By Marilyn J. Aardema, Marilyn Aardema Consulting, LLC
Rodger Curren, The Institute for InVitro Sciences
Standard testing for genotoxicity initially involves conducting Tier I in vitro assays using various mammalian cells/cell lines that are often exposed under highly non-physiological conditions. This contributes to the high rate of false positive results (results that are not confirmed in Tier II in vivo genotoxicity and/or rodent carcinogenicity tests) that can be up to 95% in the standard battery (1,2). The use of 3D human tissue models to achieve more physiologically relevant genotoxicity testing may provide an important paradigm shift in the field of genetic toxicology. We believe this conclusion is supported by recent encouraging results obtained during the development of a novel in vitro 3D human reconstructed skin micronucleus (RSMN) assay (3,4,5).
Why a New Genotoxicity Assay in a 3D Skin Model?
Since skin is the site of maximum exposure to many products including cosmetics, dermal drugs, and general chemicals, and is also a metabolically competent organ involved in the activation and detoxification of chemicals, we focused on 3D human reconstructed skin (RS) models for development of what we hope is a more physiologically relevant and therefore hopefully more predictive in vitro micronucleus assay.
We selected the EpiDermTM model (MatTek Corporation) which is constructed from primary neonatal epidermal foreskin-derived keratinocytes. Since the model is prepared from primary cells, it is expected to have more normal DNA repair and cell cycle control than the transformed cell lines used in the standard mammalian cell genotoxicity assays. RS is also expected to have a human metabolic capability that is more relevant for dermally applied chemicals than the exogenous rodent metabolizing enzymes (S9 mix) currently added in standard in vitro genotoxicity assays. These features theoretically could eliminate many of the suspected causes of false positive results in standard in vitro genotoxicity assays.
Metabolic Capacity of the EpiDerm TM Model
Very recent studies have supported our assumption that the EpiDerm TM model may have metabolic capacity that is very similar to normal human skin. EpiDerm TM has been shown to N-acetylate (a detoxification step) aromatic amine hair dye constituents p-aminophenol and p-phenylenediamine (6) consistent with what has been shown in normal human scalp (7). A comparison of expression of 139 xenobiotic metabolism genes (using microarray analysis) in the EpiDerm TM model to that of biopsied human skin demonstrated many similarities, with excellent agreement (87%) in the presence/absence of XB metabolism genes as well as the similarity in signal levels of the genes expressed in both systems (8). Overall, the expression of Phase II enzymes appeared to be more pronounced compared to Phase I enzymes both in the EpiDerm TM model and human skin, consistent with a role of skin in detoxification of xenobiotics.
Intra- and Interlaboratory Reproducibility of the RSMN Assay
Importantly, for a variety of chemicals, the RSMN assay has been shown to produce comparable results across multiple laboratories in the United States (5) [The Institute for In Vitro Sciences (IIVS), The Procter & Gamble Company (P&G), and the MatTek Corporation] as well as Europe [L'Oreal (France), and Henkel (Germany)]. Most of these studies were supported by contracts from The European Cosmetics Association (COLIPA), with contribution from the European Center for Validation of Alternative Methods (ECVAM) (9). To date, about 20 chemicals have been evaluated including genotoxic rodent skin carcinogens, genotoxic rodent non-skin carcinogens, and systemic rodent carcinogens/non-carcinogens. All chemicals were correctly identified by the laboratories as either genotoxic or nongenotoxic, and the data constitute the initial evaluation of international inter-laboratory and inter-experimental reproducibility of the assay (4,5,9).
Conclusion
The results to date support the conclusion that the RSMN assay in the EpiDerm TM 3D human skin model appears to be a valuable in vitro method for genotoxicity assessment of dermally applied chemicals. Currently, the RSMN assay would be useful as a follow-up test for dermally applied chemicals that are positive in current in vitro genotoxicity tests, especially for cosmetic ingredients (10) that can no longer be tested in vivo owing to the EU 7th Amendment to the Cosmetics Directive March 2009 ban on in vivo genotoxicity testing of cosmetics ingredients for products marketed in Europe (11). The RSMN assay could also be useful for large scale chemical evaluation programs such as REACh (Registration, Evaluation, Authorisation and Restriction of Chemical substances) (12) for which the conduct of in vivo assays on tens of thousands of chemicals is impractical.
If the performance of the RSMN assay is demonstrated to be better than current in vitro cytogenetic assays (i.e. similar high sensitivity but greater specificity than the in vitro micronucleus or in vitro chromosome aberration assays), it is envisaged that the RSMN assay could even be a replacement for these tests in Tier I batteries in the future. Further work is needed to more fully evaluate this promising new assay and other laboratories are invited to apply the technique to further learnings with the model.
References
Author Biography
©2010 Marilyn J. Aardema & Rodger Curren
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