4-allyl-2-methoxyphenyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to limit concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

- Unscheduled DNA synthesis assay (OECD 482, GLP, K, rel.2) : no induction of UDS in rats hepatocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 December 2001 to 03 June 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted according to OECD Guideline 482, however a repeat experiment was not performed

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

1986

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

other: in vitro UDS assay

Target gene:

Not applicable

Species / strain / cell type:

hepatocytes: rat

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Preliminary toxicity test: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/mL
Main test: 1, 2.5, 5, 10, 15, 20, 25, 50, 75 and 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was determined to be the solvent of choice based on solubility or miscibility of the test item and compatibility with the target cells. The test item was soluble or miscible in DMSO at ca. 500 mg/mL, the maximum concentration prepared.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

Details on test system and experimental conditions:

TEST SYSTEM:
- Source of cells and cell culture: Adult male Sprague-Dawley rats obtained from Harlan Sprague-Dawley, Inc. (Frederick, MD). The procedure used for obtaining rat hepatocyte cultures was essentially that of Williams (1977 and 1979). The rats was anesthetised with metofane and a midventral incision was made to expose the liver. The liver was perfused with 0.5 mM ethylene glycol-bis (β-aminoethyl ether)N, N, N’, N’-tetraacetic acid (EGTA) solution followed by collagenase solution (80-100 units Type I collagenase/mL culture medium). The liver was removed, transected, and shaken in a dilute collagenase solution to release the hepatocytes. The cells were pelleted by centrifugation, resuspended in complete WME (William’s Media E buffered with 0.01 M HEPES, supplemented with 2 mM L-glutamine, 50 µg/mL gentamicin and 10% fetal bovine serum) and approximately 5 x 10^5 cells were seeded into 35 mm tissue culture dishes containing complete WME. The cultures were incubated at 37 ± 1 °C in a humidified 5 ± 1% CO2 incubator for 90 to 180 minutes, washed with complete medium, refed with serum-free medium and used in the test. The dishes designated for autoradiography were also treated with 10 µci 3H-Thymidine/mL.

METHOD OF APPLICATION: in medium; Complete WME (William’s Media E buffered with 0.01 M HEPES, supplemented with 2 mM L-glutamine, 50 µg/mL gentamicin and 10% fetal bovine serum)

DURATION
- Exposure duration: 18-20 h

ASSESSMENT OF CYTOTOXICITY
After 18-20 h of exposure, a portion of the medium from two of each of the three culture dishes per treatment group was removed for LDH determinations. Additionally, the cultures of the highest dose and the vehicle control which were seeded in dishes without coverslips were lysed with Triton X-100 to release 100% of the LDH in the cultures.

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Duplicate cultures/dose for test item and vehicle control
Main test: Triplicate cultures/dose for test item, vehicle and positive controls

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- After 18-20 h of exposure, the cells in the autoradiography dishes were washed in serum free WME, swelled in 1% sodium citrate and fixed in ethanol-glacial acetic acid fixative. The coverslip were air-dried, mounted cell side up on glass slides, and allowed to dry. The slides were coated with KodakNTB-2 emulsion (diluted 1:1 in deionized water) and stored in a refrigerator for 5 to 12 days in light tight boxes with desiccant. The slides were then developed in Kodak D-19 developer (diluted 1:1 in deionized water), fixed in Kodak fixer and stained in hematoxylin-eosin stain.

NUMBER OF CELLS EVALUATED:
- The slides were counted using automated colony counter interfaced with microscope.
- Where possible, nuclear grains were counted in 50 cells in random areas on each of the three coverslips for a total of 150 cells per treatment.

CRITERIA FOR IDENTIFICATION OF NUCLEI:
The net nuclear counts were determined by counting three nucleus-sized areas adjacent to each nucleus and subtracting the average cytoplasmic count from the nuclear count. Replicate synthesis was identified by nuclei completely blackened with grains and such cells were not counted. Nuclei exhibiting toxic effects of treatment, such as dark or uneven staining, disrupted membranes, or irregular shape, were not counted.

DETERMINATION OF CYTOTOXICITY
- In the cytotoxicity assays, relative LDH activities were obtained by subtracting the LDH activity of the negative control cultures from the LDH activity of the treated cultures. Relative toxicities were obtained by comparing the relative LDH activities of the treated cultures to the relative LDH activity of the 100% lysis control cultures.

OTHER:
For each treatment slide in the UDS assay, the net nuclear counts were averaged and the mean ± SD reported. Also reported are the grand mean and SD for each dose level as well as the per cent of cells in repair (cells with ≥ 5 net nuclear grains).

