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\begin{document}

\title{HepaRG Cells as a New Approach Methodology Follow Up to a Positive
Response in Human TK6 Cell Micronucleus Assay: Naphthalene Case Study}



\author[1]{Les Recio}%
\author[2]{Jasmine Fowler}%
\author[2]{Lincoln Martin}%
\author[2]{Carol Swartz}%
\affil[1]{ScitoVation}%
\affil[2]{Integrated Laboratory Systems Inc}%


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  \date{June 08, 2024}


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\begin{abstract}
We are evaluating the use of metabolically competent HepaRG cells
combined with CometChip? for DNA damage and the micronucleus (MN) assay
as a follow up for in vitro positive genotoxic response as alternatives
to in vivo genotoxicity testing.. Naphthalene is genotoxic with rat
liver S9 in human TK6 cells inducing a nonlinear dose-response for the
induction of micronuclei in the presence of rat liver S9. To follow up
this response, we used metabolically competent HepaRG cells as a New
Approach Methodology (NAM) alternative to animals for genotoxicity
assessment of naphthalene. In HepaRG cells, naphthalene genotoxicity
was assessed using 12 concentrations of naphthalene with the top dose
used for assessment of genotoxicity of 1.7 mM corresponding to 45\% cell
survival. In contrast to human TK6 cell with S9, Naphthalene was not
genotoxic in either the HepaRG MN Assay or the Comet Assay using
CometChip?. The lack of genotoxicity in both the MN and comet assays in
HepaRG cells is likely due to Phase II enzymes removing phenols
preventing further bioactivation to quinones and efficient detoxication
of naphthalene quinones or epoxides by glutathione conjugation. In
contrast to CYP450 mediated metabolism, these Phase II enzymes are
inactive in rat liver S9 due to lack of appropriate cofactors causing a
positive genotoxic response. This data indicates that rat liver
S9-derived BMD10 over-predicts naphthalene genotoxicity BMD calculations
when compared to hepatocytes. Metabolically competent hepatocyte models
like HepaRG cells should be considered as human-relevant NAMs for use
genotoxicity assessments to reduce reliance on rodents.%
\end{abstract}%



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