Bicyclo[2.2.1]heptanebis(methylamine)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test

Negative with and without metabolic activation (S. typhimurium TA100. TA1535, TA98, TA1537 and E Coli WP2 uvrA), Japanese Guidelilnes on Industrial Chemicals (Kanpogyo No.700, Yakuhatsu No.1039,61 Kikyoku No.1014, 1986), Nishitomi, T. (1999).

Chromosome Aberration

Negative with and without metabolic activation, Japanese Guidelines on Industrial Chemicals (Kanpogyo No.700, Yakuhatsu No.1039,61 Kikyoku No.1014, 1986), Nishitomi, T. (1991).

Gene Mutation in Mammalian Cells

Negative with and without metabolic activation (mouse lymphoma L5177Y cells), OECD 476, EU Method B.17, EPA OPPTS 870.5300, Gilby, B. (2016).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 November 2015 - 05 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD and EC guidelines and in compliance with GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604, 1 November 1999.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Purchased from a reputable supplier
- Cells are heterozygous at the thymidine kinase locus, TK +/-.
- Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO. Cultures were used within ten days of recovery from frozen stock
- Cell stocks are periodically checked for freedom from mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was stored at ca -80°C.

Test concentrations with justification for top dose:

Preliminary toxicity test: 3.01, 6.03, 12.05, 24.1, 48.2, 96.41, 192.81 µg/mL

Mutation test: 50.54, 63.18, 78.97, 98.72, 123.4, 154.25, 192.81 µg/mL - With and without S9 mix (3 hours)
50.54, 63.18, 78.97, 98.72, 123.4, 154.25, 192.81 µg/mL - Without S9 mix (24 hours)

Vehicle / solvent:

Purified water (purified by reverse-osmosis)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

Preliminary toxicity test procedure
Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix.
For 3-hour exposures, cultures contained a total of 6 x 106 cells. The final volume of the cultures was 5 mL and the final concentration of the S9 fraction was 2% v/v, if present.
For 24-hour exposures, cultures contained a total of 1.5 x 106 cells in a total volume of 5 mL. One culture was prepared for each concentration of the test substance for each test condition.
Vehicle controls were tested in duplicate for each test condition.

The test substance was formulated and serially diluted in the solvent. Aliquots of 50 µL of test substance dilution (at 100 times the desired final concentration) or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 37°C) or 24 hours (static incubator, at 37°C, 5% (v/v) CO2). At the end of the 3-hour exposure period, the cells were washed once, resuspended in R10p to nominally 2 x 10E5 cells/mL (assuming no cell loss), incubated (at 37°C, 5% (v/v) CO2) and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary. At the end of the 24-hour exposure period, the cells were
washed once, resuspended in 5 mL R10p and counted, to ascertain treatment growth. The cultures were then diluted to 2 x 10E5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary.
The RSG was used to determine the concentrations of test substance used in the main test; ideally the maximum concentration should reduce RTG to approximately 10 to 20% of the concurrent vehicle control value. There was some evidence of toxicity in the preliminary toxicity test, so the maximumconcentration tested in the main mutation test in the 3-hour exposure in the absence and presence of S9 mix and in the 24-hour exposure in the absence of S9 mix was 192.81 g/mL. The formulations were added at 1% final volume in medium.

Mutation test procedure
3-hour treatment in the absence and presence of S9 mix
The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 10E7 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls. Aliquots of 100 µL of test substance dilution
(at 100 times the desired final concentration), vehicle or positive control were added, then all cultures were incubated, with continuous shaking, for 3 hours at 37°C. At least four serial dilutions of the test substance were tested.
Following the 3-hour treatment, the cells were washed once, resuspended in R10p to nominally 2 x 10E5 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 105 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 103 cells/well in
selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37C in an atmosphere of 5% CO2 in air.
After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates, the number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF). The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The maximum concentration assessed for mutant frequency in the main test was 192.81 g/mL in the absence and presence of S9 mix respectively.

24-hour treatment in the absence of S9 mix
A test with a 24-hour treatment in the absence of S9 mix was carried out. Duplicate 10 mL cultures containing 3 x 10E6 cells were treated for 24 hours with 100 µL of test substance or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, resuspended in 10 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to 2 x 105 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10E5 cells/mL with R10p where necessary. Following this, the procedure was the same as in the 3-hour treatment. The maximum concentration assessed for mutant frequency in the main test was 192.81 g/mL.

Evaluation criteria:

GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6
The assay was considered valid in accordance with the assay acceptance criteria.

The test agent was regarded as negative if:
The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.

