Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (OECD 471; GLP compliant)

In vitro mammalian cell micronucleus test: negative (OECD 487; GLP compliant)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012-10-23 to 2012-12-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-03-30
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
not applicable
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital and by peroral administrations of β-naphthoflavone each, on three consecutive days.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital and by peroral administrations of β-naphthoflavone each, on three consecutive days.
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (all strains; with and without metabolic activation)
Experiment Ia and II: 0.1; 0.3; 1; 3; 10; 33; 100; and 333 µg/plate (Salmonella strains; without metabolic activation)
Experiment II: 0.3; 1; 3; 10; 33; 100; 333; and 1000 µg/plate (Salmonella strains; with metabolic activation)
Experiment II: 3; 10; 33; 100; 333; 1000; 2500 and 5000 µg/plate (E. coli strain; with and without metabolic activation)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO (first experiment:MERCK, 64293 Darmstadt/Germany; purity > 99 %; all further experiments: Acros, New Jersey, USA, purity > 99 %).
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria (Maron et al., 1981)*.

*Reference:
- Maron D.M., J. Katzenellenbogen, and B.N. Ames (1981) Compatibility of organic solvents with the Salmonella/Microsome Test Mutation Res. 88, 343-350
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control without metabolic activation: dissolved in water; concentration: 10 µg/plate; strains: TA1535 and TA100
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Positive control without metabolic activation: dissolved in DMSO; concentration: 10 µg/plate (strain TA98) and 50 µg/plate (strain TA1537); strains: TA98 and TA1537
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Positive control without metabolic activation: dissolved in deionised water; concentration: 3 µL/plate; strain: WP2 uvrA
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control with metabolic activation: dissolved in DMSO; concentration: 2.5 µg/plate (strains TA1535, TA1537, TA98, TA100) and 10 µg/plate (strain WP2 uvrA); strains: TA1535, TA1537, TA98, TA100 and WP2 uvrA
Details on test system and conditions:
METHOD OF APPLICATION: plate incorporation (experiment I and Ia) and preincubation (experiment II)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours at 37°C

NUMBER OF REPLICATIONS: for each strain and dose level including the controls, three plates were used.

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager. Due to precipitation of the test item and reduced background growth the revertant colonies were partly counted manually.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (Hollstein et al., 1979)*.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (de Serres & Shelby, 1979)*.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. Whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

*References:
- de Serres F.J. and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay. Mutation Res. 64, 159-165
- Hollstein,M., J. McCann, F.A. Angelosanto, and W.W. Nichols (1979) Short-term tests for carcinogens and mutagens. Mutation Res. 65, 133-226
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate. The undissolved particles had no influence on the data recording.

CYTOTOXICITY:
- plates incubated with the test item showed reduced background growth with and without metabolic activation in all experiments (please refer to the table 1 in the field "Any other information on results incl. tables" below).
- toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation (please refer to the table 2 in the field "Any other information on results incl. tables" below).

MAIN EXPERIMENTS
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Table 1: Plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate)

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

33 - 5000

33 – 333 (HV1a)

333 - 5000

33 - 333

333 - 1000

TA 1537

33 - 5000

33 - 333 (HV1a)

333 - 5000

33 - 333

333 - 1000

TA 98

100 - 5000

333 - 5000

33 - 333

333 - 1000

TA 100

33 - 5000

333 - 5000

100 - 333

333 - 1000

WP2 uvrA

5000

1000 - 5000

2500 - 5000

5000

HV1a: Results for experiment Ia. Due to strong toxic effects in strains TA1535 and TA1537 in experiment I without S9 mix, this part was repeated with lower concentrations.

Table 2:

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

100 – 5000

100 – 333 (HV1a)

333 - 5000

33 - 333

333 - 1000

TA 1537

33 – 5000

100 – 333 (HV1a)

333 - 5000

10 - 333

333 - 1000

TA 98

100 - 5000

333 - 5000

33 - 333

333 - 1000

TA 100

100 - 5000

333 - 5000

100 - 333

333 - 1000

WP2 uvrA

2500 - 5000

1000 - 5000

2500 - 5000

2500 - 5000

HV1a: Results for experiment Ia. Due to strong toxic effects in strains TA1535 and TA1537 in experiment I without S9 mix, this part was repeated with lower concentrations.

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
According to Directive 67/548 /EEC and its subsequent amendments and according to Regulation (EC) No 1272/2008 and subsequent regulations, the test substance should not be considered to have a mutagenic potential, and hence no classification or labelling is required.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-08-31 to 2016-11-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2014-09-26
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-09-14
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Target gene:
not applicable
Species / strain:
lymphocytes: human (peripheral)
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: healthy human donors (non-smoking; not receiving medication)

- Sex, age and number of blood donors: one male donor (24 years old; Experiment 1); one female donor (28 years old; Experiment 2)

- Suitability of cells: lymphocytes of the donors have been shown to respond well to stimultation of proliferation with phytohemeagglutinine (PHA) and to positive control substance. All donors had previously established low incidence of micronuclei in their peripheral blood lymphocytes.

- Whether whole blood or separated lymphocytes were used: blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hours after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (mixture 1:1) supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin, the mitogen PHA, 10 % fetal bovine serum, 10 mM HEPES and the anticoagulant heparin.
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
Cytokinesis block (if used):
4 µg/mL cytochalasin B (exposure duration: 20 hours)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix: S9 supernatant, MgCl2 (8 mM), KCL (33 mM), glucose-6-phosphate (5 mM), and NADP (4 mM) in sodium-ortho-phosphate buffer (100 mM, pH 7.4)
Test concentrations with justification for top dose:
Preliminary cytotoxicity test/Experiment 1: 2.4, 4.1, 7.3, 12.7, 22.2, 38.9, 68.0, 119, 208, 625, and 1875 µg/mL (with and without metabolic activation; 4 hours exposure)
Experiment 2: 1.3, 2.3, 4.0, 7.0, 12.2, 21.3, 37.3, 65.3, 114, and 200 µg/mL (without metabolic activation; 20 hour exposure)
Please refer to the field "Rationale for test conditions" below.
Vehicle:
- Vehicle(s)/solvent(s) used: acetone (final concentration in culture medium: 0.5 %)
- Justification for choice of solvent/vehicle: the solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Negative controls:
no
Solvent controls:
yes
Remarks:
culture medium with 0.5 % acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcin
Details on test system and conditions:
DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the reduced CBPI in comparison with the controls by counting 500 cells per culture.

EXPERIMENTAL DESIGN
1) Pre-experiment/Experiment 1 (pulse treatment):
- the preliminary cytotoxicity test was designated Experiment 1, since the cultures fulfilled the requirements for cytogenetic evaluation.
- 11 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control were used.
- all cell cultures were set up in duplicate.
- exposure time was 4 hours (with and without S9 mix) and the preparation interval was 40 hours after start of the exposure.

- about 48 hours after seeding 2 blood cultures (10 mL each) were set up for each test item concentration.
- culture medium was replaced with serum-free medium containing the test item.
- for the treatment with metabolic activation 50 μL S9 mix/mL culture medium was added.
- after 4 hours the cells were spun down by centrifugation for 5 minutes and the supernatant was discarded.
- cells were resuspended in and washed with "saline G" (pH 7.2, containing NaCl, KCl, glucose • H2O, Na2HPO4 • 2 H2O and KH2PO4).
- washing procedure was repeated once as described.
- cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period.
- Cytochalasin B (4 μg/mL) was added and the cells were cultured another approx. 20 hours until preparation.

