Please indicate your confidence rating on the weight of evidence supporting/refuting the potential mode of action for the carcinogenic effects of 1,4-DX in the rat nasal cavity squamous cell carcinoma? (-5=strong refuting evidence, 0=lack of or equivocal evidence, +5=strong supporting evidence)

Answer Explanations

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  Expert 4

  	-5	-4	-3	-2	-1	0	1	2	3	4	5	I cannot answer
  Direct genotoxicity	0	0	1	0	0	0	0	0	0	0	0	0
  Indirect genotoxicity	0	0	0	0	0	0	0	0	1	0	0	0
  Proliferative regeneration induced by cytotoxicity	0	0	0	0	0	0	0	1	0	0	0	0
  Oxidative stress	0	0	0	0	0	1	0	0	0	0	0	0
  Activation of nuclear receptors and associated transcription factors (e.g., CAR, PXR)	0	0	0	0	1	0	0	0	0	0	0	0
  Other (Please explain)	0	0	0	0	0	1	0	0	0	0	0	0

  The mode of action is likely mediated through indirect genotoxicity based on observation in a relatively robust dataset in both in vitro and in vivo assays that 1,2-DX is not mutagenic and only weakly genotoxic with responses being positive generally only in the presence of toxicity.  However, the data supporting an hypothesized mode of action for rat nasal squamous cell carcinomas with key events of metabolic induction, cytotoxicity, and regenerative cell proliferation (Kasai et al., 2009) are more limited than those for liver cancer with several identified uncertainties. 
  
  Uncertainties in the most extensive analysis of MOA for nasal tumours (i.e., U.S. EPA, 2013) in the references identified for this poll  include that at least one of the key events is missing in two of the long term studies resulting in nasal tumours in which non-neoplastic nasal effects were examined. Cell proliferation was observed prior to tumour induction following 1,4-dioxane exposure in both a 2-year inhalation study in male rats (1,250 ppm) (Kasai et al., 2009) and a 2-year drinking water study in male (274 mg/kg-day) and female rats (429 mg/kg-day). However, there was no evidence of cytotoxicity in the nasal cavity in these studies (Kasai et al., 2009).  
  
  Relevant toxicokinetic data include a concentration-dependent increase of 1,4-dioxane in the blood of rats in a related 13 week study (Kasai et al., 2008); there is also evidence for induction of Cyp2E1 in rat nasal mucosa following acute oral administration of 1,4-dioxane by gavage or drinking water (Nannelli et al., 2005).
  
  Additional relevant data include the attribution of squamous cell carcinomas in the nasal cavity in male and female rats exposed to ≥240 and ≥350 mg/kg bw per day, respectively in the NTP bioassays (NTP, 1978), to the inhalation of water droplets based on histopathological re-examination of the nasal tissue sections (Goldsworthy et al., 1991).  Sweeney et al. (2008)  also concluded that rat nasal tissues are exposed by direct contact with drinking water under bioassay conditions, based on studies with fluorescent dyes.
  
  None of the studies was designed to investigate specifically the key events in the potential mode of action for nasal tumours. A targetted short term investigation with multiple dose levels sufficient to calculate benchmark doses for the hypothesized key events would be helpful to provide empirical support (e.g., suggestion of Expert 5 for investigation re liver tumours - i.e., use the chronic bioassay strains with 1,4 DX - control and at least 4 doses including the tumorigenic dose and one dose higher and two doses lower for 7 and 28 days continuous) including toxicokinetic data (e.g., suggestion of Expert 3) along with investigation, if possible, of the essentiality of cyp 2E1 induction (i.e., through use of knockout models). 
  
  
  
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  Expert 2

  	-5	-4	-3	-2	-1	0	1	2	3	4	5	I cannot answer
  Direct genotoxicity	1	0	0	0	0	0	0	0	0	0	0	0
  Indirect genotoxicity	0	0	0	0	0	0	0	0	0	0	1	0
  Proliferative regeneration induced by cytotoxicity	0	0	0	0	0	0	0	0	0	0	1	0
  Oxidative stress	0	0	0	0	0	0	0	0	0	0	1	0
  Activation of nuclear receptors and associated transcription factors (e.g., CAR, PXR)	0	0	0	0	0	1	0	0	0	0	0	0
  Other (Please explain)	0	0	0	0	0	0	0	0	0	0	1	0

  Secondary mutagenic effects on putative stem cell populations activated after damaged cells are unable to resume cycling.  
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  Expert 3