If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Statistics:

Statistical analysis performed using: Fluctuation application SAFEStat (SAS statistical applications for end users)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies, spontaneous and positive control MF, and percent small colonies in positive control cultures.
The criteria used to assess whether an assay was valid are based on the recommendations of the Plymouth and Aberdeen papers (Robinson et al., 1989; Moore et al., 2000; Moore et al., 2002; Moore et al., 2003; Moore et al., 2006 and Moore et al., 2007).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

It was concluded that NBDA did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

The test material was assessed for mutagenic potential in an in vitro mammalian cell mutation assay. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300.

This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

The test material was soluble at 154.25 mg/mL in water. A final concentration of 192.81 µg/mL, dosed at 1%v/v, was the highest concentration assessed in the preliminary toxicity test as higher concentrations altered the pH of the media by more than 1.0 unit.

Toxicity was observed in the preliminary toxicity test. Following a 3-hour exposure to the test material at concentrations from 3.01 to 192.81 µg/mL, relative suspension growth (RSG) was reduced from 134 to 6% and from 101 to 57% in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 93 to 68%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess exposure up to the highest concentration at which the pH of the media was altered by less than 1.0 unit.

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3-hour treatment in both the absence and presence of S9 mix was 192.81 µg/mL, there were no significant reductions in RTG at any concentration tested.

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 192.81 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. There were no significant reductions in RTG.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 January 1991 - 26 June 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD and EC guidelines and in compliance with GLP

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelilnes on Industrial Chemicals (Kanpogyo No.700, Yakuhatsu No.1039,61 Kikyoku No.1014, 1986)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

S9 Mix

Test concentrations with justification for top dose:

Test conditions:
Bacterial suspension: 0.1 mL
Test substance solution: 0.1 mL
Na-phosphate buffer: 0.5 mL
S9 Mix: 0.5 mL
Top agar solution: 2 mL

Pre-incubation:
Temperature: 37 °C
Time: 20 min

Incubation:
Temperature: 37 °C
Time: 48 hours

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-(2-Furyl-3-(5-nitro-2-furyl)acrylamide, 2-Aminoanthracene

Details on test system and experimental conditions:

Strains:
TA100. TA1535, TA98 & TA1537
WP2 uvrA
Purchased from reputable suppliers.

Test substance preparation: DMSO
Period of pre-culture: Approximately 10 hours
Minimum Glucose Agar plate Medium: Purchased from a reputable supplier
Storage: -80 °C

S9 Mix
Purchased from a reputable supplier

Composition of S9 Mix (Amount in 1mL S9 Mix):
S9: 0.1 mL
MgCl2 · 6H2O: 8 µmoles
KCl: 33 µmoles
ß-NADPH: 4 µmoles
ß-NADH: 4 µmoles
Na-phosphate buffer: 100 µmoles

Species, strain: Sprague-Dawley Rat
Sex: Male
Age: 7 weeks old
Weight: 195-229g
Inducing substances: Phenobarbital, 5,6-benzoflavone
Administration method: Intra-peritoneal
Administration period and amount (g/kg weight): Phenobarbital; 4 days (0.03 - 0.06), 5,6-benzoflavone; 1 day (0.08)

Test substance concentrations

Dose-finding - With and without S9 Mix;
156, 313, 625, 1250, 2500, 5000

Main test - Without S9 Mix:
39, 78, 156, 313, 625, 1250, 5000

Main test - With S9 Mix:
78, 156, 313, 625, 1250, 2500, 5000

Statistics:

No statistical methods were used.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Main test was performed twice because microbial growth inhibition was observed at low concentration in dose-finding test.
Microbial growth inhibition was observed at a few concentrations including maximum concentration with and without 59 mix in all strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The test substance did not induce a significant increase of revertant colonies in any tester strains either with or without 59 mix.
From these results, the test substance was not mutagenic under the test conditions employed in this study.

Executive summary:

The mutagenic potential of the test material was investigated in the bacterial reverse mutation assay in accordance with the Japanese Guidelines on Industrial Chemicals (Kanpogyo No.700, Yakuhatsu No.1039,61 Kikyoku No.1014, 1986) and under GLP conditions.

Four strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537 and TA 98) were exposed to the test material in DMSO at concentrations up to 5000 µg/plate in both the presence and absence of metabolic activation (S-9 mix). A pre-incubation assay was carried out and following incubation the plates were assessed for cytotoxicity and an increase in the number of his+ revertants.

In the pre-incubation test both with and without S-9 mix there was no increase in the number of his+ revertants in any tester strain.

No bacteriotoxic effect (reduced his- background growth) was observed.

Under the conditions of this study, the test material is not mutagenic in the Ames test with and without metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 November 1990 - 26 June 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD and EC guidelines and in compliance with GLP

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemicals (Kanpogyo No.700, Yakuhatsu No.1039,61 Kikyoku No.1014, 1986)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix using male rat, Sprague- Dawley and Phenobarbital and 5,6-benzoflavone inducing substances.

