Potassium trifluorozincate

Endpoint summary

Additional information

In vitro genotoxicity

In vitro: Reverse mutation assay:

In a GLP compliant Ames test, performed according to OECD guideline 471, four Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and one E. Coli strain (WP2 uvrA) were used to test the mutagenic potential of Nocolok Zn Flux both with and without metabolic activation. The test substance was suspended in dimethylsulphoxide (DMSO) at five concentrations of the test substance, ranging from 62 to 5000 µg/plate. The test substance was toxic to all strains, in both the absence and presence of S9-mix. A decrease in the mean number of revertants and/or a (slightly) less dense background lawn of bacterial growth was observed at and above 556 µg/plate in the absence of S9-mix and at and above 1667 µg/plate in the presence of S9-mix. As a result, in the absence of S9-mix, less than three non-toxic concentrations were tested which is considered to be the minimum for a valid test result. In all strains, in both the absence and presence of S9-mix, Nocolok Zn Flux did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. In conclusion, in the presence of the S9-mix Nocolok Zn Flux is not mutagenic and in the absence of S9-mix the results are insufficient to judge the ability of the test substance Nocolok Zn Flux to induce gene mutations.

In vitro: Gene mutation assay:

In a GLP compliant gene mutation test performed according to OECD guideline 476, Nocolok Zn flux was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). One test was conducted. In this test 13 and 14 single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively. The test substance was dissolved in dimethylsuphoxide (DMSO). The highest concentrations evaluated for mutagenicity were 17 μg/mL and 31 μg/mL in the absence and presence of S9-mix, respectively. In both the absence and presence of S9-mix the test substance was toxic to the cells resulting in a reduction in initial cell yield and suspension growth. The relative total growth (RTG) at the highest concentration evaluated in the absence (17 μg/mL) and presence of S9-mix (31 μg/mL) was 2% and 5%, respectively. In the absence of S9-mix, at a concentration of 17 and 10 μg/mL, the mutant frequency was increased by 205 and 96 mutants per 1,000,000 clonable cells compared to the negative control, respectively. Since the increase at 17 μg/mL was observed at a concentration causing more than 90% cytotoxicity (RTG 2%) it was considered to be an artefact and not biological relevant. The increase at 10 μg/mL was considered to be equivocal. At all other concentrations no increase in mutant frequency (MF) was observed. In the presence of S9-mix, dose related significant increases in mutant frequency were observed; at 16, 22, 26 and 31 μg/ml the MF was increased by 251, 371, 446 and 707 mutants per 1,000,000 clonable cells compared to the negative control. The RTG were 42%, 39%, 14% and 5%, respectively. In the presence of S9-mix at the concentrations causing an increase in mutant frequency, more small then large colonies were formed. The mean percentage of small colonies formed was 71%. It is concluded that Nocolok Zn Flux is mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the presence of metabolic activation (S9-mix). However, since relatively more small than large colonies were formed it might be concluded that the mechanism of mutation induction is clastogenicity than mutagenicity. In the absence of S9-mix equivocal results were obtained.

In vitro: Micronucleus test:

In a GLP compliant In vitro Mammalian Cell Micronucleus Test, performed according to OECD guideline 487 the test substance Nocolok Zn Flux was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes in both the absence and presence of a metabolic activation system (S9 -mix). Two independent in vitro micronucleus tests were conducted for which blood was obtained from two different donors. Dimethylsulfoxide (DMSO) was used as solvent. Dose levels ranging from 0.25 to 200 μg/mL were tested as final concentrations in the culture medium. Cytotoxicity was calculated from the Cytokinesis-Block Proliferation Index (CBPI). In the first test and second test, in the presence and absence of metabolic activation (S9-mix) the treatment/recovery time was 4/20 hours (pulse treatment). In the second test in the absence of S9-mix, the treatment/recovery time of the continuous treatment group was 20/28 hours. In the first test, in both pulse treatment groups with and without metabolic activation, the highest three dose levels (200, 100 and 50 μg/mL) were severely cytotoxic to the cells (only dead cells). The lower dose levels (25 and 12.5 mg/mL) were strongly cytotoxic and clearly cytotoxic to the cells, respectively. However, the cytotoxicity criterion of 55 ± 5% according to the OECD guideline 487 was not met in either pulse treatment group. Therefore, analysis of micronuclei formation was not performed. In the second test, in both pulse treatment groups with and without metabolic activation, the highest two dose levels (50 and 40 μg/mL) were severely cytotoxic to the cells. At the lower dose levels, a dose-related cytotoxicity was observed. In both pulse treatment groups, analysis of micronuclei formation was carried out in the cultures of three dose levels (20, 15 and 10 μg/mL) of the test substance. In the second test, in the pulse treatment group with metabolic activation, the test substance induced a significant dose-related increase in the number of binucleated cells containing micronuclei when compared to the numbers found in the concurrent control cultures. In the pulse treatment group without metabolic activation, the test substance induced a clear significant increase in the number of binucleated cells containing micronuclei when compared to the numbers found in the concurrent control cultures. Due to the clear positive response (clear increase in induction of micronuclei) in the pulse treatment groups, CBPI analysis and analysis of micronuclei formation was not performed in the continuous treatment group. From the results obtained, it is concluded that Nocolok Zn Flux was clastogenic and/or aneugenic to cultured human lymphocytes.

