tert-butyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

- Ames: bacterial reverse mutation test according to OECD TG 471, GLP compliant, K1, with and without metabolic activation, Salmonella typhimurium (TA100, TA1535, TA98, TA1537) and Escherichia coli (WP2 uvrA-), not mutagenic

- CA: mammalian chromosome aberration test similar to OECD TG 473, GLP not specified, K2, Chinese hamster lung (CHL/IU), with and without metabolic activation, negative/ambiguous

- HPRT: Mammalian Cell Gene Mutation Tests according to OECD TG 476, GLP compliant, K1, Chinese hamster lung fibroblasts (V79, with and without metabolic activation, negative

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 01 to July 22, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF
- Batch number of test material: 010365eda0
- Purity: 99.86 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the refrigerator protected from light
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test substance under storage conditions over the test period was guaranteed by
the sponsor, and the sponsor holds this responsibility; Homogeneity: given

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid: 11.1, 22.2, 44.4, 88.9, 177.8, 355.5, 711.0 and 1422.0 μg/ml

FORM AS APPLIED IN THE TEST
- suspension

OTHER SPECIFICS
- Physical state: liquid, colorless, clear

Target gene:

Hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line, Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line.

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of the testing facility allowing the repeated use of the same cell culture batch in experiments.
- Cell cycle length, doubling time or proliferation index : 12 - 16 h in stock cultures
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1 %).
- During 4 hours treatment no FBS was added to the medium. During 24 hours treatment the medium was supplemented with 10% FBS.
- For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as metabolic activation system.
- method of preparation of S9 mix: The S9 was prepared and stored according to the currently valid version of the ICCR SOP for rat liver S9 preparation.
- concentration or volume of S9 mix and S9 in the final culture medium: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- The protein concentration of the S9 preparation was 33.8 mg/ml in the pre- and main experiment.
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

The highest applied concentration in the pre-test on toxicity (1422 µg/mL) was chosen corresponding to 10 mM, with respect to the OECD guideline 476 (2016).
Based on the results of the pre-experiment the maximal concentration of the main experiment was 1422 µg/mL as well.

- Doses applied in the gene mutation assay: 11.1, 22.2, 44.4, 88.9, 177.8, 355.5, 711.0 and 1422.0 μg/ml
- concentrations chosen for the mutation rate analysis: 88.9, 177.8, 355.5, 711.0 and 1422.0 μg/ml

Vehicle / solvent:

- Vehicle/solvent used: DMSO (purity: c≥ 99.9)
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- Percentage of solvent in the final culture medium: The final concentration of DMSO in the culture medium will not exceed 0.5% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

positive controls: 7,12-dimethylbenz[a]anthracene (DMBA) was tested with S9 Mix, ethyl methanesulfonate (EMS) without S9 Mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: approx. 0.7 to 1.2x10^7 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h

FOR GENE MUTATION:
- Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0x10^6 cells per experimental point (concentration series plus controls) were subcultivated in 175 cm² flasks containing 30 ml medium. Two additional 25 cm² flasks were seeded per experimental point with approximately 500 cells each to determine the relative survival (RS) as measure of test item induced cytotoxicity. The colonies used to determine the relative survival (RS) are fixed and stained approximately 6 to 8 days after treatment as described below.Three or four days after the first sub-cultivation, at least 2.0x10^6 cells per experimental point are again sub-cultivated in 175 cm² flasks containing 30 ml medium. Following the expression time of approximately 7 days five 75 cm² cell culture flasks are seeded with about 4 - 5x10^5 cells each in medium containing 6-TG (11 µg/ml). Two additional 25 cm² flasks are seeded with approx. 500 cells each in non-selective medium to determine the viability. After approx. 8 days (evaluation for viability) and approx. 9 ± 2 days (mutation analysis) the colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY AND GENOTOXICIY:

Pre-test:
- cloning efficiency, absolute = mean number of cells per ml divided by the number of cells seeded x 100
- cloning efficiency, relative = (mean number of cells per ml divided by the mean number of cells per ml of the corresponding control) x 100

Main test:
- cloning efficiency I (survival) = cloning efficiency determined immediately after treatment to measure toxicity.
- cloning efficiency II (viability) = cloning efficiency determined after the expression period to measure viability of the cells without selective agent.
- cloning efficiency I (survival, absolute)= mean number of colonies per flask divided by the number of cells seeded per test point
- cloning efficiency I (survival, relative) = cloning efficiency I absolute divided by the cloning efficiency I absolute of the corresponding control x 100
- relative cell density % of control = (cell density at 1st subcultivation divided by the cell density at 1st subcultivation of the corresponding control) x 100
- cloning efficiency II (viability, absolute) = mean number of colonies per flask divided by the number of cells seeded
- cloning efficiency II (viability, relative) = cloning efficiency II absolute divided by the cloning efficiency II absolute of the corresponding control x 100
- cells survived (after plating in TG containing medium) = number of cells seeded x cloning efficiency II absolute
- mutant colonies / 10^6 cells = mean number of mutant colonies per flask found after plating in TG medium x 10^6 divided by the number of cells survived
- relative adjusted cloning efficiency I = relative cloning efficiency I x relative cell density at first subcultivation / 100

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:

a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:

a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b)there is no concentration-related increase when evaluated with an appropriate trend test,
c)all results are inside the distribution of the historical negative control data (based 95% control limits).

