Evaluation of multi-organ DNA damage by comet assay from 28 days repeated dose oral toxicity test in mice: A practical approach for test integration in regulatory toxicity testing S. Kushwaha, D.N. Tripathi, A. Vikram, P. Ramarao, G.B. Jena * Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar, Punjab 160062, India article info Article history: Received 1 October 2009 Available online 19 May 2010 Keywords: Test integration DNA damage Comet assay Micronucleus assay Methotrexate Mice abstract The use of comet assay is not new in the evaluation of genotoxic potential of different agents; however, its broad use in product safety for regulatory testing is a relatively new approach. The present study was aimed to integrate genotoxicity tests (micronucleus and comet assay) in 28 days repeated dose oral tox- icity of methotrexate (MTX) in mice. MTX was administered at the dose of 0.5, 1 and 2 mg/kg per oral repeatedly for 28 days in mice. The endpoints of evaluation for routine toxicity testing included body weight, organ weight, food intake, water intake, hematology and histology, while for the genotoxicity testing micronucleus and comet assay were used. There were no significant changes in food intake, water intake and organ weight; however, the body weight significantly decreased at the highest dose of MTX treatment as compared to control group. Histological data revealed the morphological alterations in the liver and lung cells at the highest dose of MTX treatment. Micronucleus assay results indicated that the highest dose of MTX led to significant increase in MNERTs/1000ERTs (P < 0.001) as compared to con- trol group. Further, percentage of reticulocytes (% RETs) was significantly decreased at the highest dose of MTX as compared to control group. Comet assay results indicate significant DNA damage in different organs induced by MTX as compared to control group. The results of the present study successfully dem- onstrates the integration of genotoxicity tests using comet and micronucleus assay in 28 days repeated dose oral toxicity test. Integration of genotoxicity test with routine toxicity test would reduce the cost of additional animals, test item and provide further information at an early stage of product development. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Test integration is a novel effort in the repeated-dose toxicity testing, but least exploration has been done in this area of scientific research. Genetic toxicity screening plays a vital role in the product development and marketing of new chemicals for pharmaceutical, agricultural, industrial and consumer use. MacGregor et al. recom- mended the integration of cytogenetic analysis for DNA damage in the repeated dose toxicological testing for industrial setting (MacGregor et al., 1995). Krishna et al. enriched the concept of test integration in as early as 1998 and advocated that the micronu- cleus (MN) test can be successfully carried out along with routine 2–4 weeks toxicokinetic studies. Successful integration of genotox- icity test into the routine toxicology testing in the early stage of product development has several advantages (Krishna et al., 1998). Efforts are being made by the International Workshop on Genotoxicity Test Procedures (IWGTP) to design the practical as- pects of protocol including repeated dose treatments, integration with other routine toxicity testing and automated scoring of in vivo rodent erythrocytes MN assay (Hayashi et al., 2000). The Registration, Evaluation, Authorization and Restriction of Chemi- cals (REACH) legislation emphasizes that the new technologies re- quire a shift on the way in which safety assessment is conducted for different chemicals. Further, it has been realized that the Inte- grated Testing Strategies (ITSs) are already in use for toxicological evaluation, but the concept of their integration and application in a regulatory setting have yet to be fully implemented (Hoffmann et al., 2008). The Joint Research Centre of the European Commis- sion estimated that the highest number of in vivo genotoxicity tests are essential to evaluate the chemicals under REACH program (Van der Jagt et al., 2004). In a recent workshop hosted by the European Centre for the Validation of Alternative Methods (ECVAM) held from 24 to 25 June 2008, it was emphasized that integration of 0273-2300/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2010.05.004 Abbreviations: MTX, methotrexate; MN, micronucleus; IWGTP, International Workshop on Genotoxicity Test Procedures; REACH, Registration Evaluation Authorization Restriction of Chemicals; MDA, malondialdehyde; GSH, glutathione; RETs, reticulocytes; NCEs, normochromatic erythrocytes; ERTs, erythrocytes. * Corresponding author. E-mail addresses: gbjena@yahoo.com, gbjena@gmail.com (G.B. Jena). Regulatory Toxicology and Pharmacology 58 (2010) 145–154 Contents lists available at ScienceDirect Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph