Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Mutation Assay

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TAl53 5, TA1537, TA98 and TAl00 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co—factors). The dose range for the range-finding test was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterialstrains and fresh test item formulations. The dose range was amended, following the results of the range-findi ng test, and was 50 to 5000 µg/plate.

Seven test item dose levels were selected in the main test in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the results from the first mutation test.

Results

The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first mutation test was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test there was no visible reduction in the growth of the bacterial background lawns noted at any dose level, either in the presence or absence of S9-mix. Consequently the same maximum dose level was used in the second mutation test.

Results from the second mutation test showed the test item inducing no toxicity. A test item precipitate (globular in appearance) was observed under an inverted microscope at 1500 µg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the second mutation test.

Conclusion

The test substance was considered to be non-mutagenic under the conditions of this test.

Mammalian Cell Chromosome Aberration Test

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations.

Method

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4-hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test.

Results

All vehicle (acetone) 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. The metabolic activation system was therefore shown to be functional and the test method operating as expected. The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that was the maximum practicable dose level.

Conclusion

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

Justification for selection of genetic toxicity endpoint

No mutagenic or genotoxic positive results.

Short description of key information:

- Ames Test 2014 (OECD 471, GLP, K, rel. 1): non mutagenic up to 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & WP2uvrA-

- Human lymphocyte chromosome aberration test 2014 (OECD 473, GLP, K, rel. 1): non clastogenic up to solubility limit

Endpoint Conclusion:No adverse effect observed (negative)

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:
key study
Study period:
02 July 2014 to 11 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline. There were no deviations (unplanned changes) from the study plan.
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
OPPTS harmonised guidelines
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Not required
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Non-mammalian study
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Non-mammalian study
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1: Range-finding test: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 2: Main test: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: The substance was not misicible in water, DMSO or Acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent - Tetrahydrofuran
True negative controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2, 3, 5 µg/plate respectively for WP2uvrA, TA100, TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent - Tetrahydrofuran
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent - Tetrahydrofuran
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent - Tetrahydrofuran
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
1, 2, 10 µg/plate for TA100, TA1535&TA1537, WP2uvrA respectively
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent - Tetrahydrofuran
True negative controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) at multiple dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors).

RANGE FINDING
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.025 mL of the appropriate concentration of test item or 0.1 mL of solvent or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously (see 3.5.1.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 °C +/- 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

MAIN TEST
Dose selection
The dose range used for the main test was determined by the results of the range-finding test and was 50 to 5000 µg/plate.
Seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the results from the first mutation test.
Without Metabolic Activation
The procedure was the same as described previously
With Metabolic Activation
The procedure was the same as described previously
Incubation and Scoring
All of the plates were incubated at 37 °C +/- 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

DURATION
- Preincubation period: N/A
- Exposure duration: Approximately 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A


SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9

NUMBER OF CELLS EVALUATED:
All strains 0.9 to 9 * 10>9

DETERMINATION OF CYTOTOXICITY
- Method: N/A

OTHER EXAMINATIONS:
N/A


OTHER:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material.

All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535: 7 to 40
TA100: 60 to 200
TA1537: 2 to 30
TA98: 8 to 60
WP2uvrA: 10 to 60
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1 . A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
MAHON, G.A.T., et al (1989). Analysis of data from microbial colony assays. In: KIRKLAND D.J., (eds.). Statistical Evaluation of Mutagenicity Test Data: UKEMS sub-committee on guidelines for mutagenicity testing. Cambridge University Press Report, pp. 26-65.
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
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The maximum dose level of the test item in the first mutation test was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test there was no visible reduction in the growth of the bacterial background lawns noted at any dose level, either in the presence or absence of S9-mix. Consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial backgroiund lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test. A test item precipitate (globular in appearance) was observed under an inverted microscope at 1500 µg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the second mutation test.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:
Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TAl53 5, TA1537, TA98 and TAl00 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co—factors). The dose range for the range-finding test was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of the range-findi ng test, and was 50 to 5000 µg/plate.

Seven test item dose levels were selected in the main test in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the results from the first mutation test.