There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.

In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiments.

A linear regression analysis (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

A t-test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics to evaluate a significant increase of the mutation frequency at all test points where the mutation frequency is above the corresponding solvent control. A t-test is judged as significant if the p-value is <0.05.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: The osmolarity and the pH value were determined in culture medium of the solvent control and of the highest concentration in the pre-experiment without metabolic activation.
- Osmolarity [mOsm] test substance (1422 µg/mL): 380
- Osmolarity [mOsm] solvent control: 403
- pH-value test substance (1422 µg/mL): 7.42
- pH-value solvent control: 7.43
- Precipitation and time of the determination: Pre-experiment: The test medium was checked for phase separation and precipitation at the end of the treatment period (4 h) before the test item was removed. Phase separation occurred at 711 µg/mL and above in the presence of S9 mix.
- Main Experiment: Phase separation occurred at 1422 µg/mL after four hours treatment.

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the toxicity of the test item. In this pre-test approx. 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test substance was added and the treatment proceeds for 4 hours (with and without metabolic activation) (duplicate cultures per concentration level).

STUDY RESULTS
- Concurrent vehicle negative and positive control data: EMS (300 µg/ml) and DMBA (2.3 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies. The MF of the positive control with S9 mix (52.8) was slightly below the historical control data (54.5 – 347.1). This minor deviation was judged to represent fluctuations. A clear significancy was observed and it was above the 95% control limit of the solvent control and therefore acceptable and the test was considered valid.

Pre-Experiment
- No relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed either with or without metabolic activation. There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test item measured in the pre-experiment.

Main Experiment
- No cytotoxicity was observed up the highest evaluated concentration, which showed phase separation.The observed mean mutant frequency (MF) of the solvent control and all evaluated concentrations was within the 95% control limits of the solvent historical control data. Linear regression analysis showed no statistically significant trend. A t-test was performed for all concentrations with a MF above the MF of the solvent control and did not show a statistical significance.

For more detailed information please refer to table 2 in section "Any other information on results incl. tables".

HISTORICAL CONTROL DATA
- The historical control data were generated in accordance with the OECD Guideline 476. For the solvent controls data range (min-max) and data distribution (standard deviation) are calculated for each experimental part of at least 20 experiments. The calculated 95% control limit of the solvent controls (realized as 95% confidence interval) are applied for the evaluation of acceptability and interpretation of the data. Control charts of the corresponding experiments are added as quality control method. For the positive controls data range (min-max) and data distribution (standard deviation) are calculated for each experimental part of at least 20 experiments. The min-max range of the positive controls are applied for the evaluation of acceptability. Control charts of the corresponding experiments are added as quality control method.
For more detailed information please refer to table 1 in section "Any other information on materials and methods incl. tables".

  Summary of results

				relative	relative	rel. adjusted	(MF)	95%	statistical
	conc.	PS	S9	cloning	cell	cloning	mutant	control	analysis*
	µg/mL		mix	efficiency I	density	efficiency I	colonies/	limit	t-test	linear
				%	%	%	10^6cells			regression
Column	1	2	3	4	5	6	7	8	9	10
Main Experiment / 4 h treatment				mean values of culture I and II
Solvent control with DMSO			-	100.0	100.0	100.0	9.9	2.9 – 22.4
Positive control (EMS)	300		-	73.3	103.5	74.6	271.6		0.000^S
Test item	11.1	-	-	98.6	95.7	94.4	#	2.9 – 22.4
Test item	22.2	-	-	100.7	97.7	98.4	#	2.9 – 22.4		0.266
Test item	44.4	-	-	95.8	88.2	84.2	#	2.9 – 22.4
Test item	88.9	-	-	104.2	80.4	84.0	4.4	2.9 – 22.4	n.c.
Test item	177.8	-	-	104.4	81.6	85.1	9.2	2.9 – 22.4	n.c.
Test item	355.5	-	-	99.8	75.6	75.5	7.8	2.9 – 22.4	n.c.
Test item	711.0	-	-	95.4	72.7	70.2	8.3	2.9 – 22.4	n.c.
Test item	1422.0	PS	-	88.7	74.3	65.3	11.3	2.9 – 22.4	0.623
Solvent control with DMSO			+	100.0	100.0	100.0	7.9	2.9 – 23.7
Positive control (DMBA)	2.3		+	90.4	107.7	98.5	52.8		0.011^S
Test item	11.1	-	+	87.2	98.5	85.8	#	2.9 – 23.7
Test item	22.2	-	+	87.3	106.1	92.7	#	2.9 – 23.7		0.148
Test item	44.4	-	+	89.9	95.5	86.3	#	2.9 – 23.7
Test item	88.9	-	+	94.4	101.0	96.0	12.7	2.9 – 23.7	0.034^S
Test item	177.8	-	+	88.6	105.4	93.3	7.6	2.9 – 23.7	n.c.
Test item	355.5	-	+	93.2	115.3	108.3	7.9	2.9 – 23.7	n.c.
Test item	711.0	-	+	78.8	96.0	75.8	8.6	2.9 – 23.7	0.392
Test item	1422.0	PS	+	54.4	102.2	55.7	4.7	2.9 – 23.7	n.c.

