Table 6.

RA2. Key events in the proposed mode of action of benzo(a)pyrene for rodents and their human relevance.

Key event	Evidence in rodents		References	Evidence in humans		References
	Toxicogenomic endpoints	Apical endpoints		Toxicogenomic endpoints	Apical endpoints
1.BaP binding to AHR (molecular initiating event)	Indirect evidence: upregulation of AHR-responsive genes.	YES: BaP is a well-established AHR ligand.	Malik et al. (2013); Ovesen et al. (2011). Table 5.	Cyp1b1 gene expression is induced by BaP in normal mammary epithelial cells.	YES: BaP binds to human AHR and activates AHR-dependent gene expression.	DiNatale et al. (2010; Gwinn et al. (2005)
2.Induction of cytochrome P450 enzymes (CYPs)	YES: Multiple P450 genes (e.g. Cyp1a1, Cyp1a7, Cyp1b1, Cyp2b10, Cyp2b13, Cyp3a44, Cyp2b9, Cyp2c38 and Cyp2c40) are induced by BaP in mouse liver, lung and forestomach.	YES: induction of EROD in diverse tissues following BaP exposure.	Halappanavar et al. (2011); Malik et al. (2012); Yauk et al. (2011); IARC (2010)	YES: BaP induces genes coding for P450 enzymes (Cyp1a1, Cyp1a2, Cyp2b6, Cyp2e1) in BaP-treated primary human hepatocytes.	YES: BaP increases the activity of P450 enzymes (EROD, ECOD assays) in BaP-treated primary human hepatocytes.	Wilkening et al. (2003)
3. BaP metabolism to reactive metabolites	YES: BaP induced phase II enzymes (e.g. Nrf2, Nqo1, Ugdh, Srxn1, Akr1b15, Ugt2b15, Gstm3, Gstm4 and Gstm7) in mouse liver, lung and forestomach to remove reactive metabolites.	YES: BaP metabolites have been isolated from animals exposed to BaP and studied in great detail.	Halappanavar et al. (2011); IARC (2010); Malik et al. (2012); Yauk et al. (2011)	YES: BaP induces Ephx1 and Nqo1, genes, coding for the enzymes involved in BaP metabolism, in BaP-treated primary human hepatocytes.	YES: BaP metabolites, including BaP-7,8-diol (a precursor to BPDE), were detected in human liver and lung microsomes.	Shimada et al. (1989); Wilkening et al. (2003)
4. DNA adducts and DNA damage	YES: Increased expression of genes regulated by p53 in lung, lung and forestomach from BaP-treated mice. Upregulation of Polκ and Ddit4 in mouse liver and lung indicates DNA damage repair.	YES: Adducts of BaP metabolites with DNA have been measured directly in several tissues in rats and mice.	Denissenko et al. (1996); Halappanavar et al. (2011); Labib et al. (2012); Malik et al. (2012); Wester et al. (2012)	YES: p53 pathway is activated by BaP in cultured human TK6 cells; stress response and DNA repair response are activated by BaP in human lymphoblastoidcell line.	YES: BaP metabolites (BPDE) form adducts with DNA, and BPDE–DNA adducts are detected in human white blood cells.	J. Buick et al. (Submitted); DeMarini et al. (2001); Liu et al. (2005); Perera et al. (1988)
5. Mutations	Indirect evidence: the induction of gene expression changes in DNA damage response pathways is highly correlated with genotoxicity and mutagenicity in rodents.	YES: Mutations were reported in multiple studies in rodents following BaP exposure.	Sakai et al. (2014); Malik et al. (2012); Labib et al. (2012); IARC (2010)	Indirect evidence: the induction of gene expression changes in DNA damage response pathways is highly correlated with genotoxicity and mutagenicity in human TK6 cells.	YES: BPDE–DNA adducts detected at mutational “hotspots” of p53 (G in codons 157, 248 and 273). Proto-oncogene K-Ras and p53 mutations found in lung tumors of nonsmokers exposed to BaP-containing PAH mixtures. Mutations in tumor suppressor gene p53 in BaP-exposed nonsmokers were detected.	Derk et al. (2014); Waters et al. (2010); J. Buick et al. (unpublished data); Denissenko et al. (1996); Rojas et al. (2000); DeMarini et al. (2001)
6. Altered cell signaling, proliferation and apoptosis	YES: A number of signaling pathways (including cancer pathways) were perturbed by BaP treatment in mice. Mdm2, an inhibitor of p53, was induced in the lung and liver of BaP-treated mice at high doses.	YES: Multiple in vitro studies using cultured and animal cells identified increased cell proliferation due to BaP or its metabolites. Mutations in BaP-treated mice were increased compared with controls.	Brandon et al. (2009); Burdick et al. (2003); IARC (2010); Labib et al. (2012); Wester et al. (2012)	YES: Anti-apoptotic signaling gene expression was increased in MCF-7 human breast carcinoma and HepG2 human hepatocarcinoma cells.	Data gap.	Hockley et al. (2006)
Tumorigenesis (adverse outcome)	YES: Gene expression of putative hepatocarcinogenes is markers were altered in liver, lung and forestomach of BaP-treated mice (e.g. Cdkn1a, Mdm2, Cbr1, Notch1, Nqo1, Nfe2l2 and Ephx1).	YES: Liver and lung tumors were observed in several other species.	Horikawa et al. (1991); IARC (2010); Lavoie et al. (1987); Park et al. (2011); Wester et al. (2012); Wislocki et al. (1986)	Alteration of Cdk, Mdm2, Nqo1transcript levels	NO: No direct evidence, but mutational pattern of p53 in human lung cancer is very similar to that of BPDE-treated human HeLa cells and bronchial epithelial cells.	Denissenko et al. (1996); Buick et al. (submitted); Hockley et al. (2006)

ECOD, ethoxycoumarin O-deethylase; EROD, ethylresorufin O-dealkylase