Reaction mass of (4RS,4aRS,8RS,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aRS)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one and (4RS,4aSR,8SR,8aSR)-4-ethyl-8-methyloctahydronaphthalen-1(2H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2013

negative, in vitro chromosome aberration test with human lymphocytes (with and without S-9 activation), OECD TG 473, 2014

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-07-2013 to 29-07-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised November, 2006)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine or tryptophan locus

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Range finding test: 3, 10, 33, 100, 333, 1000, 3330, 5000 μg/plate
Experiment 1: 1, 3, 10, 33, 100, 200, 333 μg/plate
Experiment 2 * : 1, 3, 10, 33, 100, 333, 666, 1000, 3330, 5000 μg/plate
* certain dose levels depending on strain; all strains tested up to 333 μg/plate
Experiment 3 (TA98 strain only) : 100, 333, 1000, 3330, 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide was used in the assay. The test formulations were used within 4 hours for the assay.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

methylmethanesulfonate

other: ICR-191; 2-nitrofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: The plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted. The revertant colonies were counted automatically with a Colony Counter.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth (bacterial background lawn) and reduction in the number of revertants

Evaluation criteria:

See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.

Statistics:

No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at > 333 or 666 or 1000 μg/plate in all strains/cell types tested ; depending on strain/cell type, where necessary the test item was tested up to the 5000 μg/plate limit dose.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of test substance on the plates was observed at the start of the incubation period at 1000 µg/plate and above in the range finding test/first mutation experiment, at the start and end of the incubation period at 5000 µg/plate in the second and third mutation experiment.

RANGE-FINDING/SCREENING STUDIES: A range-finding test was conducted on TA100 and WP2uvrA strains as part of the current assay

COMPARISON WITH HISTORICAL CONTROL DATA: The negative control values and strain specific positive controls were within the laboratory historical control data ranges, with the following exceptions:

The mean plate counts of the positive control of TA1535 in the absence of S9-mix (first experiment), WP2uvrA in the presence of S9-mix (first experiment) and TA100 in the presence of S9-mix (second experiment) were not within the laboratory historical range. However, these values were more than 3 times greater than the concurrent solvent control values, therefore this deviation in the mean plate count of the positive controls had no effect on the results of the study. Additionally, in the second experiment, the mean plate count of the solvent control of TA1535 in the absence of S9-mix was not within the laboratory historical range. Historical control data from experiments was presented within the study report.

Conclusions:

Interpretation of results:
negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to OECD TG 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP to evaluate the mutagenic activity of the test item in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat).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. The test item did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 100 μg/plate and above in the absence of S9-mix and at 333 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 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 ß-naphthoflavone). The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants or a reduction of the bacterial background lawn, was observed in all tester strains, except in the tester strains TA98 (presence of S9-mix) and WP2uvrA. Since in tester strain TA98 in the presence of S9-mix (first experiment) no toxicity or precipitate on the plates was observed and the recommended dose level of 5000 μg/plate was not selected, an additional third mutation experiment was performed utilising this strain only. The test item precipitated on the plates at the top dose of 5000 μg/plate in the second and third experiment. 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 independently repeated experiments. The negative control values were within the laboratory historical control data ranges, except the response for TA1535 in the absence of S9-mix (second experiment). However, since this value was just without the limit of the range, the validity of the test was considered to be not affected. The 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, except the response for TA1535 in the absence of S9-mix (first experiment), WP2uvrA in the presence of S9-mix (first experiment) and TA100 in the presence of S9-mix (second experiment). The values were above the limit of the range. However since more than a three-fold increase was observed compared to the solvent control, the validity of the test was considered to be not affected. Under the conditions of this study, it is concluded that that the test item was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-08-2013 to 09-11-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

: Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 20 November 2006

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable (chromosome aberration test)

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines OECD, EC, METI). Whole blood samples obtained from healthy male subjects were treated with an anti-coagulant (heparin) and cultured in the presence of a mitogen (phytohaemagglutinin). These stimulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals. Blood was collected from healthy adult, non-smoking, male volunteers. The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined. Further details on the donors is available in the full study report. Therefore the exposure time for the experiments was ca. 1.5 to 1.8 x AGT at 24 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fraction: produced in-house and/or characterised by exposure to in TA98 with the mutagens Benzo-(a)-pyrene (Sigma) and 2-aminoanthracene (Sigma) at concentrations of 5 μg/plate and 1 μg/plate, respectively

Test concentrations with justification for top dose:

See 'any other information on materials and methods incl. tables'.
The highest tested concentration was determined by the solubility of test item in the culture medium at the 3 h exposure time. At the 24 and 48 h exposure time, test item was tested beyond the limit of solubility to obtain adequate toxicity data.

I. Preliminary toxicity test:
a. 3 hour exposure 0 (control), 10, 33, 100 and 333 μg/mL with and without S9-fraction.
b. 24 h and 48 h continuous exposure time blood cultures were treated with 10, 33, 100, 333, 1000 and 3330 μg/mL without S9-fraction.

