Fatty acids, tallow, compds. with triethanolamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation

The potential of the test material to cause mutagenic effects in bacteria was assessed. Under the conditions of the study, the test material was considered to be non-mutagenic.

Chromosome Aberration

The potential of the test material to induce structural chromosomal aberrations was determined. 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

A study was conducted to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. Under the conditions of this study the test material is not mutagenic in the presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 January 2016 to 25 January 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: mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- 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.
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: 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.
- 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: 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:

Preliminary Cytotoxicity Test:
0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, and 2500 µg/mL for 4-hour treatment without S9, 4-hour treatment with S9 (2 %) and 24-hour treatment without S9.
Main Mutagenicity Test:
2.5, 5, 10, 20, 40 and 80 µg/mL for 4-hour treatment without S9, 4-hour treatment with S9 (2 %) and 24-hour treatment without S9.
The maximum dose level in the main Mutagenicity Test was limited by the onset of precipitate in the Preliminary Cytotoxicity Test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Following solubility checks performed on the test material, the test material was accurately weighed and formulated in acetone prior to serial dilutions being prepared.
Acetone is toxic to L5178Y cells at dose volumes greater than 0.5 % of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5 % to give a maximum achievable dose level of 2500 µg/mL. There was no marked change in pH when the test item was dosed into media in the solubility test 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 item formulation. The test item 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:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate (EMS) at 400 µg/mL and 150 µg/mL in the 4- and 24-h exposure groups without metabolic activation. Cyclophosphamide (CP) at 1.5 µg/mL was used in the presence of metabolic activation. Both were formulated in DMSO.

Details on test system and experimental conditions:

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 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation.

DURATION
- Pre-incubation period:
- Exposure duration: 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 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.
- 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.
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.

NUMBER OF REPLICATIONS: Performed in duplicate

- 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 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. 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 color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

- CALCULATION OF PERCENTAGE RELATIVE SUSPENSION GROWTH (%RSG)
The cell counts obtained immediately post treatment 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 = (-1n 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 = 5V / 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.

- OTHER: 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 9.77 to 2500 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 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.

Rationale for test conditions:

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) For non-toxic test items the upper test item concentrations was 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration was 5 mg/mL.
ii) Precipitating dose levels are not 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).

Evaluation criteria:

See below.

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. The statistical package used indicates the presence of statistically significant increases and linear-trend eve

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 examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared; acetone is toxic to L5178Y cells at dose volumes greater than 0.5 % of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5 % to give a maximum achievable dose level of 2500 µg/mL.
- Precipitation: The precipitate of the test item was observed at and above 39.06 µg/mL at the end of the preliminary cytotoxicity test exposure period. Therefore, the maximum dose level in the subsequent Mutagenicity Test was limited by the onset of precipitate.
In the Main Study, precipitate of the test item was observed at 40 and 80 µg/mL at the end of the exposure period.

RANGE-FINDING/SCREENING STUDIES: The dose range of the test material used in the preliminary toxicity test was 9.77 to 2500 µg/mL. There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls in all three of the exposure groups. However, the most marked reductions were only observed at dose levels at and / or above the onset of test material precipitate. The precipitate of the test material was observed at and above 39.06 µg/mL at the end of the exposure period. Therefore, the maximum dose level in the subsequent Mutagenicity Test was limited by the onset of precipitate.

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

- OTHER OBSERVATIONS: 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 or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels, in any of the three exposure groups.
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.

Conclusions:

Under the conditions of this study, the test material is not mutagenic in the presence or absence of metabolic activation.

Executive summary:

A study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was conducted in accordance with the standardised guidelines OECD 490, EU Method B.17 and EPA OPPTS 870.5300 under GLP conditions.

One main mutagenicity test was performed. In the 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 (acetone), 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. The dose levels plated for viability and expression of mutant colonies were 2.5, 5, 10, 20, 30, 40 and 80 µg/mL for 4-hour treatment without S9, 4-hour treatment with S9 (2 %) and 24-hour treatment without S9. The maximum dose level used in the Mutagenicity Test was limited by the presence of precipitate of the test material. Precipitate of the test material was observed at 40 and 80 µg/mL in the 4-hour exposure groups, and at 40 µg/mL in the 24-hour exposure group, at the end of the exposure period in the Mutagenicity 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 material did not induce any toxicologically significant 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 this study the test material is not mutagenic in the presence and absence of metabolic activation.

