Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: not mutagenic

Chromosome aberration test: not clastogenic

Mouse lymphoma test (OECDTG490): not mutagenic

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:
1 December 1998 - 5 January 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
other: GLP standards for toxicity testing of chemicals that describing the testing facility require for testing new and specific chemical compounds (Precept No.4, Kankiken No. 233, Eisei No. 38, 63 Kikyoku No.823: revised on November 18, 1988)
Qualifier:
according to guideline
Guideline:
other: GLP standards for toxicity testing of chemicals, Ministry of Labour, Japan (Notification No .76, September 1, 1988).
Qualifier:
according to guideline
Guideline:
other: the Guidelines for screening toxicity testing of chemicals describing the partially revised test procedures require for testing new chemical compounds (Kanpoan No. 287, Eisei No. 127: October 31, 1997; Kikyoku No. 2: October 31, 1997)
Qualifier:
according to guideline
Guideline:
other: the Guidelines for the standards of bacterial mutagenicity testing, Ministry of Labour, Japan (Notification No. 67, June 2, 1997).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenate S9 mix of rat treated with phenobarbital and 5,6-benzoflavone.
- method of preparation of S9 mix: To prepare S9mix, G-6-P and NADH and NADPH were dissolved in distilled water. Then 0.4M MgCl2, 1.65M KCI and phosphate buffer solution were added. The mix was filtered for sterilization and then S9 was added. The S9mix was kept in ice water bath during use. S9-mix contained per 1 mL: 4 µmol NADPH, 4 µmol NADH, 5 µmol glucose-6-phosphate, 100 µmol sodium phosphate buffer pH 7.4; 8 µmol MgCl2 ; 0.33 µmol KCl and 0.1 mL S9.
Test concentrations with justification for top dose:
Pre-incubation assay:
Experiment 1 (without and with S9) TA100, TA1535, TA1537 and TA98 and WP2uvrA: 5, 10, 50, 100 , 500, 1000, 5000,μg/plate
Experiment 2 (without and with S9) TA100, TA1535, TA1537 and TA98 and WP2uvrA: 156.3, 312.5, 625, 1250, 2500, 5000 μg/plate

The top dose was the highest dose required to be tested.
Vehicle / solvent:
Solvent: dimethylsulphoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide in DMSO 0.1 μg/plate, 0.01 μg/plate, 0.01 μg/plate for TA98, TA100 and WP2uvrA, resp.
Remarks:
Without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA) in DMSO, 0.5 μg/plate for TA98, 1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2uvrA
Remarks:
With S9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in duplicate in each strain for the test item and the positive control and in triplicate for the negative contro group. Two independent experiments were conducted.

METHOD OF TREATMENT/ EXPOSURE:
in agar; preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Examining the reduction of the bacterial background lawn using a microscope.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Amount of revertant colonies.

OTHER:
- The presence of precipitation of the test compound on the plates was determined.
- Concentration of the test substance resulting in precipitation: 2500 µg/plate and higher
Evaluation criteria:
A result was judged positive (+) when the number of revertant colonies had a 2-fold or greater increase as compared to that in the negative control group, and when the result was reproducible and dependent upon doses of the test substance; and the other cases were judged negative (-). Bacterial growth inhibition was judged positive when the background lawn of the test substance group was sparse or thin as compared to that of the negative control group.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
(> 5000 µg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
See attachement for detailed results

TEST CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the substance on the plates was observed at 2500 μg/plate and higher in the experiments with and without metabolic activation

The negative and positive control group were within the historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Conclusions:
In an Ames test, performed in accordance with GLP principles, the substance was found not to be mutagenic with or without metabolic activation.
Executive summary:

An Ames performed in accordance with GLP principles. The test item was tested up to concentrations of 5000 μg/plate with and without metabolic activation in duplicate in two independent experiments (pre-incubation). Precipitation of the substance was observed at 2500 μg/plate and higher in the experiments with and without metabolic activation. No cytotoxicity was observed. The negative and positive control group were within the historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not show a 2-fold or greater increase as compared to that in the negative control group in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in both experiments. In conclusion, based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 December 1998 - 10 February 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
other: see Version/remarks
Version / remarks:
Guidelines for screening toxicity testing of chemicals that describing the partially revised test procedures require for testing new chemical compounds (establishment of the method for screening toxicity testing) (Kanpogyo No. 700, Yakuhatsu No. 1039, 61 Kikyoku No. 1014: December 5, 1986, the GLP standards for toxicity testing of chemicals, Ministry of Labour, Japan (Notification No. 76, September 1, 1988)
Qualifier:
according to guideline
Guideline:
other: see Version/remarks
Version / remarks:
Guidelines for revisions in the standards for the chromosomal aberration test using mammalian cells in culture and in the form of reporting of test results (Kihatsu No. 652: September 29, 1997)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHL/IU, a fibroblast cell line, were obtained from the Cell Bank of National Institute of Health Sciences on October 31, 1991.

