o-TOLUIDINE CAS N°: 95-53-4 - UNEP Chemicals

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OECD SIDS o-TOLUIDINE 5. TOXICITY ID: 95-53-4 DATE: 07.01.2005 260 Test substance : other TS: no data on purity Remark : morphological transformation and ouabain resistance Result : In the presence of an exogenous metabolic activation (primary rat liver cells) o-Toluidine induced significant levels of transformation of 3T3 cells relative to the solvent control. This chemical also induced significant increases in the frequency of Ouabain resistance mutants. Test condition : Nonactivation transformation assay: The transformation assay experimental design was adapted from that reported by Kakunage, 1973 and involved treating 24-h-old cultures seeded with 3 x 10E4 cells with 5 preselected doses of the test chemical, a positive control and a solvent control. The dishes were incubated for 72h, then washed and incubation continued for approx. 24 days with twiceweekly refeedings. The assays were terminated by fixation with methanol and staining with Giemsa. Transformed foci were scored microscopically and consisted of foci of denely piled-up cells with a desoriented criss-cross pattern that was invasive into a uniformly stained, contiguous monolayer of cells. Activation transformation assay: Lethally x-radiated primary rat-liver cells were incubated for 3h with 3T3 cells and then treated with the test chemical for a total of 48h. Beginning 1- 2 days after the test chemical treatment had been completed, the cocultures were treated with 0.05 µg/ml 12-o-tetradecenoyl-phorbol-13acetate (TPA). The TPA posttreatment were continued biweekly for a total of 3 weeks and discontinued one feeding interval prior to the termination of the assay. Ouabain resistance mutation assay: 24h-old-3T3 cell cultures with and without S9-mix (Aroclor induced rat-liver cells) were incubated with the test chemical for 24h (4h for the S9 activation assays). Test chemical was removed and the cultures were refed and maintained for 5-6 day expression period. At the end of this period cells were trypsinzed, counted and repeated. Selection media containing 2 mM ouabain was added 1, 8, and 16 days after replaiting and the resultant Quar colonies were fixed, stained and counted at 19-21 days post plating. Reliability : (2) valid with restrictions method evaluation program 01.12.2003 (377) (378) Species : other: mouse; mammalian cell transformation assay Sex : Strain : other: embryonic mouse fibroblasts (C3H/10T1/2 Clone 8) Route of admin. : Exposure period : 24 hours Frequency of treatm. : Post exposure period : Doses : 0, 250, 500, 750, 1000 µg/ml in culture medium Result : Control group : yes Method : other: other: according Reznikoff, C.A. et al. (1973): Cancer Res. 33, 3231- 3238 Year : 1984 GLP : no data Test substance : other TS: no data on purity Result : Cytotoxicity increased dose-related: survival at 500 µg/ml: 86 % of control; at 1000 µg/ml 52 of control o-toluidine induced significant transformation at 500 µg/ml UNEP PUBLICATIONS
OECD SIDS o-TOLUIDINE 5. TOXICITY ID: 95-53-4 DATE: 07.01.2005 The positive control benzo(a)pyren was functional Test condition : TRANSFORMATION ASSAY (in brief) Chemicals were administered to the cells in sealed flasks. After a 24h treatment period the cells were washed free of the chemicals and maintained through a series of fresh media changes for 6 weeks at which time the induction of Type 1, 2 and 3 morphological transformants were scored. Tests were performed without exogenous metabolic activation. Concurrent cytotoxicity determinations were performed. CONTROLS Positive controls with benzo[a]pyrene 1µg/ml Negative controls with 1% DMSO STATISTICAL ANALYSIS Fisher's exact test Reliability : (2) valid with restrictions special investigation 13.02.2004 (379) (380) Species : other: mouse; mammalian cell transformation assay Sex : Strain : other: embryonic mouse fibroblasts (C3H/10T1/2 Clone 8) Route of admin. : Exposure period : Frequency of treatm. : Post exposure period : Doses : 0, 150, 300, 600 µg/ml in culture medium Result : positive Control group : yes Method : other: Reznikoff, C.A. et al. (1973): Cancer Res. 33, 3231-3238 Year : 1985 GLP : no data Test substance : other TS: no data on purity Result : In the presence of S9 mix, no transformed foci were recorded at 150 µg otoluidine/ml, one Type II focus was identified at 300 µg/ml and 3 Type II transformed foci observed at 600 µg/ml. In the absence of S9 mix no transformed foci were recorded at any concentration of o-toluidine. In the presence of S9 mix the frequency of transformation and the distribution of transformed foci among 4 flasks is indicative of a weak celltransforming potential. The positive controls were functional. Test condition : Preliminary Cytotoxicity determinations were performed to establish the range of concentration. CELL TRANSFORMATION ASSAY Chemicals were administered to the C3H/10T1/2 Clone 8 cells in flasks. After a 24h treatment period the cells were washed free of the chemicals and maintained through a series of fresh media changes for 8 weeks at which time the induction of Type 1, 2 and 3 morphological transformants were scored. --- CONTROLS Positive controls with benzo[a]pyrene, 3-methylcholanthrene, 4nitroquinoline-N-oxide, cyclophosphamide, ethylmethanesulfonate and 2acetylaminofluorene (2-AAF) Negative controls with 1% DMSO UNEP PUBLICATIONS 261
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o-TOLUIDINE CAS N°: 95-53-4 - UNEP Chemicals

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