 

*       statistical analysis based on the mean values of culture I and II

n.c.   not calculated (mean MF below MF of the solvent control)

PS =  Phase separation at the end of treatment

S =    significant trend (p < 0.05)

MF   Mutant frequency

#       cultures not continued as a minimum of only four analyzable concentrations are required

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

This GLP compliant study (according to OECD TG 476) was performed to investigate the potential of the test substance, dissolved in DMSO, to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in one experiment using two parallel cultures. The main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The highest applied concentration in the pre-test on toxicity (1422 µg/mL) was chosen corresponding to 10 mM, with respect to the OECD guideline 476 (2016). Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 11.1, 22.2, 44.4, 88.9, 177.8, 355.5, 711, and 1422 µg/ml.

In the main experiment in the absence and presence of S9 mix, no relevant cytotoxicity was observed up to the highest applied concentration, which showed phase separation.

In the main experiment in the absence and presence of S9 mix, no relevant increases in the numbers of mutant colonies were observed after treatment with the test substance.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): not specified
- Batch number of test material: not specifed
- Purity: 99.8 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specifed
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable under normal handling conditions
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: soluble in acetone, dimethylsulfoxide and 0.3% in water (20°C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration: 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA100) and 19.5, 39.1, 78.1, 156, 313, 625 μg/plate (TA1535, TA98, TA1537, WP2 uvrA-)

FORM AS APPLIED IN THE TEST
- solution in acetone

OTHER SPECIFICS
- colorless transparent liquid having an ester odor

Target gene:

trp/his

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Purchased S9 (Lot No. RAA-316, Kikkoman Corp., manufactured on October 6, 1994) was used.
- method of preparation of S9 mix:This S9 was a 9000 x g centrifugal supernatant fraction of liver homogenate produced by the following procedure: phenobarbital was administered intraperitoneally to 7-week old male Sprague-Dawley rats once at a dose of 30 mg/kg and 3 times at a dose of 60 mg/kg at 24 hour intervals, and 80 mg/kg 5,6-benzofurabon (sic) was administered once concomitantly with phenobarbital at its third administration. The S9 was stored at ≤ -80°C.
- concentration or volume of S9 mix and S9 in the final culture medium:
- S9 0.1 ml per 1 ml S9 Mix
- test substance solution (0.05 ml) was aliquoted into a sterilized test tube fitted with a screw cap, and thereto were then added 0.5 ml S9 Mix and 0.1 ml strain suspension
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not specified
- The S9 Mix was prepared with the following composition per 1 ml S9 Mix, and stored in ice until use:
• S9 0.1 ml
• Magnesium chloride hexahydrate 8 μmol
• Potassium chloride 33 μmol
• D-glucose 6-phosphate 5 μmol
• ß-NADPH 4 μmol
• ß-NADH 4 μmol
• Sodium-phosphate buffer solution (pH 7.4) 100 μmol
• Sterilized purified water Balance

Test concentrations with justification for top dose:

A preliminary study was conducted with concentrations of 5000, 2500, 1250, 625, 313, 156, 78.1 and 39.1 μg/plate, whereupon antibacterial properties were observed in all the strains in the ≥ 313 μg/plates. The antibacterial properties were noted to be stronger in the 313 μg/plate in the case of TA100 than in the other strains. Accordingly, in the present study, following concentrations were established:

-S9 mix: 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA100)
19.5, 39.1, 78.1, 156, 313, 625 μg/plate (TA1535, TA98, TA1537, WP2 uvrA-)

+S9 mix: 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA100)
19.5, 39.1, 78.1, 156, 313, 625 μg/plate (TA1535, TA98, TA1537, WP2 uvrA-)

- Concentration analysis was performed for the test substance solutions at the maximum and minimum concentrations used when the present study was carried out for the first time, and said concentrations were confirmed to be within 100 ± 5% of the respective nominal concentrations

Vehicle / solvent:

- Aceton (Acquired from: Wako Pure Chemicals Ind., Ltd.; Lot No.: KCF1401; Purity: 99.5%)
- The positive control substance NaN3 was dissolved in water for injection, other control substances were dissolved in DMSO.
- Justification for choice of solvent/vehicle: not specified
- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: Sodium azide, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 plates per dose
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; preincubation
- Type and composition of media:
- Complete liquid medium: The medium used for propagating each strain was obtained by dissolving Oxoid Nutrient Broth No.2 25 g in 1L purified water and then sterilizing the solution obtained in an autoclave (121°C for 15 minutes).
- Minimum glucose agar plate medium: CLIMEDIA AM-N medium was purchased
and used. This medium was used for detection of reverse mutation bacteria.
- Top agar: As aqueous amino acid solution, a 0.5 mM mixed aqueous solution of D-biotin, L-histidine and L-tryptophan was prepared using purified water, and this was then subjected to filtration sterilization using a micro filter (pore diameter 0.45 μm), and then stored in a refrigerator. To 100 ml purified water were added powdered agar and sodium chloride in amounts of 0.6 g and 0.5 g respectively, and sterilized in an autoclave and caused to undergo complete dissolution. Thereafter the aforesaid aqueous amino acid solution was admixed in an amount of one tenth by volume, and the mixture stored at about 45°C. This medium following mixing with bacterial suspension, test substance solution and S9 Mix, was used for overlaying on minimal glucose agar plate medium.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: to investigate the presentation or not of antibacterial properties by the test substance, any growth of background bacteria seen under a stereomicroscope was noted

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- the number of revertant colonies was counted using an automatic colony counter

OTHER:
- the test was conducted by an appropriate method for volatile substances (usage of sealed vessels); for the positive control substance, a normal test tube fitted with a cap was used, and because of the different solvents, a 0.1 ml volume was used. Moreover, sealing in plastic bag was not carried out. The other conditions used were the same as those procedures used for the test substance.
- For each test strain, the genetic characteristics such as amino acid requirements, UV sensitivity, membrane mutation, drug resistance, etc. were examined beforehand for each lot, and strains having these characteristics were used.