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: Upon receipt they were cultured and stored frozen in liquid nitrogen according to the method of Ishidate et al.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
A 9.6 g of powdered Eagle's MEM (Lot No. 1012299, Gibco co.) was dissolved in 950 mL of ultra purified water (containing 2.2 g NaHC03), pH was adjusted to 7.2 with 0.1 N HCI, and the solution was filtered for sterilization using a syringe filter (0.2 µm, Corning Co.). A 900 mL of the solution was collected and 100 mL of heat inactivated fetal calf serum (Calf serum, Lot No. 1010641, Gibco Co.) was added. A CO2 incubator was used for culture, which was conditioned at a temperature of 37°C, a humidity of 95% , and CO2 concentration of 5%.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : The S9 (Lot No.RAA-392) induced by Phenobarbital and 5,6-Benzoflavone was purchased from Kikkoman Corporation.
- method of preparation of S9 mix: To prepare S9 mix, G-6-P ( Lot No. 115802) and NADP (Lot No.040714) obtained from Oriental Yeast Co., Ltd. were dissolved in distilled water. Then solutions of 50mM MgC12, 330M KCI and HEPES buffer solution were added. The mixture was filtered for sterilization and then S9 was added. The S9mix was left in ice water bath until use.
- concentration or volume of S9 mix and S9 in the final culture medium: final concentration of 5%
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 6hr, 24hr and 48hr exposure; 24hr and 48hr fixation: 7, 21, 62, 185, 556, 1667 and 5000 µg/mL
With S9-mix, 6hr exposure; 24hr fixation: 7, 21, 62, 185, 556, 1667 and 5000 µg/mL
Cytogenetic test:
Without and with S9-mix, 6hr exposure time, 24hr fixation time: 175, 350 and 700 µg/mL
Without S9-mix, 24hr exposure time, 24hr fixation time: 175, 350 and 700 µg/mL
Without S9-mix, 48hr exposure time, 48hr fixation time: 125, 250 and 500 µg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The test substance was dissolved in DMSO.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: growth inhibition test: duplicate; cytogenic assay: single
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10^4 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 72 hr
- Exposure duration/duration of treatment: 6 hr (with and without S9-mix), 24 hr and 48 hr (without S9-mix)
- Harvest time after the end of treatment (sampling/recovery times): 24 hr and 48 hr

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): At 2 hours prior to the end of incubation, colcemid solution was added to each petri dish to a final concentration of 0.2 µg/mL
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After the incubation was completed, cells were separated with 0.25% trypsin which had been maintained at 370 collected by centrifugation (1000rpm, 5 min), and treated in hypotonic condition with 0.075M KCI solution at 370 for 15 min. Cooled Carnoy's fluid (a mixture of methanol and acetic acid at a ratio of 3: 1) was added in a volume of 0.5-1 mL and centrifuged, the resulting supernatant was removed, and the cells were again fixed with 5 mL of Carnoy's fluid. This procedure was then repeated twice. The cell suspension obtained was dropped onto the slide glass (2 places) that had placed on moistened gauze. To prepare slide specimens, the slide glasses were dried over a night, stained for approximately 15 min with Giemsa stain (Lot No. 640181934, Merck Co.) which was adjusted to 1.5% with phosphate buffer (pH 6.8), and then washed with water and dried.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): well-spread 100 cells (200 cells/dose) in metaphase per plate were observed
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): no data
- Determination of polyploidy: yes
- Determination of endoreplication: no

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cells were fixed with 10% formalin solution and stained with 0.1% crystal violet for 10 min. The cell growth rate was indirectly determined by measuring the rate of staining of the petri dishes using a monocellator (Olympus Co., Ltd.). The results obtained were shown by diagrams, and approximate 50% cell growth inhibition rate was calculated.
Evaluation criteria:
The result was judged positive when the number of cells with chromosomal aberrations increased dose-dependently compared to that of the concurrent negative control, or reproducibly increased at one or more doses, and the others were judged negative.
Statistics:
Not performed.
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was observed at a dose of 350 µg/mL or higher.