- Experiment 2 (continuous treatment):
- about 48 hours after seeding 2 blood cultures (10 mL each) were set up for each test item concentration.
- culture medium was replaced with complete medium (with 10 % FBS) containing the test item.
- after 20 hours the cells were spun down by centrifugation and the supernatant was discarded.
- cells were re-suspended in and washed with "saline G".
- washing procedure was repeated once.
- cells were re-suspended in complete culture medium containing 10 % FBS (v/v).
- Cytochalasin B (4 μg/mL) was added and the cells were cultured another approx. 20 hours until preparation.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cultures were harvested by centrifugation 40 hours after beginning of treatment.
- cells were spun down by centrifugation for 5 minutes and the supernatant was discarded.
- cells were re-suspended in approx. 5 mL saline G and spun down by centrifugation.
- cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes.
- 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts:1 part) was added to the hypotonic solution and cells were resuspended.
- after removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold.
- slides were prepared by dropping the cell suspension in fresh fixative onto a microscope slide.
- cells were stained with Giemsa.
- evaluation of the slides was performed using microscopes with 40 x objectives.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976)*. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

NUMBER OF CELLS EVALUATED:
- at least 1000 binucleate cells/culture were scored for cytogenetic damage (% micronucleated cells)
- 500 cells/cuture were scored for CBPI (% cytostasis)

*Reference:
- Countryman P.I. and Heddle J.A. (1976) The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.
Rationale for test conditions:
The highest treatment concentration in this study (experiment 1), 1875 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight and the purity (96.07%) of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

In Experiment 1, phase separation of the test item was observed at the end of treatment at 68.0 μg/mL and above in the absence and presence of S9 mix. Furthermore, toxic effects were observed after 4 hours treatment in a range from 38.9 to 208 μg/mL in the absence of S9 mix and at 1875 μg/mL in the presence of S9 mix. Considering the toxicity and phase separation data of Experiment 1, 200 μg/mL (without S9 mix) were chosen as top concentration in Experiment 2.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be negative if, in all of the experimental conditions examined:
− none of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− there is no concentration-related increase
− the results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval)

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be positive if, in any of the experimental conditions examined:
− at least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− the increase is concentration-related in at least one experimental condition
− the results are outside the range of the laboratory historical solvent control data
Statistics:
Chi square test (α < 0.05).
Key result
Species / strain:
lymphocytes: human (peripheral)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
NOTE: the following concentrations were evaluated for micronucleated cells:
Experiment 1 :12.7, 22.2, and 38.9 µg/mL (without metabolic activation; 4 hour exposure)
Experiment 1 :22.2, 38.9, and 68.0 µg/mL (with metabolic activation; 4 hour exposure)
Experiment 2: 7.0, 12.2, and 21.3 µg/mL (without metabolic activation; 20 hour exposure)

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant influence on pH was observed
- Effects of osmolarity: no relevant influence on osmolarity was observed
- Phase separation:
Experiment 1: phase separation of the test item in the culture medium was observed at 68.0 μg/mL and above in the absence and presence of S9 mix.
Experiment 2: phase separation occurred in the absence of S9 mix at 114 μg/mL and above at the end of treatment.

INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
Experiment 1: in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.
Experiment 2: in the absence of S9 mix, concentrations showing cytotoxic effects were not evaluable for cytogenetic damage.

CYTOGENETIC:
In this study in the absence and presence of S9 mix, no relevant increases in the number of micronucleate cells were observed after treatment with the test item.

Please also refer for further information on results to the field "Attached background material" below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to the field "Any other information on results incl. tables" below



Historical laboratory control data

Percentage of micronucleated cells in human lymphocyte cultures (2014-2015)

Solvent Control without S9

Micronucleated cells in %

 

Pulse treatment

(4/40)

Continous treatment

(20/40)

No. of experiments

50*

54**

Mean

0.61

0.55

95% Ctrl limit

0.07 - 1.15

0.05 - 1.05

 

 

 

1x SD

0.27

0.25

2x SD

0.54

0.50

Min

0.15

0.05

Max

1.25

1.43

 

*Aqueous solvents – 23 Experiments; Organic solvents – 27 Experiments

**Aqueous solvents – 24 Experiments; Organic solvents – 30 Experiments

 