In vivo genotoxicity

As indicated in the REACH Regulation (Annex VIII), an in-vivo mutagenicity test shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII. In addition, in the REACH guidance (Chapter R.7A – Endpoint Specific Guidance version 2.0, November 2012 page 345) it is stated that an in vivo test should be initiated as soon as possible following a positive result in an in vitro mammalian cell mutagenicity test. For the substance KZnF3, in-vitro, positive results were observed in the MLA test in the presence of S9 mix (in the absence of S9 mix equivocal results were obtained) and the Micronucleus test (mouse lymphoma cells). Based on these positive results, it was decided to perform the in-vivo mutagenicity test for cytogenicity (UDS) and the in-vivo chromosome aberration test (MN test) as part of a short-term repeated dose toxicity test (28 days).

In vivo: Unscheduled DNA synthesis test

In a GLP compliant unscheduled DNA synthesis-test performed according to OECD Guideline 486, Nocolok Zn Flux was examined for its potential to damage DNA as reflected by induced DNA synthesis to repair the damage using liver hepatocytes harvested from exposed male Wistar rats. The study comprised of four groups, viz. two test groups (2 and 8 mg/m3) exposed to Nocolok Zn flux at two concentrations, one negative control group (clean air) and one positive control group. The mean NNG determined for the mid and high dose group of the test substance (-10.09 and -12.48, respectively) were comparable to the mean NNG determined for the negative control (-11.81) and clearly lower than the mean NNG found in the positive control group (+14.40). The percentage ‘cells in repair’ of the mid and high dose group of the test substance (0.40% and 0.20%) was comparable to the negative control group (0.20 % ‘cells in repair’) and clearly lower than the percentage found in the positive control group (89.60 %). Analysis of fluoride levels (as a marker for the test substance) in urine indicated systemic availability of the test compound upon inhalation exposure. It is therefore concluded that the test substance Nocolok Zn flux did not induce unscheduled DNA synthesis (UDS) in hepatocytes of male rats, exposed to the test substance by inhalation, under the conditions used in this study.

In vivo: Micronucleus test:

In a GLP-compliant erythrocyte micronucleus test, tested according OECD guideline 474, five groups, viz. three groups exposed to Nocolok Zn flux at four concentrations (0, 0.5, 2, 8 mg/m3 dosed via inhalation for five days a week for four week) and one positive control group (Mitomycin C). The group mean numbers of MPE per 2000 PE in the positive control group (Mitomycin C; 1.5 mg/kg bw, administered once intraperitoneally) showed a statistically significant increase compared to the negative control group. The mean numbers of MPE per 2000 PE in the negative control group were within the historical control data range. Therefore, the study was considered valid. The results showed no statistically significant differences in the number of polychromatic erythrocytes (PE) per 200 erythrocytes (E) and the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) between the Nocolok Zn flux treated groups and the negative control group (clean air). Analysis of fluoride levels (as a marker for the test substance) in urine indicated systemic availability of the test compound upon inhalation exposure. It was therefore concluded that, under the conditions used in this study, Nocolok Zn flux, at exposure levels up to 8 mg/m3, did not induce chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in male rats.

Short description of key information:
Based on the available in vivo genotoxicity studies, the test substance Nocolok Zn Flux does not induce unscheduled DNA synthesis (UDS) in hepatocytes chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in male rats.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available in vivo genotoxicity studies, the test substance does not need to be classified for genotoxicity according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008