Table 7.

RA2. Evaluation of proposed mode of action by Bradford Hill criteria. As originally proposed by Hill (1965) and simplified by Meek et al. (2003).

Bradford Hill criteria	Questions asked	Evidence in animals	References
1. Dose–response relationships	Is increasing exposure to the compound associated with increased magnitude of adverse outcome?	YES: increased BaP doses were associated with increased tumor incidence in mice and rats.	Lavoie et al. (1987); Wester et al. (2012); Wislocki et al. (1986)
	Are key events observed at doses below or similar to those associated with the adverse outcome?	YES: total cumulative doses in the toxicogenomics experiments were below the cumulative doses in the cancer bioassay.
2. Temporal association between key events and adverse outcome and reversibility	Is sequence of events logical?	YES	Halappanavar et al. (2011); Labib et al. (2012); Malik et al. (2012); Yauk et al. (2011)
	Do key events and adverse outcome occur in expected order?	YES
	Does cessation of exposure (or any interference with the proposed key events) ameliorate adverse outcome?	YES: cessation of exposure (sacrificing animals 24 hours after the exposure) markedly affected BaP-perturbed gene expression, restoring the expression of many genes affected 4 hours after the treatment in mouse lung and liver.
3. Strength, consistency and specificity of association between key events and adverse outcome	Is the evidence linking exposure to a chemical, key events and adverse outcome strong?	YES	Reviewed in IARC (2010)
	Is it consistent in repeated experiments in the same laboratory and other laboratories?	YES
	Are the associations between exposure, key events and adverse outcome specific?	YES: the specificity of associations has been confirmed by several apical endpoints, including BaP metabolite detection in animal samples, DNA adducts, cell proliferation, immunoblotting and immunocytochemistry.
4. Biological plausibility and coherence of the database	Does hypothesized MOA make sense based on broader knowledge about the chemical and the biology associated with adverse outcome (e.g. chemical properties, disease etiology)?	AHR-mediated metabolism of BaP is consistent with what is known about other PAH ligands of AHR. Genotoxic carcinogens are well known, and DNA–BaP adducts are well documented. BaP is positive in mutagenicity tests.	Reviewed in IARC (2010)