Under the conditions of this study, the test material is not mutagenic in the presence and absence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 November 2015 to 18 December 2015

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

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy
Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of
31 March 2011.

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
- Treatment of test material prior to testing:
The test item was insoluble in Minimum Essential Media (MEM) and Dimethyl sulphoxide (DMSO) at 50 and 500 mg/mL respectively but was soluble in Acetone at 500 mg/mL in solubility checks performed in house. With acetone being toxic to human lymphocytes when dosed at 1 % v/v the dose volume was lowered to 0.05 % which resulted in the maximum practical dose level being reduced to 2500 µg/mL. Prior to each experiment, the test item was accurately weighed, formulated in acetone and appropriate serial dilutions prepared.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.
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. 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.

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, a non-smoking volunteer 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.
- Sex, age and number of blood donors:
Preliminary toxicity test: One female aged 23 years and one male aged 30 years.
Main experiment: One female aged 29 years.
- Whether whole blood or separated lymphocytes were used: Sufficient whole blood was drawn from the peripheral circulation.

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. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Properly maintained: Yes

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 Toxicity Test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL for 4 hour exposure with and without S9 and 24-hour without S9
Chromosome Aberration Main Experiment: 0, 2.5, 5, 10, 20, 40 and 80 µg/mL for 4 hour exposure with and without S9, and 24-hour without S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was considered incompatible with aqueous media and immiscible in dimethyl sulphoxide at 200 mg/mL. However, the test material was fully miscible in acetone at 500 mg/mL in solubility checks performed in-house.

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:

Without S9, mitomycin C was used at 0.4 and 0.1 µg/mL for 4(20)-hour cultures and 24-hour continuous exposure cultures, respectively, dissolved in Minimal Essential Medium. With S9, cyclophosphamide (CP) was used at 2 µg/mL in dimethyl sulphoxide.

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium
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:
8.05-9.05 mL MEM, 10 % (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

- With Metabolic Activation (S9) Treatment
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 0.1 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. 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.

- Without Metabolic Activation (S9) Treatment
For the 4(20)-hour exposure in the absence of 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 resuspended 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 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.
For the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 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 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.

DURATION
- Exposure duration: 4 hours for Experiment 1 with and without S9 and Experiment 2 without S9. 24 hours for Experiment 2 with S9.
- Expression time (cells in growth medium): 20 hours for Experiment 1 with and without S9 and Experiment 2 without S9. Experiment 2 with S9 was exposed to the test material concentrations for 24 hours continuously.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours for all experiments.

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
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 labeled with the appropriate identification data. 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: Performed in duplicate.

NUMBER OF CELLS EVALUATED:
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. The slides were coded using a computerized random number generator. Supplementary slides were coded manually.
Where possible the first 150 consecutive well-spread metaphases from each culture were counted; 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. 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. A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

OTHER EXAMINATIONS:
- Determination of polyploidy: 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.

Evaluation criteria:

EVALUATION CRITERIA
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 analyzed.

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

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

When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploid and endoreduplications.

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:

Remarks:

Human peripheral blood lymphocytes

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 test material was considered to have no significant effect on change in pH when the test material was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Precipitation: The qualitative assessment of the slides determined that precipitate was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum dose level of test item (80 µg/mL) in all three exposure groups.
Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 40 µg/mL in all three exposure groups.The maximum dose level selected for metaphase analysis was, therefore, the lowest precipitating dose level (40 µg/mL). Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 40 µg/mL levels in the 4(20)-hour exposure group in the absence of S9 and in the 24-hour continuous exposure group and at 80 µg/mL, in the 4(20)-hour exposure group in the presence of S9.

RANGE-FINDING/SCREENING STUDIES:
A cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 39.06 µg/mL, in all three exposure groups. Greasy/oily precipitate was observed at and above 312.5 µg/mL in the 4(20)-hour exposure group in the absence of S9, at and above 39.06 µg/mL in the 4(20)-hour exposure group in the presence of S9 and at and above 625 µg/mL in the continuous exposure group.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2500 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was also 2500 µg/mL.

The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level for all three exposure groups.

MAIN CHROMOSOME ABERRATION STUDY:
The mitotic index data for the Main Experiment confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, no mitotic inhibition was achieved. In the presence of S9, a dose-related inhibition of mitotic index of 48 % and 10 % was observed at 40 and 80 µg/mL respectively. An inhibition of mitotic index of 24 % was noted at 40 µg/mL in the 24-hour continuous exposure group.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
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 groups.

