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Additional information

Ames-Test

As reported in the scientific publication by Dean et al. in Mutation Research, 153 (1985), pages 57-77, the mutagenic potential of the test substance, identified by the common name 2 -hydroxy-5 -tert-nonyl acetophenone oxime, was evaluated in a study similar to OECD Test Guideline 471 (Ames).

The substance as 50% active component (in hydrocarbon diluent) was dissolved in DMSO and applied to four Salmonella typhimurium strains (TA 1535, 1538, 98, 100) using the plate incorporation application method as described by Ames, B.N. et al. (1975), Mutation Research, 31, 347-364, with and without metabolic activation (S9). Vehicle and positive controls were included. The concentrations 0.2, 2, 20, 500, 2000 µg/plate were tested. The results were negative in the strains used. Therefore, the test substance is considered not to be mutagenic.

The suitable read across substance Benzaldehyde, dodecylhydroxy-, oxime, branched (solvent-free and dodecylphenol-depleted), which has a branched C12 instead of a branched C9 side chain and lacks a methyl group at the oxime carbon atom (CAS # 1233873 -37 -4) was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The test was conducted according to OECD 471 guideline and GLP (BASF, 2013).

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5 000 μg/plate (SPT); 33 μg - 5 000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: Precipitation of the test substance was found from about 2 500 μg/plate onward with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about

1 000 μg/plate onward.

MUTAGENICITY:

A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

CONCLUSION:

Thus, under the experimental conditions of this study, the read across substance Benzaldehyde, dodecylhydroxy-, oxime, branched (solvent-free and dodecylphenol-depleted) is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

This study was performed to investigate the potential of the suitable read across substance Benzaldehyde, 2-hydroxy-5-nonyl-, oxime, branched, which lacks a methyl group at the oxime carbon atom (CAS # 174333 -80-3)

to induce gene mutations according to

the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. Due to strong toxic effects in experiment II without S9 mix in strains TA 1535, TA 1537, TA 98 and TA 100 this part was

repeated with reduced concentrations (reported as experiment Ila). The test item was tested at the following concentrations:

Pre-Experimen/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II a: 0.03; 0. 1; 0.3; 1; 3; 10; 33; and 100 μg/plate

The plates incubated with the test item showed reduced background growth with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was

observed following treatment with Benzaldehyde, 2-hydroxy-5-nonyl-, oxime, branched at any dose level, neither in the

presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

In vitro chromosome aberration test

The test substance, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in five independent experiments.

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment IA without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentrations of 500.0 ug/mL (without S9 mix) and 1000.0 ug/mL (with S9 mix) were chosen due to strong cytotoxic effects obtained in an HPRT study, which was performed in parallel with the test item and the same cellular system.

Dose selection for the cytogenetic experiments was performed considering the toxicity data.

No clear toxic effects indicated by reduced mitotic indices or reduced cell numbers of about or below 50 % of control were observed after treatment with the test item, except in Experiment IC in the presence of S9 mix. In this experimental part the mitotic index was reduced to 52.8 % of control at the highest evaluated concentration of 25.0 ug/mL. In Experiments IA and IIA the mitotic indices were reduced (68.6 and 65.2 % of control, respectively) at the highest evaluated concentrations in the absence of S9 mix.

No clastogenicity was observed without S9 mix. Statistically significant increases in the number aberrant cells obtained in Experiment IB in the presence of S9 mix. As these increases were not reproducible in the confirmatory Experiment IC this effect is regarded as

biologically irrelevant.

No biologically relevant increase in polyploid or endomitotic metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.

Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic or the highest evaluable concentrations.

HPRT gene mutation assay

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a

treatment time of 4 hours with and 24 hours without metabolic activation.

The highest concentration (3340.0 ug/ml approx. 10 mM) used in the range finding pre-expenment was chosen with respect to the purity of the test item and the current OECD guideline 476. The dose range of the main experiments was limited by test item induced

cytotoxicity.

The dose ranges of the first main experiment (with and without S9 mix) and the second main experiment (without S9 mix) were limited by test item induced cytotoxicity. After metabolic activation the dose range of the second main experiment was limited by precipitation of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the test substance is considered to be non·mutagenic in this HPRT assay.


Short description of key information:
The registered substance is considered to be non-genotoxic based on the available data (Ames-Test, in vitro HPRT gene mutation assay and in vitro chromosome aberration assay).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In view of the negative outcome of the Ames tests, the in vitro chromosome aberration assay and an HPRT gene mutation assay, the registered substances does not to be labelled with regard to genetic toxicity.