehensive information on activation and detoxification rates in potential target tissues in the human.

60 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens Phase I: Metabolism Styrene is metabolically activated by cytochrome P450 monooxygenase (CYP450)-dependent oxidation on the side chain to form styrene-7,8-oxide (see Figure 3-1). Because this molecule contains an asymmetric carbon, there are two enantiomers, R- and S-styrene-7,8-oxide. Styrene aromatic ring metabolites are also formed, including 4-vinylphenol (presumably through styrene-1,2-oxide) and 2-vinylphenol (presumably through styrene-2,3-oxide). Of those pathways, sty- rene-7,8-oxide and 4-vinylphenol have been the most studied. It is possible that styrene-7,8-oxide and its initial detoxification products are further metabolized, perhaps through ring oxidation, and that 4-vinylphenol is also metabolized (Carl- son et al. 2001; Carlson 2004, 2012; Cruzan et al. 2012, 2013). In the rodent, 14C- labeled CO2 is exhaled after 14C-styrene administration; this suggests that aromatic ring–opened metabolites are formed (Boogaard et al. 2000a). The precise struc- tures and toxicologic roles of those downstream metabolites are not fully charac- terized. In humans, styrene-7,8-oxide-based metabolic products form over 90% of the excreted metabolites of styrene (see below). That is clear evidence that sty- rene-7,8-oxide is the primary phase I metabolite in humans. Ring oxidation to 4- vinylphenol also occurs but to a much smaller extent. Sulfate and glucuronide conjugates of 4-vinylphenol have been identified in urine of humans but at low concentrations (less than 1% of the excreted metabolites) (IARC 2002; Vodicka et al. 2002a; NTP 2008). Aromatic ring metabolites may be critical with respect to cytotoxic or genotoxic effects in specific target organs even though they con- stitute only a minor pathway with respect to whole-body metabolism of styrene. Multiple forms of human CYP450 are reported to catalyze styrene oxida- tion, albeit with different activities, including CYP1A2, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2E1, CYP2F1, CYP2S1, CYP3A3, CYP3A4, CYP3A5, and CYP4B1 (Nakajima et al. 1994; Vodicka et al. 2006; Carlson 2008; Fukami et al. 2008; NRC USCG 2008; Bui and Hankinson 2009). Multi- ple recent studies have shown associations of polymorphisms in selected CYPs with the pattern of urinary styrene metabolite excretion; although the studies suggest a role of CYPs in styrene metabolism, they did not examine associations with disease outcome. Two CYP2E1 binding sites with allosteric interactions result in a shift to more efficient metabolism as styrene concentration increases. Many of the aforementioned CYPs are expressed in nonhepatic tissues, and this indicates that styrene may be metabolically activated in multiple organs in hu- mans. 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) by multiple forms of CYP, including CYP2E1 and CYP2F2 (Carlson 2004, 2008, 2012; Shen et al. 2010; Cruzan et al. 2012, 2013). Both side chain and aromatic ring metabolites are probably formed in the lung. In the mouse, CYP2F2 may be particularly im- portant in activation of styrene (Cruzan et al. 2012, 2013). Investigations of the role of CYP2F2 in the pulmonary response to styrene have focused solely on

Independent Assessment of Styrene 61 cytotoxicity and short-term cell proliferation, and its role is critical in both these processes in the mouse lung. It is important to consider, however, that cytotoxi- city and short-term cell proliferation responses may not be the sole determinants of the ultimate tumorigenic response and, in this context, the role of this CYP in lung tumorigenicity in mice remains uncertain. OH Conjugates CH CH3 1-Phenylethanol CH CH2 CH CH2 CH CH2 H2O O + H H Styrene CH2 CH2OH O H 2-Phenylethanol Styrene-1,2-oxide Styrene-3,4-oxide H H CH CH2 CH CH2 C CH2 CH2 C O O OH HO 2-Vinylphenol 4-Vinylphenol Styrene-7,8-oxide Phenylacetaldehyde ? GS OH CH2 COOH CH CH2 CH CH2 OH + GS N OH Phenylacetic acid C CH2 GSH conjugate 1 GSH conjugate 2 OH Phenylethylene glycol O 1-Phenyl- 2-Phenyl- (styrene glycol) 2-hydroxy 2-hydroxy CH2 C N CH2 COOH ethylmercapturic ethylmercapturic acid acid Glucuronide acetate H H Phenylaceturic acid C COOH COOH OH O Mandelic acid Benzoic acid C N CH2 COOH H Hippuric acid O C COOH Phenylglyoxilic acid FIGURE 3-1 Primary metabolic pathways of styrene. Main pathways are indicated by thick arrows. Metabolites that have been extensively studied are highlighted in the boxes. This figure is not a complete depiction of all known metabolites. A similar figure can be found in the background document for styrene (NTP 2008). GSH, glutathione. Source: Adapted from IARC (2002).

62 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens Styrene-7,8-oxide is genotoxic (see below). Metabolites derived from sty- rene-7,8-oxide are excreted in urine after styrene exposure; this is a clear indica- tion that it is formed in humans. Moreover, styrene-7,8-oxide is detected in ve- nous blood in humans and rodents after styrene exposure (NTP 2008), so it can probably migrate from the organ in which it was formed. The presence of sty- rene-7,8-oxide in the blood indicates widespread exposure to this genotoxic me- tabolite throughout the body. In humans, styrene-7,8-oxide–hemoglobin adducts and styrene-7,8-oxide–DNA adducts in lymphocytes have been observed, possi- bly because of circulating styrene-7,8-oxide or the generation of styrene-7,8- oxide in circulating blood cells (NTP 2008, 2011a). Nonenzymatic epoxidation of styrene, perhaps via oxyhemoglobin, may occur in erythrocytes (Tursi et al. 1983). As in the human, styrene-7,8-oxide is present in blood of both rats and mice exposed to styrene. Styrene exposure does not induce tumors in the rat and a statistically significant increase in tumors was only observed in the lungs of mice. The reasons for this are unclear. Styrene-7,8-oxide-based DNA adducts are formed in mouse lung after styrene exposure (Boogaard et al. 2000b), but their role in lung tumorigenesis in mice is not known. The amount of DNA ad- ducts 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. 2009). However, inasmuch as adduct formation is the first of mul- tiple steps of tumor development, a direct relationship between adduct concen- trations and tumor response among species or organs is not necessary. The lack of direct concordance between styrene-7,8-oxide-adduct concentrations and tu- mor formation does not exclude the potential role of these adducts in the pulmo- nary carcinogenic response in mice. Styrene is also metabolized via oxidation of its aromatic ring. Although a minor component, 4-vinylphenol-derived metabolites are present in urine after styrene exposure (NTP 2008, 2011a; Linhart et al. 2010, 2012). The aromatic ring–derived metabolite 4-vinylphenol is more potent than styrene or styrene- 7,8-oxide in inducing pulmonary toxicity (Carlson 2002, 2004; Cruzan et al. 2005). This metabolite is thought to be formed by CYP2E1 and CYP2F2 in ro- dents (Carlson et al. 2001). 4-Vinylphenol is further metabolized by epoxidation in the rodent liver and lung (Zhang et al. 2011). Although it is not known with absolute certainty, aromatic ring–derived metabolites are likely important in the pneumotoxicity of styrene in mice (Cruzan et al. 2009, 2012, 2013). Information is not available on target organ–specific formation of aromatic ring metabolites in humans. The genotoxicity of these metabolites has not been extensively in- vestigated. Phase II: Detoxification The side-chain–based or aromatic ring–based phase I metabolic oxidative products of styrene can be detoxified by hydrolysis via epoxide hydrolase or gluta- thione conjugation. Styrene-7,8-oxide is metabolized by epoxide hydrolase to

Independent Assessment of Styrene 63 form styrene glycol, which can be converted to mandelic acid and phenylglyoxylic acid (Figure 3-1). Those two products are excreted in urine and account for more than 90% of the excreted styrene metabolites in humans (Vodicka et al. 2006; NTP 2008, 2011a). Information is not available on the pharmacogenetics of epox- ide hydrolase relative to styrene disposition in humans; however, microsomal epoxide hydrolase knockout mice are more sensitive to styrene-induced cytotoxi- city, and this highlights its potentially important role (Carlson 2010b, 2011a). Glu- tathione-based conjugates of styrene metabolites are also formed; they account for less than 10% of the excreted metabolites in humans but may account for more than 30% of the excreted metabolites in rodents (NTP 2008; Vodicka et al. 2006). Studies in mice that are deficient in glutathione-S-transferase P1P2 (-/-) suggest that this form is not important in styrene detoxification, but other forms of gluta- thione-S-transferase may still have a role in detoxification (Carlson 2011b). Some evidence suggests that expression of glutathione-S-transferase M1 and T1 in hu- mans may contribute to individual variability in the fraction of styrene-7,8-oxide that is conjugated to glutathione (Haufroid et al. 2002; Teixeira et al. 2004; Fusti- noni et al. 2008; Vodicka et al. 2006). Further information on the precise phase II activities of styrene in critical target cells is not available. Summary of Styrene Metabolism and Toxicokinetics Metabolism of styrene is key to its toxic and carcinogenic responses. The organ-specific tumorigenic responses to styrene will depend, in large part, on the balance between the rate of activation and the rate of detoxification in each or- gan. Thorough information on styrene activation and detoxification rates specif- ic to target sites, particularly in the human, is not available. Given the wide array of CYP450 isozymes that can oxidize styrene, including forms that are known to be expressed in extrahepatic tissues (for example, CYP2E1 and CYP2A13), it is not possible to exclude the possibility that styrene bioactivation can occur in multiple target tissues. The presence of styrene-7,8-oxide in blood indicates that there is widespread tissue exposure to this genotoxic metabolite even in tissues that have low capacity for styrene activation. That highlights the importance of cellular detoxification capacities relative to organ-specific effects of styrene. In tissues that have low activity of epoxide hydrolase or glutathione-S-transferase, it might take only low levels of oxidation of styrene to produce cellular effects. The absence of marked toxicity in organs other than the liver or lung of mice (see the “Cytotoxicity” section) suggests detoxification capacities in that species are sufficient to prevent overt toxicity, except in the liver and lung. 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. Therefore, the available information on styrene metabo- lism is insufficient to exclude any tissue from being a plausible target for sty- rene-induced cytotoxicity, which could contribute to carcinogenesis.

64 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens EPIDEMIOLOGIC STUDIES The literature was searched to identify relevant epidemiologic studies that had been published by November 13, 2013 (see Appendix D). The committee established exclusion criteria for the literature search and identified studies that were most informative for determining whether an association exists between styrene exposure and carcinogenesis. The committee then reviewed and evaluat- ed 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 in- formative cohort and case–control studies. The most informative cohort studies were ones that had  Large cohorts that were exposed to high and varied concentrations of styrene.  Systematically assigned exposure estimates (such as the years in which exposure began and ended or job exposure matrices that coupled work- er histories with exposure estimates according to occupation).  Styrene exposures that could be assessed apart from exposures to other potentially carcinogenic chemicals.  Systematically and reliably assigned cancer end points.  Internal comparisons, for example incidence rate ratios (IRRs) and mortality rate ratios (MRRs) that compared workers with different lev- els of exposure in the same cohort. The most informative case–control studies were ones that had  Relatively large numbers of cases and controls.  Reliable assessments of styrene exposure based on specific job histories or other sources of information.  Interviews of cases and controls or their next of kin to collect occupa- tional histories and data related to lifestyle (for example, smoking) and other important potential confounders. On the basis of those attributes, the committee identified what it judged to be the 11 informative publications: six studies that used four cohorts in the rein- forced-plastics industry that were conducted in Europe (Kogevinas et al. 1994; Kolstad et al. 1994, 1995) and the United States (Wong et al. 1994; Ruder et al. 2004; Collins et al. 2013) and five case–control studies conducted in Europe (Scélo et al. 2004; Seidler et al. 2007; Cocco et al. 2010; Karami et al. 2011) and

Independent Assessment of Styrene 65 Canada (Gerin et al. 1998). See Table 3-1 for descriptions of the studies, includ- ing the salient strengths and weaknesses of each study. It should be noted that approximately one-third of the Kolstad et al. (1994) cohort in Denmark was in- cluded in the Kogevinas et al. (1994) European cohort. However, the two studies assessed different cancer outcomes (incidence and mortality, respectively), and the Kolstad et al. (1994) study only included male workers whereas the Kogevi- nas et al. (1994) study included male and female workers. It should also be not- ed that the Collins et al. (2013) study was an extension of the study by Wong et al. (1994). However, Wong et al. (1994) assessed somewhat different exposure metrics and necessarily focused on mortality that occurred closer to the time of high exposure. The committee reviewed all epidemiologic studies that reported human exposure to styrene and an assessment of cancer end points and it identified sev- eral epidemiologic studies that it judged to be less informative compared to the 11 studies listed above. Reasons why the studies were judged as less informative studies and were excluded from Chapter 3 include small numbers of subjects, low concentrations of exposure to styrene, and simultaneous exposure of cohorts to other chemicals in addition to styrene (especially 1,2-butadiene), which pre- vented a clear characterization of styrene exposures. For an in-depth description of all epidemiology studies that were reviewed by NTP, see NTP (2008). Evaluation of Informative Studies After identifying studies that it deemed most informative, the committee evaluated the study data to inform its judgment of whether the evidence of car- cinogenesis in humans after exposure to styrene is sufficient, limited, or incon- clusive. 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), or odds ratios (ORs). The commit- tee considered a study to be particularly credible if a relative risk or its surrogate was ≥2.0 in an entire study cohort or a subset with high expo- sure (that is, a doubling of cancer mortality or incidence compared to the less exposed or unexposed comparison group). However, a relative risk estimate ≥1.5 also added credibility to the overall body of evidence, par- ticularly if the relative risk was unlikely to be due to chance on the basis of the confidence interval (CI) around the estimate.  Exposure–response relationships for any reliably established exposure metric.  Consistency of the above two types of observations among independent cohort studies of the reinforced-plastics industry or between cohort and case–control studies. Some inconsistencies among findings in different populations is typical in the epidemiologic literature and can be the

66 TABLE 3-1 Summary of Most Informative Epidemiologic Studies Related to Styrene Exposure and Cancer Study Design, Population, Outcomes, Exposure Assessment and Exposure Metrics Strengths and Limitations and Analytic Strategy Kogevinas et al. 1994 Cumulative exposure and average exposure assessed Strengths for each worker on the basis of individual job histories  Large cohort with many workers involved (Included approximately one-third of subjects and country-, period-, and job-specific exposure in lamination. from Kolstad et al. 1994). estimates from personal sampling measurements and  Relatively long duration of followup urine measurements. (period of followup and employment varied A retrospective cohort of 40,688 male and by country, average followup = 13 years, female workers ever employed in 660  16,500 personal sampling measurements from 539,479 person–years at risk), with little reinforced-plastics plants in six countries 1955 to 1990. loss to followup (3.0% of the cohort). (Denmark, Finland, Italy, Norway, Sweden,  18,500 measurements of styrene metabolites in  Cumulative exposure computed with and United Kingdom). urine conducted in the 1980s. without a 5-year lag period.  Internal comparisons made—Poisson Mortality from all causes and specific causes. Cumulative exposure (<75, 75–199, 200–499, ≥500 regression models included cumulative ppm–years). exposure, age, sex, calendar period, and SMRs and 95% CIs: External comparisons time since first exposure. based on data from WHO, standardized by Average exposure (<60, 60–99; 100–119; 120–199; sex, age (5-year age groups), and calendar ≥200 ppm). Limitations period (5-year periods).  About 60% of the cohort was employed in Longest-held job collapsed into 5 job groups the reinforced-plastics industry for <2 Rate ratios and 95% CIs from Poisson (laminators, n = 10,629; workers with unspecified years. regression: Internal comparisons limited to tasks, n = 19,408; workers in other exposed jobs, n =  No information on smoking, alcohol use, or exposed subjects. 5,406; workers not exposed to styrene, n = 4,044; and other lifestyle factors. workers with unknown job titles, n = 1,201). Duration of exposure (assessed by combining data from payroll records and plant records showing the dates of production of reinforced plastics [in Denmark, pension- fund records were used]). Time since first exposure (<10, 10–19, ≥20 years).

Kolstad et al. 1994 Pension-fund records were used to determine duration Strengths of employment (for exposed workers, only payments  Large cohort with relatively long followup A retrospective cohort study of 36,525 male recorded during exposed employment were included). (followup during 1970–1989, range of workers in 386 reinforced-plastics plants ever followup 0 to 20 years, mean = 10.9 years, employed during 1964–1988 in Denmark and Type of company (ever producing reinforced plastics, 584,556 person–years at risk) and little loss 14,254 workers not exposed to styrene in 166 never producing reinforced plastics, and unknown (<2%) to followup. industries not producing reinforced plastics, or production) and years since first employment (<10 vs  Outcome of interest was cancer incidence company unknown. ≥10 years). instead of mortality, which avoids the issues related to cause of death Incidence of all cancers and specific For workers employed in plants producing reinforced categorization and different lengths of lymphohematopoietic cancers. plastics (n=36,525): survival after cancer diagnosis.  Internal comparisons made and yielded SIRs and 95% CIs: External comparisons  First year of employment (1964–1970, 1971–1975, similar results. based on national incidence rates standardized 1976–1988) and years since first employment (<10 for sex, age, and year of diagnosis. vs ≥10). Limitations  Years since first employment (<10 vs ≥10) and  Exposure assessment at plant level, with SIRs and 95% CIs from Poisson regression years of employment (<1 vs ≥1). 12,837 workers from 287 companies in (internal comparisons to unexposed workers; which it was estimated that ≥50% of authors reported that results were similar to Analyses of a subset of workers in plants with styrene workers were involved in reinforced- results based on external comparisons but data measurements (9,335 workers employed during the plastics production (included in Kogevinas were not provided). years of sampling). 2,473 personal air samples (not et al. 1994) and 23,748 workers from 99 linked to workers or job titles) collected during companies in which 1–49% of the 1964–1988; 1,814 of which were sampled in the 128 workforce produced reinforced plastics); companies included in the study. These were averaged 60% of workers employed <1 year. by company and dichotomized as follows: <50 ppm vs  Personal sampling data available on a ≥50 ppm. subset of the cohort, but not linked to workers or job titles.  No information on smoking, alcohol use, or other lifestyle factors. (Continued) 67

68 TABLE 3-1 Continued Study Design, Population, Outcomes, Exposure Assessment and Exposure Metrics Strengths and Limitations and Analytic Strategy Wong et al. 1994 Because of scant historical monitoring data (Wong Strengths 1990), contractors collected monitoring data around  307,932 person–years at risk in the cohort Update of Wong 1990. 1980. Coupling the monitoring data to information during the followup period (through 1989), about process changes, engineering controls, and with little loss (3.5%) to followup (due to A retrospective cohort study of 15,826 male personal protective equipment and employment unknown vital status). and female workers in 30 US reinforced- histories, a job-exposure matrix (JEM) 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. Open-mold processing. variables. Mortality from all causes and specific causes. 2. Mixing- and closed-mold processing. Limitations SMRs and 95% CIs: External comparisons 3. Finish and assembly.  24% of the cohort was employed for based on US national age-, sex-, cause-, race-, 4. Plant office and support. <1 year and 27% for >5 years. and year-specific data (race missing from 5. Maintenance and preparation.  “Conservative” historical estimates of employment records, so the entire cohort was assumed to be white). 6. Supervisory and professional. styrene exposure were reported by AD Little, Inc. (Little 1981). Exposures not Time since first exposure to styrene (<10, 10–19, assessed after 1977 (affecting 27% of the Coefficients and standard deviations from ≥20 years). Cox proportional hazards model; internal cohort). No assessment of average Duration of employment (<1, 1–1.9, 2–4.9, 5–9.9, ≥10 exposure. No information on departments comparisons for selected causes of death. years). Sensitivity analyses were done for workers or jobs worked for 3% of the cohort who employed >2 years in the 6 process categories. were assigned the lowest exposure levels.  No information on smoking, alcohol use, or Duration of exposure (<1, 1–1.9, 2–4.9, 5–9.9, ≥10 other lifestyle factors. years). Cumulative exposure (<10, 10–29.9, 30–99.9, ≥100 ppm–years). Cumulative exposure and time since first exposure.

