Methyl 2-hexyl-3-oxocyclopentanecarboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Test substance storage: At room temperature in the dark
- Description: Clear colourless liquid
- Expiry date: 28 March 2009

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/b-naphthoflavone induced rat liver S9 homogenate

Test concentrations with justification for top dose:

Dose range finding test/Experiment 1:
- TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330, and 5000 μg/plate in the absence and presence of S9-mix
- TA1535, TA1537 and TA98: 10, 33, 100, 333, 1000, and 3330 μg/plate in the absence and presence of S9-mix
Experiment 2:
- TA98 and WP2uvrA: 33, 100, 333, 1000, and 3300 μg/plate in the absence and presence of S9-mix
- TA1535, TA1537 and TA100: 33, 100, 333, 1000, and 2000 μg/plate in the absence and presence of S9-mix

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

- OTHER: In the first experiment 5% (v/v) S9-mix was used and in the second experiment 10% (v/v) S9-mix was used.

Evaluation criteria:

Acceptability of the assay:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria: a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain. b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean. c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
Data evaluation:
A test substance is considered negative (not mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control. b) The negative response should be reproducible in at least one independently repeated experiment. A test substance is considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control. b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST
- Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1000 µg/plate and upwards and at 3330 and 5000 µg/plate at the end of the incubation period.
- Toxicity: In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In tester strain TA100, reduction of the bacterial background lawn was observed in the absence of S9-mix at concentrations of 333 µg/plate and upwards and in the presence of S9-mix at concentrations of 1000 µg/plate. Microcolonies were observed at concentrations of 3330 µg/plate and upwards.

EXPERIMENT 1
- Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1000 and 3330 µg/plate and at 3330 µg/plate at the end of the incubation period.
- Toxicity: In the absence of S9-mix, toxicity was observed at concentrations of 1000 µg/plate and upwards in tester strains TA1535, TA1537, and TA98. In the presence of S9-mix, toxicity was observed at concentrations of 1000 µg/plate and upwards in tester strains TA1535, TA1537 and at a concentration of 3330 µg/plate in tester strain TA98.

EXPERIMENT 2
- Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1000 and above and at 3330 µg/plate at the end of the incubation period.
- In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In the absence of S9-mix, toxicity was observed at concentrations of 1000 µg/plate and upwards in tester strains TA1535, TA1537, TA100, and TA98. In the presence of S9-mix, toxicity was observed at concentrations of 1000 µg/plate and upwards in tester strains TA1535 and TA1537, at a concentration of 3330 µg/plate in tester strain TA98, and at a concentration 333 µg/plate and upwards in tester strain TA100.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Description: Colourless to pale yellow liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020

Target gene:

hprt locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO AA8 cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: American Type Culture Collection
- Batch No.: 5000062 procured

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Alpha Minimal Essential Medium (MEM) without ribonucleosides containing 10% Fetal Bovine Serum (FBS) and antibiotics (1% Penicillin and streptomycin) was used.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenates.
- method of preparation of S9 mix: The S9 homogenate was prepared from male Wistar rats induced with an intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL, respectively, for 3 days prior to sacrifice.
- concentration or volume of S9 mix and S9 in the final culture medium: 10%
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability and streptomycin) was used: The batch of S9 homogenate was assessed for sterility, protein content (Modified Lowry assay, Sword and Thomson, 1980) and for its ability to metabolize the pro-mutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain.

Test concentrations with justification for top dose:

Based on the results of the solubility, pH and precipitation test an initial cytotoxicity test was conducted for the selection of the test concentrations for the gene mutation test. Six concentrations of 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL of the test item were tested in an initial cytotoxicity test. Since the test item showed no change in pH and precipitation at and up to 2 μL/mL, 2 μL/mL was considered as the highest concentration in the initial cytotoxicity lest.

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2E6 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Frozen stock in a cryovial was thawed immediately in the water bath. Cells were transferred into a sterile flask with culture medium containing 10% FBS with antibiotics (1% Penicillin and Streptomycin) and incubated at 37 ± 1 °C and 5 ± 1% CO2 for 2 days. The cell lines were trypsinized and were sub-cultured thrice (Initial cytotoxicity test) and four times (Gene mutation test) before using for the experiment.
- Exposure duration/duration of treatment: 4 hours (with and without S9) at 37 ± 1 °C with 5 ± 1% CO2.

FOR GENE MUTATION:
- Expression time: 8 days
- Selection time (if incubation with a selective agent): 8 days, each replicate treatment culture was pooled and sub-cultured in quintuplicate at a density of 4E5 cells per group with culture media containing 10 μM of 6-Thioguanine and 200 cells/flask in triplicate without 6- Thioguanine for determination of the cloning efficiency.
- Fixation time: 16 days
- Method used: in medium
- Method to enumerate numbers of viable and mutants cells: Post-incubation, medium from each dish was aspirated and stained with 5% Giemsa stain. The number of colonies formed were counted manually.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency and relative survival (RS)

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- The increase is concentration-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical negative/vehicle control data.

A test chemical is considered clearly negative if, in all experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- There is no concentration-related increase when evaluated with an appropriate trend test,
- All results are inside the distribution of the historical negative/vehicle control data.

Statistics:

Data of mutant frequencies were analysed for differences among vehicle control, treatment and positive control groups using SPSS Software version 22 at a 95% level (p<0.05) of significance. The statistical significance was designated by the superscripts in the report as * Statistically significant (p<0.05) change than the vehicle control group.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO AA8 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No effect at tested concentrations
- Precipitation and time of the determination: No effect at tested concentrations

RANGE-FINDING/SCREENING STUDIES:
In the initial cytotoxicity test, there was no evidence of excessive cytotoxicity (<10% RS) at and up to 0.25 μL/mL in both the presence and absence of metabolic activation. A complete absence of the cell monolayer was observed at 1 and 2 μL/mL. At concentrations ranging from 0.0625 to 0.5 μL/mL, the RS values ranged from 1.20 to 78.31% in the presence of metabolic activation and from 1.19 to 77.38% in the absence of metabolic activation. The results of the initial cytotoxicity test indicated that the Relative Survival of the treated cells at the tested concentration of 0.25 μL/mL was 21.69 and 22.62% when compared with the respective vehicle control in the presence and absence of metabolic activation, respectively.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The positive control (3 μg/mL of Benzo(a)pyrene, in the presence of metabolic activation) resulted in a RS value of 78.05% and a mutant frequency of 274.60 per 2E6 cells which was statistically significant higher when compared with the vehicle control. The positive control (1 μg/mL of 4-Nitroquinoline N-oxide, in the absence of metabolic activation) resulted in a RS value 80.72% and a mutant frequency of 270.97 per 2E6 cells which was statistically significant when compared with that of vehicle control.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: There was no evidence of excessive cytotoxicity (< 10% RS) at any of the concentrations in both the presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 20.73 to 89.02% and in the absence of metabolic activation the RS values ranged from 21.69 to 87.95 %, respectively.
- Genotoxicity results: The test item resulted in mutant frequencies of 26.87 to 31.75 per 2E6 cells in the presence of metabolic activation at the different concentrations tested. The vehicle control resulted in mutant frequencies of 24.66 per 2E6 cells. In the absence of metabolic activation, the test item resulted in mutant frequencies of 26.47 to 32.08 per 2E6 cells. The vehicle control resulted in a mutant frequency of 25.76 per 2E6 cells. There was no statistically significant increase in the number of mutant colonies at any of the concentrations tested when compared with the vehicle control.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Description: Colourless to pale yellow liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human lymphocytes
- Suitability of cells: The blood samples obtained were subjected for evaluation of blood parameters in Advia 2120. A sample was accepted when the results incline with the acceptable range of lymphocyte count.

For lymphocytes:
- Sex, age and number of blood donors: Human peripheral lymphocytes from the blood of healthy, young, non-smoking male and female donors (28, 30 and 24 years of age) with no known recent exposure to genotoxic chemicals or radiation were used.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate.
- method of preparation of S9 mix: The S9 homogenate was prepared from male Wistar rats induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL, respectively, for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80 ± 10°C until use.
- concentration or volume of S9 mix and S9 in the final culture medium: 10%
- quality controls of S9: It was assessed for sterility by streaking the supernatant fluid on Nutrient Agar plates and incubated at 37°C for 48 hours and it was found sterile. The protein content and its ability to metabolize the promutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium strain TA100 were also evaluated and the results were found to be acceptable.

Test concentrations with justification for top dose:

An initial cytotoxicity test was conducted with concentrations of 0.25, 0.5, 1 and 2 µL/mL for the selection of test concentrations for the micronucleus test (Main study). The average percent reduction of the Cytokinesis-Block Proliferation Index (CBPl) was 100 in all the concentration tested (complete cytotoxicity). As the test concentrations selected should cover a range from that producing 45 ± 5% cytotoxicity to little or no cytotoxicity, an additional cytotoxicity test was performed at the following test concentrations of 0.0078, 0.0156, 0.0312, 0.0625, and 0.125 μL/mL. Based on the cytotoxicity tests, test concentrations of 0.0078, 0.0156 and 0.0312 μL/mL were used for the main study.

