Curren, R., K. Wallace, S. Valone and J. Harbell. The Use of the Neutral Red Bioassay Using Normal Human Keratinocytes to Detect Materials Requiring Metabolic Activation. The Toxicologist 1991. 11(1): 277.

7,12-dimethylbenzanthracene - 00057-97-6; benzo(a)pyrene - 00050-32-8; 3-methylcholanthrene - 00056-49-5; cyclophosphamide - 00050-18-0; 2-aminoanthracene - 00613-13-8

Recently, several in vitro cell and/or organ based models have been proposed as potential replacement assays for acute eye and skin irritation tests. In general, validations of these systems have used direct acting toxicants - the type of materials generally encountered in the cosmetics and personal care products industries. However, unpurified industrial chemicals contain trace amounts of chemicals which require metabolic activation before becoming toxic. Therefore, we used a commercially available system, the Neutral Red Bioassay Kit supplied by Clonetic Corporation, San Diego, CA, with five model compounds requiring metabolic activation. Low passage (P2) normal human epidermal keratinocytes (NHEK) were grown in serum-free media. 7,12-dimethylbenzanthracene (DMBA), benzo(a)pyrene (B(a)P), 3-methylcholanthrene (3-MC), cyclophosphamide (CP), and 2-aminoanthracene (2AA) were applied to exponentially growing NHEKs for 48 hours. DMBA, a skin carcinogen in rodents, was very cytotoxic to the NHEK (NR50 ~0.2 ug/ml). B(a)P had an NR50 of ~10 ug/ml. The NR50's for 3MCA, 2-AA and CP were all greater than 100 ug/ml. These results show that NHEK grown under these conditions are capable of detecting a subset of compounds which require metabolic activation to exhibit toxicity. These data suggest that some of the xenobiotic metabolizing enzymes present in vivo continue to be expressed by these cells in culture.