Kolstad et al. 1995 Pension-fund records used to determine duration of Strengths employment.  Large cohort with relatively long followup A retrospective-cohort study of 36,610 male (followup during 1970–1990, 618,900 workers in 386 reinforced-plastics plants ever Type of company. Companies classified as (1) high- person–years at risk) and little loss to employed during 1964–1988 in Denmark and probable exposure to styrene (producing reinforced followup (<2.1%). 14,293 workers not exposed to styrene in plastics with ≥50% of the workforce involved in  Internal comparisons made—Poisson similar industries. production) or (2) low-probable exposure to styrene regression models included exposure (<50% of the workforce involved in production). probability (unexposed, low, and high), Mortality from nonmalignant causes and age, year of first employment, duration of incidence of total and specific solid cancers. Year of first employment (≤1970 and >1970). employment, and time since first SMRs, SIRs, and 95% CIs: External employment, but results on exposure Duration of employment (year) (<1 and ≥1). comparisons based on age and calendar- probability not reported. specific national rates. Years since first employment (<10 and ≥10).  Outcome of interest was cancer incidence instead of mortality, which avoids the MRRs, IRRs, and 95% CIs from Poisson issues related to cause of death regression: Internal comparisons based on categorization and different lengths of workers not exposed to styrene in similar survival after cancer diagnosis. industries. Limitations  Exposure assessment at plant level.  No information on smoking, alcohol use, or other lifestyle factors. Gerin et al. 1998 Detailed questionnaires on working histories, including Strengths each job held. For each job, information about the  High participation rates of cases—82% A population-based case–control study of men company’s activities, the raw materials and final of cases agreed to participate, 82% of ages 35–70 years living in the metropolitan product, the machines used and responsibility for responses were obtained from study area of Montreal, Canada (3,730 cancer cases machine maintenance, the type of room or building in subjects and the rest from next of kin. 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. 1,066 pooled controls). A team of chemists and industrial hygienists estimated exposure for each job: (Continued) 69

70 TABLE 3-1 Continued Study Design, Population, Outcomes, Exposure Assessment and Exposure Metrics Strengths and Limitations and Analytic Strategy 12 cancer sites.  Confidence that the exposure actually occurred  Detailed retrospective exposure assessment, (possible, probable, definite). chemists and industrial hygienists ORs and 95% CIs from unconditional logistic  Frequency of exposure during a normal work responsible for exposure assessments were regression. week (<5%, 5–30%, >30% of the time). blinded to case–control status.  Concentration in the environment (low, medium,  Adjustment for age, family income, ethnic high; relative to certain occupations that were group, cigarette smoking, and respondent used as reference points). status (self or proxy).  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. (generally, authors reported that results were similar). Ever vs never exposed.  Single and multiple (styrene, benzene, toluene, and xylene) exposure models. Cumulative exposure index estimated as the sum over all jobs of the product of duration, frequency, Limitations and concentration. Categorized as low, medium, or  Relatively low prevalence of exposure— high (defined by cut-off points at the 70th and 90th 2% of study population exposed to styrene. percentiles of the distribution of all subjects; medium Era of first exposure: 0.6% before 1950; and high groups were collapsed when numbers were 0.8% during 1950–1960; 0.6% after 1960. small).  Sparse numbers of cases or controls by exposure status for some outcomes. Ruder et al. 2004 Personnel records used to determine departments in Strengths which workers were employed and for which periods  135,588 person-years at risk in the cohort Update of Okun et al. 1985. (no information on job titles). Industrial hygiene during the followup period (through 1998) surveys were conducted to classify jobs and with little loss to followup (n = 72) and few A retrospective-cohort study of 5,204 male departments within plants according to level of styrene participants excluded because of missing and female workers in two US reinforced- exposure (Okun et al. 1985). data (n = 3). plastics boatbuilding plants who worked ≥1 day during 1959–1978.  Latency analysis conducted.

Mortality from all causes and specific causes High-exposure subcohort (n = 2,063) included persons Limitations (including cancers). 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). no information on previous or subsequent based on rates for Washington state and the employment. United States standardized for sex, race, age, Low-exposure subcohort (n = 3,141) included workers  Mean duration of employment for the and calendar period. SMRs reported for the who never worked in fibrous-glass or lamination entire cohort = 1.59 + 3.0 years; mean entire cohort, the high-exposure and low- departments (TWA = 5ppm/day). duration of employment for the high- exposure cohorts, and for workers who were exposure subcohort = 1.10 + 2.1 years and employed for >1 year. Latency (<15 years latency and ≥15 years latency defined as date of first exposure to date of death, date for the low-exposure subcohort = 1.80 + last known alive, or December 31,1998). 3.4 years. Years of employment (for high-exposure group,  No information on smoking, alcohol use, or <1year vs >1 year). other lifestyle factors. Cumulative exposure (5 to <500, ≥500 to <5,000, ≥5,000 ppm*). *The publication reported incorrect units for cumulative exposure. Scélo et al. 2004 In-person interviews on jobs held at ≥1 year using Strengths standardized questionnaires (specialized questionnaires  Large case–control study with prospective A multicenter case–control study in six central were used for jobs and industries likely to entail ascertainment of cases during 1998–2002 and eastern European countries and the United exposures to known or suspected lung carcinogens). from hospitals covering entire population Kingdom (Liverpool). 2,861 newly diagnosed except in Russia (exclusion of Russian cases and 3,118 hospital-based controls that Industrial hygienists at each center evaluated the cases did not alter results). were frequency-matched to cases on age and frequency and intensity of exposure to 70 agents and  Hospital controls excluded cancer and sex. Two centers (Poland and Liverpool) indicated the level of confidence in their assessment. tobacco-related diseases. recruited population-based controls.  Detailed retrospective exposure assessment Duration of exposure (not exposed and 1–6, 7–14, >14 years). with standardized protocols across centers Lung cancer. and industrial hygienists blinded to case– ORs and 95% CIs from unconditional logistic control status. regression. (Continued) 71

72 TABLE 3-1 Continued Study Design, Population, Outcomes, Exposure Assessment and Exposure Metrics Strengths and Limitations and Analytic Strategy Weighted years of exposure (weighted by frequency  Reliability study (of a small number of jobs) of exposure in each job) (not exposed and 0.01–0.50, indicated comparability among expert teams, 0.51–3.00, >3.00). although different levels of misclassification by agent. Cumulative exposure (ppm-years). Frequency and  Adjustment for center, sex, age, tobacco intensity of exposures (2.5, 26, 100 ppm) were based on consumption, vinyl chloride, acrylonitrile, assigned midinterval weightings (2.5%, 17.5%, 65.0%). formaldehyde, and inorganic pigment dust. Categorical analyses were based on tertiles of the  Analyses also conducted with a 20-year lag distribution among exposed controls (subjects never and for jobs with high-confidence exposed made up the referent category). assessments (data not shown in publication). Limitations  Use of hospital-based controls.  Low prevalence of styrene exposure (1.8% of cases and 1.5% of controls). Seidler et al. 2007 Interviewer-administered questionnaire to obtain Strengths information on jobs held for ≥ 1year; start and end  Population-based case–control study with A population-based case–control study of men dates of employment; and job title, industry, and prospective ascertainment of cases and a and women ages 18–80 years living in 6 specific job tasks. For specific occupations, job task- case participation rate of 87.4%. regions in Germany (710 lymphoma patients specific supplementary questions were administered.  Detailed retrospective exposure assessment diagnosed during 1979–1986; 710 controls Industrial physician assessed the intensity and with industrial physician responsible for matched on sex, region, and age [±1 year of frequency of exposure for each job held. exposure assessment blinded to case– birth]). control status.  Intensity of exposure assessed as low (0.5 to 5  High prevalence of styrene exposure in Lymphoma (and lymphoma subentities). ppm), medium (>5 to 50 ppm), and high (>50 ppm). controls (23.8%). ORs and 95% CIs from conditional logistics regression.

 Frequency of exposure assessed as low (1 to 5%),  Adjustment for age, sex, region, smoking medium (>5 to 30%), and high (>30%). (pack years), and alcohol consumption  Confidence of exposure assessment (possible but (g/day) in unmatched analyses and not probable, probable, certain). adjustment for smoking and alcohol consumption in matched analyses. Cumulative exposure (ppm–years). For every job held, the sum of the product of the intensity (2.5 ppm Limitations for “low” intensity; 25 ppm for “medium” intensity;  Relatively low participation rate of controls 100 ppm for “high” intensity), frequency (3% of the (44.3%). time for “low” frequency; 17.5% of the time for  Sparse numbers of cases or controls by “medium” frequency; 65% of the time for “high” exposure status for some outcomes. frequency), and duration of employment in the job. Cumulative exposure was categorized as 0; >0 to ≤1.5; 1.5 to ≤67.1; >67.1 ppm-years. Cocco et al. 2010 In-person interviews on full-time jobs held for ≥1 year; Strengths information on activity of the company, tasks  Large case–control study with high Multicenter case–control study in Czech performed, machines used, and potential exposures participation rates of cases (88%) and Republic, France, Germany, Ireland, Italy, were ascertained. There were14 modules for specific hospital controls (81%), although lower Spain (2,348 incident cases of lymphoma occupations to gather additional details. participation rates of population controls diagnosed during 1998–2004). 2,462 controls (52%). were randomly selected from the general Occupations were coded using the 1968 International  Specific histologic outcomes were studied. population (Germany and Italy) and matched Labour Organisation Standard Classification of  Detailed retrospective exposure to cases by sex, 5-year age intervals, and Occupations and 4-digit codes of the 1996 European assessment. residence areas or selected from hospital Economic Community Classification of Economic  Adjustment for age, sex, education, and controls who were limited to diagnoses other Activities, Revision 1. center. than cancer, infectious diseases, or immune- Intensity of exposure (0 = unexposed, 1 = low,  Correction for multiple comparisons. deficient disease. 2 = medium, 3 = high). Limitations Lymphoma (by subtype). Frequency of exposure (proportion of work time  A relatively low percentage of subjects ORs and 95% CIs from unconditional logistic involving contact with the agent; 0 = unexposed, had styrene exposure assessed with high regression. 1 = 1–5% work time, 2 = 5–30% work time, 3 = >30% confidence (27% of cases and 33% of work time). controls). (Continued) 73

74 TABLE 3-1 Continued Study Design, Population, Outcomes, Exposure Assessment and Exposure Metrics Strengths and Limitations and Analytic Strategy Confidence. Based on the probability of exposure  Possibility for subjects to be occupationally (1 = possible but not probable, 2 = probable, 3 = certain) exposed to multiple chemicals. and proportion of workers exposed in a  No adjustment for smoking. given job (1 = <40%; 2 = 40–90%; 3 = >90%)  Sparse numbers of cases or controls by (low, medium, high). exposure status for some outcomes. 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). Karami et al. 2011 Jobs held ≥1 year (questionnaires were used to ascertain Strengths lifetime occupational histories: job title, tasks, working  Large case–control study with high A hospital-based case–control study with environment, time spent on each task, type of employer, participation rates of cases (90–99%) and controls frequency-matched to cases on age, and starting and ending dates of employment). controls (90% –96%) across study centers, sex, place of residence in seven centers in with additional controls from a study of head central and eastern Europe (1,097 renal-cancer Specialized occupational questionnaires were and neck cancer that likely increased power. cases and 1,476 controls). administered for nine specific jobs and eight industries.  Specific histologic type of renal cancer Renal cell cancer. Industrial hygienists evaluated the frequency and was studied. intensity of exposure to PAHs and “plastics” specific to  Detailed retrospective exposure assessment ORs and 95% CIs from unconditional logistic the dates of employment. They assigned a confidence with industrial hygienists blinded to case– regression. score to their exposure assessment (possible <40%, control status. probable 40–90%, or definite >90% exposure).  Adjustment for sex, age, center, smoking status, self-reported hypertension, body mass Ever vs never exposed. index, and family history of cancer.  Assessment of lag period and sensitivity Duration of exposure (years). analysis (restricted to exposures with a high level of confidence). Cumulative exposure (ppm–years). Product of duration in each job, the midpoint of the frequency of exposure

(3%, 17.5%, 65%), and the intensity weight of the job Limitations (low, 2.5 ppm; medium, 25 ppm; high, 100 ppm)**,  Use of hospital-based controls. summed across all of the subjects’ jobs.  Relatively low prevalence of styrene exposure in cases (2.1%) and controls (1.2%). Average exposure (ppm). Computed by dividing  Additional controls from a study of head cumulative exposure by the number of years exposed. and neck cancer made it difficult to assess ** Weights not specified in Karami et al. (2011); the representativeness of the study. assumed to be the same as those reported in Scélo  Sparse numbers of cases or controls by et al. (2004). exposure status for some exposure metrics. Collins et al. 2013 See Wong et al. (1994) for details about the exposure Strengths assessment.  Long duration of followup (1948–2008) Update of Wong et al. 1994. with 561,530 person–years at risk and little Time since first exposure to styrene (<15 vs ≥15 years). loss to followup (<1%). A retrospective-cohort study of 15,826 male and female workers in 30 reinforced-plastics Cumulative exposure (0–149.9, 150–399.9, 400–  Relatively long duration of exposure (mean facilities in 16 US states who were employed 1,199.9, ≥1,200 ppm–months). = 4.3 years). in areas exposed to styrene for ≥6 months  Proportional hazards models included during 1948–1977. Peak exposure (0, 1–719***, 720–1,799, ≥1,800 days adjustment for sex, year of hire, and year with 100 ppm or higher for 15 min). of birth. Mortality from all causes and specific causes. Average exposure (cumulative exposure divided by Limitations SMRs and 95% CIs: comparisons based on duration; results reported in the text for pancreatic  Exposure not assessed after 1977 (relevant for the US population standardized for sex, age, cancer and diabetes only). 27% of the cohort); average exposure in 1977 time interval. was 25 ppm vs 34 ppm a decade earlier. Duration of exposure (years). Text indicates no  Peak exposure defined as the average Hazard ratios and 95% CIs from proportional- increasing trends for any cause of death and duration number of peaks over 100 ppm for 15 min hazards models (for internal comparisons; of exposure. of a working day with no details provided cumulative exposure only). as to whether monitoring data or expert ***Corrected from publication: “1–179”. judgment was used to construct this exposure metric.  No information on smoking, alcohol use, or other lifestyle factors. 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

76 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens result of the variability across populations, the difficulty in controlling for all potential variables that could influence an observed association or lack thereof, and methods for selecting or retaining individuals in a study and collecting information from them.  At least two informative studies in independent populations or with vary- ing study designs were needed for the committee to consider evidence for a particular cancer outcome to be credible. In the committee’s view, the presence of negative findings in other studies did not negate positive findings. The existence of conflicting findings was one reason why the committee considered the evidence for an association between styrene exposure and carcinogenesis to be limited instead of sufficient. The committee judged the evidence to be limited if the epidemiology evidence was credible but chance, bias, and confounding could not adequately be exclud- ed. The evidence was judged to be sufficient if the epidemiology evidence was credible and chance, bias, and confounding could be excluded as an alternative explanation for the observed association. In addition, the committee considered the implications of traditional statis- tical significance, which is usually thought of in terms of a p value less than 0.05 and the exclusion of 1.0 in the 95% CI around an effect estimate. The committee considered some observations of increased frequency of disease to be informa- tive in smaller studies if they did not reach traditional statistical significance but were consistent with those of other studies. For example, the National Institute for Occupational Safety and Health cohort described by Ruder et al. (2004) in- volved fewer person–years than the Kogevinas et al. (1994), Kolstad et al. (1994), and Wong et al. (1994) cohorts and in some instances found similar SMRs, albeit with wider CIs because of the lower statistical power. 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 expo- sure data in Kolstad et al. (1994); rather, assigned exposure status was based on the proportion of employees in specific plants who were working in the produc- tion of reinforced plastics. Furthermore, as in most occupational studies, it is possible that the “healthy-worker effect” influenced observations of the cohort studies in ways that cannot be determined. The committee also looked at the presence or absence of isolated associa- tions. Each of the six cohort studies that the committee determined to be most informative included a multitude of statistical analyses of exposures and cancer outcomes, as is typical in occupational cohort studies. Similarly, many different comparisons were conducted in the five case–control studies that were judged informative by the committee, often involving different exposure metrics and specific types of cancer. Because of issues potentially related to multiple com- parisons, associations that appeared to be isolated among the multiple studies were not judged to constitute evidence of human carcinogenicity. However, the committee notes that nondifferential exposure misclassification and other data errors that are independent of exposure or disease status tend to result in an at-

Independent Assessment of Styrene 77 tenuation of observed relative risks and its surrogates; therefore, any strong as- sociations repeatedly found in informative studies need to be given more weight. Findings on Different Types of Cancers In this section, the committee describes specific findings on cancers of the lymphohematopoietic system, kidney, pancreas, and esophagus. Tables 3-2 through 3-8 and the pages that follow present the salient observations in those studies on specific types of cancer. Lymphohematopoietic Cancers Combined As discussed in Chapter 2, the definition of lymphohematopoietic neoplasm has advanced in recent decades. Some of the advance has come from recognition of subtypes that were previously lumped together, some from reclassification of subtypes, and some from revisions of knowledge and capabilities for classification of the broad array of lymphohematopoietic cancers. Recognizing that the grouping of “all lymphohematopoietic cancers” includes many biologically distinct diagno- ses in humans (NRC 2011), the committee has adopted a general approach of fo- cusing on more detailed classification when it is feasible but using broader catego- ries when needed. When detailed classification is possible, looking at the finer categories may reveal specific associations if an effect is present in some subcate- gories but not others. Thus, the committee discussions below begin with the broadest classification of lymphohematopoietic cancers and follow with more de- tailed classifications. The epidemiologic data provide credible but limited evi- dence that styrene is a risk factor for lymphohematopoietic cancers on the basis of two European cohort studies (Kogevinas et al. 1994; Kolstad et al. 1994), as the role of chance, bias, or confounding cannot be adequately excluded. Kogevinas et al. (1994) studied 40,688 workers in Denmark, Finland, Ita- ly, Norway, Sweden, and the United Kingdom and followed up in various coun- tries during 1945–1991 (539,479 person–years and an average duration of fol- lowup of 13 years). The SMR for lymphohematopoietic cancers combined was 0.93 (95% CI 0.71–1.20, 60 deaths) (see Table 3-2). Their internal analysis, which used data obtained from workers within the study (instead of an external standard population) and compared workers with different levels of styrene ex- posure to each other, showed that a longer time since first exposure (at least 10 years vs less than 10 years) was associated with a significantly higher mortality due to combined lymphohematopoietic cancers (10–19 years: MRR = 2.90, 95% CI 1.29–6.48, 25 deaths; at least 20 years: MRR = 3.97, 95% CI 1.30–12.13, nine deaths; p value for the test of linear trend = 0.012). Compared with workers who had an average exposure of less than 60 ppm (seven deaths), the MRRs for those who had an average exposure of 60–99 ppm, 100–119 ppm, 120–199 ppm, and at least 200 ppm were 1.68 (95% CI 0.59–4.79, nine deaths), 3.11 (95% CI 1.07–9.06, 10 deaths), 3.08 (95% CI 1.04–9.08, 13 deaths), and 3.59 (95% CI

78 TABLE 3-2 Summary of Observations for Lymphohematopoietic Cancers Combined Reference Observations (95% CI) Reinforced-Plastics Industry Cohorts Kogevinas et al. 1994 Full study cohort: SMR = 0.93 (0.71–1.20), n = 60 Subgroups by job category: Laminators: SMR = 0.81 (0.43–1.39), n = 13 Unspecified task: SMR = 1.19 (0.80–1.70), n = 30 Other exposed jobs: SMR = 0.65 (0.26–1.34), n = 7 Unexposed: SMR = 0.91 (0.41–1.72), n = 9 Cumulative exposure (ppm–years): <75 as reference: n = 20 75–199: MRR = 0.98 (0.43–2.26), n = 8 200–499: MRR = 1.24 (0.57–2.72), n = 10 ≥ 500: MRR = 0.84 (0.35–2.02), n = 9 p for trend = 0.65 Time since first exposure (years): <10 as reference: n = 13 10–19: MRR = 2.90 (1.29–6.48), n = 25 ≥20: MRR = 3.97 (1.30–12.13), n = 9 p for trend = 0.012 Average exposure (ppm): <60 as reference: n = 7 60–99: MRR = 1.68 (0.59–4.79), n = 9 100–119: MRR = 3.11 (1.07–9.06), n = 10 120–199: MRR = 3.08 (1.04–9.08), n = 13 ≥200: MRR = 3.59 (0.98–13.14), n = 8 p for trend = 0.019 Kolstad et al. 1994 Full study cohort: SIR = 1.20 (0.98–1.44), n = 112 Employees of companies with 1–49% reinforced-plastics workers: SIR = 1.24 (0.99–1.54), n = 81 Employees of companies with 50–100% reinforced plastics workers: SIR = 1.09 (0.74–1.55), n = 31

Year of first employment: 1964–1970: SIR = 1.32 (1.02–1.67), n = 6 1971–1975: SIR = 1.12 (0.75–1.62), n = 28 1976–1988: SIR = 0.97 (0.57–1.53), n = 18 Time since first employment ≥10 years: Overall: SIR = 1.20 (0.92–1.53), n = 64 Duration of employment <1 year: SIR = 1.65 (1.18–2.26), n = 39 Duration of employment ≥1 year: SIR = 0.84 (0.54–1.24), n = 25 Wong et al. 1994 Full study cohort (CIs not reported): SMR = 0.82 (0.56–1.17), n = 31 Subgroups by latency (that is, time since first exposure in years) (CIs not reported): <10: SMR = 0.81, n = 9 10–19: SMR = 0.66, n = 10 ≥20: SMR = 1.04, n = 12 Subgroups by cumulative exposure (ppm–years) (CIs not reported): <10: SMR = 1.05, n = 9 10–29.9: SMR = 0.56, n = 5 30–99.9: SMR = 0.76, n = 8 ≥100: SMR = 0.94, n = 9 Employed for 2 years by processing category (CIs not reported): 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). (Continued) 79

80 TABLE 3-2 Continued Reference Observations (95% CI) Collins et al. 2013 Full study cohort: SMR = 0.84 (0.69–1.02), n = 106 Latency ≥15 years: SMR = 0.87 (0.70–1.07), n = 93 Subgroups by cumulative exposure (ppm–months): 0–149.9: SMR = 0.85 (0.56–1.25), n = 26 150–399.9: SMR = 0.80 (0.51–1.21), n = 23 400–1199.9: SMR = 0.90 (0.60–1.29), n = 29 ≥1,200: SMR = 0.80 (0.53–1.16), n = 28 Cumulative exposure (ppm–months) p for trend = 0.819, hazard ratio = 0.994 (0.983–1.006) Ruder et al. 2004 Full study cohort: SMR = 0.74 (0.42–1.20), n = 16 High exposure: SMR = 0.72 (0.20–1.84), n = 4 Reference population = Low exposure: SMR = 0.74 (0.38–1.30), n = 12 Washington state Workers who were employed 1 year: Overall: SMR = 0.54 (0.17–1.26), n = 5 High exposure: SMR = 0.56 (0.01–3.09), n = 1 Low exposure: SMR = 0.53 (0.15–1.37), n = 4 High exposure by duration of employment: <1 year: SMR = 0.76 (0.15–2.50), n = 3 ≥1 year: SMR = 0.58 (0.01–4.70), 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.

Independent Assessment of Styrene 81 0.98–13.14, eight deaths), respectively, with a p value of 0.019 for the test of linear trend. 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. 1994). The study by Kolstad et al. (1994) included 36,525 male workers who were employed in 386 reinforced-plastics plants in Denmark during 1964–1988 and 14,254 employees of similar industries who were not exposed to styrene, with followup from 1970 through 1989 (584,556 person–years and an average duration of followup of 10.9 years). Between this study and the one by Kogevi- nas et al. (1994), there was an overlap of 12,837 male workers who were em- ployed in 287 Danish plants where more than 50% of the workforce manufac- tured reinforced plastics. Kolstad et al. (1994) found that the SIR for combined lymphohematopoietic cancers in workers in companies that produced reinforced plastics was 1.20 (95% CI 0.98–1.44, 112 observed cases) (see Table 3-2). When the analysis was stratified by year of first employment, those who were first employed during 1964–1970 had a significantly higher incidence of com- bined lymphohematopoietic cancers (SIR = 1.32, 95% CI 1.02–1.67, 6 cases), whereas the SIR was 1.12 (95% CI 0.75–1.62, 28 cases) for those first employed during 1971–1975 and 0.97 (95% CI 0.57–1.53, 18 cases) for those first em- ployed during 1976–1988. That observation is consistent with a possible expo- sure–response relationship, inasmuch as 2,473 historical personal air samples from the cohort showed that average styrene concentrations decreased from 180 ppm in 1964–1970 to 43 ppm in 1976–1988 (Jensen et al. 1990). Workers em- ployed for less than 1 year and with at least 10 years since first employment had a significantly higher incidence than the standard population (SIR = 1.65, 95% CI 1.18–2.26, 39 cases). The SIR in those who were employed for at least 1 year with at least 10 years since first employment was not increased. The phenome- non of high disease frequency in short-term workers is a frequent finding in oc- cupational cohort studies of cancer outcomes. While Kolstad et al. (1994) acknowledged that it was possible for short-term workers to have carcinogenic exposures in other industries or less favorable lifestyle factors, they considered it “less likely since a comparison between the exposed and unexposed short-term employees does not yield lower ratios for leukemia” (p. 277). An internal analy- sis with Poisson regression was also conducted, but no details were provided except for a statement that the rate ratios were close to the results presented (Kolstad et al. 1994). Wong et al. (1994) studied a cohort of 15,826 male and female employees who were exposed to styrene for at least 6 months during 1948–1977 in 30 par- ticipating reinforced-plastics manufacturing plants in the United States and in- cluded followup through 1989 (307,932 person–years). The study observed an SMR of 0.82 (95% CI 0.56–1.17, 31 deaths) for lymphohematopoietic cancers combined in the overall cohort (see Table 3-2). Additional subgroup analyses by latency, duration of employment, duration of exposure to styrene, cumulative styrene exposure (ppm–years), and latency and cumulative styrene exposure simultaneously did not suggest an association between styrene exposure and

82 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens mortality due to lymphohematopoietic cancers. An internal analysis with Cox proportional hazard regression—including age, sex, cumulative exposure, and duration of exposure to styrene as independent variables—did not support an association either (Wong et al. 1994). The study by Collins et al. (2013) is an extension of the Wong et al. (1994) cohort with an additional 19 years of followup (through 2008) and a total number of 561,530 person–years. The SMR for lymphohematopoietic cancers combined was 0.84 (95% CI 0.69–1.02, 106 deaths). Additional analyses by latency, cumulative exposure (ppm–months), number of peak exposures, cumu- lative duration, and average exposure and an internal analysis did not indicate an association between occupational styrene exposure and mortality due to lym- phohematopoietic cancers (Collins et al. 2013). The study by Ruder et al. (2004) included 5,204 workers exposed to styrene during 1959–1978 in two reinforced-plastic boatbuilding plants in Washington state and followup through 1998 (135,707 person–years). Using the Washington state population as the standard, the investigators observed an SMR of 0.74 (95% CI 0.42–1.20, 16 deaths) for lymphohematopoietic cancers combined (see Table 3- 2). The SMRs for people who had high and low exposures were 0.72 (95% CI 0.20–1.84, four deaths) and 0.74 (95% CI 0.38–1.30, 12 deaths), respectively. An additional analysis by duration of employment and another analysis focusing on workers who were employed for over 1 year did not support an association be- tween styrene exposure and mortality due to lymphohematopoietic cancers com- bined. However, the cohort had a smaller sample and smaller number of person– years of followup than the other studies discussed here. The total number of deaths due to lymphohematopoietic cancers combined was only five in workers who were employed for more than 1 year (Ruder et al. 2004). Studies of specific types of lymphohematopoietic cancer generate SMRs, SIRs, and MRRs with wider CIs because of the smaller number of observed events (cancer incidence or deaths). Findings on leukemia and non-Hodgkin lymphoma (NHL) are discussed below. Because Hodgkin lymphoma and multi- ple myeloma are rare and there is a paucity of data from existing studies, the committee concludes that there are insufficient data to assess whether exposure to styrene is associated with the frequency of these two malignancies. Leukemia The epidemiologic data provide credible but limited evidence that styrene exposure is associated with an increase in the frequency of leukemia on the basis of two European cohort studies (Kogevinas et al. 1994; Kolstad et al. 1994), as the role of chance, bias, or confounding cannot be adequately excluded. As re- ported by Kogevinas et al. (1994), workers in the reinforced-plastics industry who had a higher average exposure to styrene or a longer time since first expo- sure appeared to have a higher probability of dying from leukemia although none of the MRRs reached statistical significance (see Table 3-3). In the Danish cohort (Kolstad et al. 1994), workers who were first employed during 1964–

TABLE 3-3 Summary of Observations for Leukemia Reference Observations (95% CI) Reinforced-Plastics Industry Cohorts Kogevinas et al. 1994 Full study cohort: SMR = 1.04 (0.69–1.50), n = 28 Subgroups by job category: Laminators: SMR = 0.48 (0.10–1.39), n = 3 Unspecified task: SMR = 1.40 (0.79–2.28), n = 16 Other exposed jobs: SMR = 0.94 (0.26–2.40), n = 4 Unexposed: SMR = 0.99 (0.27–2.54), n = 4 Cumulative exposure (ppm–years): <75 as reference: n = 11 75–199: MRR = 0.46 (0.10–2.09), n = 2 200–499: MRR = 0.69 (0.19–2.53), n = 3 ≥500: MRR = 0.86 (0.26–2.83), n = 5 p for trend > 0.52 Time since first exposure (years): <10 as reference: n = 5 10–19: MRR = 3.01 (0.90–10.08), n = 12 ≥20: MRR = 3.79 (0.70–20.59), n = 4 p for trend = 0.094 Average exposure (ppm) <60 as reference: n = 3 60–99: MRR = 1.58 (0.32–7.79), n = 4 100–119: MRR = 4.43 (0.98–20.03), n = 8 120–199: MRR = 1.36 (0.22–8.48), n = 3 ≥200: MRR = 2.16 (0.29–16.24), n = 3 p for trend = 0.47 (Continued) 83

84 TABLE 3-3 Continued Reference Observations (95% CI) Kolstad et al. 1994 Full study cohort : SIR = 1.22 (0.88–1.65), n = 42 Employees of companies with 1–49% reinforced plastic workers: SIR = 1.15 (0.77–1.67), n = 28 Employees of companies with 50–100% reinforced plastics workers: SIR = 1.38 (0.75–2.32), n = 14 Year of first employment: 1964–1970: SIR = 1.54 (1.04–2.19), n = 30 1971–1975: SIR = 1.00 (0.46–1.90), n = 9 1976–1988: SIR = 0.51 (0.11–1.50), n = 3 Time since first employment ≥10 years: Overall: SIR = 1.57 (1.07–2.22), n = 32 Duration of employment <1 year: SIR = 2.34 (1.43–3.61), n = 20 Duration of employment ≥1 year: SIR = 1.01 (0.52–1.77), n = 12 Wong et al. 1994 Full study cohort: SMR = 0.74 (0.37–1.33), n = 11 Subgroups by latency (time since first exposure in years) (CIs not reported): <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): <10: SMR = 0.30, n = 1 10–29.9: SMR = 1.12, n = 4 30–99.9: SMR = 0.73, n = 3 ≥100: SMR = 0.80, n = 3 Employed for 2 years by processing category (CIs not reported): Open-mold processing: SMR = 0.90, n = 1 Mixing and closed-mold processing: n = 0 Finish and assembly: SMR = 0.80, n = 2

Plant office and support: SMR = 0.56, n = 1 Maintenance and preparation: SMR = 0.48, n= 1 Supervisory and professional: SMR = 1.33, n = 1 In proportional-hazard models, cumulative exposure and duration of exposure to styrene were not significant (n = 11). Collins et al. 2013 Full study cohort: SMR = 0.84 (0.60–1.14), n = 40 Latency ≥15 years: SMR = 0.88 (0.61–1.22), n = 35 Subgroups by cumulative exposure (ppm–months): 0–149.9: SMR = 0.61 (0.25–1.26), n = 7 150–399.9: SMR = 1.30 (0.71–2.18), n = 14 400–1,199.9: SMR = 0.66 (0.28–1.30), n = 8 ≥1,200: SMR = 0.83 (0.42–1.49), n = 11 Cumulative exposure (ppm–months) p for trend = 0.908, hazard ratio = 0.996 (0.979–1.014) Ruder et al. 2004 Full study cohort: SMR = 0.60 (0.19–1.40), n = 5 High exposure: SMR = 0.47 (0.01–2.63), 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. Source: committee-generated. 85

86 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens 1970 had a significantly higher incidence of leukemia (SIR = 1.54, 95% CI 1.04–2.19, 30 cases) whereas the SIRs for workers first employed after 1970 were not above 1 (see Table 3-3). Given the substantial change in the concentra- tion of styrene exposure in this cohort over time (Jensen et al. 1990), such a finding is consistent with a possible exposure–response relationship. In addition, workers who had more than 10 years of followup since their first employment in a participating plant also had a significantly higher incidence of leukemia (SIR =1.57, 95% CI 1.07–2.22, 32 cases) although the observed higher leukemia inci- dence was limited to short-term workers whose duration of employment was less than 1 year (SIR = 2.34, 95% CI 1.43–3.61, 20 cases) (Kolstad et al. 1994). The findings by Wong et al. (1994) and Collins et al. (2013) did not sup- port a leukemogenic role of styrene. The SMR for leukemia was 0.74 (95% CI 0.37–1.33, 11 deaths) and 0.84 (95% CI 0.60–1.14, 40 deaths), respectively, in the studies by Wong et al. (1994) and Collins et al. (2013). Additional analyses by latency, duration of exposure, and cumulative exposure also did not suggest an association between styrene and leukemia in these two studies. There were only five deaths due to leukemia in the study by Ruder et al. (2004). The SMR for leukemia was 0.60 (95% CI 0.19–1.40) when the Wash- ington state population was used as the standard for the overall cohort and simi- lar for people who had high or low exposures. No additional analysis was con- ducted, probably because of the small number of deaths. Non-Hodgkin Lymphoma The epidemiologic data provide credible but limited evidence that styrene exposure is a risk factor for NHL on the basis of a cohort study (Kogevinas et al. 1994) and two case–control studies (Gerin et al. 1998; Cocco et al. 2010), as the role of chance, bias, or confounding cannot be adequately excluded. In the study by Kogevinas et al. (1994), the SMR for NHL was 0.77 (95% CI 0.43–1.28, 15 deaths) for the general cohort and 1.40 (95% CI 0.56–2.88, seven deaths) for laminators, who were expected to have greater exposure to styrene. Workers who had a higher average exposure to styrene or a longer time since first expo- sure consistently had higher mortality due to malignant lymphomas (the term probably meant both Hodgkin lymphoma and NHL), as reflected by MRRs in the range of 1.65–7.15 although only one of the six MRRs reached statistical significance (see Table 3-4). The p values for the test of linear trend were 0.052 and 0.072 for average exposure and time since first exposure, respectively. In the Kolstad et al. (1994) study, the SIR for NHL was 1.33 (95% CI 0.96–1.80, 42 cases) (see Table 3-4). The SIRs for different periods of first employment were all above 1 but imprecise in that all 95% CIs included 1 (Kolstad et al. 1994). The study by Wong et al. (1994) listed a total of four deaths due to lym- phosarcoma and reticulosarcoma in Table 2 of the publication but 10 deaths due to NHL in Table 9. It is unclear which subtypes of hematopoietic cancers were

TABLE 3-4 Summary of Observations for Non-Hodgkin Lymphoma Reference Observations (95% CI) Reinforced-Plastics Industry Cohorts Kogevinas et al. 1994 Full study cohort: SMR = 0.77 (0.43–1.28), n = 15 Subgroups by job category: Laminators: SMR = 1.40 (0.56–2.88), n = 7 Unspecified task: SMR = 0.55 (0.15–1.39), n = 4 Other exposed jobs: SMR = 0.30 (0.01–1.67), n = 1 Unexposed: SMR = 1.01 (0.21–2.94), n = 3 Time since first exposure (years): <10: SMR = 0.51 (0.11–1.49), n = 3 10–19: SMR = 0.76 (0.25–1.78), n = 5 ≥20: SMR = 1.55 (0.42–3.97), n = 4 Duration of exposure (years): <2: SMR = 0.60 (0.19–1.40), n = 5 ≥2: SMR = 1.05 (0.42–2.17), n = 7 Malignant lymphomas (probably include both NHL and Hodgkin lymphoma) Average exposure (ppm): <60 as reference: n = 3 60–99: MRR = 2.51 (0.49–12.87), n = 4 100–119: MRR = 1.65 (0.15–18.57), n = 1 120–199: MRR = 7.15 (1.21–42.11), n = 8 ≥200: MRR = 4.40 (0.42–45.99), n = 2 p for trend = 0.052 Kolstad et al. 1994 Full study cohort: SIR = 1.33 (0.96–1.80), n = 42 Employees of companies with 1–49% reinforced-plastic workers: SIR = 1.65 (1.15–2.28), n = 36 Employees of companies with 50–100% reinforced-plastics workers: SIR = 0.62 (0.23–1.35), n = 6 (Continued) 87

88 TABLE 3-4 Continued Reference Observations (95% CI) Year of first employment: 1964–1970: SIR = 1.28 (0.79–1.96), n = 21 1971–1975: SIR = 1.19 (0.57–2.18), n = 10 1976–1988: SIR = 1.64 (0.82–2.94), n = 11 Time since first employment ≥10 years: Overall: SIR = 1.12 (0.69–1.70), n = 21 Duration of employment <1 year: SIR = 1.27 (0.63–2.28), n = 11 Duration of employment ≥1 year: SIR = 0.98 (0.47–1.81), n = 10 Wong et al. 1994 It is unclear how many deaths due to NHL were included in this study. Table 9 of the publication indicated 10, but earlier tables did not show this. In proportional-hazard models, cumulative exposure and duration of exposure to styrene were not significant (n = 10). Collins et al. 2013 Full study cohort: SMR = 0.72 (0.50–1.00), n = 36 Latency ≥15 years: SMR = 0.75 (0.52–1.06), n = 33 Subgroups by cumulative exposure (ppm–months): 0–149.9: SMR = 1.08 (0.58–1.85), n = 13 150–399.9: SMR = 0.17 (0.02–0.64), n = 2 400–1,199.9: SMR = 0.94 (0.49–1.64), n = 12 ≥1,200: SMR = 0.65 (0.30–1.23), n = 9 Cumulative exposure (ppm–months) p for trend = 0.766, hazard ratio = 0.994 (0.976–1.013) Ruder et al. 2004 Referred to as lymphosarcoma and reticulosarcoma, not NHL Reference population = Full study cohort: SMR = 0.39 (0.01–2.19), n = 1 Washington state High exposure: n = 0 Low exposure: SMR = 0.53 (0.01–2.93), n = 1

Case–Control Studies Gerin et al. 1998 NHL, not otherwise specified. 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. Seidler et al. 2007 B-cell NHL (ppm-years): >0 to ≤1.5: adjusted OR = 0.8 (0.6–1.2), number of exposed cases = 53; unadjusted OR = 0.8 >1.5 to ≤67.1: adjusted OR = 1.2 (0.8–1.7), number of exposed cases = 62; unadjusted OR = 1.2 >67.1: adjusted OR = 0.8 (0.4–1.8), number of exposed cases = 12; unadjusted OR = 0.9 Test for trend: p = 0.18 T-cell NHL (ppm-years): >0 to ≤1.5: adjusted OR = 1.3 (0.5–3.6), number of exposed cases = 6; unadjusted OR = 1.7 >1.5 to ≤67.1: adjusted OR = 1.6 (0.5–4.8), number of exposed cases = 4; unadjusted OR = 1.4 Test for trend: p = 0.41 Large diffuse B-cell lymphoma (ppm-years): >0 to ≤1.5: adjusted OR = 0.8 (0.4–1.5), number of exposed cases = 15; unadjusted OR = 0.8 >1.5 to ≤67.1: adjusted OR = 1.3 (0.7–2.3), number of exposed cases = 19; unadjusted OR = 1.3 >67.1: adjusted OR = 1.5 (0.5–4.4), number of exposed cases = 5; unadjusted OR = 1.4 Test for trend: p = 0.03 Follicular lymphoma (ppm-years): > 0 to ≤1.5: adjusted OR =1.1 (0.5–2.1), number of exposed cases = 12; unadjusted OR = 1.3 > 1.5 to ≤ 67.1: adjusted OR = 2.2 (1.2–4.0), number of exposed cases = 17; unadjusted OR = 2.3 >67.1: adjusted OR = 1.6 (0.5–6.0), number of exposed cases = 3; unadjusted OR = 1.6 Test for trend: p = 0.20 Chronic lymphocytic leukemia (ppm-years): > 0 to ≤1.5: adjusted OR = 1.0 (0.5–2.2), number of exposed cases = 10; unadjusted OR = 0.8 > 1.5 to ≤ 67.1: adjusted OR = 1.1 (0.5–2.2), number of exposed cases = 11; unadjusted OR = 1.1 >67.1: adjusted OR = 0.5 (0.2–2.3), number of exposed cases = 2; unadjusted OR = 0.8 Test for trend: p = 0.37 (Continued) 89

90 TABLE 3-4 Summary of Observations for Non-Hodgkin Lymphoma Reference Observations (95% CI) Marginal-zone lymphoma (ppm-years): > 0 to ≤1.5: adjusted OR = 1.0 (0.3–3.0), number of exposed cases = 4; unadjusted OR = 0.8 > 1.5 to ≤ 67.1: adjusted OR = 0.8 (0.2–2.6), number of exposed cases = 3; unadjusted OR = 0.8 Test for trend: p = 0.28 All analyses used “no exposure, i.e., cumulative exposure = 0” as the reference group. All ORs adjusted for age, sex, region, smoking, and alcohol consumption. Cocco et al. 2010 B-cell NHL: Ever exposed to styrene occupationally: Adjusted OR = 1.6 (1.1–2.3), 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: committee- generated.

