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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, therefore the test material was considered to be non-mutagenic.

Chromosome Aberration

The test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.

Mouse Lymphoma Assay

Under the conditions of the study, the material did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 February 2016 to 23 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro cytogenicity / chromosome aberration study in mammalian cells
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Prior to each experiment, the test item was accurately weighed, formulated in acetone and appropriate serial dilutions prepared.
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement
Species / strain / cell type:
lymphocytes:
Remarks:
Human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Cell cycle length, doubling time or proliferation index: The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
- Sex, age and number of blood donors if applicable:
Preliminary Toxicity Test: Female, aged 21 years
Main Experiment: Male, aged 32 years
- Whether whole blood or separated lymphocytes were used if applicable: Whole blood cultures
- Methods for maintenance in cell culture if applicable: The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
PRELIMINARY STUDY
0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1 250 and 2 500 μg/mL for 4 hour exposure with and without S9 and 24-hour without S9

MAIN STUDY
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 10, 20, 40, 80, 120, 160 and 240 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 10, 20, 40, 60, 80, 120 and 160 μg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 10, 20, 40, 60, 80, 120 and 160 μg/mL.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity.
Vehicle / solvent:
- Vehicle/solvent used: Acetone
- Justification for choice of solvent/vehicle: The test material was insoluble in culture medium at 50 mg/mL and dimethyl sulfoxide at 500 mg/mL but was soluble in acetone at 500 mg/mL in solubility checks performed in-house.

The test material was considered to be a UVCB and therefore the maximum recommended dose was 5 000 μg/mL. However, due to the necessity of using acetone as the solvent the maximum dose level that could be achieved was 2 500 μg/mL. Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed at 0.5 % in 50 μL aliquots. Consequently, the maximum achievable concentration was 2 500 μg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Absence of S9-mix: Mitomycin C (Minimal Essential Medium) 0.2 μg/mL for 4(20)-h exposure and 0.1 μg/mL for 24-hour continuous exposure. Presence of S9-mix: Cyclophosphamide (DMSO) at 2 mg/mL for 4(20)-h exposure.
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION:
- Exposure duration: 4 or 24 hours
- Expression time:
4 h exposure: 20 hours
24 h exposure: None

SPINDLE INHIBITOR: 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 re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

STAIN: When the slides were dry they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF REPLICATIONS: Duplicate lymphocyte cultures (A and B) were established for each dose level.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension 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.
- The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.
- Coding: The slides were coded using a computerized random number generator. Supplementary slides were coded manually.

NUMBER OF CELLS EVALUATED: A total of 2 000 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.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate); where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. 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 and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence endoreduplicated cells) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index


- OTHER: CULTURE CONDITIONS
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10 % (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

- 4-Hour Exposure with Metabolic Activation (S9):
After approximately 48 hours incubation at approximately 37 ºC, 5 % CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20 % S9-mix (i.e. 2 % final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.
After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.

- 4-Hour Exposure without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5 % CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then re-suspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

- 24-Hour Exposure without Metabolic Activation (S9)
As the exposure was continuous the cultures were established, at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5 % CO2 in humidified air for 24 hours. The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

- Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2 %), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix. The dose range of test item used was 9.77 to 2500 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.
Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.
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.
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Key result
Species / strain:
lymphocytes: Human peripheral blood 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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Precipitation: In the preliminary toxicity test, a precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure in the 4(20)-hour exposure groups, at and above 312.5 μg/mL in the absence of S9 and at and above 625 μg/mL in the presence of S9. In the continuous exposure group a precipitate of the test item was observed at and above 312.5 μg/mL.
In the main test, no precipitate was observed at the end of the exposure period in any of the three exposure groups.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1 250 and 2 500 μg/mL. The maximum dose was the maximum achievable dose level due to formulation difficulties.
Haemolysis was observed following exposure to the test item at and above 39.06 μg/mL in the 4(20)-hour exposure group in the absence of S9 and at and above 78.13 μg/mL in the presence of S9. In the 24-hour continuous exposure group haemolysis was seen from 9.77 μg/mL to 625 μg/mL at the end of the exposure period. Haemolysis is an indication of a toxic response of the erythrocytes and not indicative of any genotoxic response of the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 156.25 μg/mL in the 4(20)-hour exposure in the absence of metabolic activation (S9) and in the 24-hour continuous exposure group. The maximum dose with metaphases present in the 4(20)-hour exposure group in the presence of S9 was 78.13 μg/mL.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 240 μg/mL for the 4(20)-hour exposure group in the absence of S9, and 160 μg/mL for both the 4(20)-hour exposure group in the presence of S9 and the 24-hour continuous exposure group.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Preliminary Toxicity Test: The test item induced marked evidence of toxicity in all three exposure groups.
- Main Test: The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 160 μg/mL in the 4(20)-hour exposure groups in the absence and presence of S9. In the 24-hour exposure group the maximum test item dose level with metaphases suitable for scoring was 80 μg/mL.

ADDITIONAL INFORMATION ON GENOTOXICITY
The mitotic index data for the Main Experiment confirms the qualitative observations in that a dose-related inhibition of mitotic index was observed in all three exposure groups. In the 4(20)-hour exposure group in the absence of S9, marginally greater than acceptable toxicity was achieved at 160 μg/mL with 67 % mitotic index inhibition. In the presence of S9, the toxicity was seen to plateau at 120 μg/mL and 160 μg/mL with 44 % and 43 % mitotic index inhibition, respectively. Although the 160 μg/mL dose level did not demonstrate optimum toxicity, there was a reduction in the number of cells on the slides demonstrating overall toxicity to the cell population. In the ‘B’ replicate of the 160 μg/mL dose level only 93 metaphases were available for scoring as a result of toxicity and if taken in isolation this replicate did achieve optimum toxicity of 50 %. In the 24-hour continuous exposure group an inhibition of mitotic index of 43 % and 68 % was noted at 60 μg/mL and 80 μg/mL, respectively. Extreme toxicity was noted in the ‘B’ replicate of the 80 μg/mL dose level as only 103 metaphases were available for scoring; this replicate exceeded acceptable toxicity, although the dose level achieved the upper limit of acceptable toxicity. The deficiency in metaphases in the 4(20)-hour exposure group in the presence of S9 at 160 μg/mL and in the 24-hour exposure group at 80 μg/mL was due to toxicity and it is considered that sufficient metaphases were evaluated to demonstrate the effects of the test item on the frequency of cells with chromosome aberrations.
The maximum dose level selected for metaphase analysis was based on toxicity and was 160 μg/mL for the 4(20)-hour exposure groups and 80 μg/mL for the 24-hour exposure group.
The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
The required number of cells and concentrations were analysed.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the 4(20)-hour exposure group in the presence of metabolic activation or in the 24-hour continuous exposure group. In the 4(20)-hour exposure group in the absence of S9 there was a small but statistically significant increase in the frequency of cells with aberrations at 160 μg/mL. Since this dose level marginally exceeded acceptable toxicity the response was considered to be of no biological relevance and due to cytotoxicity rather than any clastogenic effect.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure conditions.
Conclusions:
Under the conditions of the study, the test material was considered to be non-clastogenic.
Executive summary:

The potential of the test material to induce structural chromosomal aberrations was determined in a GLP study which was conducted in accordance with standardised guidelines OECD 473 and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure 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, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Experiment were 10, 20, 40, 80, 120 and 160 μg/mL for 4 hour exposure without S9, 4 hour exposure with S9 and 24 hour exposure without S9. An additional dose of 240 μg/mL was tested for 4 hour exposure without S9.

All vehicle (acetone) 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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material demonstrated marked toxicity in all three exposure groups. The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour exposure group in the presence of S9 or in the 24-hour exposure group, using a dose range that included a dose level that achieved near optimum toxicity. In the 4-hour exposure group in the absence of S9 there was a small but statistically significant increase in the frequency of aberrations at 160 μg/mL, but this was considered to be of no biological relevance since it exceeded acceptable toxicity and it was therefore considered that the response was as a result of cytotoxicity.

Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 January 2016 to 12 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment. No correction was made for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Target gene:
- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).
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):
CELLS USED
- Source of cells: The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• Syngenta CTL, Alderley Edge, as frozen vials, on 20 March 2007.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.

