Trisodium 5-[[4-chloro-6-[(o-tolyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames

The substance was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5 mg/plate in the absence and presence of S9 metabolic activation (OECD TG471).

Chromosome aberration

Based on the results of each treatment group, the test material was considered “negative” for the chromosome aberration-inducing potential.

Mouse Lymphoma Assay

Under the conditions of the study the test material was not found to have the potential to induce gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 03, 2016 to December 22, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-Aminoanthracene

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative 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:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Untreated negative 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:

not determined

Untreated negative 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:

not determined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Characteristics of five Salmonella typhimurium strains

Test strain	Histidine requirement	uvrB mutation	rfa mutation	Ampicillin resistance
TA98	+	+	+	+
TA100	+	+	+	+
TA102	+	ϴ	+	+
TA1535	+	+	+	ϴ
TA1537	+	+	+	ϴ

+ means had the characteristic; ϴ means did not have the characteristic

Table 2. Toxicity of the test article of Salmonella typhimurium TA100

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)
With S9 Mix	Negative control group	226
	Positive control group^a	1125
	Test group
	5	118
	2.5	171
	1.25	194
	0.625	200
	0.3125	207
Without S9 Mix	Negative control group	185
	Positive control group	657
	Test group
	5	160
	2.5	133
	1.25	179
	0.625	214
	0.3125	255

^a Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

 

Table 3. Reverse mutation test of Salmonella typhimurium TA98

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	42	32	49	41 ± 9
	Positive control group^b	284	314	344	314 ± 30*
	Test group
	5	55	44	53	51 ± 6
	2.5	44	65	42	50 ± 13
	1.25	42	42	33	39 ± 5
	0.625	54	49	53	52 ± 3
	0.3125	43	56	71	57 ± 14
Without S9 Mix	Negative control group	41	33	30	35 ± 6
	Positive control group	233	208	225	222 ± 13*
	Test group
	5	24	17	23	21 ± 4
	2.5	22	25	39	29 ± 9
	1.25	23	26	21	23 ± 3
	0.625	32	16	23	24 ± 8
	0.3125	30	40	26	32 ± 7

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: Benzo [a] pyrene (4.0μg/plate).

Without S9 Mix: 4-Nitroquinoline-N-oxide (0.5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 4. Reverse mutation test of Salmonella typhimurium TA100

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	219	295	205	240 ± 48
	Positive control group^b	1208	1429	1475	1371 ± 143*
	Test group
	5	203	195	218	205 ± 12
	2.5	237	247	197	227 ± 26
	1.25	194	200	196	197 ± 3
	0.625	159	257	235	217 ± 51
	0.3125	212	256	285	251 ± 37
Without S9 Mix	Negative control group	140	185	180	168 ± 25
	Positive control group	789	720	771	760 ± 36*
	Test group
	5	130	175	171	159 ± 25
	2.5	152	194	166	171 ± 21
	1.25	166	139	208	171 ± 35
	0.625	171	178	205	185 ± 18
	0.3125	186	154	223	188 ± 35

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 5. Reverse mutation test of Salmonella typhimurium TA102

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	434	392	422	416 ± 22
	Positive control group^b	897	881	911	896 ± 15*
	Test group
	5	443	520	460	474 ± 40
	2.5	425	437	443	435 ± 9
	1.25	461	465	439	455 ± 14
	0.625	460	379	425	421 ± 41
	0.3125	422	463	388	424 ± 38
Without S9 Mix	Negative control group	411	381	412	401 ± 18
	Positive control group	866	840	859	855 ± 13*
	Test group
	5	315	396	414	375 ± 53
	2.5	381	394	433	403 ± 27
	1.25	389	446	401	412 ± 30
	0.625	364	422	369	385 ± 32
	0.3125	435	397	402	411 ± 21

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (10μg/plate).

Without S9 Mix: Mitomycin C (0.5μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 6. Reverse mutation test of Salmonella typhimurium TA1535

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	19	25	27	24 ± 4
	Positive control group^b	276	307	300	294 ± 16*
	Test group
	5	29	25	19	24 ± 5
	2.5	23	20	16	20 ± 4
	1.25	14	19	24	19 ± 5
	0.625	28	18	16	21 ± 6
	0.3125	15	19	18	17 ± 2
Without S9 Mix	Negative control group	15	17	19	17 ± 2
	Positive control group	240	299	276	272 ± 30*
	Test group
	5	11	13	16	13 ± 3
	2.5	19	23	21	21 ± 2
	1.25	22	18	24	21 ± 3
	0.625	13	21	20	18 ± 4
	0.3125	21	19	18	19 ± 2

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: Sodium azide (0.4μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Table 7. Reverse mutation test of Salmonella typhimurium TA1537

Group	Test article (mg/plate)	Reverse mutant colony number (CFU/plate)			Average of colonies^a
		1	2	3
With S9 Mix	Negative control group	9	12	13	11 ± 2
	Positive control group^b	417	451	409	426 ± 22*
	Test group
	5	10	8	15	11 ± 4
	2.5	9	15	14	13 ± 3
	1.25	14	17	13	15 ± 2
	0.625	17	11	14	14 ± 3
	0.3125	21	16	18	18 ± 3
Without S9 Mix	Negative control group	14	12	10	12 ± 2
	Positive control group	1019	947	1207	1058 ± 134*
	Test group
	5	10	12	10	11 ± 1
	2.5	8	8	15	10 ± 4
	1.25	7	9	10	9 ± 2
	0.625	14	9	9	11 ± 3
	0.3125	16	8	7	10 ± 5

^a Average of colonies was shown as Mean ± S.D., the data were triplicate.

^b Positive control group: With S9 Mix: 2-Aminoanthracene (4.0μg/plate).

Without S9 Mix: 9-Aminoacridine (50.0μg/plate).

^*Reverse mutant colony number were twice more than negative control group, ρ < 0.05 (One-Way ANOVA).

 

Conclusions:

According to OECD 471 test method, the substance was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5 mg/plate.

Executive summary:

This test using the procedures outlined in the SuperLub Study Plan for M62-151100096001EN which is based on the SOP for the OECD 471 (SOPF-203) and OECD 471 (OECD, 1997).The results of this OECD 471 test for the substance show that test validity criteria was met.

Based on the preliminary assay results, 5mg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of the substance at 0.3125, 0.625, 1.25, 2.5 and 5mg/plate, concurrent negative and strain-specific positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 Mix activation. No genotoxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that the substance did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5mg/plate either in the absence or in the presence of metabolite activation.

Based on the data obtained from this study, it was concluded that under the test condition, the substance was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5mg/plate in the absence and presence of S9 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 October 2019 to 13 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Solubility and stability in solvents:
- Saline: Soluble at 20.0 mg/mL (Stable)
- DMSO: Soluble at 200 mg/mL (Stable)
- Acetone: Solubility ≤0.001 g/L
(The test material was considered to be stable in the solvent if no changes including discolouration, heat generation, and foam formation were observed in the prepared solution for 2 hours)

Correction for test material purity was made in preparation of the test solutions (made by stirring, and serial dilution to the specified concentration). Test soutions were stored at 23-24°C and used within 30 minutes of preparation.

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Health Science Research Resources Bank, Japan Health Science Foundation
- Suitability of cells: CHL/IU cells had relatively larger and fewer chromosomes, which were suitable for observation. There are also abundant background data on the effects of chemicals on CHL/IU cells.

For cell lines:
- Freeze preservation conditions: Tubes cryopreservation solution for cells and cells at a density of 1.0 × 10^6 cells/mL were stored in liquid nitrogen.
- Absence of Mycoplasma contamination: yes (Mycoplasma contamination was not detected)
- Number of passages if applicable: The passage number of cells was 5 at the time of receipt. Cells passaged 11 times in total were frozen for preservation (Freeze Lot No. 171124). After thawing, cells whose passage number was 30 or less were used for the test.
- Cell characterisation: No morphological aberration was detected. The modal chromosome number was 25. The cell doubling time was approximately 14 hours.
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

MEDIA USED
Eagle's MEM supplemented with Foetal bovine serum at a concentration of 10% was used as a culture medium. Foetal bovine serum was inactivated at the Test Facility.

CULTURE CONDITIONS
- Culture dish: 60-mm plastic Petri dish (effective culture area = 20.8 cm²)
- Culture temperature and CO2 concentration: 37°C and 5%
- Preculture conditions: 2 × 10^4 cells were inoculated in the Petri dish containing 5 mL of medium and cultured for 3 days.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : manufactured by Oriental Yest Co., Ltd. (prepared from 7 week old male Sprague-Dawley derived rats, dosed i.p. with phenobarbital (30 mg/kg 4 days before killing and 60 mg/kg 1, 2 & 3 days before killing) and 5,6-benzoflavone (80 mg/kg 2 days before killing) and stored at approximately -80 °C)
- method of preparation of S9 mix: The S9 mix (1 mL) contained: S9 (0.3 mL), MgCl2 (5 µmol), KCl (33 µmol), glucose-6-phosphate (5 µmol), NADP (4 µmol), HEPES (4 µmol) in purified water (0.7 mL).
- concentration or volume of S9 in the final culture medium : 5% (1.0 mg/mL)

Test concentrations with justification for top dose:

Short-term treatment process (6-18 h)
- Cell growth inhibition test: 0, 0.0313, 0.0625, 0.125, 0.250, 0.500, 1.00, 2.00 mg/mL (with and without metabolic activation)
- Chromosome aberration test: 0, 0.500, 1.00, 2.00 mg/mL (without metabolic activation); 0, 0.354, 0.500, 0.707, 1.00, 1.41, 2.00 mg/mL (with metabolic activation)

Continuous treatment process (24-0 h)
- Cell growth inhibition test: 0, 0.0313, 0.0625, 0.125, 0.250, 0.500, 1.00, 2.00 mg/mL (without metabolic activation)
- Chromosome aberration test: 0, 0.125, 0.177, 0.250, 0.354, 0.500, 0.70 mg/mL (without metabolic activation; first test); 0, 0.199, 0.250, 0.315, 0.397, 0.500, 0.630, 0.794, 1.00 mg/mL (without metabolic activation; second test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle: The molecular weight of the test material is 788.06, thus, the solubility of the test material was checked at the final concentration of 2.00 mg/mL in the media. The test material was soluble at 20.0 mg/mL in saline and 200 mg/mL in DMSO; therefore saline was selected as a solvent.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

no

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
Cell growth inhibition tests: single
Chromosome aberration tests: duplicate
- Number of independent experiments
Cell growth inhibition tests: single test using short-term treatment process (with and without metabolic activation); single test using continuous treatment process (without metabolic activation)
Chromosome aberration tests: single test using short-term treatment process (with and without metabolic activation); two tests using continuous treatment process (without metabolic activation)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: each dish was inoculated with 2 × 10^4 cells after 3 days of precultivation
- Test material added in medium (0.3 mL for short-term treatment process; 0.5 mL for continuous treatment process)

TREATMENT PROCESS
- Cell growth inhibition tests: Test material solution was mixed with medium. Test tubes used for the test with metabolic activation were also supplemented with 0.5 mL of S9 mix and mixed the medium. Immediately the medium in culture dishes was replaced with those in test tubes, and these dishes were incubated in the CO2 incubator for 6 hours (short-term treatment process) or 24 hours (continuous treatment process). Each culture dish contained 3 mL (short-term treatment process) or 5 mL (continuous treatment process) of the medium in total during treatment with the test material. After treatment, dishes were checked for the precipitation of test material.
In the short term treatment process, dishes were washed with PBS to remove the test material, added with 5 mL of the fresh medium, and incubated for 18 hours. After incubation, the status of cells was observed under the inverted phase-contrast microscope, and then the cytotoxicity of test material was evaluated.
In the continuous treatment process the status of cells was observed under the inverted phase-contrast microscope, and then the cytotoxicity of test material was evaluated directly following the check for test material precipitation.
- Chromosome aberration tests: Preculture and treatment with the test material were performed in the same manner as the cell growth inhibition test, and chromosome specimens were prepared.
Since elongation of cell cycle was not observed at continuous treatment process for 24 hours, cell growth inhibition test and chromosome aberration test using continuous treatment process for 48 hours were not carried out.