Independent Assessment of Styrene 91 considered NHL. In the proportional-hazard model that included 10 NHL deaths (Table 9 of the publication), duration of styrene exposure and cumulative expo- sure to styrene were not associated with mortality due to NHL (Wong et al. 1994). In the study by Collins et al. (2013), the SMR for NHL was 0.72 (95% CI 0.50–1.00, 36 deaths). Additional analyses by latency, cumulative exposure (ppm–months), number of peak exposures, cumulative duration, and average exposure and an internal analysis that used Cox proportional-hazard regression did not suggest an association between occupational styrene exposure and mor- tality due to NHL. The cohort study by Ruder et al. (2004) had only one ob- served death due to lymphosarcoma and reticulosarcoma and did not use the term non-Hodgkin lymphoma. The SMR for lymphosarcoma and reticulosar- coma was 0.39 (95% CI 0.01–2.19) when the Washington state population was used as the standard. No additional analysis was conducted. Gerin et al. (1998) conducted a population-based case–control study in Montreal, Canada, that ascertained newly diagnosed incident cancer cases from 19 sites between 1979 and 1986 and population controls. For cases with a spe- cific type of cancer (for example, 215 cases of NHL), three different control groups were used: cancer controls who had cancers other than NHL (n = 2,341), population controls (n = 533), and pooled controls (n = 1,066) that consisted of 533 cancer controls randomly selected from the total of 2,341 cancer controls and 533 population controls. All three control groups were used for comparisons with cases, but most of the findings presented by the authors were derived from the comparisons between cases and pooled controls. Based on a logistic regres- sion model adjusted for age, family income, ethnic group, cigarette smoking, and respondent status, subjects who had occupational exposure to styrene ap- peared to have a higher odds of NHL than those who were not occupationally exposed to styrene (adjusted odds ratio [OR] = 2.0), although the association did not reach statistical significance (95% CI 0.8–4.8). It should be noted that the crude, unadjusted OR was 2.1, which was very close to the adjusted OR derived from the model that controlled for multiple covariates. A population-based case–control study of lymphoma was conducted in six regions of Germany during 1998–2003 (Becker et al. 2004). Seidler et al. (2007) analyzed the relationship between solvent exposure and malignant lymphoma in the Becker et al. (2004) study, which included 554 incident cases of B-cell NHL and 35 incident cases of T-cell NHL who were diagnosed at the age of 18–80 years and an equal number of gender-, region-, and age-matched population con- trols (Seidler et al. 2007). Using job task-specific supplementary questionnaires, a trained industrial physician assessed the exposure to styrene and other sol- vents. The intensity and frequency of exposure to styrene were categorized semi-quantitatively as low, medium, and high, and a cumulative exposure was calculated by incorporating duration, intensity, and frequency of exposure. Compared with subjects without occupational styrene exposure, those who had varying levels of cumulative exposure to styrene had similar frequencies of B- cell NHL and T-cell NHL based on an unconditional logistic regression model that adjusted for age, sex, region, smoking, and alcohol consumption (Table 3-

92 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens 4). Additional analyses by specific subtypes of B-cell NHL produced similar findings in general, although the trend test for diffuse large B-cell lymphoma (n = 158) was significant (p = 0.03), and elevated ORs for follicular lymphoma were observed for subjects with higher levels of cumulative exposure to styrene (Table 3-4). It should be noted that multiple myeloma was included in this study as a subtype of B-cell NHL, while it is usually considered a separate entity and a distinct type of cancer by itself. Cocco et al. (2010) conducted a multicenter case–control study of lym- phomas in the Czech Republic, France, Germany, Ireland, Italy, and Spain from 1998 to 2004. The study included 1,127 cases of B-cell NHL; 66 of the cases had occupational exposure to styrene. Three independent exposure metrics were used: intensity, frequency, and duration of exposure. Statistical analyses adjust- ed for age, sex, education, and center. Compared with people who had no expo- sure to styrene in an occupational setting, those who were exposed to styrene at work had a higher odds of B-cell NHL (adjusted OR = 1.6, 95% CI 1.1–2.3). The test for trend by increasing levels of the three independent exposure metrics yielded a p value of 0.000096, which was lower than a preset p value of 0.000125 chosen by the authors as the threshold for rejecting the null hypothe- sis. The authors chose a lower threshold than the usual 0.05 to account for mul- tiple comparisons. Supplementary tables available online showed significant trends with frequency and duration of exposure to styrene (p = 0.04 and p = 0.03, respectively) (Cocco et al. 2010). It should be noted that subjects exposed to styrene could have been exposed to other solvents, but of the different sub- groups of solvents evaluated in this study, exposure to styrene showed the high- est OR of 1.6, with the ORs for exposure to other groups of solvents ranging from 1.0 to 1.2. In addition, the list of covariates adjusted for in Cocco et al. (2010) was not as extensive as in Gerin et al. (1998) and Seidler et al. (2007), but the unadjusted and adjusted ORs in the latter studies were very close for NHL (Gerin et al. 1998) or B-cell NHL (Seidler et al. 2007) (Table 3-4), which suggests that the covariates adjusted for did not have a strong confounding ef- fect. Overall, the findings of Cocco et al. (2010) are consistent with an associa- tion between occupational exposure to styrene and B-cell NHL. 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. The US study of Wong et al. (1994) found a kidney-cancer SMR of 1.75 (95% CI 0.98–2.89); the strongest association was in workers who had been exposed for at least 2 years to open-mold processing (SMR = 4.57, CI not given, three cases). Collins et al. (2013) published an update of the Wong et al. (1994) study with about twice as many person–years of followup. The authors analyzed cumulative exposures (ppm–months), duration of exposure,

TABLE 3-5 Summary of Observations for Kidney Cancer Reference Observations (95% CI) Reinforced-Plastics Industry Cohorts Kogevinas et al. 1994 Full study cohort: SMR = 0.77 (0.44–1.25), n=16 Industrial process SMRs (95% CI) Laminators: 0.90 (0.25–2.32), n = 4 Unspecified task: 0.75 (0.30–1.54), n = 7 Other exposed jobs: 0.29 (0.01–1.61), n = 1 Unexposed: 0.69 (0.08–2.51), n = 2 Cumulative exposure (ppm–years): <75 reference: n=2 100–199: MRR = 4.40 (0.71–27.2), n = 3 200–499: MRR = 3.30 (0.42–25.6), n = 2 ≥500: MRR = 6.04 (0.74–49.5), n = 3 trend p = 0.12 Wong et al. 1994 Full study cohort: SMR= 1.75 (0.99–2.89), n = 15 SMRs by latency (years) (CIs not reported): <10: 1.67, n = 3 10–19: 1.41, n = 5 ≥20: 2.18, n = 7 SMRs by duration of exposure to styrene (years) (CIs not reported): <1: 1.89, n = 3 1–1.9: 1.96, n = 3 2–2.9: 1.51, n = 3 5–9.9: 1.26, n = 2 ≥10: 2.15, n = 4 SMRs by cumulative styrene exposure (ppm–years) (CIs not reported): <10: 0.54, n = 1 10–29.9: 2.06, n = 4 30–99.9: 1.67, n = 4 ≥100: 2.55, n = 6 (Continued) 93

94 TABLE 3-5 Continued Reference Observations (95% CI) SMRs employed 2 years in processing categories (CIs not reported): Open-mold processing: 4.57, n = 3 Mixing and closed-mold processing: n = 0 Finish and assembly: 1.94, n = 3 Plant office and support: 1.72, n = 2 Maintenance and preparation: 2.14, n = 3 Supervisory and professional: 1.85, n = 1 Proportional-hazard models, cumulative exposure, duration of exposure to styrene were not significant. Kolstad et al. 1995 Full study cohort: SIR = 0.93 (0.65–1.28), n = 37 Ruder et al. 2004 Full study cohort: SMR = 1.43 (0.57–2.95), n = 7 High exposure: SMR = 3.60 (0.98–9.20), n = 4 reference population = Low exposure: SMR = 0.80 (0.16–2.33), n = 3 Washington state SMRs for those employed >1 year: Total: 1.38 (0.28–4.04), n = 3 High exposure: 5.11 (0.62–18.4 ), n = 2 Low exposure: 0.56 (0.01–3.12), n = 1 SMRs for duration of exposure in high-exposure department (year) <1: 2.35 (0.26–10.2), n = 2 >1: 4.91 (0.55–21.3), n = 2 Collins et al. 2013 Total cohort: SMR = 1.18 (0.83–1.62), n = 38 ≥15 years latency: SMR= 1.18 (0.82–1.65), n = 34 Cumulative exposure SMRs (ppm–months): 0.0–149.9: 0.76 (0.28–1.66), n = 6 150–399.9: 1.09 (0.47–2.15), n = 8 400–1,199.9: 0.98 (0.42–1.94), n = 8 ≥1,200: 1.79 (1.02–2.91), n = 16 Test for trend: p = 0.045 Proportional-hazard model: hazard ratio for styrene exposure = 1.009 (1.000–1.017) SMR (days with ≥15 min of styrene at >100 ppm):

0: 0.88 (0.50–1.42), n = 16 1–719*: 1.08 (0.49–2.04), n = 9 720–1,799: 2.73 (1.17–5.38), n = 8 ≥1,800: 1.82 (0.59–4.24), n = 5 Test for trend: p = 0.054 *Corrected from publication: “1–179”. Case–Control Studies Gerin et al. 1998 Kidney cancer: all histologies combined Ever exposed: Adjusted OR = 0.3 (0.0-2.0), n=1; unadjusted OR = 0.3 Adjusted for age, family, income, ethnic group, cigarette smoking, and respondent status. Karami et al. 2011 Renal-cell carcinoma All exposed cases: Adjusted OR = 1.7 (0.8–3.6), n = 17; unadjusted OR = 1.76 Cumulative exposure** (years x frequency x ppm): ≤1.40: adjusted OR = 0.56 (0.19-1.67), n = 5; unadjusted OR = 0.66 >1.40: adjusted OR = 6.65 (1.82–24.27), n = 12; unadjusted OR = 5.79 Average exposure** (frequency x ppm): ≤0.175:adjusted OR = 1.09 (0.41–2.93), n = 8; unadjusted OR = 1.29 >0.175: adjusted OR = 3.05 (0.99–9.42), n = 9; unadjusted OR = 2.61 Duration of exposure** (years): ≤10:adjusted OR = 1.26 (1.47–3.39), n = 8; unadjusted OR = 1.29 >10: adjusted OR = 2.57 (0.83–7.95), n = 9; unadjusted OR = 2.61 Adjusted for center, sex, age, body-mass index, self-reported hypertension, smoking status (ever/never), and family history of cancer. **OR obtained from S. 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. Source: committee-generated. 95

96 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens average exposure, and number of days with at least 15 min exceeding 100 ppm (“number of peak exposure days”) and calculated proportional hazard ratios. Alt- hough the SMR in the combined cohort of Collins et al. (2013) was 1.18 (95% CI 0.83–1.62), the authors observed an increased and positive association between styrene exposure and kidney cancer (proportional hazards ratio = 1.009, 95% CI 1.000–1.017) and exposure–response trends for cumulative exposure (ppm– months) (p = 0.045) and for number of peak exposure days (p = 0.054). The rela- tively small study of Ruder et al. (2004) in Washington state found an SMR of 1.43 with a wide 95% CI (0.57–2.95) on the basis of seven cases. However, the SMRs for the high-exposure subsets were 3.60 (95% CI 0.98–9.20) overall and 5.11 (95% CI 0.62–18.4) for workers who had been employed for more than 1 year. The European cohort studies (Kogevinas et al. 1994; Kolstad et al. 1995) were either inconsistent with the above observations or had sparse but sugges- tive data that were based on cumulative exposure–response analysis, as noted in Table 3-5. An interview-based case–control study focusing on occupational exposure to polycyclic aromatic hydrocarbons and plastics was published by Karami et al. (2011) as part of the Central and Eastern European Renal Cell Carcinoma study. It fell outside the time window for NTP’s background document for styrene (NTP 2008) and was not reviewed in the substance profile for styrene (NTP 2011a). The interviews that were part of this study design enabled the authors to control for factors, such as smoking, which could not be controlled for in the cohort studies described above. The study also had the advantage that it included only renal-cell carcinomas whereas the cohort studies described previously in this section apparently also included other histologic types of kidney cancer, such as transitional-cell carcinomas. For study participants who were ever ex- posed vs never exposed to styrene, an increased OR was observed (OR = 1.7, 95% CI 0.8–3.6). Relative to the unexposed group and after adjustment for loca- tion, smoking, family history, hypertension, body-mass index, sex, and age, the ORs were 0.6 (95% CI 0.2–1.7) 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. Analysis by average concentration and by duration also yielded higher associations with greater styrene exposure (Table 3-5), including some associations that approached or reached statistical significance and some associations with high ORs. The committee notes that the adjusted ORs, includ- ing the one with adjustment for smoking, were similar to or higher than the un- adjusted ORs. Limitations of the study included the use of hospital-based con- trols, excluding patients who had other urologic conditions or diagnoses related to smoking; past cancer diagnosis of nonurologic cancer in the controls was pos- sible. Kidney-cancer survival rates are relatively high and have been increasing over the last several decades (NCI 2014), so studies based on death certificates have the limitation that they do not capture some incident cases, and this can introduce a bias if case survival rates vary among groups with varied degrees of exposure. A smaller population-based case–control study in a Canadian popula-

Independent Assessment of Styrene 97 tion included 177 cases in all of various histological types of kidney cancer and found no association with styrene exposure. The adjusted OR was the same as the unadjusted OR. Overall, the observations for kidney cancer include repeated observations of associations with styrene exposure for various metrics, including independent populations and contrasting study designs, high estimates of risk, and exposure– response relationships. However, the role of chance, bias, or confounding cannot be adequately excluded. Therefore, the evidence fulfills NTP’s listing criteria for limited evidence and not sufficient evidence for an association between expo- sure to styrene and kidney cancer. Pancreatic Cancer The epidemiologic data on pancreatic cancer constitute credible but lim- ited evidence that styrene exposure is associated with pancreatic cancer on the basis of four cohort studies (see Table 3-6). High case-fatality rates in pancreatic cancer make mortality a reliable index of incidence. Kogevinas et al. (1994) found an SMR of 1.48 (95% CI 0.76–2.58) for the highest-exposure group (lam- inators). The cumulative ppm–years analysis showed an exposure–response trend of p = 0.068 (MRR = 2.56 for exposures greater than 500 ppm, 95% CI 0.90–7.31, 10 cases). Kolstad et al. (1995) found a statistically significant pan- creatic-cancer excess incidence in the subgroup that had the highest probability of exposure (IRR = 2.2, 95% CI 1.1–4.5). Ruder et al. (2004) found an SMR of 1.43 (95% CI 0.78–2.41) in the overall cohort and 1.88 (95% CI 0.51–4.81) in the high-exposure subgroup. The Wong et al. (1994) study did not find an asso- ciation of styrene with pancreatic cancer. The update by Collins et al. (2013) found an overall SMR close to expected (SMR = 0.96, 95% CI 0.73–1.22), but found a significantly increased proportional hazard ratio of 1.008 (95% CI 1.002–1.015) that was based on cumulative exposure and a monotonic “increas- ing risk with increasing average exposure…with SMRs of 0.75, 0.83, 1.46, and 1.52” (Collins et al. 2013, p. 201). No large case–control studies of pancreatic cancer that include an assessment of styrene have been reported, but the com- mittee did review a small population-based case–control study in Canada that included 116 cases of pancreatic cancer (Gerin et al. 1998). The authors did not find an association of cancer with exposure to styrene. Overall, the observations for pancreatic cancer demonstrated exposure– response relationships with styrene exposure estimates in cohort mortality stud- ies conducted in both the United States and Europe and in the Danish incidence study. However, the role of chance, bias, or confounding cannot be adequately excluded. Therefore, the evidence fulfills NTP’s listing criteria of limited evi- dence, not sufficient evidence, for an association between exposure to styrene and pancreatic cancer. The committee notes that the study by Collins et al. (2013) has been recently published and was not included in the background doc- ument or substance profile.

98 TABLE 3-6 Summary of Observations for Pancreatic Cancer Reference Observations (95% CI) Reinforced-Plastics Industry Cohorts Kogevinas et al. 1994 Full study cohort: SMR = 1.00 (0.71–1.38), n = 37 ≥20 years after first exposure: SMR = 2.05 (0.58-7.29), n = 9 Industrial process SMRs: Laminators: 1.48 (0.76–258), n = 12 Unspecified task: 1.17 (0.68–1.88), n = 17 Other exposed jobs: 0.30 (0.04–1.10), n = 2 Unexposed: 0.79 (0.26–1.86), n = 5 Cumulative exposure (ppm–years): <75 reference: n = 9 100–199: MRR = 1.44 (0.48–4.34) n = 5 200–499: MRR = 1.90 (0.65–5.53), n = 6 ≥500: MRR = 2.56 (0.90–7.31), n = 10 Trend p = 0.068 Wong et al. 1994 Full study cohort: SMR = 1.13 (0.68–1.77), n = 19 SMRs by latency (years) (CIs not reported): <10: 1.45, n = 5 10–19: 0.87, n = 6 ≥20: 1.25, n = 8 SMRs by duration of exposure to styrene (years) (CIs not reported): <1: 2.03, n = 6 1–1.9: 1.04, n = 3 2–4.9: 1.29, n = 5 5–9.9: n = 0 ≥10: 1.30, n = 5

SMRs by cumulative styrene exposure (ppm–years) (CIs not reported): <10: 1.40, n = 5 10–29.9: 1.61, n = 6 30–99.9: 0.63, n = 3 ≥100: 1.06, n = 5 SMRs employed 2 years in processing categories (CIs not reported): Open-mold processing: 0.80 n = 1 Mixing and closed-mold processing: 1.57, n = 2 Finish and assembly: 0.93, n = 3 Plant office support: 0.44, n = 1 Maintenance and preparation: 0.34, n = 1 Supervisory and professional: n = 0 Kolstad et al. 1995 Full study cohort: SIR = 1.20 (0.86–1.63), n = 41 Incidence rate ratio based on exposure probability: Low: 1.1(0.6–2.2), n = 24 High: 2.2 (1.1–4.5), n = 17 Ruder et al. 2004 Full study cohort: SMR = 1.43 (0.78–2.41), n = 14 High exposure: SMR = 1.88 (0.51–4.81), n = 4 Low exposure: SMR = 1.31 (0.63–2.41), n = 10 SMRs for those employed >1 year: Total: 1.54 (0.62–3.17), n = 7 High exposure: 1.23 (0.03–6.85), n = 1 Low exposure: 1.60 (0.59–3.49), n = 6 Collins et al. 2013 Total cohort: SMR = 0.96 (0.73–1.22), n = 63 ≥15 years latency: SMR= 0.90 (0.67–1.17), n = 53 Cumulative exposure SMRs (ppm-months): 0.0–149.9: 0.90 (0.49–1.51), n = 14 150–399.9: 1.15 (0.67–1.84), n = 17 (Continued) 99

100 TABLE 3-6 Summary of Observations for Pancreatic Cancer Reference Observations (95% CI) 400–1,199.9: 0.53 (0.24–1.01), n = 9 ≥1,200: 1.24 (0.78–1.86), n = 23 Test for trend: p = 0.274 Proportional-hazards model: hazard ratio for styrene exposure = 1.008 (1.002–1.015) SMR (number of days with ≥15 min of styrene at >100 ppm): None: 0.84 (0.58–1.19), n = 32 1–719*: 1.21 (0.74–1.87), n = 20 720–1,799: 0.52 (0.11–1.51), n = 3 ≥1,800: 1.45 (0.63–2.85), n = 8 Test for trend: p = 0.337 *Corrected from publication “1–179”. SMRs, with increasing average exposure = 0.75, 0.83, 1.46, 1.52. Case–Control Study Gerin et al. 1998 Ever exposed: OR = 0.3 (0.0-2.6), 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. Source: committee-generated.