II. Main Test:
Experiment 1
i) 3-hours exposure to the test item without S9-mix, followed by a 24-hour fixation period in treatment-free media, 3(24)-hour exposure.
3(24)-hour without S9: 0, 3, 10, 30, 50, 100 and 200 μg/mL
ii) 3-hours exposure to the test item with S9-mix (1.8%), followed by a 24-hour recovery period in treatment-free media, 3(24)-hour exposure.
3(24)-hour with S9: 0, 3, 10, 30, 50, 100 and 200 μg/mL

Cytogenetic assay 1A:
Due to no scorable dose levels the following further assay was conducted: 0, 1, 10, 100, 120, 140 and 160 μg/mL with and without S9-fraction.
i) 3-hours exposure to the test item without S9-mix : 1*, 10*, 100, 120*, 140 and 160 μg/mL
ii) 3-hours exposure to the test item with S9-mix (1.8%): 1*, 10, 100*, 120, 140* and 160 μg/mL μg/mL

Experiment 2
(i) 24(24)-hour without S9: 3, 10, 30, 50*, 75* and 100* μg/mL
(ii) 48(48)-hour without S9: 3*, 10, 30*, 50, 75* and 100 μg/mL
(iii) 3(48)-hour with S9: 3, 10, 30, 50*, 100*, 150* and 200 μg/mL

where:
* = dose levels selected for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was soluble up to 33 mg/mL but formed a suspension at concentrations of 100 mg/mL DMSO vehicle pre-test solubility checks.

Untreated negative controls:

other:

Remarks:

Vehicle control served as the negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation, 0.5 and 0.75 μg/ml for a 3 h, 0.2 and 0.3 μg/ml for a 24 h exposure and 0.1 and 0.15 μg/ml for the 48 h exposure periods, respectively

Untreated negative controls:

other:

Remarks:

Vehicle control served as the negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation 10 μg/ml for a 3 h exposure period

Details on test system and experimental conditions:

METHOD OF APPLICATION: Other:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components: Lymphocytes (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin). Cultured for 48 hours and thereafter exposed to respective doses of test item for the defined exposure durations with fixation consisting of centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for the defined fixation period.

DURATION
- Preincubation period: Not reported.
- Exposure duration: See ‘any other information on materials and methods incl. tables’.

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 μg/mL)

NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.

NUMBER OF CELLS EVALUATED: A total of 1000 lymphocyte cell nuclei per culture were counted and the number of cells in metaphase per 1000 cells recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes. Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) including endoreduplicated cells, reported.
- Other: Scoring: Where possible, the first 100 consecutive well-spread metaphases from each concentration (200 per duplicate) were assessed for observations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:

Positive response criteria
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Negative response criteria
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistical analysis is also performed (see: ‘Statistics’). The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

Statistics:

Statistical analysis was performed. The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

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
- Effects of pH: Not reported.
- Effects of osmolality: Not reported.
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation: In the preliminary test: A precipitate of the test item was observed in the greater than or equal to 333 μg/mL test cultures. In the first cytogenetic assay: A precipitate of the test item was observed in the greater than or equal to 200 μg/mL test cultures. In the followup it was determined that precipitate occurred at greater than or equal to 160 μg/mL in test culture.

- Other confounding effects: None reported.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 3330 μg/mL. The maximum dose was limited based on solubility and cytotoxicity in culture media. The selection of the maximum dose level was based on toxicity for the main test.

COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY: For each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Conclusions:

Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The study was performed to the requirements of OECD TG 473 and EU Method B.10 under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of peripheral human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, multiple exposure conditions were investigated; a 3-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 1.8% final concentration with cell harvest after a 24-hour fixation period, 3-hour exposure in the absence of metabolic activation (S9) with a 24-hour fixation period. A 24-hour exposure in the absence of metabolic activation with a 24-hour fixation period and a 48-h exposure 48-hour fixation period in the absence of metabolic activation (S9). In the first cytogenetic assay, the test item was tested up to 120 and 140 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, the test item was tested up to 100 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 75 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of S9-mix, the test item was tested up to 150 μg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels. The highest concentration analysed was selected based on toxicity, inhibition of the mitotic index of about 50% or greater. The vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of from the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key study : OECD TG 471, 2013 : The study was performed to OECD TG 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP to evaluate the mutagenic activity of the test item in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat).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. The test item did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 100 μg/plate and above in the absence of S9-mix and at 333 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 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 ß-naphthoflavone). The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants or a reduction of the bacterial background lawn, was observed in all tester strains, except in the tester strains TA98 (presence of S9-mix) and WP2uvrA. Since in tester strain TA98 in the presence of S9-mix (first experiment) no toxicity or precipitate on the plates was observed and the recommended dose level of 5000 μg/plate was not selected, an additional third mutation experiment was performed utilising this strain only. The test item precipitated on the plates at the top dose of 5000 μg/plate in the second and third experiment. 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 independently repeated experiments. The negative control values were within the laboratory historical control data ranges, except the response for TA1535 in the absence of S9-mix (second experiment). However, since this value was just without the limit of the range, the validity of the test was considered to be not affected. The 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, except the response for TA1535 in the absence of S9-mix (first experiment), WP2uvrA in the presence of S9-mix (first experiment) and TA100 in the presence of S9-mix (second experiment). The values were above the limit of the range. However since more than a three-fold increase was observed compared to the solvent control, the validity of the test was considered to be not affected. Under the conditions of this study, it is concluded that that the test item was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Key study : OECD TG 473, 2014 : The study was performed to the requirements of OECD TG 473 and EU Method B.10 under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of peripheral human lymphocyte cultured mammalian cells. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, multiple exposure conditions were investigated; a 3-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 1.8% final concentration with cell harvest after a 24-hour fixation period, 3-hour exposure in the absence of metabolic activation (S9) with a 24-hour fixation period. A 24-hour exposure in the absence of metabolic activation with a 24-hour fixation period and a 48-h exposure 48-hour fixation period in the absence of metabolic activation (S9). In the first cytogenetic assay, the test item was tested up to 120 and 140 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, the test item was tested up to 100 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 75 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. In the presence of S9-mix, the test item was tested up to 150 μg/ml for a 3 h exposure time with a 48 h fixation time. Appropriate toxicity was reached at these dose levels. The highest concentration analysed was selected based on toxicity, inhibition of the mitotic index of about 50% or greater. The vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of from the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described. Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity