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Genetic toxicity in vitro

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April-May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: Salmonella typhimurium: TA100, TA98, TA97, TA1535, TA102 and the strain E.coli WP2
Metabolic activation:
with and without
Metabolic activation system:
For the assay with metabolic activation, 0.5 mL of metabolic activation mixture containing 10% of postmitochondrial fraction (S9) together with the bacteria and the test item
Test concentrations with justification for top dose:
Main Assay:
Concentrations of test item ranged between 0.05 – 5.0 mg/plate.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
9-aminoacridine
2-nitrofluorene
sodium azide
ethylmethanesulphonate
mitomycin C

The results indicate that test item Zinc Hexacyanocobaltate dodecahydrate neither produced a statistically significant dose-related increase in the number of revertants nor a statistically significant and reproducible positive response at any of the test points. According to the results obtained in this test system the test item is considered non-mutagenic.

Salmonella typhimurium TA100:

Four independent experiments (including range-finding assays) were performed, two without metabolic activation, two with metabolic activation. The mean levels of spontaneous mutations were within the range of 143-185 revertants/plate. In experiments with and without activation Zinc Hexacyanocobaltate dodecahydrate was tested in concentration range from 0.001 to 5.0 mg/plate. The mutation factor MF did not exceed the value of 2 (MF<2) at any concentration level of the test item. Positive control (sodium azide) induced statistically significant increase (p<0.001) of revertant frequency under experimental conditions without activation (MF = 3.18 and 3.31). The positive response on the treatment with 2AAF showed ability of S9 system in activating of promutagen (MF = 5.8 and 6.14 ).

Salmonella typhimurium TA1535:

Four independent experiments (including range-finding assays) were performed, two without metabolic activation, two with metabolic activation. The mean level of spontaneous mutations was within the range of 18-37 revertants/plate. In experiments without metabolic activation (including assay with preincubation) Zinc Hexacyanocobaltate dodecahydrate was tested in concentration range from 0.001 to 5.0 mg/plate.In experiments without metabolic activation, the level of revertant frequency was not increased in comparison with negative (solvent) control. In experiments with metabolic activation, Zinc Hexacyanocobaltate dodecahydrate was tested in concentration range from 0.01 to 5.0 mg/plate. Test item did not induce any statistically significant increases of revertant frequency in experiments with metabolic activation. Positive control (sodium azide) induced statistically significant (p<0.001) increase of revertant frequency (MF = 22.21, 8.09, 12.36 and 25.35). The ability of S9 system to activate the promutagen was evaluated in each experiment with the use of other Salmonella typhimurium strains (TA100 and TA98 with positive control 2AAF).

Salmonella typhimurium TA97:

Four independent experiments (including range-finding assays) were performed, two without metabolic activation, two with metabolic activation. The mean level of spontaneous mutation was within the range of 140-198 revertants/plate. In experiments without activation Zinc Hexacyanocobaltate dodecahydrate was tested in concentration range from 0.001 to 5.0 mg/plate. The statistically significant increase of mutation frequency against solvent control was not detected at any concentration of the test item. The experiments with metabolic activation were performed in the concentration range from 0.01 to 5.0 mg/plate. The test item did not induce statistically significant increase of revertant frequency in any of the experiments with metabolic activation. Positive control (9-aminoanthracene) induced statistically significant increase of revertant frequency (MF = 4.77, p<0.001, MF = 3.35, p<0.001,MF = 4.71, p<0.001 and MF = 3.34, p<0.001). The efficacy of metabolic activation was confirmed in activation of promutagen 2AAF in experiment with Salmonella typhimurium TA100, TA98 performed under the same conditions.

Salmonella typhimurium TA98:

Four independent experiments (including range-finding assays) were performed, two without metabolic activation, two with metabolic activation. The mean level of spontaneous mutation was within the range of 21- 34 revertants/plate. Zinc Hexacyanocobaltate dodecahydrate was tested in concentrations ranging from 0.01 to 5.0 mg/plate with and without metabolic activation. In experiments without metabolic activation, at each concentration level of the test item, the level of revertant frequency was not increased in comparison with negative (solvent) control. Positive control (2NF) induced statistically significant increase (p<0.001) of revertant frequency (MF=26.3 and 9.33). In experiments with S9 fraction isolated from rat liver after induction with Aroclor or 20-methylcholanthere, Zinc Hexacyanocobaltate dodecahydrate did not induce thestatistically significant increase of revertant frequency at any concentration level. The positive response to the treatment with 2AAF showed the ability of S9 system in activating of promutagen (MF=20.16, p<0.01 and MF=9.83, p<0.001).

Salmonella typhimurium TA102:

Four independent experiments were performed, two without metabolic activation, two with metabolic activation. The mean level of spontaneous mutation was within the range of 184 -224 revertants/plate. In experiments without activation, Zinc Hexacyanocobaltate dodecahydrate was tested in concentration range from 0.01 to 5.0 mg/plate. Within these experiments the level of revertant frequency was not increased in comparison with negative (solvent) control at any concentration level of test item. In the experiment with S9 fraction isolated from rat liver after induction with Aroclor and another with S9 fraction isolated from rat liver after induction with 20-methylcholanthere, Zinc Hexacyanocobaltate dodecahydrate (concentration range from 0.01 to 5.0 mg/plate) did not induce the statistically significant increase of revertant frequency in experiments with metabolic activation. Positive control (mitomycin C) induced statistically significant increase of revertant frequency MF = 2.28, p<0.01, MF = 2.82, p<0.001 and MF = 3.36 , p<0.001). The positive response demonstrating the ability of the S9 system to activate of promutagen was confirmed with positive control 2AAF on other Salmonella strains: TA100 and TA98.

E.coli WP 2:

Two independent experiments were performed, one without metabolic activation, one with metabolic activation. The mean level of spontaneous mutation was within range 15-25 revertants/plate. In experiments without activation, Zinc Hexacyanocobaltate dodecahydrate was tested in concentration range from 0.01 to 5.0 mg/plate. In experiments without metabolic activation, the level of revertant frequency was not increased in comparison with negative (solvent) control. Positive controls (mitomycin C, EMS) induced statistically significant increase of revertant frequency MF = 7.19 and 13.89 respectively, (p<0.001). In the experiment with S9 fraction isolated from rat liver after induction with Aroclor 1254, Zinc Hexacyanocobaltate dodecahydrate (concentration range from 0.01 to 5.0 mg/plate) did not induce the statistically significant increase of revertant frequency in experiments with metabolic activation. The positive response demonstrating the ability of the S9 system to activate of promutagen was confirmed with positive control 2AAF on other Salmonella strains: TA100 and TA98.

Conclusions:
Interpretation of results (migrated information):
negative

No significant increase in the mutant frequency was observed for the five tester strains of Salmonella typhimurium TA100, TA98, TA97, TA1535, TA102 and one strain of E.coli WP2 after treatment with Zinc Hexacyanocobaltate dodecahydrate in the standard plate incorporation either with or without metabolic activation.
It is concluded that Zinc Hexacyanocobaltate dodecahydrate did not exert mutagenic activity under the conditions of the performed tests.
Executive summary:

Zinc Hexacyanocobaltate dodecahydrate did not produce any significant increases of mutation frequency in these strains up to the maximum dose of 5.0 mg/plate both in the absence and presence of metabolic activation. Zinc Hexacyanocobaltate dodecahydrate did

not exert mutagenic activity under the conditions of the performed tests.

Zinc Hexacyanocobaltate dodecahydrate in accordance with these results is considered to be non-mutagenic in bacterial gene mutation test system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
One study available

Justification for classification or non-classification

Zinc Hexacyanocobaltate dodecahydrate did not produce any significant increases of mutation frequency in these strains up to the maximum dose of 5.0 mg/plate both in the absence and presence of metabolic activation. Zinc Hexacyanocobaltate dodecahydrate did

not exert mutagenic activity under the conditions of the performed tests.

Zinc Hexacyanocobaltate dodecahydrate in accordance with these results is considered to be non-mutagenic in bacterial gene mutation test system.