Independent Assessment of Styrene 101 Esophageal Cancer The committee judged there to be credible but limited evidence that high exposure to styrene in workers is associated with esophageal cancer on the basis of observations from Kogevinas et al. (1994), Wong et al. (1994), and Ruder et al. (2004) (see Table 3-7). For esophageal cancer, as for pancreatic cancer, mor- tality is a relatively reliable index of incidence because of the typically high mortality and short survival of patients. An internal analysis in the Kogevinas et al. (1994) mortality study found exposure–response patterns among the higher-exposed subjects and an SMR of 5.8 (1.0–34) after 20 years following the first exposure on the basis of six cases. The Kolstad et al. (1994) study did not find any association in the full cohort and did not report on highly exposed subgroups. Wong et al. (1994) observed an SMR of 1.92 (95% CI 1.05–3.22) that was based on 14 cases, but subgroup analyses, all with small numbers, did not show clear patterns. The updated pub- lication of Collins et al. (2013) did not include data on esophageal cancer; its unpublished background report (Collins et al. 2012) indicated that no increased SMRs were found for esophageal cancer. No proportional hazard analyses like those reported for pancreatic cancer and kidney cancer were included. The Rud- er et al. (2004) study found an SMR of 2.30 (95% CI 1.19–4.02) with 12 cases in the full cohort (the highest-exposure subgroup had a similar SMR but only two cases). The committee did not identify any large case–control studies of esophageal cancer that included an assessment of styrene exposure, but it did identify a Canadian population-based case–control study that reported 99 cases of esophageal cancer (Gerin et al. 1998). The study authors did not report an association between esophageal cancer and styrene exposure. Overall, while the epidemiologic evidence is weaker for esophageal cancer than for kidney or pancreatic cancer, there are nevertheless repeated observa- tions of mortality associations with styrene among two independent U.S. co- horts, each with relative risk estimates of approximately double what was ex- pected in the comparison group. 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 evi- dence of an association of styrene exposure and lung or breast cancer (Table 3- 8). However, it decided to summarize the data from the most informative studies because lung cancers and breast cancers have been observed in experimental animals after treatment with styrene (see review below). For lung cancer (that is, cancer of the lung, bronchus, or trachea), the Wong et al. (1994) and Collins et al. (2013) studies found statistically significant increases in their combined

102 TABLE 3-7 Summary of Observations for Esophageal Cancer Reference Observations (95% CI) Reinforced-Plastics Industry Cohorts Kogevinas et al. 1994 Full study cohort: SMR = 0.82 (0.47–1.31), n = 1 ≥20 years after first exposure: SMR = 5.82 (1.0-33.91), n = 6 Industrial process SMRs: Laminators: 1.81 (0.87–3.34), n = 0 Unspecified task: 0.83 (0.27–1.93), n = 5 Other exposed jobs: no = 0 Unexposed: 0.82 (0.47–1.31), n = 17 Cumulative exposure (ppm–years): <75 reference: n = 5 100–199: MRR = 1.01 (0.20–5.23), n = 2 200–499: MRR = 1.67 (0.39–7.18), n = 3 ≥500: MRR = 1.76 (0.42–7.30), n = 4 Test for trend p = 0.31 Wong et al. 1994 Full study cohort: SMR = 1.92 (1.05–3.22), n = 14 SMRs by latency (years) (CIs not reported): <10: 1.43, n = 2 10–19: 2.66, n = 8 ≥20: 1.38, n = 4 SMRs by duration of exposure to styrene (years) (CIs not reported): <1: 1.55, n = 2 1–1.9: 2.37, n = 3 2–4.9: 2.41, n = 4 5–9.9: 0.73, n = 1 ≥10: 2.34, n = 4 SMRs by cumulative styrene exposure (ppm–years) (CIs not reported): <10: 2.51, n = 4

10–29.9: 1.24, n = 2 30–99.9: 2.95, n = 6, p < 0.05 ≥100: 0.97, n = 2 SMRs employed 2 years in processing categories (CIs not reported): Open-mold processing: 3.57, n = 2 Mixing and closed-mold processing: n = 0 Finish and assembly: 3.01, n = 4 Plant office support: 0.98, n = 1 Maintenance and preparation: 2.30, n = 3 Supervisory and professional: 1.99, n = 1 Proportional-hazard models, cumulative exposure, duration of exposure to styrene were not significant. Kolstad et al. 1995 Full study cohort: SIR = 0.92 (0.50–1.57), n = 13 Ruder et al. 2004 Full study cohort: SMR = 2.30 (1.19–4.02), n = 12 High exposure: SMR = 1.85 (0.22–6.67), n = 2 Low exposure: SMR = 2.42 (1.16–4.44), n = 10 SMRs for those employed >1 year: Total: 1.27 (0.26–3.72), n = 3 High exposure: 2.71 (0.07–15.0), n = 1 Low exposure: 1.01 (0.12–3.64), n = 2 SMRs for duration of exposure in high-exposure department (years) <1: 1.18 (0.02–9.61), n = 1 >1: 2.74 (0.04–22.3), n = 1 Case–Control Study Gerin et al. 1998 Ever exposed: OR = 1.0 (0.0–3.5), n = 3 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. Source: committee-generated. 103

104 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens cohort on the basis of 162 and 556 cases, respectively. In the analysis by Wong et al. (1994), the most highly exposed subgroup, which consisted of people who worked in open-mold processing for at least 2 years, did not have an excess SMR (eight cases). A previous nested case–control analysis by Wong (1990) that was based on the same cohort found a strong association with smoking (Mantel-Haenszel Relative Risk = 7.33, chi-square = 4.27, p = 0.04) but no as- sociation with styrene exposure (Mantel-Haenszel Relative Risk = 0.63, chi- square = 1.11, p = 0.29). Collins et al. (2013) observed an increased SMR for lung cancer (SMR = 1.34, 95% CI 1.23–1.46) but reported inverse linear trends for cumulative exposure (p < 0.001). The proportional hazard ratio was below 1.0, and the 95% CI included 1.0. Ruder et al. (2004) found marginally in- creased lung-cancer SMRs (see Table 3-8). The Danish and European cohort studies found no evidence of an association between styrene exposure and lung cancer. Scélo et al. (2004) conducted a case–control study of industrial expo- sures and lung cancer in which there were no increased ORs associated with having been ever exposed to styrene or with the highest category of exposure duration, duration weighted by frequency, or cumulative ppm–years. For breast cancer, no increase in the frequency of disease was observed in the reinforced-plastics industry; all three cohorts that included women (Kogevi- nas et al. 1994; Wong et al. 1994; Ruder et al. 2004; Collins et al. 2013) found lower than expected SMRs for breast cancer. When high-exposure subsets were analyzed for breast cancer, no indication of an exposure–response relationship was observed. The paucity of exposed women in the cohorts limits the conclu- sions that can be drawn. No pertinent case–control studies have been identified. In addition, the mortality data used in those studies comprise a less reliable in- dex of breast cancer compared to incidence data. Conclusion on Epidemiologic Literature After identifying the most informative epidemiologic studies and evaluat- ing their results, the committee found that there is limited evidence for the car- cinogenicity of styrene on the basis of epidemiologic studies. A causal interpre- tation is credible, but alternative explanations—such as chance, bias, and confounding factors—cannot adequately be excluded. CANCER STUDIES IN EXPERIMENTAL ANIMALS Several studies have been published in which the tumor response to sty- rene-exposed animals has been measured, but as described in Appendix D, no relevant studies were identified after publication of the RoC. In general, the committee considered studies to be more informative when they included more than one dose, well-matched controls, chronic exposure, treatment groups of

TABLE 3-8 Summary of Observations for Lung, Bronchial, and Tracheal Cancers (Unless Otherwise Indicated) Reference Observations (95% CI) Reinforced-Plastics Industry Cohorts Kogevinas et al. 1994 Full study cohort: SMR = 0.99 (0.87–1.13), n = 235 Industrial process SMRs: Laminators: 1.06 (0.81–1.36), n = 60 Unspecified task: 0.99 (0.78–1.24), n = 78 Other exposed jobs: 0.89 (0.65–1.21), n = 42 Unexposed: 0.84 (0.58–1.16), n = 37 Cumulative exposure (ppm–years): <75 reference: n = 73 100–199: MRR = 0.75 (0.47–1.19), n = 25 200–499: MRR = 0.74 (0.47–1.16), n = 26 ≥500: MRR = 0.90 (0.58–1.38), n = 37 Test for trend p < 0.43 (sic) Wong et al. 1994 Full study cohort: SMR = 1.41 (1.20–1.64), n = 162 SMRs by latency (years) (CIs not reported): <10: 1.07, n = 23 10–19: 1.46, n = 70, p < 0.01 ≥20: 1.51, n = 69, p < 0.01 SMRs by duration of exposure to styrene (years) (CIs not reported): <1: 1.83, n = 37, p < 0.01 1–1.9: 1.25, n = 25 2–4.9: 1.68, n = 44, p < 0.01 5–9.9: 1.37, n = 30 ≥10: 0.97, n = 26 SMRs by cumulative styrene exposure (ppm-years) (CIs not reported): <10: 1.50, n = 37, p < 0.05 (Continued) 105

106 TABLE 3-8 Continued Reference Observations (95% CI) 10–29.9: 1.88, n = 48, p < 0.01 30–99.9: 1.33, n = 43 ≥100: 1.04, n = 34 SMRs employed 2 years in processing categories (CIs not reported): 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): Direct exposure to styrene: Mantel-Haenszel Relative Risk = 0.63, n exposed cases = 15, p = 0.29 Smoking: Mantel-Haenszel Relative Risk = 7.33, n exposed cases = 30, p = 0.04 Kolstad et al. 1995 Full study cohort: SIR = 1.12 (0.98–1.26), n = 248 Incidence Rate Ratio by exposure probability: Unexposed controls: n = 123 Low probability: 0.9 (0.7–1.1), n = 176 High probability: 1.0 (0.7–1.3), n = 72 All reinforced-plastics workers: 0.9 (0.7–1.1), n = 248 Ruder et al. 2004 Full study cohort: SMR = 1.14 (0.90–1.43), n = 76 High exposure: SMR = 1.29 (0.76–2.04), n = 18 Low exposure: SMR = 1.10 (0.84–1.43), n = 58 SMRs for those employed >1 year: Total: 0.99 (0.67–1.41), n = 31 High exposure: 1.11 (0.40–2.41), n = 6 Low exposure: 0.97 (0.62–1.43), n = 25

SMRs for duration of exposure in high-exposure department (years): <1: 1.40 (0.77–2.39), n = 14 >1: 0.73 (0.20–2.03), n = 4 Collins et al. 2013 Total cohort: SMR = 1.34 (1.23–1.46), n = 556 ≥15 years latency: SMR= 1.35 (1.24–1.48), n = 501 Cumulative exposure SMRs (ppm–months): 0.0–149.9: 1.60 (1.36–1.87), n = 157 150–399.9: 1.41 (1.18–1.67), n = 131 400–1,199.9: 1.31 ( 1.10–1.55), n = 138 ≥1,200: 1.10 (0.92–1.31), n = 130 Test for trend: p = 0.003 for inverse trend Proportional-hazard model: hazard ratio for styrene exposure = 0.997 (0.993–1.002) SMR (number of days with ≥15 min of styrene at >100 ppm): None: 1.32 (1.18–1.47), n = 314 1–719*: 1.50 (1.28–1.76), n = 154 720–1799: 1.34 (1.00–1.77), n = 49 ≥1800: 1.06 (0.76–1.46), n = 39 Test for trend: p = 0.201 for inverse trend *Corrected from publication, which printed “1–179”. Case–Control Study Gerin et al. 1998 Low exposure to styrene: OR = 0.3 (0.1–1.9), n = 5 Medium / high exposure to styrene: OR = 0.9 (0.2–3.3), n = 5 Adjusted for age, family income, ethnic group, cigarette smoking, respondent status, arsenic, asbestos, chromium VI, nickel, crystalline silica, beryllium, cadmium, and polycyclic aromatic hydrocarbons. Scélo et al. 2004 Ever exposed to styrene: OR = 0.70 (0.42–1.18), n = 51 Exposure duration OR (years): 1–6: 0.98 (0.37–2.61), n = 13 7–14: 0.72 (0.33–1.59), n = 19 ≥14: 0.59 (0.26–1.34), n = 19 107 (Continued)

108 TABLE 3-8 Continued Reference Observations (95% CI) Duration x frequency of exposure OR (years): 0.01–0.50: 0.67 (0.28–1.56), n = 13 0.51–3.00: 1.19 (0.52–2.73), n = 21 ≥3.00: 0.38 (0.13–1.03), n = 17 Cumulative exposure (years x frequency x ppm) OR: 0.01–2.75: 1.15 (0.55–2.41), n = 22 2.76–12.50: 0.37 (0.13–1.08), n = 9 ≥12.50: 0.53 (0.20–1.43), n = 20 All ORs adjusted for center, sex, age, tobacco consumption, vinyl chloride, acrylonitrile, formaldehyde, and inorganic pigment dust. 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.

Independent Assessment of Styrene 109 adequate size, the use of well-characterized test material of high purity, thor- ough necropsy and pathologic evaluation of tissues according to established criteria, and statistical evaluation of tumor data with accepted methods. The quality of the studies varied considerably; the value of some of them is limited by the numbers of animals treated, exposure duration, observation period, dose selection, or incomplete reporting of methods or results. Studies were considered less informative if any of those attributes were missing or could not be verified from the study description. Studies of Styrene Despite weaknesses in some individual studies, the overall body of evi- dence is sufficient to permit an evaluation of evidence on carcinogenicity. The strongest evidence of a tumorigenic response to styrene is in the mouse lung. Inhalation exposure to styrene has been observed to produce significant increas- es in alveolar and bronchiolar tumors in both male and female CD-1 mice (Cru- zan et al. 2001; Cohen et al. 2002), including a significant increase in malignant tumors in females at the highest dose. CD-1 mice (70 males and females) were exposed to styrene vapor 5 days/week for 6 hours/day. Two groups of 10 mice were sacrificed after 52 and 78 weeks, and the remaining 50 were exposed for 104 weeks (males) or 98 weeks (females). Styrene concentrations were 0, 20, 40, 80, or 160 ppm. Complete necropsies were performed on all animals. The major organs in all organ systems (respiratory, gastrointestinal, cardiovascular, urinary, reproductive, nervous, lymphohematopoietic, endocrine, musculoskele- tal, and cutaneous) and grossly abnormal tissues in the control and high-dose groups were examined histopathologically. Selected organs and grossly abnor- mal tissues were examined histopathologically in intermediate-dose groups. Af- ter 24 months, the incidence of bronchiolar and alveolar adenomas was in- creased significantly in males exposed to styrene at 40 ppm or higher and in females exposed at 20, 40, or 160 ppm (see Table 3-9). The incidence of carci- nomas alone was significantly increased only in females exposed at 160 ppm. 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 bron- chiolar tumors combined was observed in male mice at the highest dose, and there was a significant dose-related trend (NCI 1979a). B6C3F1 mice (50 males and 50 females) were given styrene in a corn-oil vehicle by oral gavage 5 days/week for 78 weeks in doses of 150 or 300 mg/kg. Mice (20 males and 20 females) given corn-oil vehicle alone served as controls. Mice in all treatment groups were euthanized 13 weeks after the last dose. Complete necropsies were performed on all animals. The major organs in all organ systems (respiratory, gastrointestinal, cardiovascular, urinary, reproductive, nervous, lymphohemato- poietic, endocrine, musculoskeletal, and cutaneous) and grossly abnormal tis- sues were examined histopathologically in all groups with the exception of some

110 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens TABLE 3-9 Lung-Tumor Incidence in CD-1 Mice Exposed to Styrene by Inhalation1 Tumor Incidence by Styrene Concentration Sex Lung Tumor Type 0 ppm 20 ppm 40 ppm 80 ppm 160 ppm Males Bronchioalveolar 15/50 (30%) 21/50 (42%) 35/50 (70%)2 30/50 (60%)2 33/50 (66%)2 adenoma Bronchioloalveolar 4/50 (8%) 5/50 (10%) 3/50 (6%) 6/50 (12%) 7/50 (14%) carcinoma Females Bronchioalveolar 6/50 (12%) 16/50 (32%)2 16/50 (32%)2 11/50 (22%) 24/50 (48%)2 adenoma Bronchioloalveolar 0/50 (0%) 0/50 (0%) 2/50 (4%) 0/50 (0%) 7/50 (14%)2 carcinoma 1 Source: Data from Tables 5 and 6 in NCI (1979a). Observations are expressed as num- ber of animals with the indicated tumor over the number of animals examined. 2 p < 0.05. Source: Data from Table 4 in Cruzan et al. 2001. moribund animals. A significant increase in combined adenoma and carcinoma of the lung was observed in male mice at the higher styrene dose compared with controls (Table 3-10). The authors of this National Cancer Institute (NCI 1979a) study also compared their results with those in historical controls that were treated differently (dietary controls rather than mice treated with corn-oil vehi- cle) and found that the lung-tumor incidences were similar (an average of 12%—incidences were as high as 20% in two studies). That appears to have led the authors to discount to some extent the male mouse lung-tumor findings. The authors concluded that “the findings of an increased incidence of a combination of adenomas and carcinomas of the lung provided suggestive evidence for the carcinogenicity of styrene in male B6C3F1 mice” but also stated, “However, it is concluded that, under the conditions of this bioassay, no convincing evidence for the carcinogenicity of the compound was obtained in Fischer 344 rats or B6C3F1 mice of either sex” (p. VIII). With respect to the male mouse lung-tumor findings in the NCI (1979a) study, the committee considers the use of the historical controls to be inappro- priate 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. They can include such details as the strain and sub- strain of experimental animal, the specific supplier, and even the subpopulation within the colony from which the animals were derived. Caging conditions, ven- tilation, diet, drinking water, and treatment vehicle are also important (Haseman et al. 1984; Festing and Altman 2002; Keenan et al. 2009). For those reasons, controls must be carefully matched, and the best opportunity to do this is almost always with concurrent controls. Although attempts were made by both NCI

Independent Assessment of Styrene 111 TABLE 3-10 Lung-Tumor Incidence in B6C3F1 Mice Exposed to Styrene by Gavage1 Alveolar and Bronchiolar Tumor Incidence Sex Vehicle Control 150 mg/kg 300 mg/kg Male Adenoma 0/20 (0%) 3/44 (7%) 4/43 (9%) Carcinoma 0/20 (0%) 3/44 (7%) 5/43 (11%) Combined 0/20 (0%), 6/44 (14%) 9/43 (20%), p = 0.023 p = 0.024 Female Adenoma 0/20 (0%) 1/43 (2%) 3/43 (7%) Carcinoma 0/20 (0%) 0/43 (0) 0/43 (0) Combined 0/20 (0%) 1/43 (2%) 3/43 (7%) 1 Source: Data from Tables 5 and 6 in NCI (1979a). Control data from concurrent con- trols. Initial numbers of mice: 20 controls and 50 in each dose group. Statistical compari- son results in each treatment group were from comparison with controls by one-tailed Fischer’s exact test. Results were not significant unless otherwise stated. Probability val- ue for trend from Cochran Armitage test is shown below vehicle control results. No re- sults for trend were presented for adenomas alone. (1979a) and NTP (2008) to compare results with historical controls, neither doc- umented the extent to which experimental conditions of the historical controls varied from those of the treated groups in the NCI study. The NTP historical- control group included studies in different laboratories, and this raises the possi- bility that genetic and husbandry factors were not well matched; the NCI histori- cal control comparison included animals treated differently (that is, without corn-oil vehicle treatment). The committee views concurrent controls as an ap- propriate basis of comparison and interprets the findings of the NCI study, with respect to lung tumors after oral exposure in male mice, as being positive. 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) study, the committee took the additional step of confirming that the increases would be statistically significant if contemporary statistic tests were applied. With input from a statistical con- sultant, the committee applied an age-adjusted analysis of alveolar and bron- chiolar tumors for male and female mice using information provided in the NCI report. Tumor incidences in styrene-treated mice were compared with concur- rent study controls. On the basis of data included in the report, it was difficult to determine the time of death for animals that did not reach the end of the study, which precluded the use of the poly-3 trend test. However, given that all tumor- bearing mice were identified at terminal sacrifice, the Peto test could be applied using survival curve data and estimates of the numbers of missing or unex- amined animals in each treatment group. For some groups, it was possible to infer the number of animals examined in specific treatment groups on the basis

112 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens of the data presented in the publication. In other groups, there were several pos- sibilities for the number of animals examined in specific treatment groups, and each possibility was considered. As a result, comparisons between groups re- sulted in a range of p-values rather than a single value. The p-values obtained from the Peto test are summarized in Table 3-11. Alveolar and bronchiolar ade- nomas or carcinomas were significantly increased in the high-dose group, and the trend with dose was also statistically significant. Those results are consistent with the results obtained using the statistical test in the original NCI (1979a) report and indicate that the same conclusion is reached using a more modern statistical approach. Studies of short-term exposure of pregnant mice and their progeny to high doses of styrene (Ponomarkov and Tomatis 1978) are not as well suited as the longer-term studies discussed above to assess tumor induction caused by chronic exposure. However, the short-term studies do provide some limited support for a tumorigenic effect of styrene on the lung. O20 and C57BL pregnant mice were orally exposed to styrene, and carcinogenic and developmental toxic effects were investigated (Ponomarkov and Tomatis 1978). O20 (29 treated) and C57BL (15 treated) mice were exposed at 1,350 mg/kg and 300 mg/kg, respec- tively, on gestation day 17. After weaning, progeny were exposed once a week at the same doses as dams for each strain; O20 mice were exposed for 16 weeks (discontinued because of toxicity), and C57BL for 120 weeks or until death. The authors indicate that all surviving animals were necropsied with histopathologic examination of all major organs, but the specific organs that were included in the examination were not stated. The preweaning mortality of the O20 mice (43%) was significantly increased compared with controls in which an olive-oil vehicle was used (22%); no difference in postweaning mortality was observed. The O20 strain had a significantly increased total lung-tumor incidence in both sexes compared with vehicle controls (p < 0.01). There was no significant in- crease in preweaning or postweaning mortality or tumor incidence in the C57BL mice in either dams or male and female progeny. 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. Control 0.110 – 0.115 Trend 0.057 – 0.067 Alveolar/Bronchiolar Adenoma Low Dose vs. Control 0.081 – 0.094 or Carcinoma High Dose vs. Control 0.015 – 0.017 Trend 0.011 – 0.014 Females Alveolar/Bronchiolar Low Dose vs. Control 0.684 – 0.690 Adenoma High Dose vs. Control 0.304 – 0.304 Trend 0.124 – 0.126 Alveolar/Bronchiolar Carcinoma —1 —1 1 No alveolar/bronchiolar carcinomas observed. Source: committee generated.

