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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test

Under the conditions of the test the test material was not mutagenic in any of the strains tested with and without metabolic activation.

In VitroMammalian Chromosome Aberration Test

Under the conditions of this study, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

Mouse Lymphoma Assay

Under the conditions of this study, the test 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 in this assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 September 2017 to 05 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: The appropriate vehicle (solvent) and the behaviour of the test material formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. DMSO was used as solvent to prepare the stock solution of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 4 hours after preparation. Analytical determination of the test material concentration, stability and homogeneity was not performed because of the character and the short period of study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The stock solution was placed in an ultrasonic water bath for 2 minutes before use.
Target gene:
S. typhimurium: Histidine
E. coli: Tryptophan
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
- Methods for growing in culture: The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum was used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10 - 14 hours at 37°C in a Gyrotory water bath shaker.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
The Typical Composition (g/1 000 mL) of Minimal Glucose Agar
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g
Distilled water q.s. ad 1 000 mL
Minimal glucose agar plates (Batch number: 145659, Expiry date: 20 December 2017) were provided by Merck. Certificates of Analysis were obtained from the Supplier.

Nutrient Broth No.2:
Nutrient Broth No.2 25.0 g
Distilled water q.s. ad 1 000 mL
Sterilization was performed at 121 °C in an autoclave.

Nutrient Agar
Nutrient Agar 20.0 g
Distilled water q.s. ad 1 000 mL
Sterilization was performed at 121 °C in an autoclave.

Top Agar for Salmonella typhimurium Strains
Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Distilled water q.s. ad 1 000 mL
Sterilization was performed at 121 °C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin (F.W. 244.31) 122.2 mg
L-Histidine x·HCl x H2O (F.W. 209.63) 104.8 mg
Distilled water q.s. ad 1 000 mL
Sterilization was performed by filtration using a 0.22 μm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100 mL
Agar solution 900 mL

Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/mL):
L-Tryptophan (F.W. 204.23) 2 000 mg
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration using a 0.22 μm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth 50 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution 947.5 mL

- Properly maintained: Yes.
The strains are stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of Citoxlab Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures, to which DMSO was added as a cryoprotective agent.
- Periodically checked for phenotype stability: The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames. Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of Citoxlab Hungary Ltd.
- Periodically 'cleansed' against high spontaneous background: Yes. Each test strain reverts spontaneously at a frequency that is characteristic of the strain.
Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011-2016 were (as guide) as follows: Salmonella typhimurium TA98: 9-50, TA100: 54-210, TA1535: 1-46, TA1537: 1-24, Escherichia coli WP2 uvrA: 11-82.
- Viability: The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Examined concentrations in the Initial Mutation Test were 5 000, 1 581, 500, 158.1, 50 and 15.81 μg/plate.
Examined concentrations in the Confirmatory Mutation Test were 5 000, 1 581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the different concentrations were used.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: In a preliminary compatibility test the solubility of the test material was examined using Distilled water, N,N-Dimethylformamide (DMF), Dimethyl sulfoxide (DMSO) and Acetone. Partial dissolution was observed at the 100 mg/mL concentration using Distilled water and Acetone. At this concentration suspension with test material particles was observed using DMSO and DMF.
At 50 mg/mL concentration there was no change for Distilled water or Acetone, but a suspension of smaller test material particles was observed far DMSO and DMF. After 2 minutes ultrasonic water bath a homogeneous suspension was obtained for DMSO and DMF. Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. The obtained stock solution (100 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.
Untreated negative controls:
yes
Remarks:
An untreated control was used to demonstrate that the chosen vehicle induced no deleterious or mutagenic effects. If the solvent of the positive control substance differed from the solvent of the test material, both solvents were run in the assay.
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO) and distilled water, depending on the solubility of the test material and the solubility of strain specific positive control chemicals.
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine and 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION
Initial mutation test: plate incorporation method
Confirmatory mutation test: pre-incubation method

DURATION
- Pre-incubation period: 20 minutes
- Selection time:
Initial mutation test: 48 ± 1 h.
Confirmatory mutation test: 48 ± 1 h.

SELECTION AGENT
S. typhimurium: Histidine
E. coli: Tryptophan

NUMBER OF REPLICATIONS
Initial mutation test: Triplicate for each control or concentration level.
Confirmatory mutation test: Triplicate for each control or concentration level.

- PRELIMINARY CONCENTRATION RANGE-FINDING TEST (INFORMATORY TOXIICTY TEST).
Based on the solubility test, a 50 mg/mL stock solution of the test material was prepared in DMSO. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate of the test material, in the absence and presence of metabolic activation. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

PROCEDURE FOR EXPOSURE IN THE INITIAL MUTATION TEST
The Initial Mutation Test followed the standard plate incorporation procedure.
Bacteria (cultured in Nutrient Broth No.2) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test material and other components were prepared freshly and added to the overlay (45°C).

The content of the tubes:
Top agar 2 000 μL
Vehicle or test material formulation (or reference controls) 100 (50)* μL
Overnight culture of test strain 100 μL
Phosphate buffer (pH 7.4) or S9 mix 500 μL
*Treatment volume was 100 μL for test material formulations and its solvent; treatment volume was 50 μL for positive control substances and their solvents.

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

PROCEDURE FOR EXPOSURE IN THE CONFIRMATORY MUTATION TEST
The Confirmatory Mutation Test followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the Initial Mutation Test.
Bacteria (cultured in Nutrient Broth No.2) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled.
Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.
Rationale for test conditions:
Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the different concentrations were used.
Evaluation criteria:
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests;

Criteria for a Positive Response:
A test material was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
The test material was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The colony numbers on the untreated / negative (solvent) / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test material and for the controls using Microsoft Excel™ software.
* Mutation factor (MF): Mean number of revertants on the test material plate / mean number of revertants on the vehicle control plate.
Key result
Species / strain:
other: Salmonella typhimurium TA 98, TA100, TA1535, TA1537; Escherichia coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate of the test material was detected in all examined bacterial strains with and without metabolic activation in the Initial Mutation Test on the plates at 5 000 μg/plate concentration. The same effect was observed in the Confirmatory Mutation Test in all Salmonella typhimurium bacterial strains with and without metabolic activation on the plates at 5 000 and 1 581 μg/plate concentrations and in Escherichia coli WP2 uvrA strain with and without metabolic activation at 5 000 μg/plate concentration. In these concentrations, the background lawn development could not be assessed due to the strong precipitate, but the colony counting was not affected.


RANGE-FINDING/SCREENING STUDIES
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (± S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
Precipitate/slight precipitate was detected on the plates in the Preliminary experiment in Salmonella typhimurium TA98 strain with and without metabolic activation at 5 000 and 2 500 μg/plate concentrations and in Salmonella typhimurium TA100 strain with and without metabolic activation at 5 000 μg/plate concentration.
Inhibitory or toxic effects of the test material were not detected in the preliminary experiment.
Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5 000 μg/plate


ADDITIONAL INFORMATION ON CYTOTOXICITY
Inhibitory, cytotoxic effect of the test material was not detected in the Initial Mutation Test and Confirmatory Mutation Test.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 15.81 μg/plate concentration without metabolic activation (the observed mutation factor value was:
MF: 1.38). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
In the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 5 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.31). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Validity of the Test

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Conclusions:
Under the conditions of the test the test material was not mutagenic in any of the strains tested with and without metabolic activation.
Executive summary:

The test material was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test material was dissolved in DMSO at a concentration of 50 mg/mL. Concentrations of 5 000; 2 500; 1 000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test were 5 000, 1 581, 500, 158.1, 50 and 15.81 μg/plate, and in the Confirmatory Mutation Test were 5 000, 1 581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Precipitate of the test material was detected in all examined bacterial strains with and without metabolic activation in the Initial Mutation Test on the plates at 5 000 μg/plate concentration. The same effect was observed in the Confirmatory Mutation Test in all Salmonella typhimurium bacterial strains with and without metabolic activation on the plates at 5 000 and 1 581 μg/plate concentrations and in Escherichia coli WP2 uvrA strain with and without metabolic activation at 5 000 μg/plate concentration.

Note: In these concentrations, the background lawn development could not be assessed due to the strong precipitate, but the colony counting was not affected. Inhibitory, cytotoxic effect of the test material was not detected in the Initial Mutation Test and Confirmatory Mutation Test.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test material has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May 2018 to 13 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
other: The test method is designed to be in alignment with the following Japanese Guidelines:
Version / remarks:
- Kanpoan No. 287 -- Environment Protection Agency“
- Eisei No. 127 -- Ministry of Health & Welfare
- Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
TK+/- 3.7.2c
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: 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).
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Methods for maintenance in cell culture if applicable: The stocks of cells are stored in liquid nitrogen at approximately -196 °C. 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) at 37 °C with 5 % CO2 in air.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 with 20 % donor horse serum (R20), 10 % donor horse serum (R10), and without serum (R0), are used during the course of the study. R10 media was used as described above.
- 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 specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 1.25. 2.5, 5, 10, 20, 40, 80, 160 and 320 μg/mL. These dose levels were selected to avoid the excessive precipitate that was observed in the solubility test.

Results from the preliminary toxicity test were used to set the test material dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test materials the upper test material concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test material is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test: 0, 0.31, 0.63, 1.25, 2.5, 5, 10, 20 and 30 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Following solubility checks performed in-house the test material was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The formulation formed a fine white suspension suitable for dosing into the cultures. Solubility in RO media and acetone was investigated, but shown to be unsuitable for dosing. The test material was considered to be a complex mixture and therefore the maximum recommended dose (MRD) was initially set at 5 000 μg/mL. The purity of the test material was treated as 100 %. The test material would not formulate suitably at the MRD but formed a suitable fine white suspension for dosing at 250 mg/mL to achieve 2 500 μg/mL. There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm.
- No analysis was carried out to determine the homogeneity, concentration or stability of the test material formulation. The test material was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration.
Untreated negative controls:
no
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:
PRELIMINARY TOXICITY TEST
- 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 1.25 to 320 μg/mL for all three of the exposure groups. These dose levels were selected to avoid the excessive precipitate that was observed in the solubility test. Following the exposure periods the cells were washed twice with R10, re-suspended in R20 medium, counted and then serially diluted 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.
- 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, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. 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 exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

METHOD OF APPLICATION: In medium.
- Cell density at seeding: 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 cm^2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation.

DURATION
- Exposure duration: The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.
- Expression time: At the end of the exposure periods, 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 dilution 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.
- Selection time: On Day 2 of the experiment, the cells were counted, diluted to 10^4 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.
SELECTION AGENT: 4 μg/mL 5-trifluorothymidine (TFT)


NUMBER OF REPLICATIONS
The exposures were performed in duplicate (A + B), both with and without metabolic activation (2 % S9 final concentration) at eight dose levels of the test material 0.31 to 30 μg/mL for all three exposure groups, vehicle and positive controls.


NUMBER OF CELLS EVALUATED
Measurement of Survival, Viability and Mutant Frequency
- The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure 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.

Plate Scoring
- 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 material (Cole et al., 1990). 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. 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.


DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth

Calculation of Percentage Relative Suspension Growth (%RSG)
- The cell counts obtained immediately post exposure and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth:

4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)

24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)

Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count

%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

Calculation of Day 2 Viability (%V)
- Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method:

P(0) = number of negative wells / total wells plated

%V = (-ln P(0) x 100) / number of cells/well

Calculation of Relative Total Growth (RTG)
- For each culture, the relative cloning efficiency, RCE, was calculated:

RCE = %V / Mean Solvent Control %V

- Finally, for each culture RTG is calculated:

RTG = (RCE x RSG)/100

Calculation of Mutation Frequency (MF)
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
Evaluation criteria:
DATA EVALUATION
- 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 by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). 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:
no cytotoxicity, but tested up to precipitating concentrations
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 material was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm when the test material was dosed into the media.
- Precipitation: The concentration of 30 μg/mL in all three exposure groups were not plated out for 5-TFT resistance and viability due to excessive precipitate (only one precipitating dose level required under OECD 490 guideline) Precipitate was observed at and above 20 μg/mL in all three exposure groups.


RANGE-FINDING/SCREENING STUDIES:
There was evidence of slight dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material in all of the three exposure groups when compared to the concurrent vehicle control groups. Precipitate of the test material was observed at and above 20 μg/mL in all three exposure groups. Therefore, the dose levels selected for the main test were based on precipitate.


CONTROL DATA
- Positive control data: 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.
- Negative (solvent) control data: The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: %RSG. In the concentrations analysed, there was no evidence of marked dose related toxicity following exposure to the test material in either of the three exposure groups, as indicated by the %RSG and RTG values. There was no evidence of any marked reductions in viability (%V) in either of the three exposure groups, indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with the positive control substances.

RESULTS
- The test material did not induce any toxicologically significant or dose related increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels, in any of the three exposure groups.

Table 1: Results of the Preliminary Cytotoxicity Test

Dose (µg/mL)

%RSG (-S9) 4-Hour Exposure

%RSG (+S9) 4-Hour Exposure

%RSG (-S9) 24-Hour Exposure

0

100

100

100

1.25

90

89

103

2.5

77

99

108

5

80

103

99

10

88

85

102

20 p

78

103

90

40 p

80

85

88

80 p

80

90

92

160 p

76

78

89

320 p

74

68

70

p = Precipitate

Conclusions:
Under the conditions of this study, the test 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 in this assay.
Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 490, EU Method B17, OPPTS 870.5300 and in alignment with other Japanese guidelines, under GLP conditions.

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.

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 material at eight dose levels in duplicate, together with vehicle (DMSO), 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 material used in the main test was selected following the results of a preliminary toxicity test at a concentration range of 1.25 to 320 μg/mL. The maximum dose level used in the Mutagenicity Test was limited by precipitate. The dose levels plated for viability and expression of mutant colonies were 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL.

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 within the historical control data range, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant or dose related (linear-trend) increases in either of the three exposure groups. The GEF value was not exceeded at any test material concentration.

Under the conditions of this study, the test 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 in this assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 May 2018 to 08 August 2018
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)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines.
Version / remarks:
31 March 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Metaphase chromosomes of normal human lymphocytes
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human non-smoking volunteers 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.
- Sex, age and number of blood donors:
Preliminary Toxicity Test: Female, aged 24 years.
Main Experiment: Female, aged 27 years.
- Whether whole blood or separated lymphocytes were used: Whole blood drawn from the peripheral circulation.
- Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

MEDIA USED
- Type and identity of media including CO2 concentration: 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 °C with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0.625, 1.25, 2.5, 5. 10, 20, 40, 80 and 160 µg/mL
Main Experiment (4(20)-hour Exposure Groups): 1.25, 2.5, 3.75, 5, 7.5, 10, 20, 30, and 40 µg/mL
Main Experiment (24-hour Exposure Group): 0.313, 0.625, 1.25, 2.5, 3.75, 5 and 10 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
The test material was considered to be a mixture and therefore the maximum recommended dose was initially set at 5 000 µg/mL. The test material was insoluble in tissue culture media at 50 mg/mL but was soluble in dimethyl sulfoxide at 250 mg/mL in solubility checks performed in house within study project number PD75TM, and the data from this was considered suitable for use in the study. Within the solubility check a precipitate of the test material was observed at and above 156.3 µg/mL, and based on this the maximum dose level was reduced to 160 µg/mL in the preliminary toxicity test. The solubility of the test material was investigated in the Envigo Research Limited Mouse Lymphoma assay, Study number PD75TM.
There was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
The test material was formulated within two hours of it being applied to the test system; the test material formulations were assumed to be stable.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- 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 and 0.75 mL heparinized whole blood.

DURATION
- Preincubation period: 48 hours
- Exposure duration: 2 or 24 hours
- 4-Hour Exposure With Metabolic Activation (S9): After approximately 48 hours incubation at approximately 37 °C, 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 0.1 mL of the appropriate solution of vehicle control or test material was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL 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 °C, 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 °C in 5 % CO2 in humidified air. The dose range of test material used was 1.25 to 40 µg/mL.
- 4-Hour Exposure Without Metabolic Activation (S9): After approximately 48 hours incubation at approximately 37 °C 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 0.1 mL of the appropriate vehicle control, test material 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 °C, 5 % CO2 in humidified air, the cultures were centrifuged and 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. The dose range of test material used was 1.25 to 20 µg/mL.
- 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 0.1 mL of vehicle control, test material 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 °C, 5 % CO2 in humidified air for 24 hours. The dose range of test material used was 0.313 to 10 µg/mL.

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 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 °C to ensure complete fixation prior to slide preparation.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- Preparation of Metaphase Spreads: 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.
– Staining: When the slides were dry they were stained in 5 % Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

EVALUATION OF RESPONSE
- 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.
- 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 of cells with endoreduplicated chromosomes) was also reported. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.


OTHER:
PRELIMINARY TOXICITY TEST
- The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.
- Three exposure groups were used:
i) 4-hour exposure to the test material 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 material 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 material without S9-mix.
- The dose range of test material used was 0.625 to 160 µg/mL.
- Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test material 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 material toxicity and for selection of the dose levels for the Main Experiment.
Evaluation criteria:
DATA EVALUATION
The following criteria were used to determine a valid assay:
-The frequency of cells with structural 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 analysed.

CRITERIA FOR DETERMINING STUDY CONCLUSION
Providing that all of the acceptability criteria are fulfilled, a test material can be considered to be clearly negative if, in any of the experimental conditions examined:
- The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
- No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
- There is no concentration-related increase at any dose level.

A test material can be classified as genotoxic if:
- The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
- At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
- The observed increase in the frequency of cells with structural aberrations is considered to be dose-related

When all of the above criteria are met, the test material can be considered able to induce chromosomal aberrations in human lymphocytes.
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 (Richardson et al. 1989).
- 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
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- The dose range for the Preliminary Toxicity Test was 0.625 to 160 µg/mL. The maximum dose was the lowest precipitating dose level.
- A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 20 µg/mL, in the 4(20)-hour exposure group in the presence of metabolic activation (S9), and at 160 µg/mL in the 4(20)-hour exposure group and the 24 hour continuous exposure group in the absence of metabolic activation.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 160 µg/mL in all three exposure groups. The test material induced no marked dose-related toxicity in any of the exposure groups.
- The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level was 40 and 20 µg/ml for the 4(20)-hour exposure groups in the absence an presence of S9 respectively, and was 10 µg/mL for the continuous exposure group.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate observations were made at the end of exposure in blood-free cultures and there was none noted at any level tested in all exposure groups. During slide assessment clear precipitate was observed on the slides at the maximum dose level tested in all three exposure groups.

