Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item diluted in sterile water, tested up to concentrations of 5 mg/plate in the in vitro bacterial mutagenicity assay (Salmonella reverse mutation Assay) was not mutagenic in both the presence and absence of metabolic activation (SafePharm, 2003).

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. The test material did not induce a statistically significant increase in the numbers of polyploid cells or a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system at any dose level in either of two separate experiments (SafePharm, 2004).

The third in vitro study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (HPRT, OECD 476). The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment (Harlan, 2011).

In conclusion, the available in vitro genotoxicity studies with the test item showed no mutagenic effects.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-07-09 to 2010-10-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
The test material was accurately weighed, formulated in RO medium and serial dilutions prepared.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr J Cole of the
MRC Cell Mutation Unit at the University of Sussex, Brighton, UK
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1: 4 hours exposure, +/- S9: 0, 250, 500, 750, 1000, 1250, 1500, 1750,2000 µg/ml
Experiment 2: 4 hours exposure with S9: 0, 50,100, 200, 300, 400, 500, 600, 700 µg/ml
24 hours without S9: 0, 125, 250, 500, 750, 1000, 1250, 1500, 1750 µg/ml
There was no marked increase in osmolality greater than 50 mOsm when the test material was dosed into media in the solubility test. However, a marked increase in pH greater than 1 pH unit was observed at and above 2500 μg/ml. Therefore, the maximum dose level was reduced to 2000 μg/ml in the subsequent preliminary toxicity test (Scott et al 1991 ).
Vehicle / solvent:
RO medium
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Mouse lymphoma assay
METHOD OF APPLICATION:
- Metabolic activation system: male Sprague Dawley rats are induced with phenobarbital/ β-naphthoflavone
- Dosing:   
Experiment 1: 4 hours exposure, +/- S9: 0, 250, 500, 750, 1000, 1250, 1500, 1750,2000 µg/mL
Experiment 2: 4 hours exposure with S9: 0, 50,100, 200, 300, 400, 500, 600, 700 µg/mL
24 hours without S9: 0, 125, 250, 500, 750, 1000, 1250, 1500, 1750 µg/mL
- Solvent: RO medium
- Controls
Positive without metabolic activation:
- Ethylmethanesulfonate (EMS) at 400 µg/mL and 150 µg/mL
Positive with metabolic activation:
- Cyclophosphamide (CP) 2 µg/mL

DURATION
- Exposure duration:
Experiment 1: 4 hours exposure, +/- S9
Experiment 2: 4 hours exposure with S9
24 hours without S9
Expression time (cells in growth medium): 2 days in total at 37 °C in 5% CO2/95% humidified air, the cell density was determined each day and adjusted to 2 x 10E5 cells/mL.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. +
Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value.
Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation at 37°C with 5% C02 in air.
The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate).
The numbers of small and large colonies seen in the TFT mutation plates were also recorded.
Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately % to % of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates.
The plates were incubated for approximately two to four hours.
MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

- Cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated
for at least 6 days at 37 °C in a humidified atmosphere with 5% CO2


SELECTION AGENT (mutation assays): selected for 5-trifluoro-thymidine (TFT) resistance



Rationale for test conditions:
The dose range of test material was selected following the results of a preliminary toxicity test and for the first experiment was 250 to 2000 μg/ml in both the absence and presence of metabolic activation. For the second experiment the dose range was 50 to 700 μg/ml in the absence of metabolic activation, and 125 to 1750 μg/ml in the presence of metabolic activation.
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-200 x 10-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10-6 mutant frequency per survivor are not normally acceptable and will be repeated.

Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance.

Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.
Statistics:
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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:
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 1 o-6 viable cells.
Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Conclusions:
negative with and without metabolic activation. Test item is not mutagenic.
Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-,

locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method 817 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Methods.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and for the first experiment was 250 to 2000 μg/ml in both the absence and presence of metabolic activation. For the second experiment the dose range was 50 to 700 μg/ml in the absence of metabolic activation, and 125 to 1750 μg/ml in the presence of metabolic activation. Results. The maximum dose level used in the mutagenicity test was limited by a combination of an excessive increase in pH at dose levels higher than 2000 μg/ml and test material-induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion:

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

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:
2003-10-01 to 2003-10-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The test material was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated impurity content (3%) of the test material.
Target gene:
mutated gene loci resposible for histidine auxotropy
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst Escherichia coli strain WP2uvrA' was obtained from the British Industrial Biological Research Association on 17 August 1987.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Plate incorporation test: 50, 150, 500, 1500 and 5000 ug/plate
Preincubation test: 50, 150, 500, 1500 and 5000 ug/plate
Vehicle / solvent:
Sterile distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
Details on test system and experimental conditions:
Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five 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 was determined in a preliminary toxicity assay and was 50 to 5000 mg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control and the positive control materials used in the series of plates
without S9-mix were as follows:
N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 3 ug/plate for TA100, 5 ug/plate for TA1535 and 2 ug/plate for WP2uvrA
9-Aminoacridine (9AA): 80 ug/plate for TA1537
4-Nitroquinoline-l-oxide (4NQO): 0.2 ug/plate for TA98
In addition, 2-Aminoanthracene (2AA) and Benzo(a)pyrene (BP), which are non-mutagenic in the absence of metabolising enzymes, were used in the series of plates with S9-mix at the following concentrations:
2AA at 1 ug/plate for TAl 100
2AA at 2 ug/plate for TA1535 and TA1537
BP at 5 ug/plate for TA98
2AA at 10 ug/plate for WP2uvrA
Rationale for test conditions:
- Regulation (EC) No. 440/2008 method B.13/14: Mutagenicity: Reverse Mutation Test Using Bacteria, adopted May 30, 2008;
- OECD Guideline for Testing of Chemicals, No. 471: Bacterial Reverse Mutation Test, adopted July 21, 1997;
Evaluation criteria:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2001 and 2002.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 X 10E9 bacteria per ml.
Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.and the integrity of the S9-mix.
Statistics:
According to the OECD Guideline 471, a statistical analysis of the data is not mandatory
Key result
Species / strain:
other: Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA-
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
Additional information on results:
The vehicle (sterile distilled water) 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 test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.
The test material was, therefore, tested up to the maximum recommended dose level of 5000 mg/plate.
No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Test item was not mutagenic under the test conditions used in this bacterial assay.
Executive summary:

Introduction. The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS hannonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors) . The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results. The vehicle (sterile distilled water) 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 test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore , tested up to the maximum recommended dose level of 5000 ug/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

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.

Conclusion:

Test item diluted in sterile water was examined for mutagenic activity in aSalmonella typhimurium-Escherichia colidirect plate incorporation assay. The assay was performed using S. typhimurium strains TA1535, TA1537, TA98, and TA100 and E. coli strain WP2uvrA-over a dose range of 50 to 5,000 ug/plate in both the presence and absence of a phenobarbitone/β-naphthoflavone-induced rat-liver S9 metabolic activation system.

Test item was not mutagenic under the test conditions used in this bacterial assay.

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:
2003-09-29 to 2004-01-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
The test material was accurately weighed, dissolved in Minimal Essential Media (MEM) and serial dilutions prepared.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
lymphocytes of fresh heparinised whole blood
Additional strain / cell type characteristics:
other: Cultured human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
S-9
Test concentrations with justification for top dose:
Final concentration of test item (μg/ml) without S9: 0*, 117.19, 234.38, 468.75*, 937.5*, 1875*, 2500, MMC 0.4*
Final concentration of test item (μg/ml) with S9 . 0*, 117.19, 234.38, 468.75*, 937.5*, 1875*, 2500, CP 10*
* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide
There was an increase in pH that was greater than 1 pH unit at 5000 µg/ml compared to the vehicle control when the test material was dosed into culture media. Consequently the maximum
dose level for the subsequent phase of the study was limited to 2500 µg/ml.
Vehicle / solvent:
Minimal Essential Media (MEM
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
Minimal Essential Medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls.
Four treatment conditions were used for the study i.e. 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-hour 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.

Experiment 1
i) 4-hour exposure to the test material without S9-mix followed by 20-hour culture in treatmentfree media prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatmentfree media prior to cell harvest.
Experiment 2
i) 24-hour continuous exposure to the test material prior to cell harvest.
ii) 4-hour exposure to the test material with S9-mix followed by 20-hour culture in treatmentfree media prior to cell harvest

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/ml) two hours before the required harvest time.
After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in 0.075M hypotonic KC1.
After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded.
The cells were resuspended and then fixed by dropping the KC1 cell suspension into fiesh methanoVglacial acetic acid (3:l vlv).
The fixative was changed at least three times and the cells stored at 4OC for at least four hours to ensure complete fixation.

Preparation of Metaphase Spreads
The lymphocytes were resuspended in several ml of fiesh fixative before centrifugation and resuspension in a small amount of fixative.
Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry.
Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Gurrs Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

Qualitative Slide Assessment
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 material.
These observations were used to select the dose levels for mitotic index evaluation.

Mitotic Index
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.

Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test material used was 9.75 to 2500 µg/ml.
Rationale for test conditions:
Justification for the Selection of the Test System
The OECD Guideline for Testing of Chemicals Section 4, No 473 – “In Vitro Mammalian Chromosome Aberration Test” adopted 26 September, 2014 – recommends using a variety of cell lines or primary cell cultures (e.g. Chinese hamster fibroblasts, human or other mammalian peripheral blood lymphocytes).
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a
clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
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.
Key result
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:
All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.
All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolizing system.
The test material was toxic but 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 induced mitotic inhibition.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Interpretation of results (migrated information): negative
Test item was non-clastogenic to human lymphocytes in vitro

Executive summary:

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 (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 2000/32/EC. The study design also meets the requirements of the UK Department of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals.

Methods.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising 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-hour 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.

 

Results.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was 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 induced mitotic inhibition.

 

Conclusion.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In Experiment 1, cells were treated for 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 second group of cells were treated for 4 hours in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hour 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 test material did not induce a statistically significant increase in the numbers of polyploid cells or a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system at any dose level in either of two separate experiments.

 

Using a dose range that included a dose level that induced mitotic inhibition, test item was 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

Justification for classification or non-classification

Based on the results of the in vitro genotoxicity studies and according to the criteria of EC Regulation 1272/2008 the test item is not mutagenic and therefore must not be classified.