Remarks on result:

other: The test material was considered not to induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the presence or absence of a liver enzyme metabolising system.

Assay Validity

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 analyzed.

 

 

Conclusions:

Under the conditions of the study, the test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either with or without metabolic activation and was therefore 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 were treated with the test material and evaluated for chromosome aberrations. The dose levels selected for the main mutagenic test were were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected were 0, 2.5, 5, 10, 20, 40 and 80 µg/mL for 4 hour exposure with and without S9 and 24-hour 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.

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

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November 2015 to 30 November 2015

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

Version / remarks:

Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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 University of California, Berkeley and the British Industrial Biological Research Association.
- Methods for maintenance in cell culture: 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 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.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cultures were maintained in nutrient broth supplied by Oxoid Limited. Plate incorporation and pre-incubation methods used trace amino-acid supplemented liquid media overlayed onto Vogel-Bonner agar plates.
- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate. All were found to be satisfactory.

Additional strain / cell type characteristics:

other: S. typhimurium: all strains possess rfa- and uvrB-; TA98 and TA100 also possess the R-factor plasmid pKM101. E. coli strain possesses the uvrA- mutation

Metabolic activation:

with and without

Metabolic activation system:

S9-mix - rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors).

Test concentrations with justification for top dose:

Experiment 1 - Plate Incorporation Method
1.5, 5, 15, 50, 150, 500, 1 500 and 5 000 µg/plate. The maximum concentration is the maximum recommended dose level.

Experiment 2 - Pre-Incubation Method
5, 15, 50, 150, 500, 1 500 and 5 000 µg/plate. The dose range used for Experiment 2 was determined by the results of Experiment 1 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone.
- Justification for choice of solvent/vehicle: Solubility checks performed in-house determined that the test material was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 50 and 100 mg/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

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:

- EXPERIMENT 1
METHOD OF APPLICATION: Plate Incorporation Method
Without metabolic activation: 0.1 mL of the appropriate concentration of test material, 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.
With Metabolic Activation: The procedure was the same as described above except that following the addition of the test material formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: Each concentration of the test material, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

DETERMINATION OF CYTOTOXICITY
- Method: Examined for effects on the background lawn of bacterial growth.


- EXPERIMENT 2
METHOD OF APPLICATION: Pre-Incubation Method
Without metabolic activation: 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the test material formulation or 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.
With metabolic activation: The procedure was the same as described previously except that following the addition of the test material 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.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: Each concentration of the test material, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

DETERMINATION OF CYTOTOXICITY
- Method: Examined for effects on the background lawn of bacterial growth.

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.

Acceptance Criteria
See below.

Statistics:

Statistical analysis of data was performed in line with the recommendations of UKEMS.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

E. coli WP2 uvr A

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:

TEST-SPECIFIC CONFOUNDING FACTORS
A test material film (cream coloured in appearance) was noted at 5000 µg/plate following visual assessment of the plates in the first mutation test after employing the plate incorporation method. However, in the second mutation test (pre-incubation methodology) a globular precipitate was noted at and above 1500 µg/plate with an associated film observed at 5000 µg/plate. None of these observations prevented the scoring of revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of Salmonella strains TA100 and TA1535 in the absence and presence of S9 mix and TA1537 in the absence of S9-mix at 5000 µg/plate. No toxicity was noted to Salmonella strains TA1537 in the presence of S9-mix, or TA98 and Escherichia coli strain WP2uvrA in the absence or presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second mutation test. Toxicity results from Experiment 2 (pre-incubation method) were very similar to the first mutation test with visible reductions in the growth of the bacterial background lawns noted at 5000 µg/plate to Salmonella strains TA100, TA1537 and TA1535 in the absence and presence of S9 mix. No toxicity was noted to Salmonella strain TA98 and Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.

There were no toxicologically 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 toxicologically 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). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 50 µg/plate (WP2uvrA dosed in the absence of S9-mix and TA100 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.

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, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation method (Experiment 1) and pre-incubation methods (Experiment 2) with and without metabolic activation. The dose levels assessed were 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate for Experiment 1. The dose range for Experiment 2 was amended to 15, 15, 50, 150, 500, 1500 and 5000 µg/plate following the results of Experiment 1.

The vehicle (acetone) 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. In Experiment 1, the test material induced a visible reduction in the growth of the bacterial background lawns of Salmonella strains TA100 and TA1535 in the absence and presence of S9-mix and TA1537 in the absence of S9-mix at 5000 µg/plate. No toxicity was noted to Salmonella strains TA1537 in the presence of S9-mix, or TA98 and Escherichia coli strain WP2uvrA in the absence or presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate in experiment 2. Toxicity results from Experiment 2 were very similar to experiment 1 with visible reductions in the growth of the bacterial background lawns noted at 5000 µg/plate to Salmonella strains TA100, TA1537 and TA1535 in the absence and presence of S9-mix. No toxicity was noted to Salmonella strain TA98 and Escherichia coli strain WP2uvrA in the absence and presence of S9-mix. A test material film (cream coloured in appearance) was noted at 5000 µg/plate following visual assessment of the plates in the first mutation test after employing the plate incorporation method. However, in the second mutation test (pre-incubation methodology) a globular precipitate was noted at and above 1500 µg/plate with an associated film observed at 5000 µg/plate. None of these observations prevented the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2. Small, statistically significant increases in revertant colony frequency were observed in experiment 1 at 50 µg/plate (WP2uvrA dosed in the absence of S9-mix and TA100 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a doseresponse relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.

Under the conditions of this study, the test material was 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

In vitro data.

The following studies were all awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Bacterial reverse mutation

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, TA100 and Escherichia coli strain WP2uvrA were treated with the test material, using the plate incorporation method (Experiment 1) and pre-incubation methods (Experiment 2) with and without metabolic activation. The dose levels assessed were 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate for Experiment 1. The dose range for Experiment 2 was amended to 15, 15, 50, 150, 500, 1500 and 5000 µg/plate following the results of Experiment 1. The vehicle (acetone) 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. In Experiment 1, the test material induced a visible reduction in the growth of the bacterial background lawns of Salmonella strains TA100 and TA1535 in the absence and presence of S9-mix and TA1537 in the absence of S9-mix at 5000 µg/plate. No toxicity was noted to Salmonella strains TA1537 in the presence of S9-mix, or TA98 and Escherichia coli strain WP2uvrA in the absence or presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate in Experiment 2. Toxicity results from Experiment 2 were very similar to Experiment 1 with visible reductions in the growth of the bacterial background lawns noted at 5000 µg/plate to Salmonella strains TA100, TA1537 and TA1535 in the absence and presence of S9-mix. No toxicity was noted to Salmonella strain TA98 and Escherichia coli strain WP2uvrA in the absence and presence of S9-mix. A test material film (cream coloured in appearance) was noted at 5000 µg/plate following visual assessment of the plates in the first mutation test after employing the plate incorporation method. However, in the second mutation test (pre-incubation methodology) a globular precipitate was noted at and above 1500 µg/plate with an associated film observed at 5000 µg/plate. None of these observations prevented the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation (S9-mix) in Experiment 2. Small, statistically significant increases in revertant colony frequency were observed in Experiment 1 at 50 µg/plate (WP2uvrA dosed in the absence of S9-mix and TA100 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.

Under the conditions of this study, 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 and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals.

Duplicate cultures of human lymphocytes were treated with the test material and evaluated for chromosome aberrations. The dose levels selected for the main mutagenic test were were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected were 0, 2.5, 5, 10, 20, 40 and 80 µg/mL for 4 hour exposure with and without S9 and 24-hour 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.

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

A study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was conducted in accordance with the standardised guidelines OECD 490, EU Method B.17 and EPA OPPTS 870.5300 under GLP conditions.

One main mutagenicity test was performed. In the 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 (acetone), 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. The dose levels plated for viability and expression of mutant colonies were 2.5, 5, 10, 20, 30, 40 and 80 µg/mL for 4-hour treatment without S9, 4-hour treatment with S9 (2 %) and 24-hour treatment without S9. The maximum dose level used in the Mutagenicity Test was limited by the presence of precipitate of the test material. Precipitate of the test material was observed at 40 and 80 µg/mL in the 4-hour exposure groups, and at 40 µg/mL in the 24-hour exposure group, at the end of the exposure period in the Mutagenicity 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 material did not induce any toxicologically significant 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 this study the test material is not mutagenic in the presence or absence of metabolic activation.

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation 1272/2008, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available genetic toxicity studies.