Independent Assessment of Styrene 113 A/J mice (25 females) were given an intraperitoneal injection three times a week for a total of 20 doses, which equaled a total exposure of 200 µmol of sty- rene (about 100 mg/kg); 4-(N-Nitrosomethylamino-)-1-(3-pyridyl)-1-butanone was used as a positive control (Brunnemann et al. 1992). Mice were sacrificed 20 weeks after their last injection. Complete necropsies were performed on all animals. Some of the major organs and sites (head, lung, heart, liver, spleen, pancreas, kidney, and adrenal) and grossly abnormal tissues were preserved and examined histopathologically in all groups. Lung adenomas were observed in three styrene-treated mice and one control; the difference was not statistically significant. In contrast with the positive findings of lung tumors in mice, styrene ex- posure of rats by both oral and inhalation routes has had consistently negative results except for mammary tumors. Sprague-Dawley rats (30 males and 30 fe- males in each dose group and 60 male and 60 female controls) were exposed to styrene vapor by inhalation (at 25, 50, 100, 200, and 300 ppm) 4 hours/day, 5 days/week for 52 weeks and then observed until death (Conti et al. 1988). Com- plete necropsies were performed on all animals. 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. There was no significant difference in mortality or body weight between any group and controls. A nonsignificant increase in total malignant tumors was observed in both males and females at 100 ppm but not at the higher concentrations tested. There was a significant increase in malignant mammary tumors in females in all exposure groups. Cruzan et al. (1998) exposed Sprague-Dawley rats (70 males and 70 fe- males) to styrene vapor at 0, 50, 200, 500, and 1,000 ppm 6 hours/day, 5 days/week for 104 weeks. Rats were sacrificed at 105 and 107 weeks. Complete necropsies were performed on all animals. The major organs in all organ sys- tems (respiratory, gastrointestinal, cardiovascular, urinary, reproductive, nerv- ous, lymphohematopoietic, endocrine, musculoskeletal, and cutaneous) and grossly abnormal tissues were examined histopathologically in the control and high-dose groups. Selected organs and grossly abnormal tissues were examined histopathologically in the intermediate-dose groups. Body-weight gains were lower in the male 500- and 1,000-ppm groups and in the female 200-, 500-, and 1,000-ppm groups compared with controls. There were no significant increases in any tumor type in males or females. Ponomarkov and Tomatis (1978) gave 21 pregnant BD IV rats an oral ga- vage with 1,350 mg/kg of styrene in olive oil on gestation day 17. After wean- ing, progeny (73 males and 71 females) were given styrene at 500 mg/kg via a gastric tube once a week throughout their lifespan or until 120 weeks, when they were sacrificed. The authors indicated that all surviving animals were necrop- sied with histopathologic examination of all major organs, but the specific or- gans that were examined were not stated. There was no difference in litter size, body weight, or tumor incidence between the treated and control groups.

114 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens NCI (1979a) gave F344 rats (50 males and 50 females) an oral gavage 5 days/week for 103 weeks at 500 mg/kg or for 78 weeks at 1,000 or 2,000 mg/kg. Complete necropsies were performed on all animals. The major organs in all organ systems (respiratory, gastrointestinal, cardiovascular, urinary, reproduc- tive, nervous, lymphohematopoietic, endocrine, musculoskeletal, and cutaneous) and grossly abnormal tissues were examined histopathologically in all groups except for some moribund animals. The 53-week survival of the males in the high-dose group was six of 50, and the 70-week survival of the females in the high-dose group was seven of 50. The 90-week survival in the medium-dose group was 44 of 50 in both males and females and in the low-dose group 47 of 50 in both sexes. No differences in tumor incidence were observed in either sex at any of the doses compared with the corn-oil vehicle controls. Conti et al. (1988) used Sprague-Dawley rats (40 males and 40 females) to test oral exposure to styrene at 50 and 250 mg/kg via a gastric tube. Rats were exposed 4 or 5 days/week for 52 weeks and then observed until death. Complete necropsies were performed on all animals. The major organs in all organ sys- tems (respiratory, gastrointestinal, cardiovascular, urinary, reproductive, nerv- ous, lymphohematopoietic, endocrine, musculoskeletal, and cutaneous) and grossly abnormal tissues were examined histopathologically in all groups. In- creased mortality was observed in the females in the high-dose group compared with the olive-oil vehicle controls; no significant difference was observed in males. There was no significant difference in body weight or tumor incidences in either sex. Beliles et al. (1985) used Sprague-Dawley rats (50 treated males, 70 treat- ed females, 76 control males, and 106 control females) to study drinking-water exposure to styrene. Rats were exposed via drinking water for 2 years at 125 or 250 ppm. In males, the daily doses were estimated to be 7.7 and 14.0 mg/kg for the low and high doses, respectively. In females, the daily doses were estimated to be 12.0 and 21.0 mg/kg for the low and high doses, respectively. Complete necropsies were performed on all animals. The major organs in all organ sys- tems (respiratory, gastrointestinal, cardiovascular, urinary, reproductive, nerv- ous, lymphohematopoietic, endocrine, musculoskeletal, and cutaneous) and grossly abnormal tissues were examined histopathologically in the control and high-dose groups, including moribund animals. No observed effects on mortali- ty, tumor rates, or type of tumors were reported. A separate analysis of the data (Huff 1984) found a dose-related increase in combined mammary tumors in fe- males that was significant in terms of trend (p = 0.032) and when the high-dose group was compared to controls (p = 0.039). Conti et al. (1988) gave Sprague-Dawley rats (40 males and 40 females) four intraperitoneal injections at 2-month intervals (200 mg total); no significant differences in tumor incidence were observed when the exposed rats were com- pared with the olive-oil vehicle controls. In the same study, the researchers gave

Independent Assessment of Styrene 115 Sprague-Dawley rats (40 males and 40 females) a single subcutaneous injection of 50 mg of styrene at the age of 13 weeks and observed them until death; no significant differences were observed when the exposed rats were compared with the olive-oil vehicle controls. As evident from the study descriptions above, a styrene effect on mamma- ry tumors in rats is contradictory. Rats exposed to styrene by inhalation were reported to have a significant increase in malignant mammary tumors (Conti et al.1988) although inconsistencies in reporting of the data render this observation inconclusive (IARC 1994a). Huff (1984) analyzed data from oral exposure of Sprague-Dawley rats to styrene in the same study and found a significant in- crease in combined mammary tumors in high-dose females and a significant trend with dose. In contrast with those suggestive findings, no increase in mam- mary tumors was observed in the NCI (1979a) oral study of styrene-exposed rats, and the inhalation study by Cruzan et al. (1998) found a significant inverse trend between dose and mammary gland carcinoma after styrene inhalation in Sprague-Dawley rats (that is, the incidence decreased with increasing styrene concentration in air). There is no evidence from mouse and rat bioassays of in- creased cancer incidence at other sites. Studies of Styrene-7,8-oxide and Styrene Mixtures A bioassay was conducted with a mixture of 70% styrene and 30% beta- nitrostyrene in B6C3F1 mice and F344 rats (NCI 1979b). The mixture was ad- ministered to male rats by gavage in corn oil at 150 or 300 mg/kg and to female rats at 75 or150 mg/kg. The mixture was administered to mice by gavage in corn oil at 87.5 and 175 mg/kg for mice of both sexes. Each dose group consisted of 50 males and 50 females, and the controls were 20 males and 20 females that were given only corn oil. Rats were exposed for 79 weeks and observed for an additional 29 weeks, and mice were exposed for 78 weeks and observed for an additional 14 weeks. No significant increases in tumors at any site were ob- served in rats. Significant increases in alveolar or bronchiolar carcinoma and alveolar and bronchiolar adenoma were observed in the low-dose male mice but not the high-dose male mice, which experienced high mortality. Because the bioassay involved a combination of styrene with another substance (beta- nitrostyrene), the interpretation of findings with respect to potential styrene car- cinogenicity is confounded, and the committee did not include it in its evalua- tion. Styrene-7,8-oxide, a metabolite of styrene, was evaluated in a cancer bio- assay. Styrene-7,8-oxide is the principal metabolite of styrene in rodents and humans, and its carcinogenicity is potentially relevant as supporting information in a determination of the carcinogenicity of styrene in humans. Four studies

116 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens evaluated tumor response to administration by gavage. In one, B6C3F1 mice and F344 rats (52 males and 52 females per treatment group per species) were given styrene-7,8-oxide by gavage in corn oil 3 days/week for 104 weeks (Lijin- sky 1986). Doses were 0, 375, and 750 mg/kg for mice and 0, 275, and 550 mg/kg for rats. Significant increases in forestomach tumors were observed after both high and low doses in both species and sexes. Similarly, significant in- creases in forestomach tumors were observed in male and female Sprague- Dawley rats (40 per treatment group per sex) that were given styrene-7,8-oxide by gavage in corn oil at 50 or 250 mg/kg for 52 weeks (Conti et al. 1988). Sig- nificant increases in forestomach tumors were also observed in BD IV rats given styrene-7,8-oxide by gavage in olive oil at 200 mg/kg (Ponomarkov et al. 1984). The only other tumor response observed was a significant increase in hepatocel- lular neoplasms in B6C3F1 mice given styrene-7,8-oxide by gavage at 375 mg/kg (but not 750 mg/kg) (Lijinsky 1986). Styrene-7,8-oxide is a reactive compound, so it is not surprising that tu- mors occurred primarily in tissues proximal to the site of administration—in the case of gavage, the forestomach. If administered by a different route, or when styrene-7,8-oxide is formed from metabolism of styrene, the distribution of sty- rene-7,8-oxide in the body would probably be substantially different and would plausibly lead to different sites of tumorigenesis. In view of that, despite the discordant sites of tumors between styrene (lung) and styrene-7,8-oxide (forestomach), positive findings with styrene-7,8-oxide are considered support- ing evidence of the carcinogenicity of styrene. Summary of Evidence from Studies in Animals In summary, the committee identified studies that showed positive find- ings of lung tumors in mice after both inhalation and oral administration of sty- rene in well-conducted chronic bioassays (NCI 1979a; Cruzan et al. 2001). Re- sults of another study that is more limited in value for assessing carcinogenicity (Ponomarkov and Tomatis 1978) are also reasonably consistent with the produc- tion of lung tumors in mice after styrene exposure. Contradictory findings on mammary tumors have been observed in rats. For other tumor sites, rats exposed to styrene by both oral and inhalation routes have been consistently negative (Jersey et al. 1978; NCI 1979a; Beliles et al. 1985; Conti et al. 1988; Cruzan et al. 1998); the tumorigenic response appears to be species-specific. MECHANISTIC AND OTHER RELEVANT DATA Genotoxicity Styrene is a highly reactive chemical whose potential for genotoxicity has been investigated for 3 decades. Many studies have been designed to determine

Independent Assessment of Styrene 117 whether styrene or styrene-7,8-oxide—its reactive epoxide metabolic product— elicits DNA damage that leads to mutagenic and clastogenic events in animal and human cells or in animals and humans exposed to styrene or styrene-7,8- oxide. A comprehensive review of data with respect to carcinogenicity of sty- rene-7,8-oxide was conducted by the International Agency for Research on Can- cer (IARC 1994b). The formal evaluation at that time was that there was suffi- cient evidence of the carcinogenicity of styrene-7,8-oxide in experimental animals. A later review by IARC (2002) concluded that exposure of humans to styrene leads to the generation of styrene-7,8-oxide-induced DNA adducts and other forms of DNA damage. The overall evaluation by IARC, which attached heavy weight to the evidence of genotoxicity, was that styrene is possibly car- cinogenic in humans. NTP also reviewed styrene-7,8-oxide and in 2002 listed it as “reasonably anticipated to be a human carcinogen” (NTP 2002). DNA adducts are considered mechanism-based biomarkers of exposure to chemical carcinogens and have been used to identify people and populations at risk for cancer and to set exposure limits for occupational carcinogens (Swen- berg et al. 2008; Jarabek et al. 2009). The presence of DNA adducts in target tissues reflects the formation of reactive metabolites, such as styrene-7,8-oxide, that bind covalently to DNA and to proteins (Poirier 2012). The presence of structurally modified DNA bases substantially increases the probability that pol- ymerase errors during DNA synthesis will create mutations in genes that may lead to cancer (Knobel and Marti 2011). There are also reports that oxidative DNA damage, mediated by reactive oxygen species, is caused by exposure of tissues to styrene-7,8-oxide and con- tributes to its genotoxic effects. A study by Laffon et al. (2002a) that suggested exposure to styrene may result in oxidative DNA damage was cited in the back- ground document for styrene; however, Gamer et al. (2004), using 8-oxoguanine as a biomarker, found no evidence of oxidative stress. In a comparative study of styrene-exposed workers and unexposed clerks (Manini et al. 2009), exposed workers showed lower concentrations of 8-oxoguanine adducts in white blood cell DNA but higher concentrations of 8-oxoguanine in urine. Similar results were obtained by Wongvijitsuk et al. (2011). Considering that 8-oxoguanine is a weak mutagen and is efficiently repaired by base-excision repair, it seems un- likely that oxidative DNA damage plays a strong role in styrene-associated gen- otoxicity. 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 oxida- tive DNA damage, and nucleotide-excision repair of adducts derived from sty- rene). Thus, susceptibility to styrene genotoxicity may be affected at the indi- vidual level by DNA-repair capacity, some aspects of which may be inducible (Vodicka et al. 2004a, 2006).The effects of single-nucleotide polymorphisms on styrene genotoxicity in vivo have been comprehensively reviewed by Vodicka et al. (2006).

118 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens Evaluation of Genotoxicity Evidence The committee’s charge was to “integrate the level-of-evidence conclu- sions, and consider…all relevant information in accordance with RoC listing criteria” (see Appendix B). The RoC includes “studies on genotoxicity (ability to damage genes) and biological mechanisms” for each substance listed (NTP 2011c, p. 3). That information is evaluated with other relevant evidence to ad- dress the RoC listing criteria. Specifically, “data derived from studies of tissues or cells obtained from humans exposed to the substance in question, which is particularly valuable in evaluating whether a relevant cancer mechanism is oper- ating in humans,” constitute one of several lines of evidence used to establish whether there is sufficient or limited evidence of carcinogenicity from studies in humans (NTP 2011c). The committee reviewed the relevant literature, including all recently pub- lished studies, with the goal of determining whether it is biologically plausible for styrene to act as a carcinogen through a genotoxic mechanism. The commit- tee’s comprehensive review of scientific peer-reviewed literature on the geno- toxicity and mutagenicity of styrene and the dates covered by the search are de- scribed in Appendix D. As noted in the Environmental Protection Agency Cancer Guidelines (EPA 2005), one must go beyond simply counting the num- bers of studies that report statistically significant results or statistically nonsig- nificant results on carcinogenesis and related modes of action to reach credible conclusions about the relative strength of the evidence and the likelihood of cau- sality. Accordingly, the committee first categorized evidence pertaining to sty- rene and styrene-7,8-oxide genotoxic and clastogenic mechanistic events into tables on DNA damage (Table 3-12), sister-chromatid exchanges (Table 3-13), micronuclei (Table 3-14), and chromosomal aberrations (Table 3-15). Studies in each table were categorized as positive if a statistically significant effect was observed. Studies were categorized as negative if they reported an absence of a particular effect (that is, no statistically significant difference from the appropri- ate control group). Committee members exercised their scientific judgment in categorizing studies, but they did not perform a formal quality assessment of each individual study or make critical judgments regarding study design or methodology, recognizing that all studies cited have been subjected to some form of peer review. Table 3-16 summarizes the evidence. For each mechanistic event (Tables 3-13 to 3-15), the committee used a set of causal criteria (EPA 2005) as general guidance to determine the strength of the overall evidence of causality.

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. 1995 — Vodicka et al. 1996 Marczynski et al. 1997b Pauwels and Veulemans 1998 Laffon et al. 2001b Laffon et al. 2002b Vodicka et al. 2002a Laffon et al. 2003b Cemeli et al. 2009c Fabiani et al. 2012c In vivob Brenner et al. 1991 Holz et al. 1995 — — Maki-Paakkanen et al. 1991 Vodicka et al. 2004a Human Vodicka et al. 1993 Godderis et al. 2004 Vodicka et al. 1994 Hanova et al. 2010c Vodicka et al. 1995 Teixeira et al. 2010c Marczynski et al. 1997a Hanova et al. 2011c Somorovska et al. 1999 Vodicka et al. 1999 Laffon et al. 2002a Migliore et al. 2002 Shamy et al. 2002 Buschini et al. 2003 Fracasso et al. 2009c Manini et al. 2009c Mikes et al. 2010c Wongvijitsuk et al. 2011c Costa et al. 2012c (Continued) 119

120 TABLE 3-12 Continued Styrene Styrene-7,8-oxide Positive Negative Positive Negative In vitro Sina et al. 1983 — Sina et al. 1983 — Liu et al. 1988a Dypbukt et al. 1992 Bjørge et al. 1996 Herrero et al. 1997 In vivob Walles and Orsen 1983d Kligerman et al. 1993 Walles and Orsen 1983d Gate et al. 2012c Byfält -Nordqvist et al. 1985d Byfalt-Nordqvist et al. 1985d Rodent Cantoreggi and Lutz 1993 Lutz et al. 1993e Pauwels et al. 1996d Sasaki et al. 1997d Vaghef and Hellman 1998d Vaghef and Hellman 1998d Boogaard et al. 2000b Tsuda et al. 2000d Vodicka et al. 2001b Otteneder et al. 2002 Mikes et al. 2009c Gate et al. 2012c a Studies were categorized as positive if a statistically significant effect was observed. Studies were categorized as negative if there was an ab- sence 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.

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. 1980a — Norrpa et al. 1983a Norppa et al. 1983a Norppa and Vainio 1983 Pohlova et al. 1984 Norppa and Tursi 1984 Pohlova and Sram 1985 Chakrabarti et al. 1993 Zhang et al. 1993 In vitro Lee and Norppa 1995 Lee and Norppa 1995 Uüskula et al. 1995 Chakrabarti et al. 1997 Ollikainen et al. 1998 Laffon et al. 2001b Human Andersson et al. 1980 Meretoja et al. 1978a — — Camurri et al. 1983 Watanabe et al. 1981 Camurri et al. 1984 Watanabe et al. 1983 Yager et al. 1993 Hansteen et al. 1984 Hallier et al. 1994 Maki-Paakkanen 1987 Tates et al. 1994 Kelsey et al. 1990 In vivob Artuso et al. 1995 Brenner et al. 1991 Karakaya et al. 1997 Maki-Paakkanen et al. 1991 Biro et al. 2002 Sorsa et al. 1991 Laffon et al. 2002a Van Hummelen et al. 1994 Teixeira et al. 2004 Holz et al. 1995 Teixeira et al. 2010e Rappaport et al. 1996 Costa et al. 2012e De Raat 1978 — De Raat 1978 — Rodent Norppa and Tursi 1984 Nishi et al. 1984 In vitro Norppa et al. 1983b Von der Hude et al. 1991 (Continued) 121

122 TABLE 3-13 Continued Styrene Styrene-7,8-oxide Positive Negative Positive Negative Conner et al. 1979 Preston and Abernethy 1993d Conner et al. 1982c Norppa et al. 1979c Conner et al. 1980 Sinsheimer et al. 1993c,f Conner et al. 1982c Rodent In vivob Sharief et al. 1986f Kligerman et al. 1992 a Studies were categorized as positive if a statistically significant effect was observed. Studies were categorized as negative if there was an ab- sence 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 IARC (1994b); dIdentified from IARC (2002); eIdentified through committee’s literature search; f Denotes chemical administration through intraperitoneal injection.

