CXR is pleased to announce publication of a review entitled “Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator” in the journal Critical Reviews in Toxicology.
The review is based on the conclusions of a workshop organised by Toxicology Excellence for Risk Assessment (TERA) and attended by experts from regulators, industry and academia.
Many non-genotoxic pharmaceuticals, agrochemicals and chemicals increase the incidence of rodent liver tumours during long term carcinogenicity studies. Faced with a compound that has caused (or is expected to cause) rodent tumours, it is necessary to demonstrate the relevance or irrelevance to humans to the satisfaction of regulatory bodies. The activation of nuclear receptors, including the Constitutive Androstane Receptor (CAR), is a common Mode of Action (MOA) for chemicals that exhibit non-genotoxic hepatocarcinogenicity in rodents, as typified by the rodent liver tumours caused by long term administration of phenobarbital.
Experimental approaches to determine human relevance
A deeper understanding of the molecular mechanisms and the dose responsive key events involved in this MOA can help inform a model that is most appropriate for use in risk assessment. The workshop concluded that the MOA for phenobarbiotal-induced rodent liver tumour formation was considered to be qualitatively not plausible for humans.
Therefore, the workshop recommended that if data indicates a compound induces liver tumours (or could be reasonably expected to induce liver tumours, based on a likelihood that it is a nuclear receptor ligand), the following framework can be suggested to specifically confirm a nuclear receptor mediated mode of action:
- Confirm non-genotoxicty.
- Exclude cytotoxicity.
- Exclude non-CAR receptor MOAs.
- Perform appropriate rodent studies to examine endpoints including:
- Early, observable key and associative events (e.g. cell proliferation, CYP2B induction, apoptosis suppression, hypertrophy, liver weight).
- Evaluate dose–response
- CAR null animal study to show lack of effects.
- CAR activation/nuclear translocation assays – if suitable models are available and valid.
- Confirmation of lack of human relevance via CAR MOA, e.g. use of primary human hepatocytes and when appropriate humanized models.
- If more than one nuclear receptor is activated, use selective receptor knockout animals.
The public workshop on nuclear receptor mode of action and dose-response was held at the National Institute of Environmental Health Sciences (NIEHS) in Research Triangle Park, North Carolina on September 27-29, 2010. Case studies were used to address activation of the aryl hydrocarbon receptor (AHR); the constitutive androstane receptor/Pregnane X receptor (CAR/PXR), and the peroxisome proliferator-activated receptor-alpha (PPARα). The CAR/PXR workshop was co-chaired by CXR Research Director Cliff Elcombe and Douglas Wolf of the US Environmental Protection Agency. Other panel participnts were drawn from Syngenta Crop Protection, Dow Agrosciences, Bayer CropScience, the European Chemicals Agency, Auburn University, CellzDirect/Life Technologies, Dow Chemical Company, Michigan State University, the U.S. Food and Drug Administration-CDER, Penn State University, the University of Pittsburgh, and the University of Surrey.
CXRs experimental capabilities
CXR Biosciences uses a multidisciplinary approach, consistent with that advocated by the workshop, to evaluate the risk to humans of chemicals and pharmaceuticals that elicit a carcinogenic response in rodents, developed over many years of assisting global pharmaceutical, chemical and agrochemical companies.
CXR has pioneered the use of knockout (KO) rat and mouse models to aid in understanding the relevance of specific modes-of-action for human risk assessment (2, 3), and these studies have been included in regulatory submissions (4).
Once the mode-of-action of rodent non-genotoxic carcinogenicity has been demonstrated, appropriate in-vitro studies are used to assess human relevance. Liver carcinogenicity is typically modelled in primary hepatocytes from rodents and humans, and hepatocyte proliferation compared using S-phase labelling. CAR activating non-genotoxic carcinogens cause proliferation in rodent hepatocytes, but not in human hepatocytes. CXR’s hepatocyte proliferation studies are frequently included in regulatory submissions (5).
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Founded in 2001, CXR Biosciences® uses its collaborative approach and toxicological expertise to help customers of all sizes solve issues relating to the safety of compounds or selection of research candidates. CXR Biosciences® offers tailored preclinical services in the areas of investigative & mechanistic toxicology, exploratory & discovery toxicology and PK & metabolism. Our customers include leading pharmaceutical, agrochemical, chemical and biotechnology companies. CXR Biosciences® is located in Dundee, Scotland, United Kingdom.
(1) Elcombe et al. (2013). Crit Rev Toxicol. 2013 Nov 4. “Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator.”
(2) Ross et al. (2010) Toxicol Sci. 116(2):452-66. “Human constitutive androstane receptor (CAR) and pregnane X receptor (PXR) support the hypertrophic but not the hyperplastic response to the murine non-genotoxic hepatocarcinogens phenobarbital and chlordane in vivo.”
(4) CLH report: Proposal for Harmonised Classification and Labelling. Based on Regulation (EC) No 1272/2008 (CLP Regulation), Annex VI, Part 2. Substance Name: SULFOXAFLOR. https://echa.europa.eu/documents/10162/6acb4d37-01ed-484b-981e-7319d9290af3
(5) For example, see Draft Assessment Report for Penflufen, October 2011