Evaluation criteria:

All conclusions were based on scientific judgement; however, as a guide to interpretation of the data, the following criteria were used.
- If the mean net nuclear count was increased by at least 5 counts over the negative control, the results for that dose level were considered significant. A test item was judged positive if it induced a dose-related response and at least one dose produced a significant increase in the average net nuclear grains when compared to that of the negative control. In the absence of a dose response, a test item which showed a significant increase in the mean net nuclear grain count in at least two successive doses was considered positive.
-If a test item showed a significant increase in the mean net nuclear grain count at one dose level without a dose response, the activity of the test item was considered to be equivocal.
- The test item was considered negative if no significant increase in the mean net nuclear grain counts was observed.

Statistics:

None

Key result

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Yes; At the initiation of treatment, precipitation was observed at ≥ 333 µg/mL. At the termination of the treatment, precipitation was observed at ≥ 667 µg/mL.
- Other confounding effects: None

PRELIMINARY TOXICITY TEST:
- At the initiation of treatment, precipitation was observed at ≥ 333 µg/mL. At the termination of the treatment, precipitation was observed at ≥ 667 µg/mL.
- Measurement of released LDH in the hepatocyte cultures treated with test item resulted in relative toxicities of > 100% for cultures treated with ≥ 667 µg/mL. For cultures treated with 100 and 333 µg/mL, the relative toxicity was 53.5 and 59.7%, respectively. For cultures treated with 33 and 67 µg/mL, the relative toxicity was 28.9 and 25.8%, respectively. Relative toxicities of the remaining cultures were < 9.3%.
- A microscopic examination of the hepatocyte cultures at the termination of the treatment indicated high toxicity in cultures treated with ≥ 100 µg/mL, moderate toxicity in cultures treated with 33 and 67 µg/mL, and low toxicity in cultures treated with 10 µg/mL. All other cultures appeared morphologically normal.
- Based on the results of the toxicity assay, the highest concentration of the test item selected for UDS assay was 100 µg/mL.

UDS ASSAY
- At the initiation and at the termination of the treatment, precipitation was not observed in any of the test item treated cultures.
- Measurement of released LDH in the hepatocyte cultures treated with test item resulted in relative toxicities of > 100% for cultures treated with ≥ 50 µg/mL. For cultures treated with 20 and 25 µg/mL, the relative toxicity was 73.3 and 84.5%, respectively. For cultures treated with 15 µg/mL, the relative toxicity was 33.7%. For cultures treated with 5 and 10 µg/mL, the relative toxicity was 14.1 and 10.7%, respectively. All other relative toxicities were ≤ 7.5%.
- A microscopic examination of the hepatocyte cultures at the termination of the treatment indicated high toxicity in cultures treated with ≥ 50 µg/mL, moderate toxicity in cultures treated with 20 and 25 µg/mL, and low toxicity in cultures treated with 10 and 15 µg/mL. All other cultures appeared morphologically normal.
- Test item did not induce a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the negative control) at any dose level in isolated rat hepatocytes.

None

Conclusions:

Under the test conditions, the test item did not induce a significant increase in the mean number of net nuclear grain counts at any dose level in isolated rat hepatocytes. Therefore, the test item is considered to be negative in the UDS assay.

Executive summary:

In an in vitro UDS (unscheduled DNA synthesis) assay performed according to the OECD test guideline No. 482 and in compliance with GLP, primary cultures of rat hepatocytes were exposed to test item for 18-20 h at the following concentrations:

Preliminary toxicity test: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/mL

Main test: 1, 2.5, 5, 10, 15, 20, 25, 50, 75 and 100 µg/mL

Vehicle (DMSO) and positive control groups were also included in the test.

Preliminary toxicity test: At the initiation of treatment, precipitation was observed at ≥ 333 µg/mL. At the termination of the treatment, precipitation was observed at ≥ 667 µg/mL. Measurement of released LDH in the hepatocyte cultures treated with test item resulted in relative toxicities of > 100% for cultures treated with ≥ 667 µg/mL. For cultures treated with 100 and 333 µg/mL, the relative toxicity was 53.5 and 59.7%, respectively. For cultures treated with 33 and 67 µg/mL, the relative toxicity was 28.9 and 25.8%, respectively. Relative toxicities of the remaining cultures were < 9.3%. A microscopic examination of the hepatocyte cultures at the termination of the treatment indicated high toxicity in cultures treated with ≥ 100 µg/mL, moderate toxicity in cultures treated with 33 and 67 µg/mL, and low toxicity in cultures treated with 10 µg/mL. All other cultures appeared morphologically normal. Based on the results of the toxicity assay, the highest concentration of the test item selected for UDS assay was 100 µg/mL.

