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Bacterial reverse mutation assay (Ames test)

A bacterial gene mutation assay (Ames test) was performed with CAS 112-84-5 according to OECD TG 471 and in compliance with GLP (Jones, 1990). The strains Salmonella typhimurium TA 98, TA100, TA 1535, TA 1537 and TA 1538 were tested in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 2 repetitions at concentrations from 50 to 5000 µg/plate (vehicle: tetrahydrofuran). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. A preliminary dose finding study was carried out with the tester strains TA 98, TA100, TA 1535, TA 1537 and TA 1538 with dose levels from 5 to 5000 µg/plate to determine the nontoxic concentrations for the main experiments. No cytotoxicity was observed in any of the tester strains with dose levels up to 5000 µg/plate.Precipitation of the test material was observed at concentrations500 µg/plate.The included positive and negative controls showed the expected results. Under the conditions of the study, the test substance did not induce mutations in the bacterial gene mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

 

In vitro mammalian chromosome aberration (CA) test

An in vitro chromosome aberration test in Chinese hamster lung fibroblasts (V79) was performed with CAS 112-84-5 according to OECD TG 473 and in compliance with GLP (Hall, 2010). Chinese hamster lung fibroblasts (V79) were treated withthe test materialor vehicle (acetone) for 4 or 18 hoursin the absence and presence of a metabolic activation system (Aroclor-induced rat liver S9-mix)at concentrations from 4.9 to 1250 µg/mL (experiment I, -/+ S9-mix), 2.3 to 450 µg/mL (experiment II, -S9-mix) and 4.7 to 300 µg/mL (experiment II, +S9-mix). Fixation of the cells was performed 18 hours after start of exposure. No significant increase in the number of polyploid and aberrant cells was noted in any of the tested concentrations with and without metabolic activation system. The aberration rates of the cells after treatment with the test item (0.0 - 3.8% aberrant cells) were close to the range of the solvent control values (0.5 - 2.5% aberrant cells) and within the range of the laboratory’s historical control data. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the controls. In both experiments, positive controls showed distinct increases in the number of cells with structural chromosome aberrations. In experiment I in the absence and presence of S9-mix no clear cytotoxicity was observed up to the highest evaluated concentration, where test item precipitation occurred. In experiment II in the absence of S9-mix clear cytotoxicity was observed at 300 and 450 µg/mL. Test item precipitation occurred in the presence of S9 mix at the highest evaluated concentration and the cell number was reduced to 60 % and 67.8 % of control after treatment with 75 and 300 µg/mL, respectively.Under the conditions of the study,no clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

 

In vitro mammalian cell gene mutation assay (MLA)

An in vitro chromosome aberration test in mouse lymphoma L5178Y cells was performed with CAS 112-84-5 according to OECD TG 476 and in compliance with GLP (Wollny, 2010). Mouse lymphoma L5178Y cells were exposed to the test materialor vehicle (acetone) in two independent experiments for 4 or 24 hours in the absence and presence of a metabolic activation system (Aroclor-induced rat liver S9-mix) at concentrations from 9.4 to 300 µg/mL (experiment I, -/+ S9-mix) and 4.7 to 150 µg/mL (experiment II, -/+ S9-mix). After removal of the test material, further gene expression of Mouse lymphoma L5178Y cells in growth medium was allowed for 48 hours, followed by a selection time of 10 – 15 days with the selection agent trifluorothymidine (TFT, 5 μg/mL). No relevant toxic effect indicated by a relative total growth of less than 50 % of survival in both parallel cultures was observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment.No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item.The included positive controls showed the expected results.Under the experimental conditions reported the test material did not induce mutations in the mouse lymphoma L5178Y cells in the absence and presence of metabolic activation. 


Justification for selection of genetic toxicity endpoint
No study was selected as all studies were negative.

Short description of key information:
In vitro gene mutation:
Bacterial reverse mutation assay (Ames test / OECD 471): negative
In vitro mammalian chromosome aberration test (CA / OECD 473): negative
In vitro mammalian cell gene mutation assay (MLA / OECD 476): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of CAS 112-84-5 do not meet the criteria for classification according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.