- Suitability of cells: All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker. In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule

MEDIA USED
Top agar was prepared using 0.6 % Bacto agar and 0.5 % sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd.
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
- Properly maintained: Yes. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Plate Incorporation Method (Experiment 1): 1.5, 5, 15, 50, 150, 500, 1 500 and 5 000 μg/plate.
Pre-Incubation Method (Experiment 2: 15, 50, 150, 500, 1 500 and 5 000 μg/plate
The maximum dose level of the test material was selected as the maximum recommended dose level of 5 000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: Solubility checks performed in-house determined that the test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION
Experiment 1: Plate incorporation
Experiment 2: Pre-incubation

Experiment 1: Without metabolic activation
0.1 mL of the appropriate concentration of test item, solvent vehicle 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.

Experiment 1: With metabolic activation
The procedure was the same as described previously 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.

Experiment 2: Without metabolic activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

Experiment 2: With metabolic activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

DURATION
- Pre-incubation period:
Experiment 1: Not applicable
Experiment 2: 20 minutes
- Expression time:
Experiment 1: 48 hours
Experiment 2: 48 hours

SELECTION AGENT:
- Salmonella typhimurium strains: Histidine
- Escherichia coli strain: Tryptophan

NUMBER OF REPLICATIONS:
Experiment 1: Eight concentrations of the test were assayed in triplicate against each tester strain.
Experiment 2: All testing for this experiment was performed in triplicate.

DETERMINATION OF CYTOTOXICITY
Experiment 1: The plates were viewed microscopically for evidence of thinning (toxicity).
Experiment 2: The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were performed (for verification purposes) due to test item precipitation.

OTHER EXAMINATIONS:
Experiment 1: All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
Experiment 2: All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.

- OTHER:
Six test item concentrations 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 change in test methodology from plate incorporation to pre-incubation.
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.
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.
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).

A test material was considered non-mutagenic (negative) in the test system if the above criteria were 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 material activity. Results of this type are reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
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.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5 000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and 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 background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). Although none of the bacterial tester strains exhibited weakened background lawns, several (TA1535 in particular) did express decreases in revertant colony frequency at the maximum dose level.
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 metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). 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 metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix employing the plate incorporation method (Experiment 1) or the pre-incubation method (Experiment 2) in the absence of S9-mix. However, a white, fine, particulate test item precipitate was noted by eye at 5 000 μg/plate in the second mutation test in the presence of S9-mix, this observation did not prevent the scoring of revertant colonies.

CONTROL DATA
- Positive control data: 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.
- Negative (solvent/vehicle) control data: The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.
Conclusions:
Under the conditions of this study, the test material was considered to be non-mutagenic.
Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471, EU Method B.13/14., USA EPA 870.5100 and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods 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 Experiment 1 was predetermined and was 1.5 to 5 000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5 000 μg/plate.

Six test item concentrations 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 change in test methodology.

The vehicle (dimethyl sulphoxide) 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 experiment was selected as the maximum recommended dose level of 5 000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and 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 background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). Although none of the bacterial tester strains exhibited weakened background lawns, several (TA1535 in particular) did express decreases in revertant colony frequency at the maximum dose level. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix employing the plate incorporation method (Experiment 1) or the pre-incubation method (Experiment 2) in the absence of S9-mix. However, a white, fine, particulate test item precipitate was noted by eye at 5 000 μg/plate in the second mutation test in the presence of S9-mix, 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 metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). 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 metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Under the conditions of the test, the test material was considered to be non-mutagenic.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 April 2016 to 26 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
• Kanpoan No. 287 - Environment Protection Agency
• Eisei No. 127 - Ministry of Health and Welfare
• Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In vitro mammalian cell gene mutation test: Mouse Lymphoma Assay
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Following solubility checks performed in-house for the Chromosome Aberration Test performed on the same test item, the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared. The test item was considered to be a mixture, therefore the maximum proposed dose level was 5 000 μg/mL and no correction for purity was applied. Due to the sensitivity of L5178Y cells to acetone, the formulations were prepared at twice the concentration required in culture and dosed at 0.5 %. Consequently, the maximum achievable concentration was 2 500 μg/mL.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The 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. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time. The stocks of cells are stored in liquid nitrogen at approximately -196 °C.
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours and were sub-cultured accordingly.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10 % donor horse serum (giving R10 media) with 5 % CO2 in air. RPMI 1640 with 20 % donor horse serum (R20), 10 % donor horse serum (R10), and without serum (R0), were used during the course of the study.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes. Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically 'cleansed' against high spontaneous background: Yes. The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
4-hour treatment without S9: 10, 20, 30, 40, 60 and 80 μg/mL.
4-hour treatment with S9 (2 %): 5, 10, 20, 40, 60 and 80 μg/mL.
24-hour treatment without S9: 2.5, 5, 10, 20, 40 and 60 μg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Following solubility checks performed in-house for the Chromosome Aberration Test performed on the same test item, the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
Without metabolic activation: Ethylmethanesulphonate in DMSO at 400 μg/mL and 150 μg/mL in the 4-hour and 24-hour exposure groups. With metabolic activation: Cyclophosphamide in DMSO at 1.5 μg/mL.
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium.
- Cell density at seeding:
4-hour exposure group with and without metabolic activation: 1 x 10^6 cells/mL in 10 mL aliquots.
24-hour exposure group with and without metabolic activation: 0.3 x 10^6 cells/mL in 10 mL cultures.
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2 % S9 final concentration) at 8 dose levels of the test item (2.5 to 80 μg/mL for 4-hours –S9, 2.5 to 100 μg/mL for the 4-hours +S9 and 24-hours -S9), vehicle and positive controls. 2 mL of S9-mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group) were added to each universal.

DURATION
- Exposure duration: 4 or 24 hours at 37 °C with continuous shaking using an orbital shaker within an incubated hood.
- Expression time: Two days
- Selection time: 10 to 12 days

ASSESSMENTS
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then re-suspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured every 24 hours for the expression period of two days, by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2 000 cells/well) in selective medium. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

SELECTION AGENT: 4 μg/mL 5 trifluorothymidine (TFT)

STAIN: To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualization of the mutant colonies, particularly the small colonies.

NUMBER OF REPLICATIONS: Two

NUMBER OF PLATES SCORED: Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5 % CO2 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 as the additional information may contribute to an understanding of the mechanism of action of the test item. Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ 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.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth. The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
- Any supplementary information relevant to cytotoxicity:
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 9.77 to 2 500 μg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, re-suspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 10^5 cells/mL.
The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5 000 μg/mL or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20 % survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA.
Evaluation criteria:
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20 % survival (80 % toxicity), but no less than 10 % survival (90 % toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test material for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10 % are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.

An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Statistics:
The experimental data was analysed using a dedicated computer program, Mutant 240C which follows the statistical guidelines recommended by the UKEMS. The statistical package used indicates the presence of statistically significant increases and linear-trend events.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
There was evidence of toxicity following exposure to the test item in all three of the exposure groups, as indicated by the % RSG and RTG values.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: There was no marked change in pH when the test item was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Precipitation: A precipitate of the test item was observed in the preliminary cytotoxicity test at and above 156.25 μg/mL in the 4 hour –S9 exposure group, 312.5 μg/mL in the 4-hour +S9 exposure group and 625 μg/mL in the 24-hour exposure group.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The dose range of the test item used in the preliminary toxicity test was 9.77 to 2 500 μg/mL for all three exposure groups. In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was also evidence of marked reductions of %RSG values of cells treated with test item. In the subsequent mutagenicity experiments the maximum dose was limited by test item induced toxicity.

MUTAGENICITY TEST
There was evidence of toxicity following exposure to the test item in all three of the exposure groups, as indicated by the % RSG and RTG values. There was evidence of reductions in viability (%V) in both 4-hour exposure groups, therefore indicating that residual toxicity had occurred. Optimum levels of toxicity were achieved in the 4-hour –S9 and 24-hour –S9 exposure groups, with near to optimum toxicity achieved in the 4-hour + S9 exposure group. Two dose levels were excluded from analysis due to excessive toxicity; these were 80 μg/mL in both 4-hour exposure groups. Acceptable levels of toxicity were seen with both positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant increases in the mutant frequency x 10^-6 per viable cell in either of the three exposure groups. The mutant frequency values were all within the acceptable range for a vehicle control culture and the GEF was not exceeded in all three exposure groups. No precipitate was observed during the main test.