TABLE 3-14 Studies of Micronuclei Associated with Styrene or Styrene-7,8-oxidea Styrene Styrene-7,8-oxide Positive Negative Positive Negative Linnainmaa et al. 1978b — Linnainmaa et al. 1978a — Linnainmaa et al. 1978b In vitro Laffon et al. 2001b Speit et al. 2012d Meretoja et al. 1977 Maki-Paakkanen 1987 — — Hogstedt et al. 1983 Hagmar et al. 1989 Nordenson and Beckman 1984 Maki-Paakkanen et al. 1991 Human Brenner et al. 1991 Sorsa et al. 1991 Tates et al. 1994 Tomanin et al. 1992 Holz et al. 1995 Yager et al. 1993 In vivob Laffon et al. 2002a Van Hummelen et al. 1994 Godderis et al. 2004 Anwar and Shamy 1995 Teixeira et al. 2004 Karakaya et al. 1997 Vodicka et al. 2004a Hanova et al. 2010d Migliore et al. 2006a Teixeira et al. 2010d Costa et al. 2012d In vitro — — Turchi et al. 1981 — Rodent Penttila et al. 1980c Kligerman et al. 1992 — Fabry et al. 1978c b In vivo Norppa 1981c,e Gate et al. 2012d Penttila et al. 1980c Gate et al. 2012d a Studies were categorized as positive if a statistically significant effect was observed. Studies were categorized as negative if there was an ab- sence 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); dIdentified through committee’s literature search; eDenotes chemical admin- istration through intraperitoneal injection. 123

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. 1978a — Linnainmaa et al. 1978b Linnainmaa et al. 1978b Pohlova et al. 1984g Fabry et al. 1978 In vitro Pohlova and Sram et al. 1985 Norppa et al. 1981 Jantunen et al. 1986 Pohlova et al. 1984g Pohlova and Sram 1985 Meretoja et al. 1977 Thiess et al. 1980 — — Meretoja et al. 1978a Watanabe et al. 1981 Fleig and Thiess 1978 Watanabe et al. 1983 Hogstedt et al. 1979 Nordenson and Beckman 1984 Human Andersson et al. 1980 Pohlova and Sram 1985 Dolmierski et al. 1983 Maki-Paakkanen 1987 Camurri et al. 1983 Jablonicka et al. 1988 Camurri et al. 1984 Hagmar et al. 1989 In vivob Hansteen at al. 1984 Maki-Paakkanen et al. 1991 Forni et al. 1988 Sorsa et al. 1991 Tomanin et al. 1992 Oberheitmann et al. 2001 Tates et al. 1994 Biro et al. 2002 Artuso et al. 1995 Vodicka et al. 2004a Anwar and Shamy 1995 Vodicka et al. 2004c Lazutka et al. 1999 Migliore et al. 2006b Somorovska et al. 1999 Helal and Elshafy 2013f Matsuoka et al. 1979 Matsuoka et al. 1979 Turchi et al. 1981 — Rodent In vitro Ishidate andYoshikawa 1980

Meretoja et al. 1978be Loprieno et al. 1978e,h Loprieno et al. 1978e,h Fabry et al. 1978e Norppa et al. 1980be Sinsheimer et al. 1993c,e Norppa et al. 1979e Sbrana et al. 1983e,h Rodent In vivob Sinha et al. 1983e Sharief et al. 1986e Kligerman et al. 1992 Preston and Abernethy 1993d a Studies were categorized as positive if a statistically significant effect was observed. Studies were categorized as negative if there was an ab- sence 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 IARC (1994a,b); dIdentified from IARC (2002); eIdentified from Scott and Preston (1994a); fIdentified through committee’s literature search; gDenotes chemical administration through intraperitoneal injection; hDenotes chemical administration through oral gavage. 125

126 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens TABLE 3-16 Summary of Genotoxic Effects of Styrene in Humans and Rodents Sister-Chromatid Chromosomal DNA Damage Exchanges Micronuclei Abberations Styrene SO Styrene SO Styrene SO Styrene SO In vitro N/A + (10/0) + (6/0) + (10/0) + (1/0) + (4/0) + (5/0) + (6/0) Human In vivo +/- (17/6) N/A +/- (13/12) N/A -/+ (11/12) N/A +/- (17/15) N/A In vitro + (1/0) + (5/0) + (3/0) + (3/0) N/A + (1/0) +/- (2/1) + (1/0) Rodent In vivo + (10/1) + (6/1) + (4/1) +/- (2/2) +/- (2/2) - (0/3) - (1/7) +/- (2/2) “+” All or most of the studies indicate the effect. “+/-” Most of the studies indicate the effect, although many studies show lack thereof. “-/+” Most of the studies indicate lack of the effect, although many positive studies have been published. “-” All or most of the studies indicate lack of the effect. Parentheses indicate total number of studies demonstrating the effect or lack thereof. N/A, no studies identified. Abbreviations: SO, styrene-7,8-oxide. 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) are observed when human or rodent cells are incubated in the presence of styrene or styrene-7,8-oxide. Studies conducted in various in vitro model systems, including freshly isolated human blood cells and whole blood, were consistently strong (one negative study identified among dozens of positive studies). Furthermore, positive effects were observed in connection with many types of mechanistic events that pertain to genotoxicity. DNA adducts initiate carcinogenesis; however, those adducts may be un- detectable in target tissues in later stages of the carcinogenesis process. DNA adducts also form in non-target tissues and bioactivation may occur at sites other than the target organ (Swenberg et al. 2008). Additionally, mutations induced by a specific adduct may require additional mutations to produce cancer (Vogel- stein et al. 2013). The committee considered this mechanistic information in evaluating whether styrene,7-8,oxide–DNA adducts contribute to the develop- ment of human cancer. The committee made several observations about evidence in the in vitro studies that used human and rodent cells. All studies that were evaluated in this category used purified styrene or styrene-7,8-oxide; thus, chemical specificity was firmly established. Most studies used positive and negative controls, and this strengthens the chemical specificity of the associations between exposure and genotoxicity. Temporality of the observed associations between styrene, styrene-7,8-oxide, and genotoxicity was clearly established. And concentration– response relationships between genotoxic effects and styrene or styrene-7,8- oxide were observed in studies that were designed to measure such effects.

Independent Assessment of Styrene 127 The committee concludes that the evidence of genotoxicity and clastogen- icity of styrene and styrene-7,8-oxide in human and rodent cells in vitro is con- sistent, strong, and specific with respect to exposure to styrene or styrene-7,8- oxide. Temporal and exposure–response relationships have been established. These mechanistic events have been studied extensively in human cells, and the results are consistent with those found in cells obtained from rodents. Genotoxic and Clastogenic Effects in Animals or Humans Exposed to Styrene and in Rodents Exposed to Styrene-7,8-Oxide Many studies have attempted to evaluate the genotoxicity and clastogenic- ity of styrene in humans exposed in an occupational setting. Styrene-induced DNA damage was found in many of the studies (Table 3-12). Most studies in rodents demonstrate sister-chromatid exchanges after exposure to styrene or styrene-7,8-oxide, but only about half of the human studies that examined sty- rene exposure demonstrated the same effects (Table 3-13) . With respect to mi- cronuclei, studies of humans or rodents exposed to styrene are also equally di- vided; however, formation of micronuclei was not observed in three studies of rodents exposed to styrene-7,8-oxide (Table 3-14). For chromosomal aberrations (Table 3-15), about half of the studies of humans exposed to styrene show a sig- nificant effect. In exposed rodents, formation of chromosomal aberrations was not found in most of the studies of styrene exposure or in half studies of styrene- 7,8-oxide exposure. 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 num- ber 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 styrene- 7,8-oxide was associated with the positive and negative observations. Studies of rodents provided evidence of a temporal relationship for the observed associa- tion in that effects were observed only after exposure to the agents in question. Studies of rodents also provided strong evidence of a genotoxic concentration– response relationship. Evidence of genotoxicity and clastogenicity of styrene in exposed humans is generally strong, although some studies reported no effects. The strongest positive observations involved DNA-damage end points found in studies of di- verse cohorts of subjects exposed to styrene in various occupations. Various assays were used to evaluate the mechanistic events, and the statistical signifi- cance of the effects was firmly established in the positive studies. Some investi- gators established exposure to styrene through biomonitoring, but many of their studies were of occupational groups and the contributions from other toxic agents cannot be excluded. An exposure–response association for several bi- omarkers of genotoxicity and clastogenicity was demonstrated through work- place dosimetry (Fracasso et al. 2009) or the use of urinary biomarkers of expo- sure (Teixeira et al. 2010).

128 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens The committee concludes that the biologic plausibility of genotoxic and clastogenic effects of styrene, as observed in exposed animals and humans, is supported by solid and extensive observational evidence. Several negative stud- ies notwithstanding, the evidence is generally strong and specific with respect to styrene or styrene-7,8-oxide exposure. Both temporal and exposure–response relationships have been clearly established by diverse studies, including studies of exposed humans. That most of the observational evidence used in this evalua- tion is derived from studies of humans exposed to styrene substantially strength- ens the relevance of the mechanistic evidence to the epidemiologic findings. Summary of Evidence on Genotoxicity of Styrene Styrene requires metabolic activation to electrophilic intermediates (for example, styrene-7,8-oxide) for it to be able to form covalent adducts with DNA. 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 pres- ence of DNA adducts—occurring predominantly at the N7, N2, and O6 posi- tions of guanine—has been amply demonstrated in cell culture, experimental animals, and, most important, lymphocytes of workers occupationally exposed to styrene (Table 3-12). These findings have been reproduced in many laborato- ries and provide strong evidence of the genotoxic effects of styrene. Unless removed by nucleotide excision repair, styrene-7,8-oxide DNA ad- ducts invariably serve as a substrate for DNA polymerases, including special- ized lesion-bypass polymerases that may either block DNA synthesis at the le- sion site or catalyze the introduction of nucleotides that lead to mutational changes. Evidence of the mutagenicity of styrene and styrene-7,8-oxide was established early on in studies of bacteria and other nonmammalian systems (IARC 1994a, 2002). Consistently positive results, with or without metabolic activation, have been reported for gene-mutation end points in bacteria and other model organisms exposed to styrene-7,8-oxide. In studies with styrene, results were less consistent in the absence of metabolic activation; positive results were reported in Salmonella typhimurium strains TA1530 or TA1535 with the addi- tion of an exogenous metabolic activation system (IARC 2002). Additional evi- dence of the mutagenic potential of styrene or styrene-7,8-oxide includes studies of Chinese hamster (V79) cells, mouse lymphoma (L5178Y), and human T lymphocytes (HPRT locus) (Vodicka et al. 2006). Moreover, many studies of occupationally exposed workers report a positive association between styrene exposure and frequency of sister-chromatid exchanges, micronuclei, and chro- mosomal aberrations (Tables 3-13 to 3-15). Although the evidence for and against an association of clastogenic effects with styrene exposure in humans is nearly equally divided, the diversity of studies, exposure scenarios, and method- ology support the biologic plausibility of the genotoxicity of styrene in exposed humans. Even low-concentration occupational exposure to styrene was shown to result in an increase in various genotoxic effects (Wongvijitsuk et al. 2011).

Independent Assessment of Styrene 129 Genotoxic effects have been explored in comparisons with structurally related epoxides, many of which are classified as human carcinogens or as likely to be human carcinogens (Fabiani et al. 2012). The committee concludes that the genotoxicity and mutagenicity of sty- rene has been thoroughly and comprehensively investigated. The evidence re- viewed 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. For example, styrene-3,4- oxide may also be mutagenic (Watabe et al. 1982). However, to the committee’s knowledge, the potential contribution of styrene-3,4-oxide to the carcinogenic response to styrene and the potential contribution of other aromatic-ring metabo- lites of styrene in addition to styrene-7,8-oxide have not been investigated. Overall, the observations in various studies performed over the last 3 dec- ades have been consistent. Temporal and exposure–response relationships have been established. Not only is the experimental evidence extensive, it is likely to be relevant to all target tissues that have been associated with cancer after expo- sure to styrene. Causality is strengthened by the large amount of evidence ob- tained from studies of exposed humans. Immunosuppression The human immune system plays a critical role in defending the body against external pathogens and in being on perpetual alert against internally transforming (premalignant) or transformed malignant cells (cancers). The con- cept of “immune surveillance” describes those functions specifically and relies on the involvement of a network of white blood cells, also known as leukocytes. There are two basic types of leukocytes: phagocytes (including neutrophils, monocytes, and macrophages, which are important in innate immunity) and lymphocytes (including T, B, and natural killer [NK] cells that allow the im- mune system to recognize, memorize, and specifically respond to previous in- vaders). NK lymphocytes are critical for the innate and adaptive immune sys- tems because they can destroy virus-infected or malignantly transformed cells. Therefore, they are extremely important in immunosurvillance. When deficiency is present in one or more immune components as a result of congenital or ac- quired conditions, immunodeficiency or immunosuppression might occur and the incidence of malignancies might increase. For example, Kaposi sarcoma, NHL, and cervical cancers occur at a higher rate in people who have acquired immunodeficiency syndrome (AIDS) as a result of infection by human immuno- deficiency virus (HIV) (Labarga 2013). In addition, all de novo neoplasms have a greater incidence in renal-transplantation patients because of antilymphocytic treatment (Andrés 2005). As discussed in Chapter 2, NTP identified immunosuppression as a possi- ble mechanism by which styrene exposure could lead to malignancies, but the

130 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens background document and the substance profile lack strong evidence to support this mechanism. Therefore, the committee undertook an independent literature search to identify research that would inform the topic (see Appendix D). The committee considered studies relevant if they reported data on changes in basic hematologic measures, such as white blood cell (WBC) count and WBC differ- ential count.2 The committee also included studies that reported such measures as weight of lymphoid organs and expression of functional markers. Efforts were made to identify and include studies that reported effects on local, system- ic, innate, and adaptive immunity in exposed animals or humans. Studies that reported genotoxic measures were excluded from this section because they are discussed in more detail in the genotoxicity sections of Chapter 2 and the pre- sent chapter. The committee’s literature search yielded 233 results, 19 of which were relevant articles that were not already cited in the background document for styrene. Eight of the studies documented hematologic effects in experimental animal models or in animal cells (Table 3-17), and 11 described hematologic effects in humans or human cells (Table 3-18). Those studies were reviewed in detail by the committee and are discussed below. Animal Studies Leukocytopenia and Lymphocytopenia Two studies described hematologic effects in peripheral blood that were consistent with leukocytopenia and lymphocytopenia. Brondeau et al. (1990) observed a transient decrease in WBCs (leukopenia) in rats exposed to styrene for 4 hours. Seidel et al. (1990) observed decreased lymphocyte counts (lym- phocytopenia) in peripheral blood of female C57BL/6 x DBA/2 hybrid mice after exposure to styrene. Systemic vs Localized Lymphoid Organs In animal models, the response of lymphocytic organs to styrene exposure can vary at different locations. For example, the weight of the spleen was signif- icantly lower in mice exposed to styrene than in controls, whereas the weights of peripheral lymph nodes were higher in exposed mice than in controls (Dogra et al. 1989). Lymphocytic proliferation in the spleen was significantly lower in styrene-7,8-oxide-exposed C57BL/6 mice than in styrene-exposed mice; this indicates that an active intermediate form of styrene may be needed for systemic inhibition to occur (Grayson and Grill 1986). 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.

TABLE 3-17 Immune Effects of Inhalation or Intraperitoneal Exposure to Styrene in Animals Cell Type Hematologic Effects Reference RBC ↑ erythroid by inhalation Nano et al. 2000 No change by intraperitoneal administration Nano et al. 2000 WBC ↓ by 4h exposure, but innate rather than specific effects cannot be ruled out Brondeau et al. 1990 Neutrophils ↓ numbers of promyelocytes and myelocytes temporarily by inhalation (chronic) but unchanged by Nano et al. 2000 intraperitoneal administration (acute) Monocytes ↓ nitro-blue tetrazolium, monocyte attachment, phagocytic activity Dogra et al. 1989 NK cells ↓ activity by styrene, styrene-7,8-oxide Grayson and Gill 1986 Lymphocyte ↓ numbers Seidel et al. 1990 ↓ spleen weight Dogra et al. 1989 ↓ splenic lymphocyte counts vs no change in lymphocyte counts in regional or peripheral lymph Dogra et al. 1989 nodes or bone marrow T cells ↑ interferon-gamma in local lymph nodes by inhalation Ban et al. 2003 ↑ T-helper lymphocyte cytokine and interleukin level Ban et al. 2006 ↑mitogen-stimulated proliferation Sharma et al. 1981; Dogra et al. 1989 ↑ delayed-type hypersensitivity Dogra et al. 1989 B cells ↓ immunoglobulin M plaque-forming unit Dogra et al. 1989 ↑ mitogen-stimulated proliferation at lowest and middle doses Dogra et al. 1989 Stem cells Unaffected in CFU-S and CFU-C but lower in BFU-E and CFU-E although statistical difference Seidel et al. 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. 131

132 TABLE 3-18 Immune Effects of Inhalation Exposure to Styrene in Humans Cell type Hematologic Effects Reference RBC ↓ RBC and hermatocrit Checkoway and Williams 1982 WBC ↓ absolute neutrophils Checkoway and Williams 1982 Hagmar et al. 1989; Tulinska et al. 2000; Biro et al. 2002; No difference in CBC Jahnova et al. 2002 ↑ WBC Somorovska et al. 1999 Monocytes ↑ adherent molecule expression Somorovska et al. 1999 Hagmar et al. 1989; Stengel et al. 1990; Tulinska et al. 2000; ↑ percentage Jahnova et al. 2002 ↓ percentage Khristeva 1986 ↑ necrosis, apoptosis, increased bcl-2 and raf-1 proteins Diodovich et al. 2004 Lymphocyte ↓ lymphocytes Tulinska et al. 2000 No change Biro et al. 2002 ↑ lymphocytes Khristeva 1986 T cells ↓ numbers Tulinska et al. 2000 ↑ CD4+ T (Th) cells Mutti et al. 1992; Bergamaschi et al. 1995; Biro et al. 2002 No difference in mitogen-induced proliferation of lymphocytes Hagmar et al. 1989; Somorovska et al. 1999 ↓ mitogen-induced proliferation of lymphocytes Somorovska et al. 1999; Tulinska et al. 2000; Jahnova et al. 2002 ↓ large-granule lymphocytes Somorovska et al. 1999 NK cells ↑ NK cells Mutti et al. 1992; Bergamaschi et al. 1995 ↓ NK function (K562 cell lysis) Bergamaschi et al. 1995 B cells No change in numbers Mutti et al. 1992; Tulinska et al. 2000 ↑ CD25+ expression in B cells Bergamaschi et al. 1995 No change in mitogen-induced proliferation of lymphocytes Hagmar et al. 1989; Tulinska et al. 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.

Independent Assessment of Styrene 133 through such mechanisms as increased interferon-gamma, interleukin (specifi- cally IL-4, IL-5, and IL-13), and immunoglobulin E (IgE) production were ob- served more in lung and lymph nodes than in those produced from lymphocytes in the spleen of female BALB/c mice (Ban et al. 2003, 2006). Innate vs Adaptive Immunity Immune responses are typically divided into two categories—innate (non- specific) responses and adaptive (antigen-specific) responses. Monocytes, mac- rophages, neutrophils, and NK cells are the main effector cells in innate immuni- ty, 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. For example, a substantial impairment was observed in macrophage and monocyte functional studies and resulted in a reduction in nitroblue tetrazolium, changes in surface attachment, and changes in phagocytic indexes in mice exposed to styrene (Dogra et al 1989). In addition, in a dose-dependent manner, styrene and styrene-7,8-oxide were strong suppres- sors of NK-cell activity in exposed mice, whereas cytotoxic T-cell activity was not affected (Grayson and Grill 1986). In contrast, styrene exerted more stimulatory effects on adaptive cellular immunity in mice by enhancing delayed hypersensitivity (also known as type IV hypersensitivity) (Dogra et al. 1989), mitogen-stimulated lymphoblastic trans- formation (Sharma et al. 1981), increased production of interferon-gamma and cytokines, and increased production of interleukins by T-helper type 2 lympho- cytes (Sharma et al. 1981; Dogra et al. 1989; Ban et al. 2003, 2006). For B lym- phocytes, Dogra et al. (1989) observed reduced IgM plaque-forming colonies but increased liposaccharide-stimulated proliferation. Hematopoietic Malignancy The committee found two animal studies that provided information on hematopoietic measures of malignancies (Seidel et al. 1990; Nano et al. 2000). Animals exposed to styrene had reduced erythroid lineage colony-forming func- tion (specifically, burst-forming unit erythroid [BFU-E] and colony-forming unit erythroid [CFU-E]), but normal colony-forming unit function in the spleen (CFU-S) and colony-forming unit function in culture (CFU-C) (Seidel et al. 1990). Nano et al. (2000) aimed to determine whether there was a higher fre- quency of malignancies in hematopoietic tissues of rats treated with styrene by either injection or inhalation; they did not observe an increase in the frequency of preleukemic or leukemic disorders in rats exposed to styrene, although de- creased promyelocytes and myelocytes were observed after exposure by inhala- tion (Nano et al. 2000).

134 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens Human Studies Leukocytopenia and Lymphocytopenia The studies of humans exposed to styrene reported inconsistent results for WBC and lymphocyte counts. Abnormal WBC differential was identified in two studies (Somorovska et al. 1999; Jahnova et al. 2002), but more results showed either normal complete blood counts or increased WBCs in exposed people (Hagmar et al. 1989; Somorovska et al. 1999; Tulinska et al. 2000; Biro et al. 2002; Jahnova et al. 2002). Systemic vs Localized Lymphoid Organs The committee did not identify any studies of humans that reported sys- temic vs localized effects on the immune system after exposure to styrene. Innate vs Adaptive Immunity The committee identified studies that reported effects on the innate im- mune system following exposure to styrene. Increases in monocytes or monocy- tosis were reported in five of seven studies (Hagmar et al. 1989; Stengel et al. 1990; Somorovska et al. 1999; Tulinska et al. 2000; Jahnova et al. 2002). Only one study found a decrease in monocytes (Khristeva 1986) and another found that necrosis and apoptosis were increased in monocytes (Diodovich et al. 2004). For NK cells, two studies found that the number of NK cells were in- creased in workers exposed to styrene (Mutti et al. 1992; Bergamaschi et al. 1995). Bergamaschi et al. (1995) also performed a functional study of NK cells (that is, in vitro lysis of leukemia cell lines) and demonstrated that workers ex- posed to styrene had significantly lower cytotoxic activity toward leukemia cells than the control group. On the basis of those results, styrene might have sup- pressive effects on NK cells, but more studies are needed before a stronger con- clusion can be reached. Among the studies that investigated effects of styrene on adaptive im- munity, inconsistent results were observed between the number of lymphocytes in workers exposed to styrene and the number in controls. For example, Khriste- va (1986) reported an increase in the number of lymphocytes, but Tulinska et al. (2000) observed a decrease in the number of lymphocytes and Biro et al. (2002) found no change. Three studies found a decrease in mitogen-induced T-cell pro- liferation (Somorovska et al. 1999; Tulinska et al. 2000; Jahnova et al. 2002), and two found no difference between exposed and non-exposed groups (Hagmar et al. 1989; Somorovska et al. 1999). Most of the studies found no change in the number of B lymphocytes and no change in mitogen-induced proliferation of B lymphocytes in workers exposed to styrene (Hagmar et al. 1989; Mutti et al. 1992; Bergamaschi et al. 1995; Tulinska et al. 2000; Jahnova et al. 2002).