Solvent Control with S9

Micronucleated cells in %

 

Pulse treatment (04/40)

No. of experiments

67*

Mean

0.64

95% Ctrl limit

0.08 - 1.20

 

 

1x SD

0.28

2x SD

0.56

Min

0.15

Max

1.35

 *Aqueous solvents – 24 Experiments; Organic solvents – 43 Experiments

 

Aqueous solvents: DMEM/Han’s F12, Deionised water (10% v/v)

Organic solvents: DMSO (0.5 or 1.0%), Acetone, Ethanol and THF (0.5%)

Positive Control without S9

Micronucleated cells in %

 

Pulse treatment

(4/40)

Continous treatment

(20/40)

 

MMC

Demecolcin

No. of experiments

50

54

Mean

11.66

3.55

95% Ctrl limit

1.48 - 21.85

1.69 - 5.41

 

 

 

1x SD

5.09

0.93

2x SD

10.18

1.86

Min

4.15

2.10

Max

24.00

6.40

 

Positive Control with S9

Micronucleated cells in %

 

Pulse treatment (4/40)

 

CPA

No. of experiments

81

Mean

4.80

95% Ctrl limit

0.88 - 8.73

 

 

1x SD

1.96

2x SD

3.92

Min

2.25

Max

11.30

Conclusions:
The substance tested non-clastogenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.
Executive summary:

An in vitro mammalian cell micronucleus test was performed with the test item dissolved in acetone using peripheral human lymphocytes. The test procedure was according to the method described in OECD guideline 487 (2014).

The assay was performed in two independent experiments with the following test concentrations and exposure durations:

Preliminary cytotoxicity test/Experiment 1: 2.4, 4.1, 7.3, 12.7, 22.2, 38.9, 68.0, 119, 208, 625, and 1875 µg/mL (with and without metabolic activation; 4 hours exposure)

Experiment 2: 1.3, 2.3, 4.0, 7.0, 12.2, 21.3, 37.3, 65.3, 114, and 200 µg/mL (without metabolic activation; 20 hour exposure)

Positive and solvent controls were run concurrently.

The cells were prepared 40 hours after start of treatment with the test item. During the post-exposure period cytochalasin B had been added to the cell cultures to ensure that there were binucleate cells to be evaluated for micronuclei.

In each experimental group two parallel cultures were analysed. At least 1000 binucleate cells per culture were scored for cytogenetic damage (% micronucleated cells). To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.

Phase separation of the test item in the culture medium was observed at 68.0 μg/mL and above in Experiment 1 in the absence and presence of S9 mix. In addition, phase separation occurred in Experiment 2 in the absence of S9 mix at 114 μg/mL and above at the end of treatment. No relevant influence on osmolarity or pH was observed.

The following concentrations were evaluated for cytogenetic damage:

Experiment 1 :12.7, 22.2, and 38.9 µg/mL (without metabolic activation; 4 hour exposure)

Experiment 1 :22.2, 38.9, and 68.0 µg/mL (with metabolic activation; 4 hour exposure)

Experiment 2: 7.0, 12.2, and 21.3 µg/mL (without metabolic activation; 20 hour exposure)

In Experiment 1 in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment 2 in the absence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage.

In this study in the absence and presence of S9 mix, no relevant increases in the number of micronucleate cells were observed after treatment with the test item.

The positive and solvent controls were considered to be valid.

The substance tested non-clastogenic under the conditions of the study.

According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

The substance was not observed to be mutagenic in a reliable bacterial reverse mutation assay (OECD 471) or in an in vitro mammalian cell micronucleus test (OECD 487).

Justification for classification or non-classification

Genetic toxicity in vitro

The test substance should be considered void of genotoxicity based on a bacterial reverse mutation assay (OECD 471) and an in vitro mammalian cell micronucleus test (OECD 487). Thus, the test substance has no mutagenic potenital and does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.