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To assess mutagenic potential of reaction mass of isomers of: C7-9-alkyl 3-(3,5-di-tert-butyl-4 hydroxyphenyl)propionate the following genetic toxicity studies on the related substances were used:

In vitro tests:

Reverse Mutation Assay "Ames Test" using Salmonella Typhimurium and Echerichia Coli (Butyl 3,5 -bis(1,1 -dimethylethyl)-4 -hydroxybenzenepropanoate (CAS No. 52449 -44 -2)

The study was conducted according to the OECD guideline 471, EU Method B14 and the USA, EPA (TSCA) OPPTS harmonised guidelines.Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard cofactors).

The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the range-finding study. The experiment was repeated on a separate day using the same dose range as the range-finding study, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate and opaque film was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

Reverse Mutation Assay "Ames Test" usingSalmonella TyphimuriumandEcherichia Coli (Benzenepropanoic acid, 3,5 -bis(1,1 -dimethylethyl)-hydroxy-,2 -ethylhexylester CAS No. 144429 -84 -5)

The study was conducted according to the OECD Guideline 471, EU Method B14 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were valid. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A light precipitate (oily in appearance) was observed with the naked eye at 5000 µg/plate in the range-finding test and under an inverted microscope in the main test. This observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

Chromosome Aberration Test in CHL Cells in vitro (Butyl 3,5 -bis(1,1 -dimethylethyl)-4 -hydroxybenzenepropanoate (CAS No. 52449 -44 -2))

The study was conducted according to the Japanese New Chemical Substance Law (MITI) and the updated Annex V B10 Method. Duplicate cultures of Chinese Hamster Lung (CHL) cells were treated with the test material at several dose levels, together with vehicle and positive controls. Five treatment regimens were used: Experiment 1, 6(18)-hours exposure both with and without the addition of an induced rat liver homogenate metabolising system. Experiment 2, 24 hours continuous exposure, 48 hours continuous exposure and 6(18)-hours exposure with metabolic activation. The dose levels used were selected on the basis of the results of a preliminary toxicity test and were in the range of 840 to 2700 µg/mL for the 6(18)-hour treatment with metabolic activation and 2 to 12 mg/mL for the 6(18), 24 and 48-hour treatments without metabolic activation. The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control treatments gave highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any significant increases in the frequency of cells with aberrations excluding gaps in any of the treatment cases. The test material was shown to be toxic to CHL cells in vitro, particularly in the absence of metabolic activation. The test material was shown to be non-clastogenic to CHL cells in vitro.

Chromosome Aberration Test in CHL Cells in vitro (Benzenepropanoic acid, 3,5 -bis(1,1 -dimethylethyl)-hydroxy-,2 -ethylhexylester (CAS No. 144429 -84 -5))

This study was conducted according to a method that was designed to assess the potential chromosomal mutagenicity of a test material on the metaphase chromosomes of the Chinese Hamster Lung (CHL) cell line according to the requirements of the OECD guideline 473 and EU Method B10. The study was performed according to the Standard Test Method. The study consisted of a Cell Growth Inhibition Test and two separate experiments. Except in the Inhibition Test, duplicate cultures of Chinese Hamster Lung (CHL) cells that had been treated with the test material in either the presence or absence of metabolic activation, were evaluated for induction of chromosome aberrations. Duplicate vehicle and positive controls were included in parallel in both experiments. The metabolic activation system was liver S9 prepared from male rats induced with a combination of phenobarbitone/p-naphthoflavone. Four exposure conditions were used: In Experiment 1 the exposures were 6(18)-hour both with and without metabolic activation (S9, at a final concentration of 5%); Experiment 2 included a 24-hour continuous exposure in the absence of S9 and a repeat of the with-S9 exposure group (at 2% final concentration). The dose levels used were selected on the basis of molecular weight, solubility and the results of the Cell Growth Inhibition Test. The dose range for the 6(18)-hour exposures in Experiment 1 were 3.75 to 45 µg/mL in the absence of S9, and 60.94 to 1950 µg/mL in the presence of S9. In Experiment 2 the dose range was 1.88 to 30 µg/mL for the 24 hours exposure group and 30 to 480 µg/ml for the with S9 (at 2%) exposure group.

The vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for the CHL cell line. All of the positive control materials induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any statistically significant increases in the frequency of cells with aberrations in any of the exposure groups. The dose levels of the test material were shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all exposure groups. The test material was considered to be non-clastogenic to CHL cells in vitro.

L5178Y TK +/- Mouse Lymphoma Assay (Butyl 3,5 -bis(1,1 -dimethylethyl)-4 -hydroxybenzenepropanoate (CAS No. 52449 -44 -2))

The study was conducted according to a method, that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD Guideline 476.Two independent experiments were performed.

In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 6.56 to 210 µg/mL in the absence of metabolic activation and 13.13 to 420 µg/mL in the presence of metabolic activation. The dose range for Experiment 2 was 3.13 to 125 µg/mL in the absence of metabolic activation, and 13.13 to 280 µg/mL in the presence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating satisfactory performance of the test and of the activity of the metabolizing system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

In vivo tests:

Micronucleus Test in the Mouse (Butyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate (CAS No. 52449 -44 -2))

A study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method used has been designed to comply with the OECD Guideline 474, EU Method B12 and the USA EPA, TSCA and FIFRA guidelines.

A range-finding study was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes; therefore the main study was performed using only male mice. The micronucleus study was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) of 1500 mg/kg and with 750 and 375 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single oral dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively.

No statistically significant decreases in the PCE/NCE (polychromatic erythrocytes/normochromatic erythrocytes) ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. However, the presence of a premature death and clinical signs indicated that systemic absorption had occurred.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes. The test material was considered to be non-genotoxic under the conditions of the test.

Prediction by the Toxtree modelling software:

Genetic toxicity potential of nonyl 3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoate was assessed using the Toxtree (version 2.1.0.) modelling tool and the OECD QSAR Toolbox for Grouping Chemicals into Categories (version 2.1). Toxtree was developed by IDEA Consult Ltd (Sofia, Bulgaria) and is approved and recommended by the EU Joint Research Center in Ispra (Italy) (LINK:http://ecb.jrc.ec.europa.eu/qsar/qsar-tools/index.php?c=TOXTREE). OECD Toolbox has been developed in collaboration with the ECHA.

According to the modelling results of Toxtree, no functional groups were identified for Nonyl 3-(3,5-ditert-butyl-4-hydroxyphenyl)propanoate which would produce gene mutations in Salmonella typhimurium strain TA 100. One positive structural alert H-acceptor-path was identified for the micronucleus assay by this modelling software. However, the target chemical was classified as “No alerts for micronucleus assay” by the profiling method "Micronucleus alerts by Benigni/Bossa" in the OECD Toolbox.

Predictions by the QSAR OECD Toolbox software:

The chemical benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, C7-9-branched alkyl esters (CAS 125643-61-0) was evaluated by the QSAR OECD Toolbox software for its genotoxicity potential. The prediction was based on the measured values of chemicals assigned into the category. The experimental data for the related chemicals butyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate (CAS 52449 -44 -2) and benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-,2-ethylhexylester (CAS 144429 -84 -5) were added in the software manually and together with data of other structurally similar analogues were used for the read-across.

The target chemical was classified as "Phenols, Esters" by the profiling method "Aquatic toxicity classification by ECOSAR ". Therefore, at first the chemicals with the same classification were searched. 122 chemicals (inclusive manually added data of two chemicals) were assigned into the category.

The prediction based on data available for Ames Test:

There were 10 chemicals in the category for which data were available for the Bacterial Reverse Mutation Assay (Ames Test). These chemicals were scanned for their differences to the target and thereafter the category was refined eradicating chemicals with different characteristics relevant for genotoxicity endpoint. In this manner, a chemical was eliminated which possessed different to the target DNA binding mechanism (performed by the grouping method "DNA binding by OASIS" ). Furthermore, the chemicals with other organic functional groups were removed (performed by the "Organic functional groups (nested)"). The selected 5 neighbour chemicals which were used for read-across represent the same class of chemicals and are all negative inin vitroBacterial Reverse Mutation Assay.

