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Reverse Mutation Assay using Bacteria (Ames test)

SIKA Hardener LI was assessed in an Ames test according to EU method B.13/14, OECD guideline 471 and EPA OPPTS 870.5100. Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of SIKA Hardener LI. Five independent experiments were carried out, including two plate incorporation tests (Experiment I, and Experiment III) and in three pre-incubation tests (Experiment II, Confirmatory Mutation Test; Experiment IV, Complementary Confirmation Mutation Test and Experiment V, 2nd Complementary Confirmatory Mutation Test). Experiments I-IV were carried out with and without metabolic activation (S9 Mix), Experiment V was performed without metabolic activation. Concentrations up to 5000μg/plate (limit concentrations) and concurrent controls were tested in triplicates.

Using the plate incorporation method (Experiment I and III) a higher number of revertant colonies was observed compared to solvent controls, but considered to be of minor intensity and not dose related. Mutation factors were below the biological relevant threshold and the number of revertant colonies remained within the historical control range. Therefore, the observations were considered to reflect biological variability.

An inhibitory, cytotoxic effect of the test item was observed in some strains using the plate incorporation method. In Experiment I (Initial Mutation Test) strong reductions in the number of revertant colonies were observed, although no difference in background lawn development was noted. After 48 hours incubation using the plate incorporation method, microdrops were observed in all tester strains at concentrations of 5000 ug/plate, with or without metabolic activation.

Using the pre-incubation method (Experiment II, IV and V) a higher number of revertant colonies was observed compared to solvent controls. However, again the increased number of revertant colonies was of minor intensity and did not follow a dose-response relationship. Mutation factors were below the biological relevant threshold and the number of revertant colonies within the historical control range. Therefore, these observations too were considered to reflect biological variability. An inhibitory, cytotoxic effect of the test item was observed in some strains using the pre-incubation method. In Experiment II (Confirmatory Mutation Test) slightly reduced background lawn was observed. Further, numbers of revertant colonies were reduced. After 48 hours incubation microdrops were observed in all tester strains at concentrations of 5000 µg/plate with or without metabolic activation. The revertant colony numbers of solvent control plates without S9 Mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies. In each experiment the viability of the bacterial cells was checked by a plating experiment. In conclusion, the reported data of this mutagenicity assay show, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, SIKA Hardener LI was considered non-mutagenic in this bacterial reverse mutation assay.

 

In-vitro Mammalian Chromosome Aberration Test

SIKA Hardener LI was tested in a chromosome aberration assay in V79 cells according to EU method B.10, OECD guideline 473 and EPA OPPTS 870.5375. The test item was dissolved in acetone and the following concentrations (without and with metabolic activation) were selected on the basis of preliminary cytotoxicity investigations. In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time without and with S9 mix: 39.06, 78.12, 156.25 and 312.50μg/mL test item.

Experiment B with 20/28 h treatment/sampling time without S9 mix: 2.44, 4.88, 9.76, and 19.53μg/mL test item.

Experiment B with 3/28 h treatment/sampling time with S9 mix: 39.06, 78.12, 156.25 and 312.50μg/mL test item.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted.

In Experiment B, there were no biologically significant increases in the number of cells showing structural chromosome aberrations up to cytotoxic concentrations, without S9 mix over a prolonged treatment period (20 hours). Further, a three-hour treatment with SIKA Hardener LI up to cytotoxic concentrations in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations.

In experiment A and in experiment B the number of aberrant cells with and without gap exceeded the historical control variation in some cases, however these biological alteration were not considered toxicologically significant.Experiment A and in experiment B no statistically significant differences between treatment and control groups and no dose-response relationships were noted.There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The validity of the test was shown using ethylmethane sulphonate (0.4 and 1.0μg/mL) and N-nitrosodimethylamine (1.0μg/mL) as positive controls.

In conclusion, SIKA Hardener LI tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test using Chinese Hamster lung cells. Therefore, SIKA Hardener LI was considered not clastogenic in this system.

 

In-vitro Mammalian Cell Gene Mutation Test

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of SIKA Hardener LI to cause gene mutation and/or chromosome damage according to EU method B.17 and OECD guideline 476. Treatments were carried out for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation. Acetone was used as solvent. Treatment concentrations were selected based on a preliminary cytotoxicity test. The following concentrations were tested:

Assay 1: 3-hour treatment in the presence of S9-mix: 140; 130; 120; 110; 100; 75; 50; 25; 12.5 and 6.25μg/mL. 3-hour treatment in the absence of S9-mix: 80; 70; 60; 50; 40; 30; 20 and 10μg/mL.

Assay2: 3-hour treatment in the presence of S9-mix: 140; 130; 120; 110; 100; 75; 50; 25; 12.5 and 6.25μg/mL. 3-hour treatment in the absence of S9-mix: 80; 70; 60; 50; 40; 35; 30; 25; 20; 15 and 10μg/mL. 24-hour treatment in the absence of S9-mix: 50; 40; 37.5; 35; 32.5; 30; 27.5; 25; 22.5; 20; 17.5; 15; 10 and 5μg/mL.

Assay 3: 3-hour treatment in the absence of S9-mix: 80; 70; 60; 50; 40; 35; 30; 25; 20; 15 and 10μg/mL. 24-hour treatment in the absence of S9-mix: 50; 40; 37.5; 35; 32.5; 30; 27.5; 25; 22.5; 20; 17.5; 15; 12.5; 10 and 5μg/mL.

In all assays, the mutation frequencies of positive controls (Cyclophosphamide in experiments with metabolic activation system and 4-Nitroquinoline-N-oxide in experiments without metabolic activation system) were acceptable and were in accordance with historical data. The spontaneous mutation frequency of the negative (solvent) control was just below the normal range in cases of experiments without metabolic activation. However, the low mutation frequency of the solvent control plates in these assays was considered to reflect the genuine low mutation rate of the samples rather than non-optimal culture conditions. Thus, the Mouse Lymphoma Assay with SIKA Hardener LI using the L5178Y TK +/- 3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.

In all assays, treatment with the test item did not result in a statistically or biologically significant dose-dependent increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9 mix).

In conclusion, no mutagenic effect of SIKA Hardener LI was observed either in the presence or absence of metabolic activation system under the conditions of this mouse lymphoma assay.


Justification for selection of genetic toxicity endpoint
Three in vitro studies on different types of genetic toxicity were used for assessment. Therefore it is not possible to select a single study.

Short description of key information:
SIKA Hardener LI was tested in three different in vitro genetic toxicity studies, both with and without metabolic activation. SIKA Hardener LI did not induce gene mutations by frameshift or base-pair substitution in an Ames test. SIKA Hardener LI did not induce structural chromosome aberrations in Chinese Hamster lung cells and was therefore not considered clastogenic in the tested system. Additionally, SIKA Hardener LI showed no mutagenic effect in a mouse lymphoma assay. Overall, SIKA Hardener LI was considered non genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on results of three different in vitro genetic toxicity studies, SIKA Hardener LI was not classified and labelled as genotoxic according to Directive 67/548/EEC (DSD) and to Regulation (EC) No 1272/2008 (CLP).