MAIN EXPERIMENT
- The mitotic index data confirm the qualitative observations in that no marked dose-related inhibition of mitotic index was observed in any of the exposure groups. Therefore, the maximum dose level selected for metaphase analysis was the lowest precipitating dose level and maximum dose level investigated in all three exposure groups.
- 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 material did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation.
- The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The qualitative assessment of the slides determined that toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the maximum dose level tested in all three exposure groups.
Conclusions:
Under the conditions of this study, the test material was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guideline OECD 473 and Japanese guidelines, under GLP conditions with a chromosome aberration test in human lymphocytes in vitro.

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 this study, three exposure conditions were investigated; 4 hours exposure 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, 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 maximum dose investigated was limited by precipitate to 160 µg/mL. The results indicated that in the main test the maximum concentration should be limited by the presence of precipitate on the resultant slides. The dose levels selected for the Main Experiment were as follows:

- 4(20)-hour without S9: 1.25, 2.5, 5.0, 10, 20, 30 and 40 µg/mL;

- 4(20)-hour with S9 (2 %): 1.25, 2.5, 3.75, 5.0, 7.5, 10 and 20 µg/mL; and,

- 24-hour without S9: 0.313, 0.625, 1.25, 2.5, 3.75, 5.0 and 10 µg/mL.

All vehicle 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 was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

Under the conditions of this study, the test material was considered to be 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

Ames Test

The test material was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test material was dissolved in DMSO at a concentration of 50 mg/mL. Concentrations of 5 000; 2 500; 1 000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test were 5 000, 1 581, 500, 158.1, 50 and 15.81 μg/plate, and in the Confirmatory Mutation Test were 5 000, 1 581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Precipitate of the test material was detected in all examined bacterial strains with and without metabolic activation in the Initial Mutation Test on the plates at 5 000 μg/plate concentration. The same effect was observed in the Confirmatory Mutation Test in all Salmonella typhimurium bacterial strains with and without metabolic activation on the plates at 5 000 and 1 581 μg/plate concentrations and in Escherichia coli WP2 uvrA strain with and without metabolic activation at 5 000 μg/plate concentration.

Note: In these concentrations, the background lawn development could not be assessed due to the strong precipitate, but the colony counting was not affected. Inhibitory, cytotoxic effect of the test material was not detected in the Initial Mutation Test and Confirmatory Mutation Test.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test material has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

In Vitro Mammalian Chromosome Aberration Test

The genetic toxicity of the test material was investigated in accordance with the standardised guideline OECD 473 and Japanese guidelines, under GLP conditions with a chromosome aberration test in human lymphocytes in vitro. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 this study, three exposure conditions were investigated; 4 hours exposure 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, 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 maximum dose investigated was limited by precipitate to 160 µg/mL. The results indicated that in the main test the maximum concentration should be limited by the presence of precipitate on the resultant slides. The dose levels selected for the Main Experiment were as follows:

- 4(20)-hour without S9: 1.25, 2.5, 5.0, 10, 20, 30 and 40 µg/mL;

- 4(20)-hour with S9 (2 %): 1.25, 2.5, 3.75, 5.0, 7.5, 10 and 20 µg/mL; and,

- 24-hour without S9: 0.313, 0.625, 1.25, 2.5, 3.75, 5.0 and 10 µg/mL.

All vehicle 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 was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

Under the conditions of this study, the test material was considered to be non-clastogenic to human lymphocytes in vitro.

Mouse Lymphoma Assay

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 490, EU Method B17, OPPTS 870.5300 and in alignment with other Japanese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

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 material at eight dose levels in duplicate, together with vehicle (DMSO), 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 material used in the main test was selected following the results of a preliminary toxicity test at a concentration range of 1.25 to 320 μg/mL. The maximum dose level used in the Mutagenicity Test was limited by precipitate. The dose levels plated for viability and expression of mutant colonies were 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL.

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 within the historical control data range, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant or dose related (linear-trend) increases in either of the three exposure groups. The GEF value was not exceeded at any test material concentration.

Under the conditions of this study, the test 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 in this assay.

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.