The prediction based on data available forin vitro Mammalian Chromosome Aberration Test:

There were 8 chemicals in the category for which data were available for the investigated endpoint. These chemicals were scanned for their differences to the target and thereafter the category was refined eradicating chemicals with different characteristics relevant for genotoxicity endpoint. In this manner, a chemical was eliminated which possessed different organic functional groups (performed by the "Organic functional groups"). Further, structurally dissimilar chemicals (similarity level up to 30 -40%) were removed. All removed chemicals were different to the target regarding DNA binding mechanism. The remaining 2 neighbour chemicals are the chemicals whose data were added in the software. They were identified structurally similar to the target at 80 -100% level (Dice) and had no differences to the target in respect to properties relevant for genotoxicity. The target chemical was predicted to be negative.

The prediction based on data available forin vivo Chromosome Aberration Test:

The target chemical was classified as "Esters, Phenols" by Aquatic toxicity classification by ECOSAR. There were few chemicals with experimental data available for the investigated endpoint. Therefore, at first chemicals were searched by "Structure similarity" (another predefined structure based categorization method). 11879 chemicals were arranged into the category, and for 210 chemicals of them there was data available for the in vivo Chromosome Aberration test. The prediction was performed based on these data. Thereafter, the chemicals in the category were scanned for the differences to the target and the chemicals were removed which were different regarding properties relevant for genotoxicity endpoint. The target chemical was classified as "Alkyl phenols AND P450 Mediated Activation to Quinones and Quinone-type Chemicals" by the profiling method "DNA binding by OECD". Phenols can be oxidised by Cytochrom P450 to a quinone methide followed by Michael addition and has been suggested to be the primary route of DNA binding (QSAR Toolbox 2.1.), possibly leading to a mutation. Therefore, all chemicals with this classification were retrieved into a subcategory. Further, the chemicals whose chemical elements in their structure differed from those of the target were eliminated. A similar subcategory could also be built if chemicals were subcategorized according to the difference in "Micronucleus alerts by Benigni/Bossa", and in organic functional groups. The target chemical is predicted to be negative.

 


Justification for selection of genetic toxicity endpoint
No study was selected since all in vitro studies and one in vivo study were negative.

Short description of key information:
The related substances of reaction mass of isomers of: C7-9-alkyl 3-(3,5-di-tert-butyl-4 hydroxyphenyl)propionate did not induce significantly the frequencies of revertant colonies in any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The substances were toxic to CHL cells but not clastogenic. In mammalian cells, butyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate (CAS No. 52449 -44 -2) did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level with and without metabolic activation. In in vivo micronucleus test, there was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the same test material.
The chemical nonyl 3-(3,5-ditert-butyl-4-hydroxyphenyl) propanoate which represents structure with C9 alkyl chain from isomeric mixture of reaction mass of isomers of: C7-9-alkyl 3-(3,5-di-trans-butyl-4-hydroxyphenyl) propionate was predicted to be negative in in vitro gene mutation test in bacterial strains (Toxtree predictions). The target chemical under CAS 125643 -61 -0 was predicted negative in in vitro gene mutation test in bacterial strains and in in vitro Chromosome Aberration Test (QSAR OECD Toolbox prediction). One positive structural alert H-acceptor-path was identified for the Micronucleus Assay by both modelling tools. However, the prediction of in vivo Chromosome Aberration Test for the target chemical based on the experimental data of structurally similar chemicals with the same DNA binding mechanism turned out in negative result.
Based on the experimental data of analogues substances and on the prediction results of the modelling software, reaction mass of isomers of: C7-9-alkyl 3-(3,5-di-trans-butyl-4-hydroxyphenyl) propionate is considered to be non-genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The weight of evidence suggests that the test substance is not expected to present a significant risk for mutagenicity in humans, therefore classification is not required in accordance with EU CLP (Regulation (EC) No. 1272/2008).

 

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