Evaluation criteria:

In order for a result to be judged positive, the number of revertant colonies (mean value) on the plate treated with test substance must be two or more times greater than the number seen with the negative control, and there must be clear dose correlation and reproducibility.

Statistics:

No statistical analysis was conducted.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at a concentration of 313 μg/plate, with or without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Possibility of evaporation from medium: Yes

RANGE-FINDING/SCREENING STUDIES: Yes
-In the preliminary study, one plate was used for each concentration.
- The preliminary study was conducted with concentrations of 5000, 2500, 1250, 625, 313, 156, 78.1 and 39.1 μg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Yes

Ames test:
- Signs of toxicity: antibacterial properties were observed in all the strains in the ≥ 313 μg/plates
- Individual plate counts: Yes
- Mean number of revertant colonies per plate and standard deviation: Yes

For more detailed information please refer to Table 1 and 2 in the section "Any other information on results incl. tables".

Table 1: Study results (Main study 1)

 

S9 Mix	Test substance concentration (mg/plate)	Number of revertants (number of colonies/plate)
		Frameshift type						Frameshift type
		TA100		TA1535		WP2 uvrA		TA98		TA1537
	Solvent control	95		9		23		11		11
		105	( 102)	4	(     8)	32	( 29)	16	( 13)	11	( 10)
		105	(±     6)	10	(± 3)	32	(± 5)	11	(± 3)	9	(± 1)
	9.77	88
				(       )		(       )		(       )		(       )
		98	( 95)
		99	(±     6)	(       )		(       )		(       )		(       )
	19.5	107		9		28		13		11
		125	( 111)	6	(     8)	20	( 23)	16	( 13)	9	(     8)
		100	(± 13)	10	(± 2)	21	(± 4)	10	(± 3)	5	(± 3)
	39.1	113		9		31		13		7
		101	( 101)	7	(     8)	27	( 28)	8	( 10)	11	( 10)
-		90	(± 12)	7	(± 1)	25	(± 3)	8	(± 3)	13	(± 3)
	78.1	89		7		30		11		8
		76	( 82)	8	(     6)	15	( 29)	10	( 11)	10	( 11)
		82	(± 7)	4	(± 2)	41	(± 13)	12	(± 1)	15	(± 4)
	156	78		5		40		19		8
		99	( 91)	6	(     5)	32	( 35)	14	( 14)	4	(     7)
		95	(± 11)	5	(± 1)	32	(± 5)	8	(± 6)	10	(± 3)
	313	0*		2*		15*		9*		5*
		0*	(     0)	4*	(     5)	16*	( 15)	4*	(     6)	1*	(     3)
		0*	(± 0)	8*	(± 3)	14*	(± 1)	4*	(± 3)	2*	(± 2)
	625			0*		0*		0*		0*
		(       )
				0*	(     0)	0*	(     0)	0*	(     0)	0*	(     0)
		(       )		0*	(± 0)	0*	(± 0)	0*	(± 0)	0*	(± 0)
	Solvent control	94		5		32		25		15
		107	( 106)	6	(     7)	33	( 35)	14	( 20)	9	( 10)
		117	(± 12)	10	(± 3)	39	(± 4)	22	(± 6)	6	(± 5)
	9.77	128
				(       )		(       )		(       )		(       )
		115	( 116)
		104	(± 12)	(       )		(       )		(       )		(       )
	19.5	113		6		49		18		12
		115	( 106)	12	(     9)	37	( 42)	20	( 17)	9	( 12)
		90	(± 14)	9	(± 3)	41	(± 6)	13	(± 4)	15	(± 3)
	39.1	119		9		29		14		10
		87	( 101)	5	(     7)	37	( 34)	12	( 13)	10	( 11)
+		98	(± 16)	8	(± 2)	35	(± 4)	12	(± 1)	12	(± 1)
	78.1	109		10		44		18		15
		118	( 119)	9	( 11)	41	( 46)	22	( 20)	16	( 15)
		129	(± 10)	13	(± 2)	53	(± 6)	19	(± 2)	13	(± 2)
	156	101		3		36		21		13
		113	( 106)	6	(     7)	36	( 36)	16	( 17)	9	( 11)
		105	(± 6)	13	(± 5)	37	(± 1)	15	(± 3)	10	(± 2)
	313	0*		8*		27		20*		3*
		0*	(     0)	9*	(     7)	34	( 29)	15*	( 15)	4*	(     4)
		0*	(± 0)	4*	(± 3)	27	(± 4)	10*	(± 5)	4*	(± 1)
	625			0*		0*		0*		0*
		(       )
				0*	(     0)	0*	(     0)	0*	(     0)	0*	(     0)
		(       )		0*	(± 0)	0*	(± 0)	0*	(± 0)	0*	(± 0)
	Positive control	AF-2		NaN3		ENNG		AF-2		9-AA
	Concentration	0.01		0.5		2		0.1		80
-	(mg/plate)
	Number of colonies/plate	579 572 639	( 597) (± 37)	299 304 324	( 309) (± 13)	632 630 610	( 624) (± 12)	584 561 538	( 561) (± 23)	895 937 918	( 917) (± 21)
	Positive control	2-AA		2-AA		2-AA		2-AA		2-AA
	Concentration	1		2		10		0.5		2
+	(mg/plate)
	Number of colonies/plate	735 632 617	( 661) (± 64)	421 370 451	( 414) (± 41)	861 830 882	( 858) (± 26)	203 275 203	( 227) (± 42)	133 135 124	( 131) (±     6)

(Remarks) *: Antibacterial properties observed.