  	-5	-4	-3	-2	-1	0	1	2	3	4	5	I cannot answer
  Direct genotoxicity	1	0	0	0	0	0	0	0	0	0	0	0
  Indirect genotoxicity	0	0	0	0	0	1	0	0	0	0	0	0
  Proliferative regeneration induced by cytotoxicity	0	0	0	0	0	0	1	0	0	0	0	0
  Oxidative stress	0	0	0	0	0	1	0	0	0	0	0	0
  Activation of nuclear receptors and associated transcription factors (e.g., CAR, PXR)	0	0	0	0	0	1	0	0	0	0	0	0
  Other (Please explain)	0	0	0	0	0	0	1	0	0	0	0	0

  The overall MoA information regarding nasal tumors is sparse.  As has been discussed/debated through Rounds 1 and 2, nasal tumors in mice and rats were restricted to the highest tested doses suggesting that, given the lack of direct DX genotoxicity, induction of high-dose specific tumors is likely operating by an undefined but a high-dose threshold MoA.  This is further supported by the observation that DX drinking water dosing results in substantive confounding inhalation of DX-containing drinking water droplets producing a potential site-of-contact exposure not anticipated for humans, and that the tumor-inducing systemic highest-tested oral doses were all above clearly established liver metabolic saturation thresholds.  In addition, vVolunteer studies, although limited and dated, show DX as irritating to the nose, eyes and throat after short-term 1000 ppm+ inhalation exposures, suggesting that occupationally-exposed humans may not tolerate the high DX exposures in the animal bioassays.
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  Expert 6

  	-5	-4	-3	-2	-1	0	1	2	3	4	5	I cannot answer
  Direct genotoxicity	1	0	0	0	0	0	0	0	0	0	0	0
  Indirect genotoxicity	0	0	0	0	0	0	0	0	0	1	0	0
  Proliferative regeneration induced by cytotoxicity	0	0	0	0	0	0	0	0	0	1	0	0
  Oxidative stress	0	0	0	0	0	0	0	0	1	0	0	0
  Activation of nuclear receptors and associated transcription factors (e.g., CAR, PXR)	0	0	0	0	0	0	1	0	0	0	0	0
  Other (Please explain)	0	0	0	0	0	0	0	1	0	0	0	0

  There is no indication that DX is directly genotoxic. A cytotoxic effect on the nasal mucosa with regenerative proliferation appears very likely. As in the liver, the molecular events leading to cytotoxicity are not entirely clear. A receptor-mediated mechanism (vanilloid receptor?) appears feasible, while receptors typically involved in xenobiotic metabolism such as CAR, PXR do not seem to play a key role
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  Expert 5

  	-5	-4	-3	-2	-1	0	1	2	3	4	5	I cannot answer
  Direct genotoxicity	0	0	0	0	0	0	0	0	0	0	0	1
  Indirect genotoxicity	0	0	0	0	0	0	0	0	0	0	0	1
  Proliferative regeneration induced by cytotoxicity	0	0	0	0	0	0	0	0	1	0	0	0
  Oxidative stress	0	0	0	0	0	0	0	0	1	0	0	0
  Activation of nuclear receptors and associated transcription factors (e.g., CAR, PXR)	0	0	0	0	0	0	0	0	0	0	0	1
  Other (Please explain)

  A recent review of the MOA nasal tumors induce in rats by formaldehyde provide direction on the approach that one could take in addressing the 1,4 DX induced nasal tumors MOA.  (Thompson, C. M., Gentry, R., Fitch, S., Lu, K., & Clewell, H. J. (2020). Similarly other studies have examined nasal tumors in rats following chemical treatment ( Green T, Lee R, Moore RB, Ashby J, Willis GA, Lund VJ, Clapp MJ. Acetochlor-induced rat nasal tumors: further studies on the mode of action and relevance to humans. Regul Toxicol Pharmacol. 2000 Aug;32(1):127-33. doi: 10.1006/rtph.2000.1413. PMID: 11029275.: Recio L. Oncogene and tumor suppressor gene alterations in nasal tumors. Mutat Res. 1997 Oct 31;380(1-2):27-31. doi: 10.1016/s0027-5107(97)00124-3. PMID: 9385387. )  An updated mode of action and human relevance framework evaluation for Formaldehyde-Related nasal tumors. Critical Reviews in Toxicology, 50(10), 919–952. https://doi.org/10.1080/10408444.2020.1854679;  Jeffrey AM, Iatropoulos MJ, Williams GM. Nasal cytotoxic and carcinogenic activities of systemically distributed organic chemicals. Toxicol Pathol. 2006;34(7):827-52. doi: 10.1080/01926230601042494. PMID: 17178686.).  The Key events defined by other compounds for chemically induced nasal tumors (such as the case for formaldehyde)  may provide some incite on future experiemntal approahes.