Main test: At the initiation and at the termination of the treatment, precipitation was not observed in any of the test item treated cultures. Measurement of released LDH in the hepatocyte cultures treated with test item resulted in relative toxicities of > 100% for cultures treated with ≥ 50 µg/mL. For cultures treated with 20 and 25 µg/mL, the relative toxicity was 73.3 and 84.5%, respectively. For cultures treated with 15 µg/mL, the relative toxicity was 33.7%. For cultures treated with 5 and 10 µg/mL, the relative toxicity was 14.1 and 10.7%, respectively. All other relative toxicities were ≤ 7.5%. A microscopic examination of the hepatocyte cultures at the termination of the treatment indicated high toxicity in cultures treated with ≥ 50 µg/mL, moderate toxicity in cultures treated with 20 and 25 µg/mL, and low toxicity in cultures treated with 10 and 15 µg/mL. All other cultures appeared morphologically normal.

Under the test conditions, the test item did not induce a significant increase in the mean number of net nuclear grain counts at any dose level in isolated rat hepatocytes. Therefore, the test item is considered to be negative in the UDS assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May to 20 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 471 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on July 05, 2016 / Signed on October 28, 2016)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan for S. typhimurium and E. coli, respectively

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% S9: S9-mix from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).

Test concentrations with justification for top dose:

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix
Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: In solubility checks performed in house the test item was noted as immiscible in sterile distilled water at 50 mg/mL but fully miscile in DMSO at the same concentration. DMSO was therefore selected as the vehicle.
- Preparation of test formulation: The test item was accurately weighed and appropriate dilutions prepared in DMSO by mixing on a vortex mixer on the day of each experiment. No correction was required for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of of 4 x 10^-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM
- Bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Rationale for test conditions:

Maximum concentration was 5000 μg/plate (the maximum recommended dose level).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- Other confounding effects: None

HISTORICAL CONTROL DATA
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2015 and 2016 of the corresponding Testing Laboratory.

MUTATION TEST
- In the first mutation test (plate incorporation method) there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), although several tester strains exhibited slight decreases in revertant colony frequency at the maximum dose level. Consequently, the same maximum dose level (5000 µg/plate) was used as the maximum concentration in the second mutation test. The test item induced a visible reduction in the growth of the bacterial background lawns in Experiment 2 after employing the pre-incubation modification with weakened lawns noted in the absence of metabolic activation (S9-mix) initially from 1500 µg/plate (TA1535, TA98 and TA1537) and at 5000 µg/plate (TA100). No toxicity was noted to Escherichia coli strain WP2uvrA. In the presence of S9-mix, weakened lawns were noted at 5000 µg/plate to all of the tester strains. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method).

OTHERS
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

None

Conclusions:

Under the test condition, test item is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- with or without metabolic activation.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test item both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

 

Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

 

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 validated.

 

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), however several Salmonella tester strains exhibited slight decreases in revertant colony frequency at the maximum dose level. Consequently, the same maximum dose level (5000 µg/plate) was used as the maximum concentration in the second mutation test. The test item induced a visible reduction in the growth of the bacterial background lawns in Experiment 2 after employing the pre-incubation modification with weakened lawns noted in the absence of metabolic activation (S9-mix) initially from 1500 µg/plate (TA1535, TA98 and TA1537) and at 5000 µg/plate (TA100). No toxicity was noted toEscherichia colistrain WP2uvrA. In the presence of S9-mix, weakened lawns were noted at 5000 µg/plate to all of the tester strains. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method).

 

Under the test condition, test item is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- with or without metabolic activation.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Envigo, 2017	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100 E. coli WP2uvrA	-S9 +S9	Up to limit concentration	-S9 : non mutagenic +S9 : non mutagenic
2 BioReliance, 2003	UDS (OECD 482, K, rel.2)	DNA repair	Rat (hepatocytes)	N.A.	Up to cytotoxic concentration	Non-genotoxic

Gene mutation Assay (Test n° 1 ):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (Test n°1, see Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

DNA damage repaired by unscheduled DNA synthesis (Test n°2)

The ability of the substance to induce DNA damage (i. e. patch repair) in vitro was assessed using an UDS Assay (Test n°2) in primary cultures of rat hepatocytes incubated with the test item up to 100 µg/mL for 18-20 hours. Vehicle (DMSO) and positive control groups were also included in the test.

Under the test conditions, the test item did not induce a significant increase in the mean number of net nuclear grain counts at any dose level in isolated rat hepatocytes. Therefore, the test item is considered to be non-genotoxic in the UDS assay.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.