RANGE-FINDING/SCREENING STUDIES: The concentration that induces 50% cell growth inhibition was approximately 450 µg/mL for the cell lines with and without S9 and for the cell line of 24 hr incubation, and approximately 400 µg/mL for the cell line of 48 incubation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : In each positive control group, the incidence of cells with structural abnormality was markedly increased, and the values in the negative and positive control groups were approximate to those in the background data.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
The cell growth rates, which were concurrently measured in the chromosomal aberration test, were 95, 79 and 44% for respective doses of 175, 350 and 700 µg/mL in the cultured cell line without S9; 95, 76 and 45% for respective doses of 175, 350 and 700 µg/mL in the cell line with S9 in the short term incubations; 83, 71 and 40% for respective doses of 175, 350 and 700 µg/mL in the cell line of 24 hr incubation; and 98, 86 and 37% for doses of 125, 250 and 500 µg/mL in the cell line of 48 hr incubation.
- Genotoxicity results (for both cell lines and lymphocytes) :
The incidences of cells with structural and numerical abnormalities were 0-1.0 and 0-0.5% respectively for each cultured cell line, with no difference from that observed in the negative control group. The incidence of gaps was 0-1 gaps per dose, approximately the same incidence in the negative control group.

HISTORICAL CONTROL DATA: see the attached background material.
Conclusions:
A chromosome aberration study with the substance was performed according to Japanese guidelines and GLP principles, in cultured Chinese hamster lung (CHL/IU) cells in one experiment. It is concluded that the substance is not clastogenic in Chinese hamster lung (CHL/IU) cells.
Executive summary:

A chromosome aberration study with the substance was performed according to Japanese guidelines and GLP principles, in cultured Chinese hamster lung (CHL/IU) cells in one experiment. The test item precipitated at the concentrations of 350 µg/mL or higher. The cell growth rates, which were concurrently measured in the chromosomal aberration test, were 95, 79 and 44% for respective doses of 175, 350 and 700 µg/mL in the cultured cell line without S9; 95, 76 and 45% for respective doses of 175, 350 and 700 µg/mL in the cell line with S9 in the short term incubations; 83, 71 and 40% for respective doses of 175, 350 and 700 µg/mL in the cell line of 24 hr incubation; and 98, 86 and 37% for doses of 125, 250 and 500 µg/mL in the cell line of 48 hr incubation. Reliable positive and negative controls were included.

Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. No effects of the test item on the number of polyploid cells were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Based on the results it can be concluded that the substance is not clastogenic in cultured Chinese hamster lung (CHL/IU) cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Jul 2019 - 17 Sep 2019
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)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells:
L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells:
recommended test system in international guidelines

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: stock cultures of the cells were stored in liquid nitrogen (-150°C).
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: For 3 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
Selective medium:
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine (TFT).
Non-selective medium:
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).
Environmental conditions:
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 57 - 98%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 - 37.4°C).
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal enzymes (S9 homogenate) was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix:
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl ; 1.7 mg glucose-6-phosphate ; 3.4 mg NADP ; 4 μmol HEPES. The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 4% (v/v).
Test concentrations with justification for top dose:
- Dose-range finding test, with S9 (3h) and without S9 (3h): 7.8, 16, 31, 63 and 125 μg/mL
- Dose-range finding test, without S9 (24h): 7.8, 16, 31, 63 and 125 μg/mL
- Experiment 1, without S9 (3h): 0.98, 2.0, 3.9, 7.8, 16, 31, 63, and 125 μg/mL
- Experiment 1, with S9 (3h): 0.98, 2.0, 3.9, 7.8, 16, 31, 63, 125 and 250 μg/mL
- Experiment 2, without S9 (24h): 0.98, 2.0, 3.9, 7.8, 16, 31, 63, and 125 μg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: The test item formed a clearcolourless solution dimethyl sulfoxide at concentrations of 100 mg/mL and below.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): test concentrations: 1 per test concentration; positive control: 1; solvent control: 2
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Short term treatment: 3 hours (with and without S9-mix)
Prolonged treatment: 24 hours (without S9-mix)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selective agent): 11 or 12 days
- Method used: microwell plates
- Selective agent is used: 5 μg/mL trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
- Criteria for small (slow growing) and large (fast growing) colonies:
The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (see 'Any other information on materials and methods incl. tables' for details on calculations)
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more than the mutation frequency in the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of the mutation frequency in the controls + 126.

ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6)
Statistics:
Not performed
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: SPS-100 precipitated in the exposure medium at concentrations of 125 μg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- Both in the absence and presence of S9-mix, in the 3-hour and 24-hour exposure period, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 125 μg/mL compared to the suspension growth of the solvent control.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. Although in the presence of S9-mix the response of CP was just above the upper control limits, these limits are 95% control limits
and a slightly higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Gene mutation tests in mammalian cells:

- Results from cytotoxicity measurements:
No significant toxicity was observed, and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.

- Genotoxicity results:
No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with SPS-100 either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the SPS-100 treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

RESULT TABLES AND HISTORICAL CONTROL DATA
see attached background material.
Conclusions:
SPS-100 is not mutagenic in the TK mutation test system.
Executive summary:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and in accordance with GLP principles, to assess the potential of SPS-100 to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. In the first experiment, SPS-100 was tested up to concentrations of 125 μg/mL in the absence and

up to concentrations of 250 μg/mL in the presence of S9-mix. The incubation time was 3 hours. In the second experiment, SPS-100 was tested up to concentrations of 125 μg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at any dose level in the absence and presence of S9-mix. SPS-100 precipitated in the culture medium at 125 μg/mL therefore it was chosen to be the highest concentration analyzed for mutagenicty as recommended in the guideline. Solvent (negative) and positive controls were included and showed that the test system was valid. In the absence and presence of S9 -mix, SPS-100 did not induce a biologically relevant increase in the mutation frequency at any of the dose levels. In conclusion, SPS-100 is not mutagenic in the mouse lymphoma L5178Y test system.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

An Ames performed in accordance with GLP principles. The test item was tested up to concentrations of 5000 μg/plate with and without metabolic activation in duplicate in two independent experiments (pre-incubation). Precipitation of the substance was observed at 2500 μg/plate and higher in the experiments with and without metabolic activation. No cytotoxicity was observed. The negative and positive control group were within the historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not show a 2-fold or greater increase as compared to that in the negative control group in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in both experiments. In conclusion, based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Chromosome aberration test:

A chromosome aberration study with the substance was performed according to Japanese guidelines and GLP principles, in cultured Chinese hamster lung (CHL/IU) cells in one experiment. The test item precipitated at the concentrations of 350 µg/mL or higher. The cell growth rates, which were concurrently measured in the chromosomal aberration test, were 95, 79 and 44% for respective doses of 175, 350 and 700 µg/mL in the cultured cell line without S9; 95, 76 and 45% for respective doses of 175, 350 and 700 µg/mL in the cell line with S9 in the short term incubations; 83, 71 and 40% for respective doses of 175, 350 and 700 µg/mL in the cell line of 24 hr incubation; and 98, 86 and 37% for doses of 125, 250 and 500 µg/mL in the cell line of 48 hr incubation. Reliable positive and negative controls were included.

Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. No effects of the test item on the number of polyploid cells were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Based on the results it can be concluded that the substance is not clastogenic in cultured Chinese hamster lung (CHL/IU) cells.

Mouse lymphoma test:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and in accordance with GLP principles, to assess the potential of SPS-100 to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. In the first experiment, SPS-100 was tested up to concentrations of 125 μg/mL in the absence and up to concentrations of 250 μg/mL in the presence of S9-mix. The incubation time was 3 hours. In the second experiment, SPS-100 was tested up to concentrations of 125 μg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at any dose level in the absence and presence of S9-mix. SPS-100 precipitated in the culture medium at 125 μg/mL therefore it was chosen to be the highest concentration analyzed for mutagenicty as recommended in the guideline. Solvent (negative) and positive controls were included and showed that the test system was valid. In the absence and presence of S9 -mix, SPS-100 did not induce a biologically relevant increase in the mutation frequency at any of the dose levels. In conclusion, SPS-100 is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

Based on the results of the Ames test, in vitro chromosome aberration and mouse lymphoma studies, the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and its amendments.