Independent Assessment of Styrene 135 Conclusions on Immunosuppression Evidence As mentioned in Chapter 2 and above, the background document and the substance profile lack strong evidence to support immunosuppression as a po- tential mechanism of carcinogenesis, so the committee undertook a literature search (see Appendix D). No relevant studies of the immunosuppressive effects of styrene were identified that were not available to NTP (that is, all relevant articles were published by June 10, 2011). After reviewing the relevant studies, the committee determined that the evidence on immune effects after exposure to styrene varies and is inconsistent. 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. In con- trast, effects on humoral immunity in styrene-exposed animals varied. For ex- ample, IgM plaque-forming cells were decreased, but lipopolysaccharide- induced proliferation was increased. In humans exposed to styrene, effects were more varied and both suppressive and stimulatory effects were observed. Addi- tional research is needed to understand the effects of styrene on the immune system and to explore whether immunosuppression is a possible mechanism for styrene-induced carcinogenesis. Cytotoxicity The use of cytotoxic responses to investigate the mode of action by which exposure to metabolically activated compounds, such as styrene, produces tu- mors depends on a clear definition of the conditions that render a specific organ or tissue susceptible to injury. As outlined in the section “Metabolism and Toxi- cokinetics” above, many factors contribute to those conditions. Recent literature regarding cytotoxicity of styrene addresses three general questions: Which iso- zymes of the cytochrome P450 mono-oxygenase system are involved in meta- bolic activation? What are the chemical nature and reactivity of the metabolites? Which antioxidants and phase II enzymes interact with the reactive metabolites to modulate toxicity? Pulmonary and Hepatic Toxicity Recent studies of the mechanisms by which styrene produces cytotoxic in- jury have relied on the cytotoxic response in the lungs, and occasionally the liv- er, of one species: the mouse. The relevance of bioactivation (by cytochrome P450 mono-oxygenases) and detoxification (by glutathione S-transferases and epoxide hydrolases) pathways has been evaluated by using relatively nonspecif- ic assays (Carlson 2010b, 2011a,b, 2012; Meszka-Jordan et al. 2009; Shen et al.

136 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens 2010). For substances released into the airway space, bronchoalveolar-lavage fluid has been analyzed for protein, cells, and succinic dehydrogenase. For the liver, serum has been analyzed for sorbitol dehydrogenase. Although those non- specific approaches appear to provide reliable screening tools and reflect the overall responses of the organs, they lack sufficient specificity to define the re- sponse in the presumed target cells for styrene, the Clara cells. That is especially true in the mouse lung, in which cells that have high metabolic potential are not restricted to the terminal bronchioles but are distributed throughout the airway tree, with some activity in the gas-exchange area (Buckpitt et al. 1995). Studies with another CYP450-activated cytotoxic aromatic hydrocarbon, naphthalene, have also documented that acute Clara cell injury increases in terminal bronchi- oles as the intraperitoneal dose is elevated and that injury extends about as far as lobar bronchi at higher doses (Plopper et al. 1992). When naphthalene is admin- istered via inhalation, acute injury is equal to or greater in proximal bronchi than that produced in terminal bronchioles (West et al. 2001). Other studies of styrene have assessed toxicity by examining proliferation in the presumed target area (terminal bronchioles). Toxicity was determined on the basis of differential counts of cells that have undergone proliferation and that have incorporated and expressed a DNA precursor (5-bromo-2’-deoxyuridine, BrdU) (Cruzan et al. 2012, 2013). The committee notes that restricting the his- topathologic and quantitative analysis to terminal bronchioles may not accurate- ly characterize the full response, because the cells that are most likely to be un- dergoing replication, and most of their daughter cells, are also the cells that are most likely to be damaged by bioactivated cytotoxicants. In addition, at higher doses, the level of cell death may be so high that repopulation of distal airways is principally by progenitor cells that are found in more proximal airways and at airway bifurcations (Stripp et al. 1995; Lawson et al. 2002). Kaufmann et al. (2005) reported high levels of labeled cells in proximal bronchi following 3 days of exposure to styrene and two of its metabolites, sty- rene-7,8-oxide and 4-vinylphenol. Further complicating the interpretation of BrdU–incorporation studies of styrene cytotoxicity is the fact that epithelial cells injured by initial exposure to a cytotoxic agent undergo a cycle of necrosis and exfoliation of injured cells and squamation and proliferation of surviving cells, followed by migration and differentiation of newly produced cells to repopulate injured sites. The first phase is usually complete by 2 to 3 days following a sin- gle exposure and the second phase by 5 to 7 days following exposure. The tim- ing depends on the route and concentration of the toxicant exposure. This pro- cess has been well documented for the oxidant gas ozone (Paige and Plopper 1999; Plopper et al. 2001) and for naphthalene (Van Winkle et al. 1995; Lawson et al. 2002), and seems to be the case for styrene based on observations by Kaufmann et al. (2005). When this injury–repair cycle occurs in the presence of elevated levels of the cytotoxicant, as are produced by repeated daily exposures, the repaired population becomes tolerant to further injury, obviating the need for proliferation and repair, even at doses approaching the LD50 (the dose that is lethal to 50% of the test organisms). The production of tolerance has been doc-

Independent Assessment of Styrene 137 umented for ozone (Paige and Plopper 1999; Plopper et al. 2001), nitrogen diox- ide (Kubota et al. 1987), naphthalene (O’Brien et al. 1989; West et al. 2003), 4- ipomeanol (Boyd et al. 1981), and coumarin (Born et al. 1999). Assessments of metabolite reactivity and cellular antioxidant responses have relied on direct assays of relevant cellular chemicals either in isolated target cells or in organ homogenates or serum (Carlson 2010a; Harvilchuck and Carlson 2009; Harvilchuck et al. 2008, 2009). Bioactivation by Cytochrome P450 Mono-oxygenases A review cited in the background document for styrene listed a substantial number of CYP450 isozymes that have been identified in the lungs and liver of mice, rats, and humans as having the ability to metabolize styrene to styrene- 7,8-oxide and other metabolites (Vodicka et al. 2006). A more recent review summarizes the large number of isozymes found in human lung (Carlson 2008). Although the specific CYP450 mono-oxygenases capable of catalyzing the me- tabolism of styrene have been reported, the committee found almost no available information on the kinetics of the process or an evaluation of the catalytic effi- ciencies of the enzymes involved (that is, Kcat/Km). Thus, it is still not known which isozymes are critical for the generation of metabolites that result in cyto- toxicity. Of further concern is the inadequate characterization of possible com- pensatory changes in gene expression in the CYP2F2–null animals. No data were identified that demonstrated how the null animals differed from the wild- type in terms of disposition kinetics of styrene or styrene-7,8-oxide. A recent in vitro study that used lung and liver microsomes, principally from mice, has identified additional phenolic metabolites whose production ap- pears to be based primarily on the activity of two CYP450 isozymes, CYP2F2 and CYP2E1, on the basis of modestly selective CYP450 inhibitors, and on the basis of studies that used very high substrate concentrations (500 µM) (Shen et al. 2010). The study also strongly emphasized that there is active metabolism of styrene in both the liver and the lung. However, the toxicity of the metabolites was tested only in the mouse lung and only at concentrations that did not gener- ate toxicity by the parent compounds, styrene and styrene-7,8-oxide, or by any of the phenols except 4-vinylphenol. It is not clear how the liver would respond to those metabolites or to what degree production of the metabolites by the liver might contribute to toxic responses in the lung. Three studies that used the same strain of CYP2F2 knockout mice demon- strated that metabolism by the 2F2 isozyme is critical for the production of high levels of distal bronchiolar cytotoxicity in lungs of mice, whether styrene is ad- ministered via intraperitoneal injection (Carlson 2012; Cruzan et al. 2012, 2013) or orally (Cruzan et al. 2012). Liver toxicity was also present in both deficient and wild-type mice (Carlson 2012). Metabolism of styrene to the R- and S- styrene oxide enantiomers was identical in the liver of wild-type and knockout mice but markedly reduced in the lungs of knockout mice (Carlson 2012). Alt- hough CYP2F2 knockout mice appear to be insensitive to styrene exposure,

138 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens leading the authors to conclude that this is a key enzyme associated with the metabolic activation of styrene, no data were presented to demonstrate a change in the rates of metabolism in airways of knockout mice compared with wild-type mice. Furthermore, although the knockout animals were characterized to deter- mine whether there were compensatory changes in the concentration of CYP450s, no attempts were made to evaluate alterations in proteins associated with detoxification. It is not clear what the full metabolic potential of the lungs and liver of the knockout animals are for metabolizing styrene to other metabo- lites. On the basis of an assay of bronchiolar epithelial proliferation, bronchiolar toxicity, produced by both styrene-7,8-oxide enantiomers, was markedly lower in knockout than in wild-type mice and is equal to that of carrier-treated controls (Cruzan et al. 2012). Whether this was true for epithelium in other airways that were more proximal was not assessed. The study also found that the portion of bronchiolar epithelial cells, which contained BrdU labelling, actually decreased at higher doses of styrene, and this suggests that cells that have the potential for rep- lication may be lost as part of the toxic response at higher doses. In CYP2F2-/- mice in which a transgene for three human CYP450 mono-oxygenases (CYP2F1, 2A13, and 2B6) was inserted, lung toxicity, on the basis of the same proliferation assay, was observed with 4-vinylphenol but not styrene or the R- or S-styrene ox- ides (Cruzan et al. 2013). A major deficiency of these studies is that there were no quantitative measurements of the differences in metabolic capacity of the airways in wild-type and transgenic mice. How the presence of the CYP450 isozymes in the liver and other organs affected their response was not addressed. In knockout mice deficient in hepatic CYP450 reductase, which is critical for CYP450 func- tion, lavage and serum markers of lung and liver toxicity were higher than in carri- er-treated controls (Carlson 2012). Although the metabolism of styrene to the R- or S-styrene oxides was markedly reduced in the liver, production of the R-styrene oxide in the lungs doubled, and S-styrene oxide production was unchanged com- pared with controls. The literature suggests that more than one CYP450 isozyme is involved in generating cytotoxic metabolites from styrene. More organs than the lung appear to serve as sites for both metabolic activation and cytotoxic injury, and the re- sponses in different organs are unique to the organ. However, a clear under- standing of the roles of CYP450s in the cytotoxicity of styrene will require fur- ther 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 4- vinylphenol, have been used to define markers of oxidative stress and the cellu- lar stress response only in Clara cells in mice. Expression of Clara cell secretory protein (CC10) mRNA was affected differently when exposure to those com- pounds was in vitro (expression was increased by R- and S-styrene oxide and

Independent Assessment of Styrene 139 decreased by 4-vinylphenol) as opposed to in vivo by intraperitoneal injection (expression was decreased by racemic and R-styrene oxide over a time course) (Harvilchuck et al. 2008). Expression of CC10 protein followed the same pat- tern. Reactive oxygen species were increased in Clara cells by both short-term in vitro and in vivo exposure to styrene, racemic styrene oxide, R-styrene oxide, and S-styrene oxide, but not 4-vinylphenol (Harvilchuck et al. 2009). Cellular markers of oxidative stress (8-hydroxydeoxyguanosine and superoxide dis- mutase) and indicators of apoptosis (bax/bcl-2 and caspase 3) were also in- creased by styrene or R-styrene oxide. Expression of all four markers returned to control values over an extended postexposure period after R-styrene oxide treatment but not in all cases for styrene. Repeated exposures to styrene and R- styrene oxide produced different short-term responses for the expression of CC10 mRNA and bax/bcl-2 mRNA (Harvilchuck and Carlson 2009). None of those studies addressed oxidative stress in other organs created by the presence of circulating styrene and its metabolites. To define mechanisms by which the metabolites of styrene react with potential target cells more clearly, future stud- ies will need to address oxidative stress in other cell populations that have dif- ferent 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). Depletion in both bronchoalveolar lavage fluid and plasma ranged from 30% up to 90%. Replenishment to steady-state concentrations after exposure to either compound required about 24 hours. Systemic pretreatment with known antioxidants to increase cellular antioxidant pools modulated sty- rene toxicity differently in two of the principal target organs (lung and liver) (Meszka-Jordan et al. 2009). On the basis of exposure to the most toxic metabo- lite, R-styrene oxide, glutathione pretreatment reduced liver toxicity without altering toxicity in the lungs. N-Acetylcysteine pretreatment had the same effect on the liver and a partial reductive effect on lung toxicity. Administration of a synthetic tetrapeptide analogue of glutathione, 4-methoxy-L-tyrosinyl-g-L- glutamyl-L-cysteinyl-glycine (UPF1), for a week before R-styrene oxide treat- ment enhanced toxicity in both lung and liver. Glutathione appears to play a role in modulating cellular toxicity produced by styrene metabolites, but a clear defi- nition of the roles of the cellular and extracellular pools requires further studies that compare responses in a number of cell populations and organs that have different degrees of susceptibility. Detoxification Pathways The role of enzyme systems responsible for the detoxification of styrene and its metabolites has not been clearly defined for most potential target organs.

140 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens Recent work that focused on the glutathione conjugation pathway used mice that were deficient in glutathione S-transferase pi (GST P1P2-/-), one of the three classes of glutathione S-transferases (Carlson 2011b). The results demonstrated that absence of this form did not alter the liver or lung toxicity of styrene (Carl- son 2011b). The toxicity of racemic styrene oxide was not altered in the liver but was increased in the lungs of deficient mice. That was also the case for 4- vinylphenol. Depletion of glutathione in the lung and liver by styrene or 4- vinylphenol was unaltered in deficient mice. Expression of peroxiredoxin VI (a bifunctional enzyme) in the liver of GST P1P2-/- mice was substantially in- creased in comparison to wild-type mice (Kitteringham et al. 2003), and the effect of this change on metabolism and cytotoxicity, and possibly other altera- tions in protein concentration, is unclear. Microsomal epoxide hydrolase is thought to play a critical role in the de- toxification of styrene by the hydrolysis of styrene-7,8-oxide to styrene glycol. Mice deficient in microsomal epoxide hydrolase do not exhibit a difference in the metabolism of styrene to the R- or S-styrene oxides in either the liver or the lung (Carlson 2010b). Metabolism of styrene-7,8-oxide to glycol is reduced for R- and S-styrene oxide in the liver and for R-styrene oxide in the lung. The tox- icity of styrene is substantially higher in both the lung and liver of deficient mice than in wild-type mice. However, the toxic response to racemic styrene-7,8- oxide did not differ in the liver and lung between deficient and wild-type mice. 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). Neither enantiomer produced liver toxicity, but S-styrene oxide pro- duced greater lung toxicity. R-styrene oxide substantially depleted liver gluta- thione, but S-styrene oxide did not. The glutathione S-transferases and epoxide hydrolase pathways appear to play a role in the detoxification of reactive styrene metabolites. Their contribu- tions to cellular injury will require more comprehensive studies that compare activity and responses in multiple organs that have different levels of suscepti- bility. Summary of Cytotoxicity Evidence In summary, the studies cited above, in combination with those included in the background document for styrene (NTP 2008), suggest that the mode of action by which styrene produces toxicity is highly complex. The final cellular outcome associated with exposure to a metabolically activated chemical, particularly with chemicals present at relatively low concentrations in the environment, is highly dependent on both the catalytic efficiency of the enzymes involved in the activa- tion and detoxification processes and the amount of protein present in target cells. Establishing the mode of action for styrene on the basis of cytotoxicity and later proliferation at injured sites will depend on a comprehensive approach to identify

Independent Assessment of Styrene 141 the cellular, metabolic, and chemical processes involved in different organs and to define rigorously how their interactions modulate the toxic response. Although research points to the importance of CYP2F2 in biomarker alterations (that is, BrdU labeling indices) that have been observed in styrene-exposed mice, the committee judged the studies to generally lack the scientific rigor necessary to ensure the validity of the conclusions. All the studies noted above relied on the intraperitoneal injection of styrene and its metabolites into the model species, the mouse, with the exception of Cruzan et al. (2012) study, which exposed mice via gavage. Consequently, the response of the candidate target organ, the lung, is based on the concentration of the compound delivered to it by the circulation. In none of the studies were the circulating concentrations determined. Other organ systems in the animal were exposed at the same time. When the response in anoth- er organ, the liver, was compared with that in the lung, it became clear that at least two organs are targets for cytotoxicity produced by styrene and its circulating me- tabolites. Studies of workers in the styrene industry found styrene or its metabo- lites in both blood and urine and identified a number of additional target organs in at least three other systems—the lymphohematopoietic system (bone marrow, lymph nodes, and spleen), gastrointestinal system (esophagus and pancreas), and urinary system (kidney and bladder)—that should be included in mechanistic stud- ies that use animal models. The need to study other organs in addition to the lungs is especially true for studies in which metabolic capabilities of the model are al- tered by eliminating the genes for specific activation and detoxification enzymes in the animal as a whole. Additional studies that compare the kinetics of styrene metabolism using a range of recombinant P450 proteins, including CYP2F1 (the human orthologue of CYP2F2), are needed to establish the catalytic efficiency of these proteins with styrene. When both liver and lung were assessed in the studies evaluated in this section, the metabolic function and toxic response in both organs were altered. When gene manipulation was restricted to one organ, the liver, the toxic response in the other, the lung, was altered. Circulating concentrations of key compounds in the toxic response (when evaluated) were also altered. Taken as a whole, this evidence suggests that the activities and toxic responses of multiple organs may play a role in modulating the circulating concentrations of styrene, its metabolites, and other key compounds, such as glutathione, and in affecting the toxic response of other organs in the same individual. 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 recom- mendation for styrene and provide scientific justification for its recommenda- tion.” As discussed throughout this report, a substance can be categorized as reasonably anticipated to be a human carcinogen on the basis of sufficient evi- dence in animals or limited evidence in humans and a substance can be catego-

142 Review of the Styrene Assessment in the NTP 12th Report on Carcinogens rized as known to be a human carcinogen on the basis of sufficient evidence in humans (see Box 1-2). Guided by the RoC listing criteria, the committee inte- grated data from individual studies to determine whether the evidence in exper- imental animals reached the level of limited or sufficient and to determine whether the evidence in humans reached the level of limited or sufficient. Sup- porting information was provided from mechanistic studies. The RoC listing criteria do not provide guidance on the integration of information across data streams (that is, across human, experimental animal, and mechanistic infor- mation) or the reconciliation of cross-data inconsistencies, so the committee only integrated information within data streams to derive a listing recommenda- tion. The committee identified evidence of styrene exposure that would poten- tially lead to carcinogenicity through genotoxic and mutagenic mechanisms, and that evidence is considered strong, inasmuch as it has been found in vivo and in vitro in both humans and rodents. The genotoxic mechanism is probably rele- vant for all target tissues associated with cancer after exposure to styrene. Identi- fication of styrene metabolites, such as styrene-7,8-oxide, strongly supports the production of reactive intermediates in a variety of tissues in both humans and animals. The reactive metabolites, which may be produced in one organ and transported to produce toxicity in other sites, have been identified in the blood of humans exposed to styrene. Animal toxicology and carcinogenesis studies clear- ly support the possibility that multiple organs can be affected regardless of their capacity for metabolic activation. In humans, evidence of carcinogenicity in multiple organs is credible but limited. Those findings were based on large oc- cupational cohort studies in the reinforced-plastics industry and on case–control studies. In sum, the committee finds that compelling evidence exists to support a listing of styrene as, at a minimum, reasonably anticipated to be a human carcinogen. That conclusion is based on credible but limited evidence of carcinogenicity in traditional epidemiologic studies, on sufficient evidence of carcinogenicity in animals, and on convincing evidence that styrene is genotoxic in exposed humans. 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] that can be useful for evaluating whether a relevant cancer mechanism is operating in people”. The evidence of styrene genotoxicity in ex- posed humans is convincing, so a strong argument could be made to support the listing of styrene as a known human carcinogen if data derived from the study of tissues or cells from humans in and of themselves are considered sufficient for making such a determination. The committee notes that there is ambiguity with respect to weighing the mechanistic evidence in applying the listing criteria. The types of evidence that are available to determine the listing and classi- fication of substances in the RoC continue to evolve. In the future, there will

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Many people in the United States are exposed to styrene. Sources of environmental exposure included food (from migration of styrene from polymer packaging materials), cigarette smoke, vehicle exhaust and other forms of combustion and incineration of styrene polymers. Occupational exposure to humans can occur during the industrial processing of styrene. It is used to create a broad spectrum of products, including latex paints and coatings; synthetic rubbers; construction materials, such as pipes, fittings, and lighting fixtures; packaging; household goods, such as synthetic marble, flooring, and molded furnishings; and automotive parts. In 2011, the National Toxicology Program (NTP) listed styrene as "reasonably anticipated to be a human carcinogen" in its 12th Report on Carcinogens, marking the first time that the substance was listed. Congress directed the Department of Health and Human Services to arrange for the National Academy of Sciences to independently review the substance profile of styrene and it listing in the NTP report.

Review of the Styrene Assessment in the National Toxicology Program 12th Report on Carcinogens concurs with the NTP determination that there is limited but credible evidence that exposure to styrene in some occupational settings is associated with an increase in the frequency of lymphohematopoietic cancers. Additionally, the NRC report authoring committee independently reviewed the scientific evidence from studies in humans, experimental animals, and other studies relevant to the mechanisms of carcinogenesis and made level-of-evidence conclusions. Based on credible but limited evidence of carcinogenicity in traditional epidemiologic studies, on sufficient evidence of carcinogenicity in animals, and on convincing evidence that styrene is genotoxic in exposed humans, this report finds that compelling evidence exists to support a listing of styrene as, at a minimum, "reasonably anticipated to be a human carcinogen."

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