Conclusions:
Under the conditions of the study, the material did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic.
Executive summary:

The mutagenic activity of the test material was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The GLP study was conducted in accordance with the standardised guidelines OECD 490 and EU Method B.17, US EPA OPPTS 870.5300 and in alignment with Japanese guidelines for testing of new chemical substances and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels in duplicate, together with vehicle (R0 media), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9), and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item exhibited dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were 10, 20, 30, 40, 60 and 80 μg/mL for 4-hour treatment without S9, 5, 10, 20, 40, 60 and 80 μg/mL for 4-hour treatment with S9 (2 %) and 2.5, 5, 10, 20, 40 and 60 μg/mL 24-hour treatment without S9.

The maximum dose level used was limited by test item induced toxicity. No precipitate was observed during the main test. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Under the conditions of the study, the material did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The following genetic toxicity studies were assigned a reliability score of 1 in line with the principles for assessing data quality as defined by Klimisch et al. (1997).

 

Ames Test

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471, EU Method B.13/14., USA EPA 870.5100 and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods 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 Experiment 1 was predetermined and was 1.5 to 5 000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5 000 μg/plate.

Six test item concentrations 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 change in test methodology.

The vehicle (dimethyl sulphoxide) 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 experiment was selected as the maximum recommended dose level of 5 000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and 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 background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). Although none of the bacterial tester strains exhibited weakened background lawns, several (TA1535 in particular) did express decreases in revertant colony frequency at the maximum dose level. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix employing the plate incorporation method (Experiment 1) or the pre-incubation method (Experiment 2) in the absence of S9-mix. However, a white, fine, particulate test item precipitate was noted by eye at 5 000 μg/plate in the second mutation test in the presence of S9-mix, 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 metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). 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 metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Under the conditions of the test, the test material was considered to be non-mutagenic.

 

Chromosome Aberration

The potential of the test material to induce structural chromosomal aberrations was determined in a GLP study which was conducted in accordance with standardised guidelines OECD 473 andJapanese Guidelines for Screening Mutagenicity Testing Of Chemicals.

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure 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, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Experiment were 10, 20, 40, 80, 120 and 160 μg/mL for 4 hour exposure without S9, 4 hour exposure with S9 and 24 hour exposure without S9. An additional dose of 240 μg/mL was tested for 4 hour exposure without S9.

All vehicle (acetone) 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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material demonstrated marked toxicity in all three exposure groups. The test material did not induce any statistically significant increases in the frequency of cells with aberrations in the 4(20)-hour exposure group in the presence of S9 or in the 24-hour exposure group, using a dose range that included a dose level that achieved near optimum toxicity. In the 4-hour exposure group in the absence of S9 there was a small but statistically significant increase in the frequency of aberrations at 160 μg/mL, but this was considered to be of no biological relevance since it exceeded acceptable toxicity and it was therefore considered that the response was as a result of cytotoxicity.

Under the conditions of the study, the test material was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic.

 

Mouse Lymphoma Assay

The mutagenic activity of the test material was evaluated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. The GLP study was conducted in accordance with the standardised guidelines OECD 490 and EU Method B.17, US EPA OPPTS 870.5300 and in alignment with Japanese guidelines for testing of new chemical substances and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels in duplicate, together with vehicle (R0 media), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9), and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item exhibited dose-related toxicity to the cells in each of the three exposure groups of the preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were 10, 20, 30, 40, 60 and 80 μg/mL for 4-hour treatment without S9, 5, 10, 20, 40, 60 and 80 μg/mL for 4-hour treatment with S9 (2 %) and 2.5, 5, 10, 20, 40 and 60 μg/mL 24-hour treatment without S9.

The maximum dose level used was limited by test item induced toxicity. No precipitate was observed during the main test. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Under the conditions of the study, the material did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6, consequently it is considered to be non-mutagenic.

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.