Results

The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first mutation test was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test there was no visible reduction in the growth of the bacterial background lawns noted at any dose level, either in the presence or absence of S9-mix. Consequently the same maximum dose level was used in the second mutation test.

Results from the second mutation test showed the test item inducing no toxicity. A test item precipitate (globular in appearance) was observed under an inverted microscope at 1500 µg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the second mutation test.

Conclusion

The test substance was considered to be non-mutagenic under the conditions of this test.

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:
18 July 2014 to 15 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
N/A
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
N/A
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1
0, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/ml without S9
0, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/ml with S9

Experiment 2
0, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/ml without S9
0, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/ml with S9

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was insoluble at a number of concentrations up to 500 mg/ml in DMSO and acetone. The test item produced a suspension in acetone suitable for dosing at 25 mg/mL and therefore the test item could only be tested at this maximum practicable limit. Unfortunately, acetone is toxic to human lymphocytes at 100uL; therefore all of the formulations were prepared at concentrations two times greater than required in the culture flasks. To compensate, each formulation was dosed using 50 uL aliquots. Consequently, the highest achievable concentration was 125 ug/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.4 ug/ml in Expt. 1, 0.2 ug/ml in Expt. 2
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
5 ug/ml in both experiments
Details on test system and experimental conditions:
- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum,
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA

METHOD OF APPLICATION: in medium; preincubation; in suspension; as impregnation on paper disk
With Metabolic Activation
Cultures were established approximately 48 hours prior to treatment. Cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing.

Without Metabolic Activation
Cultures were established approximately 48 hours prior to treatment. In Experiment 1, cultures were incubated at 37°C for 4 hours in the presence of the test material prior to washing. In Experiment 2, the cultures were incubated in the presence of the substance at 37°c for 24 hours
DURATION
- Preincubation period: 48
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 20 or 0 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Mitosis was arrested by addition of democolcine two hours prior to the required harvest time and the cells were harvested and fixed

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 ug/ml
STAIN (for cytogenetic assays): 5% Gurrs Giemsa

NUMBER OF REPLICATIONS: Treatments performed in duplicate.

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Where possible the first 100 consecutive well-spread metaphases from each culture were counted.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes in comparison to controls
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated
Evaluation criteria:
A test iten can be classified as non-genotoxic if:

1. The number of induced chromosome aberrations in all evaluated groups is within the range of historical control data.
2. No toxicologically or statistically significant increase in the number of structural chromosome aberrations is observed following statistical analysis.

A test item can be classified as genotoxic if:

1. The number of induced chromosome aberrations in all evaluated groups is not within the range of historical control data.

and

2. Either a concentration-related or statistically significant increase in the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.

.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes:
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:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 0, 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/ml.
The maximum dose was based on the maximumpracticable dose level. Precipitate was observed at 125 µg/ml in all exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 125 ug/mL in both of the 4(20)-hour exposure groups. In the 24-hour exposure group metaphase cells were present up to 125 ug/mL . The selection of the maximum dose level was based on the maximum practicable dose level.

Experiment 1.
No dose-related inhibition of mitotic index was observed in the 4(20)-hour exposure group dosed in the absence of S9. There was a moderate decrease in mitotic index in the 4(20)-hour exposure group dosed in the presence of S9-mix (36%). Therefore, the maximum dose level selected for metaphase analysis was the maximum practicable dose level of 125 ug/mL.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. 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 any statistically significant increases in the frequency of cells with aberrations, either in the absence or presence of metabolic activation. There was no significant increase in the incidence of polyploidy.

Experiment 2.
No dose-related inhibition of mitotic index was observed in either exposure groups. The maximum dose level selected for metaphase analysis was the maximum practicable dose level of 125 ug/mL.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. 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 any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. There was no statistically significant increase in the frequency of polyploid cells in either exposure group.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See attached background material.

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce a statistically significant increase in the frequency of cells with aberrations, in either the presence or absence of a liver enzyme metabolising system, in either of two separate experiments. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations.

Method

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4-hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test.

Results

All vehicle (acetone) 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. The metabolic activation system was therefore shown to be functional and the test method operating as expected. The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that was the maximum practicable dose level.

Conclusion

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

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

Additional information

Justification for classification or non-classification

Harmonized classification:

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

Self classification:

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