Test concentrations with justification for top dose:

Without metabolic activation, 24 & 48 hours: 0, 50, 100, 200, 300, 400, 500 µg/mL
With metabolic activation: 0, 200, 400, 600, 700, 800, 900, 1000 µg/mL

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

Cell number;
Without metabolic activation = 4x10E3 (24 hrs), 2x10E3 (48 hrs)
With metabolic activation = 2x10E3

Plate;
With & without metabolic activation = 2 plates, 6cm diameter plastic dish
Solvent: DMSO
Amount of medium: 5mL/plate (Without metabolic activation), 3mL/plate (With metabolic activation)

Preparation of test substance solutions:
Concentration of the highest concentration solution: 82 mg/mL (24 & 48 hrs, without metabolic activation), 148 mg/mL (With metabolic activation)
Amount of test substance: 410 mg (24 & 48 hrs, without metabolic activation), 740 mg (with metabolic activation)
amount of solvent: 5 mL
Appearance: Solution
Storage period: Within 1 hour
Storage conditions: At room temperature
Sterilisation: Not sterilised

Treatment
Amount of test substance: 0.025 mL/plate (without metabolic activation), 0.015 mL/plate (with metabolic activation)
Treatment period: 24 & 48 hours (without metabolic activation), 6 hours (with metabolic activation)
Amount of S9 Mix: 0.5 mL/plate (With metabolic activation)

Mitotic inhibitor
Mitotic inhibitor: Colcemid
Treatment concentration: 0.1 µg/mL - final concentration
Treatment period: 2 hours

Statistics:

Statistical analysiswas performed by chi-square test to detect differences in the incidences of aberrant cells between the cells treated with the negative control and the test substance. For the structural aberration, data without gaps were used in the statistical analysis.

Species / strain:

other: Chinese Hamster

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the cell growth inhibition test, 50% cell growth inhibition concentrations (TCID50) were ca. 400 µg/mL in the treatment for 24 hours without metabolic activation, ca. 405 µg.mLin the treatment for 48 hours, and ca. 730 µg/mL in the treatment with metabolic activation with S9 Mix.
Based on this result, the highest concentration of the test substance in the chromosomal aberration test were set at 410 µg/mL in the treatment without metabolic activation and at 740 µg/mL in the treatment with metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

In the chromosomal aberration test, no significant differences compared to the negative control group wre detected on chromosomal aberrations in any test substance treatment group. Based on this result, the test substance was judged to be negative for the ability to induce chromosomal aberration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro

Ames Test

The mutagenic potential of the test material was investigated in the bacterial reverse mutation assay in accordance with the Japanese Guidelines on Industrial Chemicals (Kanpogyo No.700, Yakuhatsu No.1039,61 Kikyoku No.1014, 1986) and under GLP conditions.

Four strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537 and TA 98) were exposed to the test material in DMSO at concentrations up to 5000 µg/plate in both the presence and absence of metabolic activation (S-9 mix). A pre-incubation assay was carried out and following incubation the plates were assessed for cytotoxicity and an increase in the number of his+ revertants.

In the pre-incubation test both with and without S-9 mix there was no increase in the number of his+ revertants in any tester strain.

No bacteriotoxic effect (reduced his- background growth) was observed.

Under the conditions of this study, the test material is not mutagenic in the Ames test with and without metabolic activation.

Chromosome Aberration

In the chromosomal aberration test, no significant differences compared to the negative control group were detected on chromosomal aberrations in any test substance treatment group. Based on this result, the test substance was judged to be negative for the ability to induce chromosomal aberration.

Gene Mutation in Mammalian Cells

The test material was assessed for mutagenic potential in an in vitro mammalian cell mutation assay. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17 and EPA OPPTS 870.5300.

This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

The test material was soluble at 154.25 mg/mL in water. A final concentration of 192.81 µg/mL, dosed at 1 % v/v, was the highest concentration assessed in the preliminary toxicity test as higher concentrations altered the pH of the media by more than 1.0 unit.

Toxicity was observed in the preliminary toxicity test. Following a 3-hour exposure to the test material at concentrations from 3.01 to 192.81 µg/mL, relative suspension growth (RSG) was reduced from 134 to 6 % and from 101 to 57 % in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 93 to 68 %. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess exposure up to the highest concentration at which the pH of the media was altered by less than 1.0 unit.

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3-hour treatment in both the absence and presence of S9 mix was 192.81 µg/mL, there were no significant reductions in RTG at any concentration tested.

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 192.81 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. There were no significant reductions in RTG.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

It was concluded that the test material did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.