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: One

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 44 to 48 hours at 37 ± 1 °C at 5 ± 1% CO2.
- Exposure duration/duration of treatment: Cells were incubated both with (Set no. 1) and without (Set no. 2) metabolic activation (S9) for 3 to 6 hours and without metabolic activation for 20 to 24 hours (Set no. 3) at 37 ± 1 °C at 5 ± 1% CO2

MICRONUCLEUS:
- Cytokinesis block: Cytochalasin B at 6 µg/mL. For Set no. 1 and 2, the cells were harvested after exposure and the cell suspension in fresh media was mixed with cytochalasin B and then transferred to fresh tissue culture tubes. The tubes were later incubated for 20 to 24 hours at 37 ± 1 °C at 5 ± 1% CO2. For Set no. 3 the cytochalasin B was added during the exposure period.
- Methods of slide preparation and staining technique used including the stain used: Clean slides were stored in a container with distilled water and kept in the refrigerator for 1 hour before use. The cell suspension was mixed using a pipette and a few drops of the suspension was aspirated and dropped onto the chilled slide pre-labelled with the study number, with (+S9) or without metabolic activation (-S9), treatment/group and slide number. The slides were air dried and the smear was stained using Acridine Orange stain by allowing the stain to retain for 5 minutes.
- Number of cells spread and analysed per concentration: A minimum of 3 slides were prepared for each concentration and stained. Slides were evaluated for the presence of micronuclei in at least 1000 binucleates per culture.
- Criteria for scoring micronucleated cells: Measurement of the relative frequencies of mononucleate, binucleate and multinucleate cells in the culture was performed to determine cell proliferation and the cytostatic activity of the treatment to ensure that, only cells that divide during or after treatment were scored.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, other: Cytotoxicity was quantified from the Cytokinesis-Block Proliferation Index (CBPI). CBPI was determined from 500 cells per culture. This measurement was used to estimate cytotoxicity by comparing values in the treated and vehicle control cultures.

Evaluation criteria:

A test item is considered to be clearly positive if:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
- The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.

A test item is considered to be clearly negative if:
- None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control.
- There is no concentration-related increase when evaluated with an appropriate trend test.

Statistics:

Data were analysed using SPSS software version 22 for differences among vehicle control, positive control and test item groups using ANOYA following Dunnett's test at a 95% level of confidence (p<0.05) and the statistical significance was designated by superscripts throughout the study report as stated *: Statistically significant (p≤0.05).

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

But below 45 ± 5%

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH tested at concentrations up to 2 μL/mL was comparable to the vehicle control.
- Water solubility: The test item was miscible in dimethyl sulphoxide (DMSO) at 200 μL/mL.
- Precipitation and time of the determination: The test item did not precipitate at any of the tested concentrations up to 2 μL/mL.

RANGE-FINDING/SCREENING STUDIES:
In the initial cytotoxicity, the average percent reduction of the Cytokinesis-Block Proliferation Index (CBPI) was 100 in all the concentration tested (complete cytotoxicity). As the test concentrations selected should cover a range from that producing 45±5% cytotoxicity to little or no cytotoxicity, an additional cytotoxicity test was performed at test concentrations of 0.0078, 0.0156, 0.0312, 0.0625 and 0.125 μL/mL.
In the follow-up cytotoxicity test, the average reduction of the CBPI was in the range of 5.32 to 41.07% at the concentrations 0.0078, 0.0156 and 0.0312 μL/mL, respectively. The average percent reduction of the CBPI was not greater than 45±5% at 0.0312 μL/mL. Hence 0.0312 μL/mL is selected as the highest concentration for the micronucleus test. Other concentrations tested were 0.0078 and 0.0156 μL/L.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The positive controls resulted in a statistically significant increase in the micronuclei frequency (p<0.05) under identical conditions, when compared with the vehicle control.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: Concentrations of 0.0078, 0.0156 and 0.0312 μL/mL were tested for the presence of micronuclei in the main study. The test item did not induce cytotoxicity, both in the presence and absence of metabolic activation, with the maximum reduction in CBPI being 39.34%.
- Genotoxicity results: In the micronucleus test, concentrations of 0.0078, 0.0156 and 0.0312 μL/mL were tested for the presence of micronuclei. The test item at concentrations of 0.0078, 0.0 156 and 0.0312 μL/mL resulted in a percent micronuclei of 0.19, 0.15 and 0.30, respectively (in the presence of metabolic activation), and 0.15, 0.10 and 0.20, respectively (in the absence of metabolic activation), for the short-term treatments. Post long-term treatment (in the absence of metabolic activation) 0.10, 0.05 and 0.10 percent micronuclei were observed at 0.0078, 0.0156 and 0.0312 μL/mL, respectively. The percent micronuclei at all the treatments were not significantly different when compared to the vehicle control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

