title: Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative.
abstract: As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative International Validation Study of an in vivo rat alkaline comet assay, we examined 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) to investigate the effectiveness of the comet assay in detecting genotoxic carcinogens. Each of the three test chemicals was administered to 5 male Sprague-Dawley rats per group by oral gavage at 48, 24, and 3h before specimen preparation. Single cells were collected from the liver and glandular stomach at 3h after the final dosing, and the specimens prepared from these two organs were subjected to electrophoresis under alkaline conditions (pH>13). The percentage of DNA intensity in the comet tail was then assessed using an image analysis system. A micronucleus (MN) assay was also conducted using these three test chemicals with the bone marrow (BM) cells collected from the same animals simultaneously used in the comet assay, i.e., combination study of the comet assay and BM MN assay. A genotoxic (Ames positive) rodent carcinogen, DBE gave a positive result in the comet assay in the present study, while a genotoxic (Ames positive) non-carcinogen, ASD and a non-genotoxic (Ames negative) non-carcinogen, ANT showed negative results in the comet assay. All three chemicals produced negative results in the BM MN assay. While the comet assay findings in the present study were consistent with those obtained from the rodent carcinogenicity studies for the three test chemicals, we consider the positive result in the comet assay for DBE to be particularly meaningful, given that this chemical produced a negative result in the BM MN assay. Therefore, the combination study of the comet assay and BM MN assay is a useful method to detect genotoxic carcinogens that are undetectable with the BM MN assay alone.
authors.affiliation: LSI Medience Corporation (Old name: Mitsubishi Chemical Medience Corporation), 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan. Electronic address: Takasawa.Hironao@mg.medience.co.jp.
authors.forename: Hironao
authors.initials: H
authors.lastname: Takasawa
authors.affiliation: LSI Medience Corporation (Old name: Mitsubishi Chemical Medience Corporation), 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Takashima
authors.affiliation: LSI Medience Corporation (Old name: Mitsubishi Chemical Medience Corporation), 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan; Yakult Honsha Co., Ltd., 5-11 Izumi, Kunitachi-shi, Tokyo 186-8650, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Narumi
authors.affiliation: LSI Medience Corporation (Old name: Mitsubishi Chemical Medience Corporation), 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan.
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Kawasako
authors.affiliation: LSI Medience Corporation (Old name: Mitsubishi Chemical Medience Corporation), 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Hattori
authors.affiliation: LSI Medience Corporation (Old name: Mitsubishi Chemical Medience Corporation), 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Kawabata
authors.affiliation: LSI Medience Corporation (Old name: Mitsubishi Chemical Medience Corporation), 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hamada
mesh_terms: D000284:Administration, Oral; D000814:Aniline Compounds / Q000633:toxicity*; D000818:Animals; D001853:Bone Marrow / Q000187:drug effects; D002273:Carcinogens / Q000633:toxicity; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D015946:Ethylene Dibromide / Q000633:toxicity*; D008099:Liver / Q000187:drug effects; D008297:Male; D015162:Micronucleus Tests / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013270:Stomach / Q000187:drug effects; D062367:ortho-Aminobenzoates / Q000633:toxicity*
chemical_list: D000814:Aniline Compounds; D002273:Carcinogens; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D015946:Ethylene Dibromide; C013813:4-anisidine
keywords: 1,2-Dibromoethane; Carcinogenicity; DNA damage; In vivo comet assay; JaCVAM Validation Study; o-Anthranilic acid; p-Anisidine