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor: To obtain metaphase cells, colcemid solution was added to the culture plates at the final concentration of 0.2 μg/mL, 2 hours prior to the end of incubation period.
- Methods of slide preparation and staining technique used including the stain used: After treatment, the culture medium in the dish was transferred to a centrifuge tube. Cells attached to the dish were detached using 0.25% trypsin solution and added to the same centrifuge tube. Cells were centrifuged and the supernatant was removed. Cells were treated with the hypotonic solution of 0.075 mol/L potassium chloride solution in a thermostat water bath at 37°C for 15 minutes. Then, a few drops of fixing solution (a mixture of alcohol and acetic acid) were added to the tube for semifixation of cells. Cells were centrifuged, and the solution in the tube was replaced with the fresh fixing solution. This procedure was repeated several times to replace the fixing solution, and cells were sufficiently fixed. A cell suspension was prepared at an appropriate concentration, put onto the slide glass, and air-dried. The chromosome specimen were stained with 2% Giemsa solution.
- Method of counting cells: Cells were detached from the culture dish using 0.25% trypsin solution and a cell suspension was prepared. A part of the cell suspension was stained with trypan blue, and cells were counted using a cell counter
- Observation of the specimens of chromosome aberration test: Blind method was used in the observation of specimens. Well-spread metaphase cells (in principle, 300 cells per each dose) were examined under a microscope at a magnification of 600-1000×
- Chromosome structural aberration: As the modal number of chromosomes in the CHL/IU cells was 25, cells with a chromosome number of 25 ± 2 were regarded as the object of the analysis. Structural aberrations were recorded according to the following categories, and a cell was counted as 1 aberrant cell if it had 1 or more aberrations of any type. The types of aberrations were also recorded (and included: chromatid breaks, chromatid exchange, chromosome breaks, chromosome exchange, and others - such as fragmentation, etc.). For example, if 1 cell had 2 break-type aberrations and 3 exchange-type aberrations, the number of aberrant cells would be recorded as 1, and the number of aberrant types would be recorded as 1 for the break-type and 1 for the exchange-type aberration.
- Chromosome numerical aberration: The number of cells was recorded when cells with chromosome doubling of haploid (triploid or hyperploid; called “poly”) or endoreduplication (called “end”) occurred.
- Gap: Chromatid and chromosome gaps were recorded separately from the other aberrations and were not included in the structural aberration. Achromatic regions smaller than the chromatid width in a coaxial line were considered gaps, whereas achromatic regions larger than the chromatid width were considered break-type aberrations.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)
RICC obtained from the negative control was defined as 100%. When the calculated value is less than 0, the lower limit of the value is 0.
RICC = ( Increase in the number of cells treated with the test substance* / Increase in the number of control cells*) x 100
* Calculated as (cell count at the end of treatment) minus (cell count at the start of treatment)

Evaluation criteria:

A positive result is obtained if the following criteria are fulfilled:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- The increase is dose-related when evaluated with the trend test.
- All of the results are outside the control limits based on the laboratory historical negative control data.

A negative result is obtained if the following criteria are fulfilled:
- None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with the trend test.
- All of the results are inside the control limits based on the laboratory historical negative control data.

In case results are neither clearly negative nor clearly positive, an analysis of additional cells or a repeat experiment (possibly using modified experimental conditions) may be performed.
If, after further investigations, it is still not possible to draw a conclusion of positive or negative results, the test material response is considered to be equivocal.

Statistics:

The test concentrations were compared with the negative control by use of Fisher's exact test (two-sides, p < 0.05 is significant., GraphPad Prism 8 Software).
The trend analyses for dose-related were performed by use of Cochran-Armitage trend test (two-sides, p < 0.05 is significant., GraphPad Prism 8 Software).
The control limits of based on the laboratory historical negative control data were ≤ 95% of Poisson distribution.

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

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Short-term treatment process
- Cell growth inhibition test: Precipitation of the test material was not observed at any dose in the initiation or completion of treatment either with or without metabolic activation.
In the absence of metabolic activation RICC of 50% was not observed at any dose. Therefore, 2.00 mg/mL was determined to be the maximum concentration, and 3 concentrations (0.500, 1.00 and 2.00 mg/mL) were selected which corresponded to a geometric sequence with a common ratio of 2.
In the presence of metabolic activation, cell growth inhibition of ≥50% (100-RICC) was observed at dose of 2.00 mg/mL. Therefore, 2.00 mg/mL was determined to be the maximum concentration, and 6 concentrations (0.354, 0.500, 0.707, 1.00, 1.41 and 2.00 mg/mL) were selected which corresponded to a geometric sequence with a common ratio of 2^1/2.
- Chromosome aberration test:
In the absence of metabolic activation RICC of 50% was not observed at any dose, therefore these specimens were used for chromosome analysis. Chromosome analysis was performed at 3 doses of 0.500, 1.00 and 2.00 mg/mL. The results showed that the both chromosome structural aberration and chromosome numerical aberration were not observed to increase above concurrent negative control at any dose, therefore statistical analysis was not performed.
In the presence of metabolic activation cell growth inhibition of 55 ± 5% (100-RICC) was observed at a dose of 1.41 mg/mL, therefore these specimens were used for chromosome analysis. Chromosome analysis was performed at 3 doses of 0.707, 1.00 and 1.41 mg/mL. The results showed that the frequency of both chromosome structural aberration and chromosome numerical aberration exhibited no significant difference compared with concurrent negative control by use of Fisher's exact test. Furthermore, chromosome structural aberration and chromosome numerical aberration exhibited no significant trend by use of Cochran-Armitage trend test.

Continuous treatment process
- Cell growth inhibition test: Precipitation of the test material was not observed at any dose in the initiation or completion of treatment. Cell growth inhibition of ≥ 50% (100-RICC) was observed at and above doses of 0.500 mg/mL. Therefore, the top dose was considered as the next higher dose level of 1.00 mg/mL with a common ratio of 2^1/2, with the following settings used: top dose of 0.707 mg/mL at 6 levels in total with a common ratio of 2^1/2 (0.125, 0.177, 0.250, 0.354, 0.500 and 0.707 mg/mL).
- Chromosome aberration test:
First test: Cell growth inhibition of 55 ± 5% (100-RICC) was not observed at any dose. Cell growth inhibition of target was not observed at any dose. Therefore, a chromosome aberration test of the 2nd time was performed within the dose range of 0.199 to 1.00 mg/mL at 8 levels using a common ratio of 2^1/3.
Second test: Cell growth inhibition of 55 ± 5% (100-RICC) was observed at a dose of 0.397 mg/mL, therefore these specimens were used for chromosome analysis. Chromosome analysis was performed at 3 doses of 0.250, 0.315 and 0.397 mg/mL. The results showed that the frequency of chromosome structural aberration exhibited no significant difference compared with concurrent negative control by use of Fisher's exact test. Furthermore, the chromosome structural aberration exhibited no significant trend by use of Cochran-Armitage trend test.
The chromosome numerical aberration was not observed to increase above concurrent negative control at any dose, therefore statistical analysis was not performed.

In short-term treatment process (in the presence and absence of a metabolic activation system) and continuous treatment process (24-hours), the frequency of chromosome structural aberrations and numerical aberrations at all of the results were inside the control limits based on the laboratory historical negative control data.

Summary of chromosome aberration test results

S9 mix	Dose (mg/mL)	Number of chromosome structural aberrations and classification of structural aberration								Number of chromosome numerical aberrations				RICC (%)
		Cells	ctb	cte	csb	cse	others	Total (%)	gap	Cells	poly	others	Total (%)
short-term treatment process (6-18 h)
without	Negative control	150	1	0	0	0	0	1	0	151	1	0	1	100
		150	0	0	0	0	0	0	0	150	0	0	0	100
		300	1	0	0	0	0	1 (0.3)	0	301	1	0	1 (0.3)	100
	0.500	150	1	0	0	0	0	1	0	150	0	0	0	99
		150	0	0	0	0	0	0	0	150	0	0	0	107
		300	1	0	0	0	0	1 (0.3)	0	300	0	0	0 (0.0)	104
	1.00	150	1	0	0	0	0	1	0	150	0	0	0	92
		150	0	0	0	0	0	0	0	150	0	0	0	83
		300	1	0	0	0	0	1 (0.3)	0	300	0	0	0 (0.0)	88
	2.00	150	0	0	0	0	0	0	0	150	0	0	0	88
		150	0	0	0	0	0	0	1	150	0	0	0	95
		300	0	0	0	0	0	0 (0.0)	1	300	0	0	0 (0.0)	92
	Positive control	150	14	11	0	0	0	23	1	150	0	0	0
		150	5	13	0	0	0	18	2	150	0	0	0
		300	19	24	0	0	0	41 (13.7*)	3	300	0	0	0 (0.0)
with	Negative control	150	0	1	0	0	0	1	0	150	0	0	0	100
		150	0	0	0	0	0	0	0	150	0	0	0	100
		300	0	1	0	0	0	1 (0.3)	0	300	0	0	0 (0.0)	100
	0.707	150	2	0	0	0	0	2	0	150	0	0	0	65
		150	0	1	0	0	0	1	0	150	0	0	0	78
		300	2	1	0	0	0	3 (1.0)	0	300	0	0	0 (0.0)	72
	1.00	150	0	0	0	0	0	0	0	150	0	0	0	52
		150	0	0	0	0	0	0	0	150	0	0	0	52
		300	0	0	0	0	0	0 (0.0)	0	300	0	0	0 (0.0)	52
	1.41	150	0	0	0	0	0	0	1	151	1	0	1	47
		150	1	1	0	0	0	1	0	150	0	0	0	37
		300	1	1	0	0	0	1 (0.3)	1	301	1	0	1 (0.3)	42
	Positive control	150	5	8	0	0	0	12	1	151	1	0	1
		150	12	13	0	0	0	19	0	150	0	0	0
		300	17	21	0	0	0	31 (10.3*)	1	301	1	0	1 (0.3)
continuous treatment process (24 h) - second test
without	Negative control	150	1	0	0	0	0	1	0	150	0	0	0	100
		150	0	0	0	0	0	0	0	150	0	0	0	100
		300	1	0	0	0	0	1 (0.3)	0	300	0	0	0 (0.0)	100
	0.250	150	2	0	0	0	0	2	2	150	0	0	0	65
		150	1	1	0	0	0	2	0	150	0	0	0	60
		300	3	1	0	0	0	4 (1.3)	2	300	0	0	0 (0.0)	62
	0.315	150	0	1	0	0	0	1	2	150	0	0	0	67
		150	1	0	0	0	0	1	0	150	0	0	0	61
		300	1	1	0	0	0	2 (0.7)	2	300	0	0	0 (0.0)	64
	0.397	150	2	1	0	0	0	3	0	150	0	0	0	45
		150	0	1	0	0	0	1	1	150	0	0	0	45
		300	2	2	0	0	0	4 (1.3)	1	300	0	0	0 (0.0)	45
	Positive control	150	10	10	0	0	0	20	0	150	0	0	0
		150	5	15	0	0	0	20	1	150	0	0	0
		300	15	25	0	0	0	40 (13.3*)	1	300	0	0	0 (0.0)

ctb: chromatid break

cte: chromatid exchange

csb: chromosome break

cse: chromosome exchange

poly: cells with chromosome doubling of haploid (triploid or hyperloid)

* Statistically significant compared to to negative control (Fisher's exact test) P <0.05

Acceptance criteria

In short-term treatment process (in the presence and absence of a metabolic activation system) and continuous treatment process (24-hours), the negative controls were inside the control limits based on the laboratory historical negative control data.

In short-term treatment process (in the presence and absence of a metabolic activation system) and continuous treatment process (24-hours), the chromosome structural aberration of positive controls were inside the range based on the laboratory historical positive control data, and they were statistically significant compared to the concurrent negative control.

Based on the above results, the assay was considered valid.

Conclusions:

Based on the results of each treatment group, the test material was considered “negative” for the chromosome aberration-inducing potential.

Executive summary:

The clastogenic potential of the test material was investigated in a study which was conducted un accordance with the standardised guideline OECD 473, and under GLP conditions using CHL/IU cells.

Under the conditions of the study the frequency of chromosome structural aberration exhibited no significant difference compared with concurrent negative control in any of the tests using short-term treatment process in the presence of a metabolic activation system and continuous treatment process for 24 hours. The chromosome structural aberration was not observed to increase above concurrent negative control at any dose in short-term treatment process in the absence of a metabolic activation system.

The frequency of chromosome numerical aberration exhibited no significant difference compared with concurrent negative control in any of the tests using short-term treatment process in the presence of a metabolic activation system. The chromosome numerical aberration was not observed to increase above concurrent negative control at any dose in short-term treatment process in the absence of a metabolic activation system and continuous treatment process for 24 hours.

Since elongation of cell cycle was not observed at continuous treatment process for 24 hours, cell growth inhibition test and chromosome aberration test using continuous treatment process for 48 hours were not carried out.

Based on the results of each treatment group, the test material was considered “negative” for the chromosome aberration-inducing potential.

Results from both of the positive and negative controls under all treatment conditions were within the acceptable ranges that confirmed the validity of the tests, indicating that these tests were carried out appropriately.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 December 2019 to 12 February 2020

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

Specific details on test material used for the study:

Solubility and stability in solvents:
- Saline: Soluble at 20.0 mg/mL (Stable)
- DMSO: Soluble at 200 mg/mL (Stable)
- Acetone: Solubility ≤0.001 g/L
(The test material was considered to be stable in the solvent if no changes including discolouration, heat generation, and foam formation were observed in the prepared solution for 2 hours)

Correction for test material purity was made in preparation of the test solutions (made by stirring, and serial dilution to the specified concentration). Test soutions were stored at 21-24°C and used within 40 minutes of preparation.

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 cells supplied by National Institute of Health Sciences (NIHS)
- Suitability of cells:
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination: yes (cells that had been checked for mycoplasma contamination and other properties were used)
- Cell doubling time: 9-12 hrs
- Number of passages if applicable: Cells were cultured so as not exceed concentrations of 1.2 × 10^6 cells/mL

MEDIA USED
- RPMI-0 for dilution or washing of the cells (500 mL RPMI1640, 5 mL 10000 u/mL penicillin 10000 μg/mL streptomycin, 0.5 mL 200 mg/mL sodium pyruvate)
- RPMI-10 for normal culture (500 mL RPMI1640, 50 mL inactivated horse serum, 5 mL 10000 u/mL penicillin 10000 μg/mL streptomycin, 0.5 mL 200 mg/mL sodium pyruvate)
- RPMI-20 for colonisation in 96-well plates (500 mL RPMI1640, 100 mL inactivated horse serum, 5 mL 10000 u/mL penicillin 10000 μg/mL streptomycin, 0.5 mL 200 mg/mL sodium pyruvate)

CULTURE CONDITIONS
- Container: 75 cm² or 150 cm² cultivation flask
- Temperature and CO2 concentration: 37°C and 5%

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: manufactured by Oriental Yest Co., Ltd. (prepared from 7 week old male Sprague-Dawley derived rats, dosed i.p. with phenobarbital (30 mg/kg 4 days before killing and 60 mg/kg 1, 2 & 3 days before killing) and 5,6-benzoflavone (80 mg/kg 2 days before killing) and stored at approximately -80 °C)
- method of preparation of S9 mix: The S9 mix (10 mL) contained: 4 mL S9, 2 mL glucose-6-phosphate (180 mg/mL), 2 mL NADP (25 mg/mL), and 2 mL KCl (150mM)
- amount of S9 added per incubator: 2%
- final concentration of S9 protein per incubator: 0.39 mg/mL

Test concentrations with justification for top dose:

Dose-ranging study: 0.125, 0.250, 0.500, 1.00 and 2.00 mg/mL (short and continuous treatment, with and without metabolic activation)

Mutagenicity tests:
- Short-term treatment: 0.445, 0.500, 0561, 0.630, 0.707, 0.794, 0.891 mg/mL (test 1 without metabolic activation); 0.707, 0.841, 1.00, 1.19, 1.41, 1.68 and 2.00 mg/mL (test 2 and test 3 without metabolic activation); 0.125, 0.250, 0.500, 1.00, 2.00 mg/mL (with metabolic activation)
- Continuous treatment: 0.354, 0.421, 0.500, 0.595, 0.707, 0.841, 1.00, 1.19 mg/mL (without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle:
The molecular weight of the test material is 788.06, thus, the solubility of the test material was checked at the final concentration of 2.00 mg/mL in the media. The test material was soluble at 20.0 mg/mL in saline and 200 mg/mL in DMSO; therefore saline was selected as a solvent.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration:
Dose range-finding study: single
Gene mutation test: single for the test material treated group and the positive control group. The negative control was performed in duplicate.
- Number of independent experiments
Dose range-finding study: short-term (3-hour) treatment (with and without metabolic activation) and continuous treatment (24-hour) (without metabolic activation)
Gene mutation test: short-term treatment (with and without metabolic activation - three tests) and continuous treatment (without metabolic activation)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: cell suspension 10^6 cells/mL
- Treatment conditions: short-term treatment (3-hours). continuous treatment (24-hours)
- Test substance added in medium:
Dose range-finding study: 0.5 mL test material solution - added to 0.25 mL S9 mix (150mM KCl without metabolism activation), 1.75 mL RPMI-0, and 2.5 mL cell suspension (short-term treatment); 1.25 mL test material solution - added to 8.75 mL RPMI-10, and 2.5 mL cell suspension (continuous treatment)
Gene mutation test: 2.0 mL test material solution or 0.2 mL control substance solution - added to 1 mL S9 mix (150mM KCl without metabolism activation), 7.0 or 8.8 mL RPMI-0 (test material or control treatments, respectively), and 10 mL cell suspension (short-term treatment); 5.0 mL test material solution or 0.5 mL control substance solution - added to 35.0 or 39.5 mL RPMI-10 (test material or control treatments, respectively), and 10 mL cell suspension (short-term treatment)
- Treatment:
Dose range-finding study: the treatment solution was prepared for a cultivation container as outlined above. In the short-term treatment method, immediately after addition of the test material solution, the treatment container was sealed, and placed in the tilting shaker in the CO2 incubator. The container was gently shaken to assure that the cells did not stay in one place. In the continuous treatment method, immediately after addition of the test material solution, the cells were left standing throughout the treatment period in the CO2 incubator.
Gene mutation test: the treatment solution was prepared for a cultivation container as outlined above. The test was performed in the manner of the dose range-finding study.

REMOVAL OF TEST MATERIAL
Dose range-finding study: at the end of the treatment period, the treatment solutions were centrifuged for 5 minutes at approximately 200g and the supernatant was removed by the aspirator, or after standing for 10 minutes, the supernatant was gently removed with the pipette. Ten mL of RPMI-0 culture medium was added in the treatment container, and the cells were suspended by pipetting. The cell suspensions were centrifuged under the same conditions. The supernatant was discarded and the cells were suspended in 10 mL of RPMI-10 culture medium.
Gene mutation test: as per the dose range-finding study.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY , PLATING EFFICIENCY, RELATIVE SURVIVAL AND MUTATION FREQUENCY
Dose range-finding study: After having removed the test material, the cell suspensions were measured cell concentration. Then, the cell suspensions were prepared in RPMI-10 medium at 2 × 10^5 cells/mL. The cell suspensions were transferred to the 75 cm² flasks, and cultured in the CO2 incubator. The cell suspensions were measured, diluted and cultured daily for 2 days (expression time). From these results, the relative rate of cell multiplication (RSG: Relative Suspension Growth) was calculated in order to estimate the cytotoxicity on the Main test. No treatment to form colonies was carried out.
Gene mutation test: After having removed the test material, the cell suspensions were measured cell concentration. Then, the cell suspensions were prepared in RPMI-10 medium at 2 × 10^5 cells/mL. The cell suspensions were further diluted to 8 cells/mL, and the cell suspensions were dispensed 200 μL per well in 96-well plates [Measurement of plating efficiency (PE1) and the relative survival (RS1)]. The plates were incubated in the CO2 incubator for 10-14 days then scored. The remaining cell suspensions A were transferred to the 75 cm² flasks, and cultured in the CO2 incubator. The cell suspensions were measured, diluted and cultured daily for 2 days (expression time). From these results, the RSG was calculated.
After an expression time of 2 days following test material treatment, 50 mL of a cell suspension were prepared at 1 × 10^4 cells/mL. The cell suspensions were further diluted to 8 cells/mL, and the cell suspensions were dispensed 200 μL per well in 96-well plates [Measurement of plating efficiency (PE2) and the relative survival (RS2)]. For the remaining cell suspensions, trifluorothymidine (TFT) was added to a final concentration of 3 μg/mL. These cell suspensions were dispensed 200 μL per well in four 96-well plates per solvent control group and in two 96-well plates per each treatment group [Detection of mutation frequency (MF)].
The PE2 plates were incubated in the CO2 incubator for 10-14 days then scored. The MF plates were incubated in the CO2 incubator for 12 days then scored.

METHODS FOR OBSERVATION AND MEASUREMENT
- Method for cell counting: To measure viable cell concentration, the cells were stained with a trypan blue solution. The viable cells were determined with the cell counter (Bio-Rad, type TC20).
- Observation of PE0 and PE2 plate: After completion of the 10-14 day incubation period, the number of colonies per plate was determined by stereomicroscopic observation.
- Observation of MF plates: After completion of the 12 day incubation period, the number and size of colonies per plate was determined by stereomicroscopic observation. Each well per plate was classified by four categories based on the presence or absence of colony formation, as follows:
I. The well included only large cell colonies (L Well)
II. The well included only small cell colonies (S Well)
III. The well included cell colonies of both types (L/S Well)
IV. The well had no cell colonies
The wells contaminated with bacteria etc. were excluded from observation and the data analysis were performed excluding those wells from the total number of wells. The size classification of each colony was determined based on the following criteria:
1) Colony Size
Small colony: Colony smaller than 1/4 of the well diameter
Large colony: Colony exceeding 1/4 of the well diameter
2) Colony shapes
Small colony: Colony density is high, margin is clear
Large colony: Colony density is low, margin is indistinct

CALCULATIONS
- RCC (Relative Cell Count)
The cell survival rate immediately after test material treatment. The RCC was calculated according to the following formula:
RCC = [(Cell concentration of the dose level) / (Cell concentration of the negative control)] × 100

- SG (Suspension Growth)
The fold-increase in the number of cells over the course of the treatment and expression times. For the short-term treatment, the SG was calculated by multiplying the fold-increase on day 1 by the
fold-increase on day 2 (Daily Cell Growth-1 × Daily Cell Growth-2). For the continuous treatment, the SG was the fold-increase during the continuous treatment multiplied by the fold-increases on
expression days 1 and 2.

- PE (Plate Efficiency, PE0 and PE2)
The plating efficiency of the cells. The PE0 immediately after test material treatment and the PE2 after culturing for 2 days were calculated. The PE was calculated according to a Poisson distribution as follows:
PE (%) = [ -ln (EW / TW) / N] x 100
where:
EW: number of wells with no colonies
TW: total number of wells
N: mean number of subcultured cells/well, N = 1.6

- RS (Relative Survival, RS0 and RS2)
The RS0 (RS2), which was the ratio of the PE0 (PE2) in the test material treated groups to the PE0 (PE2) in the negative control, was calculated. The RS was calculated from the PE of each 96-well plate as follows:
RS = [PE (the treated group) / PE (the negative control)] × 100

- RSG (Relative Suspension Growth) and %RSG
The relative total two-day suspension growth of the test material treated groups compared to the total two-day suspension growth of the negative control. The RSG value was calculated by the Daily Cell Growth as follows:
RSG = [ Daily Cell Growth at day-1 Daily Cell Growth at day- 2 (the treated group) ] / [ Daily Cell Growth at day-1 Daily Cell Growth at day- 2 (the negative control) ]
%RSG = (RSG of the treated group / RSG of the negative control) x 100
The Daily Cell Growth represents the cell growth rate per day, and was calculated by the cell concentration measured on days 1 and 2 as follows:
Daily Cell Growth = ( Day 2 cell concentration / Day 1 cell concentration )

- RTG (Relative Total Growth)
The RTG is comprehensive cytotoxicity index. It expressed as the product of the RSG and the RS2.
RTG = RSG × RS2

- MF (Mutation Frequency)
The MF was determined according to a Poisson distribution as well as PE. The MF was calculated as follows:
MF = [ -ln(EW/ TW) / N ] / PE2
where:
EW: number of wells with no colonies
TW: total number of wells
N: mean number of subcultured cells/well, N = 2000
For the gene mutation frequency, the total gene mutation frequency (T-MF), the gene mutation frequency of large colonies (L-MF), and the gene mutation frequencies of small colonies (S-MF) were
calculated. Each EW was calculated as follows:
EWT = TW - (A + B + C)
EWL = TW - (A + C)
EWS = TW - (B + C)
“A”, “B” and “C” represents the number of wells with only large colonies, the number of wells with only small colonies and the number of wells with both large and small colonies, respectively.

- %SC (% of Small Colony)
Percentage of the MF that results in small colonies. The %SC was calculated as follows:
%SC = [ (S-MF) / (T -MF) ] x 100

Evaluation criteria:

The judgment of the results confirmed a meaningful MF increase and whether or not it was dose-dependent. The determination of increase in mutation frequency was evaluated by the Global Evaluation Factor (GEF) note) judgment method. Under the method, a result is judged to be negative when the values of the test groups were below the value which added 126 (× 10^-6) to the MF value of the negative control.

The test material was considered to be clearly positive if, in any of the conditions tested, the increase in MF above the concurrent background exceeded the GEF and the increase was dose-dependent. The test material was considered to be clearly negative if, in all conditions tested, there was no dose-dependent response or, if there was an increase in MF, it did not exceed the GEF. In addition, the result would not be considered positive if the increase in MF occurred only at or below 10% RTG.
When there was no culture showing an RTG value between 10 - 20% RTG, the test material was considered to be negative if the following two circumstances were observed.
i) There was no dose-dependent response and no MFs above those seen in the concurrent negative control or historical background ranges in a series of data points within 20% to 100% RTG. In addition, there was at least one data point between 20 and 25% RTG.
ii) There was no dose-dependent response and no MFs above those seen in the concurrent negative control or historical background ranges in a series of data points within 25% to 100% RTG. In addition, there was also a negative data point slightly below 10% RTG.
If the result obtained was difficult to make such clear judgment as described above, the confirmatory test was performed in principle and comprehensively judgment was made, accordingly. If there was no positive or negative conclusion in the results obtained in the confirmatory test, the test material response was concluded to be equivocal.

Statistics:

not applicable

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Dose range-finding study
In both the short-term and the continuous treatment groups, test material precipitate was not observed on the cultures at any of the doses at the end of treatment. Further, under any of the treatment conditions, coloring from the test material was observed, and therefore, the colour change of the culture solution due to the addition of the test material could not be confirmed.
Regarding cytotoxicity, in the short-term treatment without metabolic activation, the %RSG at 0.500 mg/mL was 78%, and unmeasurable excessive cytotoxicity was observed at ≥ 1.00 mg/mL.
In the short-term treatment with metabolic activation, %RSG of 20% or less was not observed at the highest dose of 2.00 mg/mL (%RSG: 47%). In the continuous treatment, the %RSG at 0.500 mg/mL was 54%, and unmeasurable excessive cytotoxicity was observed at ≥ 1.00 mg/mL.
The findings of the dose range-finding study were used to establish the concentrations for testing in the Main Gene Mutation Test.

- Gene Mutation test by short-term (3-hour) treatment process
In the short-term treatment with and without metabolic activation, test material precipitate was not observed on the cultures at all the doses at the end of treatment.
Regarding cytotoxicity, in the short-term treatment without metabolic activation, 10 to 20% RTG was not observed at any of the doses conducted. Therefore, the gene mutation test by short-term treatment without metabolic activation of the second time was carried out.
As a result of the second test, the test material precipitate was not observed on the cultures at all the doses at the end of treatment. However, at high doses, the cell-containing suspension formed the viscous suspension and was accidentally removed. Therefore, the second test was discontinued. When removing the test material, the test material-treated groups did not use the aspirator so as not to remove these viscous suspended matters, and the supernatant was gently removed with the pipette. And then, the third test was carried out.
In the results of the third test, the test material precipitate was not observed on the cultures at any dose at the end of treatment. Regarding cytotoxicity, 10 to 20% RTG was observed at 1.41 mg/mL (RTG: 19%). This dose was the highest dose. Unmeasurable excessive cytotoxicity was observed at ≥ 1.68 mg/mL.
Based on the above results, in the short-term treatment without metabolic activation, the highest dose was set at 1.41 mg/mL that showed RTG of 10-20%. And then, 5 doses of 0.707 to 1.41 mg/mL were included in the analysis of the gene mutant frequencies.
In the short-term treatment with metabolic activation, the RTG at the highest dose of 2.00 mg/mL was 85%.
In the short-term treatment with metabolic activation, 5 doses from 0.125 to 2.00 mg/mL, including the highest dose of 2.00 mg/mL, were included in the analysis of the gene mutant frequencies.
In the short-term treatment without metabolic activation, the total gene mutation frequency (T-MF) tended to show dose-dependent responses. However, at 0.707 to 1.41 mg/mL the T-MF ranged from 84 × 10^-6 to 149 × 10^-6, and did not exceed the combined value of negative control and GEF at any dose. In the short-term treatment with metabolic activation, at 0.125 to 2.00 mg/mL the T-MF ranged from 141 × 10^-6 to 219 × 10^-6, and did not exceed the combined value of negative control and GEF at any dose. In addition, no dose-dependent response was observed.
From the above results, the test material was judged to be "negative" both in the short-term treatment with and without metabolic activation.
Results from both of the positive and negative controls under treatment conditions met acceptance criteria, indicating that these tests were carried out appropriately.

- Gene Mutation test by continuous (24-hour) treatment process
In the results of the test, the test material precipitate was not observed on the cultures at any dose at the end of treatment.
Regarding cytotoxicity, 10 to 20% RTG was observed at 1.19 mg/mL (RTG: 18%).
Based on the above results, the highest dose was set at 1.19 mg/mL that showed RTG of 10-20%.
And then, 8 doses of 0.354 to 1.19 mg/mL were included in the analysis of the gene mutant frequencies.
The T-MF tended to show dose-dependent responses. However, at 0.354 to 1.19 mg/mL the T-MF ranged from 109 × 10^-6 to 216 × 10^-6, and did not exceed the combined value of negative control and GEF at any dose.
From the above results, the test material was judged to be "negative" in the continuous treatment.
Results from both of the positive and negative control under treatment condition met acceptance criteria, indicating that these tests were carried out appropriately.

Results for gene mutation test

Dose (mg/mL)	Cytotoxicity								Mutation Frequency (x10^-6)
	RCC	SG	%RSG	PE0 (%)	RS0 (%)	PE2 (%)	RS2 (%)	RTG (%)	Mutation Frequency (x10^-6)
									Total	Large	Small	%SC
	short-term treatment (3-hours) process without metabolic activation: third test
0 (negative control) (average)	100	19.3	100	82	100	112	100	100	53	34	19	36
0.707	30	11.5	60	61	75	98	87	52	84	53	27	33
0.841	20	11.4	59	52	63	11	100	59	87	65	19	22
1.00	9	9.7	50	42	51	67	60	30	105	82	20	19
1.19	2	10.8	56	71	87	67	60	33	127	91	32	25
1.41	2	6.8	35	67	82	61	55	19	149	104	39	26
1.68	0	na	na	na	na	na	na	na	na	na	na	na
2.00	0	na	na	na	na	na	na	na	na	na	na	na
Positive control	105	9.8	51	63	77	49	43	22	700	288	295	42
	short-term treatment (3-hours) process with metabolic activation
0 (negative control) (average)	100	18.5	100	78	100	94	100	100	144	87	48	33
0.125	76	22.2	120	89	114	92	98	118	141	109	26	18
0.25	87	19.1	103	56	72	108	115	119	143	105	30	21
0.500	87	21.8	118	75	96	77	82	97	219	169	38	17
1.00	54	21.9	118	87	111	89	95	113	161	116	36	22
2.00	17	15.0	81	116	148	98	105	85	180	123	44	25
Positive control	96	9.7	52	52	66	49	52	27	950	535	246	26
	continuous treatment (24-hours) process without metabolic activation
0 (negative control) (average)	100	80.8	100	85	100	114	100	100	97	73	20	21
0.354	40	35.0	108	65	76	98	86	93	109	62	41	38
0.421	30	22.1	91	67	78	120	106	96	122	68	46	37
0.500	21	13.4	79	73	85	116	102	81	158	76	68	43
0.595	18	8.4	60	45	52	105	92	55	131	78	44	34
0.707	13	6.8	67	28	33	116	102	68	118	58	52	44
0.841	8	2.1	30	40	46	89	78	24	120	71	42	35
1.00	5	1.4	32	22	26	101	89	29	178	72	93	52
1.19	3	0.5	23	25	30	87	76	18	216	81	116	54
Positive control	64	64.2	124	75	88	95	83	103	481	201	185	39

RCC: Relative Cell Count

SG: Suspension Growth

RSG: Relative Suspension Growth

PE0: Plate Efficiency at day-0 (for cytotoxicity), RS0: Relative Survival at day-0

PE2: Plate Efficiency at day-2 (to measure mutation frequency), RS2: Relative Survival at day-2

RTG: Relative Total Growth

%SC: % of Small Colony

Conclusions:

Under the conditions of the study the test material was not found to have the potential to induce gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells in vitro.

Executive summary:

The potential of the test material to induce gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells was investigated in vitro, in a study which was conducted in accordance with the standardised guideline OECD 490 and under GLP conditions.

As the result of gene mutation tests, in the short-term treatment without metabolic activation, the total gene mutation frequency (T-MF) tended to show dose-dependent responses, but the increases did not exceed the combined value of negative control and Global Evaluation Factor (GEF) at both the highest dose and lower doses. In the short-term treatment with metabolic activation, the T-MF did not show dose-dependent responses, and did not exceed the combined value of negative control and GEF at any dose. In the continuous treatment without metabolic activation, the T-MF tended to show dose-dependent responses, but the increases did not exceed the combined value of negative control and GEF at both the highest dose and lower doses.

From the results obtained in these tests, the test material was comprehensively concluded as "negative". Therefore, under these test conditions, the test material has no potential for mutagenicity in the mouse lymphoma thymidine kinase locus gene mutation test. Results from both of the positive and negative controls under all treatment conditions met acceptance criteria, indicating that these tests were carried out appropriately.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames

Based on the preliminary assay results, 5mg/plate was set as the highest dose in this study. Inthe mutagenicity assay, five doses of the substance at 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate, concurrent negative and strain-specific positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 Mix activation. No cytotoxicity was observed in all five tester strains up to 5mg/plate in the absence and presence of metabolite activations. Results showed that the substance did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 up to 5mg/plate either in the absence or in the presence of metabolite activation.

Based on the data obtained from this study, it was concluded that under the test condition, the substance was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 5 mg/plate in the absence and presence of S9 metabolic activation.

Chromosome aberration

The clastogenic potential of the test material was investigated in a study which was conducted un accordance with the standardised guideline OECD 473, and under GLP conditions using CHL/IU cells. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Under the conditions of the study the frequency of chromosome structural aberration exhibited no significant difference compared with concurrent negative control in any of the tests using short-term treatment process in the presence of a metabolic activation system and continuous treatment process for 24 hours. The chromosome structural aberration was not observed to increase above concurrent negative control at any dose in short-term treatment process in the absence of a metabolic activation system.

The frequency of chromosome numerical aberration exhibited no significant difference compared with concurrent negative control in any of the tests using short-term treatment process in the presence of a metabolic activation system. The chromosome numerical aberration was not observed to increase above concurrent negative control at any dose in short-term treatment process in the absence of a metabolic activation system and continuous treatment process for 24 hours.

Since elongation of cell cycle was not observed at continuous treatment process for 24 hours, cell growth inhibition test and chromosome aberration test using continuous treatment process for 48 hours were not carried out.

Based on the results of each treatment group, the test material was considered “negative” for the chromosome aberration-inducing potential.

Results from both of the positive and negative controls under all treatment conditions were within the acceptable ranges that confirmed the validity of the tests, indicating that these tests were carried out appropriately.

Mouse Lymphoma Assay

The potential of the test material to induce gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells was investigated in vitro, in a study which was conducted in accordance with the standardised guideline OECD 490 and under GLP conditions.

As the result of gene mutation tests, in the short-term treatment without metabolic activation, the total gene mutation frequency (T-MF) tended to show dose-dependent responses, but the increases did not exceed the combined value of negative control and Global Evaluation Factor (GEF) at both the highest dose and lower doses. In the short-term treatment with metabolic activation, the T-MF did not show dose-dependent responses, and did not exceed the combined value of negative control and GEF at any dose. In the continuous treatment without metabolic activation, the T-MF tended to show dose-dependent responses, but the increases did not exceed the combined value of negative control and GEF at both the highest dose and lower doses.

From the results obtained in these tests, the test material was comprehensively concluded as "negative". Therefore, under these test conditions, the test material has no potential for mutagenicity in the mouse lymphoma thymidine kinase locus gene mutation test. Results from both of the positive and negative controls under all treatment conditions met acceptance criteria, indicating that these tests were carried out appropriately.

Justification for classification or non-classification

Bearing in mind the three negative in vitro studies in accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.