In a bacterial reverse mutation assay, performed according to OECD Guideline 471 and following GLP, the test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and b-naphthoflavone). The test substance was dissolved in ethanol. In the dose range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. In tester strain TA100, toxicity was observed at dose levels of 333 µg/plate and above in the absence of S9-mix and at 1000 µg/plate and above in the presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. The test item precipitated on the plates at dose levels of 3330 and 5000 µg/plate. Results of this dose range finding test were reported as part of the first experiment of the mutation assay. Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 10 to 3330 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains. In an independent repeat of the assay with additional parameters, the test item was tested at a concentration range of 33 to 2000 µg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537 and TA100. The test item was tested at a concentration range of 33 to 3330 µg/plate in the absence and presence of 10% (v/v) S9 mix in tester strains TA98 and WP2uvrA. Toxicity was observed in all tester strains, except in tester strain WP2uvrA. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

In vitro gene mutation in mammalian cells

In a mammalian gene mutation in vitro test, performed according to OECD Guideline 476 and following GLP, the test substance was evaluated in a hprt gene mutation assay in CHO AA8 cells. On the basis of the results of pH and precipitation tests and initial cytotoxicity tests, 0.25 μL/mL was selected as the highest concentration for the gene mutation test. Positive controls, 3 μg/mL of Benzo(a)pyrene (with metabolic activation (+S9)) and 1 µg/mL of 4-Nitroquinoline N- oxide (without metabolic activation (-S9)) were used for the gene mutation test. Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test.

The test item resulted in mutant frequencies of 26.87 to 31.75 per 2E6 cells in the presence of metabolic activation at the different concentrations tested. The vehicle control resulted in a mutant frequency of 24.66 per 2E6 cells. In the absence of metabolic activation, the test item resulted in mutant frequencies of 26.47 to 32.08 per 2E6 cells. The vehicle control resulted in a mutant frequency of 25.76 per 2E6 cells. There was no statistically significant increase in the number of mutant colonies at any of the concentrations tested when compared with the vehicle control. Positive controls resulted in mutant frequencies of 274.60 (with metabolic activation) and 270.97 (without metabolic activation) per 2E6 cells, which was statistically significant higher when compared with the vehicle control.

In conclusion, the test item is considered as non-mutagenic at and up to the concentration of 0.25 μL/mL, both in the presence and absence of metabolic activation under the laboratory conditions tested.

In vitro micronucleus test

In this GLP compliant OECD 487 study, the test item was evaluated for the formation of micronuclei in the cytoplasm of interphase cells, using human lymphocytes. The test item was miscible in dimethyl sulphoxide (DMSO) at 200 μL/mL. The test item did not precipitate at any of the tested concentrations up to 2 μL/mL. The pH tested at concentrations up to 2 μL/mL was comparable to the vehicle control. Based on the solubility and precipitation test results, 2 µL/mL was chosen as the highest concentration for the initial cytotoxicity test. Cytokinesis was blocked using Cytochalasin B, the cells were harvested and slides were prepared. To assess the cytotoxicity of the test item, the Cytokinesis-Block Proliferation Index (CBPl) was calculated for cultures treated with the test item and vehicle control. In the micronucleus test, the test item was tested at concentrations of 0.0078, 0.0156 and 0.0312 μL/mL for short-term treatment in the presence and absence of metabolic activation and for long-term treatment in the absence of metabolic activation. These concentrations were based on two initial cytotoxicity tests.

The test item did not induce cytotoxicity of more than 45% up to 0.0312 μL/mL when compared to the vehicle control. No statistically significant increase in the percentage of micronuclei in binucleated cells was observed in any of the tested concentrations when compared to the vehicle control. The positive controls resulted in an increase of the micronuclei frequency with statistical significance (p<0.05) under identical conditions, when compared with the vehicle control. This demonstrated the sensitivity of the test system towards the positive control and confirmed that the test conditions were adequate.

In conclusion, based on the results obtained, it is concluded that the test item is non-clastogenic and/or non-aneugenic in cultured human lymphocytes at and up to 0.0312 µL/mL both after short-term and long-term treatment (both in the presence and absence of metabolic activation) as it showed no evidence of an increase in the induction of micronuclei under the test conditions.

Justification for classification or non-classification

Based on the available information classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.