(Mean value) (± Standard deviation)

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, NaN3: Sodium azide,
ENNG: N-Ethyl-N’-nitro-N-nitrosoguanidine, 9-AA: 9-Aminoacrydine, 2-AA: 2-Aminoanthracene

 

 

Table 2: Study results (Main study 2)

 

S9 Mix	Test substance concentration (mg/plate)	Number of revertants (number of colonies/plate)
		Frameshift type									Frameshift type
		TA100			TA1535			WP2 uvrA			TA98		TA1537
	Solvent control	71			8			20			14		4
		89	(     83)		5	(     8)		18	( 23)		14	( 15)	8	(      7)
		89	(± 10)		10	(±	3)	31	(± 7)		18	(± 2)	8	(±	2)
	9.77	77
					(       )			(       )			(       )		(       )
		79	( 78)
		79	(±	1)	(       )			(       )			(       )		(       )
	19.5	81			8			26			13		10
		78	( 80)		10	(     9)		25	( 26)		18	( 15)	11	( 10)
		82	(±	2)	9	(± 1)		28	(± 2)		15	(± 3)	10	(± 1)
	39.1	96			10			32			15		7
		76	( 84)		11	(     9)		25	( 26)		19	( 16)	11	(     9)
-		80	(± 11)		6	(±	3)	22	(± 5)		14	(± 3)	10	(± 2)
	78.1	82			8			22			19		5
		70	( 74)		5	(     8)		25	( 23)		10	( 15)	6	(     8)
		71	(±	7)	12	(± 4)		21	(± 2)		17	(± 5)	13	(± 4)
	156	76			7			28			19		7
		70	( 77)		12	( 10)		23	( 22)		19	( 17)	7	(     6)
		84	(±	7)	11	(±	3)	15	(± 7)		13	(± 3)	5	(± 1)
	313	0*			5*			8*			9*		4*
		0*	(     0)		2*	(     4)		16*	( 11)		7*	(     6)	6*	(     4)
		0*	(± 0)		4*	(± 2)		10*	(±	4)	2*	(± 4)	2*	(± 2)
	625				0*			0*			0*		0*
		(       )
					0*	(     0)		0*	(     0)		0*	(     0)	0*	(     0)
		(       )			0*	(± 0)		0*	(± 0)		0*	(± 0)	0*	(± 0)
	Solvent control	96			12			34			15		5
		81	( 85)		10	( 10)		37	( 36)		20	( 19)	5	(     6)
		79	(±	9)	9	(±	2)	37	(± 2)		22	(± 4)	8	(± 2)
	9.77	73
					(       )			(       )			(       )		(       )
		91	( 84)
		89	(± 10)		(       )			(       )			(       )		(       )
	19.5	77			8			40			20		7
		73	( 86)		11	( 11)		35	( 37)		26	( 24)	7	(     6)
		107	(± 19)		13	(± 3)		35	(± 3)		26	(± 3)	5	(± 1)
	39.1	81			9			40			23		5
		80	( 86)		5	(     9)		25	( 31)		23	( 20)	7	(     7)
+		98	(± 10)		14	(± 5)		29	(± 8)		14	(± 5)	10	(± 3)
	78.1	84			15			41			24		7
		89	( 87)		11	( 12)		40	( 39)		24	( 21)	4	(     6)
		89	(±	3)	9	(±	3)	37	(± 2)		16	(± 5)	7	(± 2)
	156	75			5			33			16		5
		97	( 90)		9	(      8)		34	( 33)		19	( 18)	4	(     6)
		99	(± 13)		10	(±	3)	33	(± 1)		19	(± 2)	9	(± 3)
	313	0*			9*			26			14*		2*
		0*	(     0)		3*	(     5)		36	( 30)		14*	( 14)	4*	(     3)
		0*	(± 0)		3*	(± 3)		29	(± 5)		13*	(± 1)	3*	(±	1)
	625				0*			0*			0*		0*
		(       )
					0*	(     0)		0*	(     0)		0*	(     0)	0*	(     0)
		(       )			0*	(± 0)		0*	(± 0)		0*	(± 0)	0*	(± 0)
	Positive control	AF-2			NaN3			ENNG			AF-2		9-AA
	Concentration	0.01			0.5			2			0.1		80
-	(mg/plate)
	Number of colonies/plate	581 562 572	( 572) (± 10)		208 220 218	( 215) (±     6)		427 400 441	( 423) (± 21)		484 508 525	( 506) (± 21)	507 560 522	( 530) (± 27)
	Positive control	2-AA			2-AA			2-AA			2-AA		2-AA
	Concentration	1			2			10			0.5		2
+	(mg/plate)
	Number of colonies/plate	619 630 676	( 642) (± 30)		266 250 315	( 277) (± 34)		794 790 786	( 790) (±     4)		401 376 428	( 402) (± 26)	146 135 137	( 139) (±     6)

(Remarks) *: Antibacterial properties observed.

(Mean value) (± Standard deviation)

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, NaN3: Sodium azide,
ENNG: N-Ethyl-N’-nitro-N-nitrosoguanidine, 9-AA: 9-Aminoacrydine, 2-AA: 2-Aminoanthracene

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested similar to OECD TG 471 for its mutagenic potential using the bacterial strains Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli (WP2 uvrA-), in a reverse mutation assay. Based on a preliminary study, concentrations were established for TA100, namely a 313 μg/plate for the maximum concentration, plus concentrations of 156, 78.1, 39.1, 19.5 and 9.77 μg/plate; for the other strains, six concentrations were established with 625 μg/plate as the maximum concentration, plus concentrations of 313, 156, 78.1, 39.1 and 19.5 μg/plate. The test was conducted  both in the presence and absence of metabolic activation and vehicle and positive controls were assessed concurrently with the test substance. The first and the second time the main study was carried out, the number of revertant colonies of test strain induced by the test substance was not two or more times greater than the number seen with the negative (solvent) control, either with or without S9 Mix. Antibacterial properties were observed in all the test strains with and without S9 Mix. From the above results, it was concluded that the test substance is not mutagenic.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Principles of method if other than guideline:

The study reveals the following deviations from the OECD TG 473:
- a not-well established solvent (acetone) was used
- there was no short term treatment (3-6 h) with and without metabolic activation of the cell with the test chemical
- the concentration of 400 µg/ml tested in the Chromosome aberration test showed a high celltoxicity of over 50%; in addition precipitation was observed (When determining the highest test chemical concentration, concentrations that have the capability of producing artifactual positive responses, such as those producing excessive cytotoxicity and precipitation in the culture medium should be avoided because of these factors could lead to artifactual positive results.)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): not specified
- Batch number of test material: not specified
- Purity: 99.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not specified
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable under usual handing conditions
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Its solubility in water is 0.3% (20°C), and it is soluble in dimethyl sulfoxide and acetone.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid: please, see section "Test concentrations with justification for top dose"

FORM AS APPLIED IN THE TEST
- The test substance was dissolved in acetone to prepare test substance solution at 100 times the maximum concentration. This was further diluted using the solvent to prepare test substance solutions at 100 times each concentration.

OTHER SPECIFICS
- colorless transparent liquid having an ether-like (sic) odor

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Dainippon Pharma
- Suitability of cells: not specified
- Normal cell cycle time (negative control): not specified

For cell lines:
- Number of passages if applicable: a passage number of 5 or less were used
- Methods for maintenance in cell culture: Dimethyl sulfoxide was added in a proportion of 10% with respect to the cell suspension, and the mixture was divided into 1 mL aliquots which were then stored frozen in liquid nitrogen. For the tests, this mixture was thawed and cultured. For the cell culture, a plastic petri dish (diameter 10 cm) was used; a plastic flask (culture surface area 25 cm2 or 75 cm2) was used only when the cells were treated with test substance.

MEDIA USED
- Type and composition of media: MEM: Preparation was carried out in accordance with the instructions provided with Minimum Essential Medium, wherein to 1 l thereof was added 0.35 g sodium hydrogen carbonate, the pH was adjusted to about pH 7.2-7.3, and then sterilization filtration was performed under pressure using a membrane filter with a 0.22 µm pore size.
- Culture solution: Inactivated (heat-treated at 56°C for 30 minutes) calf serum was added to MEM in a proportion of 10%.
- CO2 concentration: 5%
- humidity level: humid conditions, not further specified
- temperature: 37°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: A commercial product (Kikkoman Corp., Lot No.: RAA-318, produced on November 11, 1994) was used.
- method of preparation of S9 mix: This S9 is 9000 g supernatant fraction of liver homogenate produced by intraperitoneal administration, to 7-week-old male SD rats (body weight 192-229 g), of phenobarbital 30 mg/kg once and 60 mg/kg 3 times, at 24 -hour intervals, and concomitant administration of 5,6-benzoflavone 80 mg/kg once on the 3rd phenobarbital administration. The S9 was stored at ≤-80°C until use.
- concentration or volume of S9 mix and S9 in the final culture medium: S9 mix was prepared, at the time of use, to the following composition per 1 ml; the mixture was stored on ice until use:
S9: 0.3 mL
Magnesium chloride (hexahydrate): 5 µmol
Potassium chloride: 33 µmol
Glucose-6-phosphate: 5 µmol
ß-NADP+: 4 µmol
HEPES (pH 7.2): 4 µmol
Sterile purified water: Balance
- When metabolic activation was employed in the co-presence of S9 mix, cells were treated with 30 µl test substance solution, 0.5 ml S9 mix and 2.5 ml culture medium for 6 hours, washed 3 times using MEM, then further cultured in 5 ml of fresh culture medium for 18 hours.
- quality controls of S9: not specified

Test concentrations with justification for top dose:

In order to determine appropriate concentrations for the chromosome aberration test, a cell proliferation inhibition test was carried out. Based on these results from the cell proliferation inhibition test, a chromosome aberration test was conducted for 3 concentrations with a common ratio of 2, with a concentration exceeding TCID50 as the maximum concentration.

The test substance solutions having the highest and the lowest concentrations used in the chromosome aberration test were subjected to concentration analysis; both were confirmed to be within 100±5% of the nominal concentration.

-S9 mix (24 h treatment): 0, 100, 200, 400 μg/mL
-S9 mix (48 h treatment): 0, 50, 100, 200 μg/mL
-S9 mix (6 h pulse treatment): 0, 175, 350, 700 μg/mL
+S9 mix (6 h pulse treatment): 0, 188, 375, 750 μg/mL

The widely used concentrations of 0.03 and 10 μg/mL were chosen as the concentrations of positive controls MMC and BP, respectively.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: not specified
- Justification for percentage of solvent in the final culture medium: not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: Mitomycin C and with metablic activation: Benzo(a)pyrene

Remarks:

MMC was dissolved in Japanese Pharmacopoeia physiological saline at a concentration of 3 μg/mL, and sterilized by filtration. BP that had been dissolved in DMSO at concentration of 4 mg/mL and stored frozen, was thawed at room temperature and used.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: cell suspension of 5 mL containing 4x10^3 cells/ml
- Test substance added in medium: Yes

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 3 d
- Exposure duration/duration of treatment: 24 and 48 h
- Harvest time after the end of treatment (sampling/recovery times): 24 and 48 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: At two hours before sample production, colcemid was added to each flask so as to form a final concentration of 0.1 µg/ml, and cells in metaphase were accumulated.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells were washed once with PBS(-), thereafter, cells were released by treating with trypsin, and cells were collected by centrifuging at 1000 rpm (maximum centrifuge acceleration, 170-180g) for 5 minutes (the same hereinafter). The supernatant was discarded, then 4 ml of 0.075M potassium chloride solution was added and hypotonic treatment (37°C, 15 minutes) was allowed to proceed. Furthermore, 4 ml of chilled methanol acetic acid (3:1) mixture (Carnoy’s fixative) was added to fix the cells. After centrifugation, the fixative was discarded, and 4 ml of fresh fixative was added. This procedure was repeated 3-4 times. On completion of fixation, a small amount of fixed cell suspension was prepared, a glass slide was placed on a wet towel, and the cell suspension was dripped onto the glass slide at 2 locations; the glass slide was then dried to obtain a sample slide. Two slides per concentration were prepared. The slides were stained with 3% Giemsa solution diluted with 1/150M phosphate buffered solution (pH 6.8) for 20 minutes, washed with water, dried, then sealed with a mounting agent, to obtain samples for observation.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 100
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Metaphase images comprising 25±2 well-spread chromosomes were selected, and the presence or absence of structural aberration was investigated. The classification of aberrations was as follows:
- Gap (including chromatid and chromosome types; abbreviated as g)
- Chromatid break (abbreviated as ctb)
- Chromatid exchange (abbreviated as cte)
- Chromosome break (abbreviated as csb)
- Chromosome exchange (dicentric chromosome, ring chromosome etc; abbreviated as cse)
- Other (fragmentation)
A gap is defined as an unstained section observed on a chromatid, where said unstained section is on the vertical axis of the chromatid and is at least as wide as the chromatid but not markedly so; the unstained section has a definite shape, and is distinguishable from a break. Structural aberrations were investigated for 100 cells per flask, i.e. 200 cells per concentration.
- Determination of polyploidy and endoreplication: A metaphase investigation was undertaken in respect of 100 cells per flask, i.e., 200 cells per concentration, and the number of polyploid cells including endoreduplication cells were counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Calculation of 50% cell proliferation inhibition concentration: When cytotoxicity was observed, the 50% cell proliferation inhibition concentration (TCID50) for the test substance was calculated.
- Mitotic index: The number of mitotic cells were counted for 1000 cells per flask, i.e. 2000 cells per concentration, and the mitotic index was determined. (for more details please see table 1 in section "Any other information on results incl. tables")

- Any supplementary information relevant to cytotoxicity: Dose levels selected for scoring did not show a appropriate toxicity in every case.

METHODS FOR MEASUREMENTS OF GENOTOXICITY
Wherein, the incidence of chromosome aberration was displayed, and if the result was positive, the D20 value (test substance concentration required to induce aberration in 20% of metaphase images, mg/ml) was calculated.

Evaluation criteria:

Judgement criteria of test results: For structural aberration, cells having one or more aberrations were regarded as cells with chromosomal aberration, and the data were compiled in two ways; excluding cells having gap aberration only (-g) and including said cells (+g). The compiled + g data was subjected to a chi-squared test with the negative control. Numerical aberration was tested in the same way. For the final assessment, if the percentage of cells exhibiting structural aberration, including cells exhibiting only gaps or numerical aberration, was less than 5%, the chromosome aberration inducibility of the test substance was deemed negative (-); if said percentage was 5% or more but less than 10%, said inducibility was deemed pseudo-positive (±); and if said percentage was 10% or more, said inducibility was deemed positive (+). Wherein, the incidence of chromosome aberration was displayed, and if the result was positive, the D20 value (test substance concentration required to induce aberration in 20% of metaphase images, mg/ml) was calculated.

Statistics:

Significant difference was tested using chi-squared test.

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the study, a 24-hour treatment at 400 µg/mL without metabolic activation exhibited chromosome structural aberration induced by the test substance. It should be noted that this concentration exhibited a cytotoxicity of over 50% in a previous cytotoxicity test. In addition, precipitation (separation) of test substance was observed at the time of cell treatment. According to the OECD TG 473, when determining the highest test chemical concentration, concentrations that have the capability of producing artifactual positive responses, such as those producing excessive cytotoxicity and precipitation in the culture medium should be avoided because of these factors could lead to artifactual positive results.)
Therefore the result of the study is probably ambigous.

Table 1: Mitotic index

Without metabolic activation system
Treatment	Treatment period (hrs)	Treatment concentration (µg/mL)	Number of observed cells	Mitotic index (%)
Negative control (acetone)	24	0	2,000	4.2
Test substance	24	100	2,000	3.4
	24	200	2,000	3.8
	24	400	2,000	0.9
Positive control (MMC)	24	0.03	2,000	3.0
Negative control (acetone)	48	0	2,000	3.3
Test substance	48	50	2,000	3.7
	48	100	2,000	2.3
	48	200	2,000	1.8
Positive control (MMC)	48	0.03	2,000	3.0
With metabolic activation system
Treatment	Presence or absence of S9 mix	Treated concentration (µg/mL)	Number of observed cells	Mitotic index (%)
Negative control (acetone)	+	0	2,000	6.9
Test substance	+	188	2,000	7.7
	+	375	2,000	5.9
	+	750	2,000	5.4
Positive control (BP)	+	10	2,000	4.8
Negative control (acetone)	-	0	2,000	6.0
Test substance	-	175	2,000	7.5
	-	350	2,000	5.5
	-	700	2,000	5.1
Positive control (BP)	-	10	2,000	4.9

Conclusions:

The chromosome aberration inducibility of the test substance with respect to CHL/IU cells under the present test conditions is probably ambigous.

Executive summary:

The test substance was evaluated for genotoxic potential in an in vivo chromosome aberration study using female Chinese hamster lung-derived cell line CHL/IU similar to OECD TG 473. Cell proliferation inhibition was observed with 24-hour and 48-hour treatment without metabolic activation, and with and without S9 mix with metabolic activation; the TCID50 concentrations were 343, 184, 617 and 596 µg/mL, respectively. On 24-hour treatment at 400 µg/mL without metabolic activation, 10.5% of cells exhibited chromosome structural aberration induced by the test substance. Moreover, on 24-hour treatment less than 5% of cells exhibited numerical aberration. Under the other treatment conditions, less than 5% of cells exhibited structural aberration or numerical aberration. The D20 value calculated from the results of this test was 0.85 mg/mL for 24-hour treatment without metabolic activation. It should be noted that this concentration exhibited a cytotoxicity of over 50% in a previous cytotoxicity test. In addition, precipitation (separation) of test substance was observed at the time of cell treatment. According to the OECD TG 473, when determining the highest test chemical concentration, concentrations that have the capability of producing artifactual positive responses, such as those producing excessive cytotoxicity and precipitation in the culture medium should be avoided because of these factors could lead to artifactual positive results.) Therefore the chromosome aberration inducibility of the test substance with respect to CHL/IU cells under the present test conditions is ambigous.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo:

- MNT: mammalian in vivo micronucleus test similar to OECD TG 474, K2, Crlj:CD1(ICR) SPF mice, gavage (concentrations: 500, 1000 and 2000 mg/kg bw), negative

An according to OECD TG 474 and GLP compliant, K1 study in Swiss CD-1 mice (intraperitoneally at dose-levels of 2000, 1000 and 500 mg/kg bw) conducted on the structural analoge nBMA support these findings.

 

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Principles of method if other than guideline:

Animals are treated with the test chemical once and samples of bone marrow are taken only 24 hours after treatment (In the preliminary study the bone marrow sampling times were set at about 24, 48 and 72 hours after the administrations); Tween® 80 was used as dispersant aid to prepare dosing solutions

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (supplier): Office of Chemical Safety, Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare
- Lot/batch number of test material: not specified
- Purity: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool dark place (in a refrigerator, measured temperature range: 3-8°C).
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: The test substance was analyzed by the test substance manufacturer on completion of the animal study, and was confirmed as stable. Stability and homogeneity were confirmed for 5 and 100 mg/ml test substance suspensions (vehicle: aqueous 0.5 w/v% CMC-Na solution with 0.1 % Tween® 80 added) that had been stored at room temperature for 24 hours.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid: 25, 50 and 100 mg/ml

FORM AS APPLIED IN THE TEST
- The test substance was obtained as a homogenous suspension
- The volume administered to each animal was calculated based on the body weight on the day of administration

OTHER
Test solution of each concentration used in the main study was sampled, taking 10 ml from the upper, medium and lower layer, respectively; the concentration and homogeneity of these samples were confirmed. The results revealed that these concentrations were 95.2-98.4% of the nominal value of the respective concentrations, and the homogeneity (C.V.) was 0.1-1.3%. Thus there was no problem with concentration or homogeneity, as both were within 100 ± 10% of the nominal value.

Species:

mouse

Strain:

other: CrlJ:CD1 (ICR) SPF

Details on species / strain selection:

Mice are widely used in micronucleus studies, and so mouse was selected as the animal species; the strain of mouse used in the present study was chosen because the characteristics of the strain are well known, and there is a lot of background data.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 31.9-35.7 g
- Assigned to test groups randomly: Animals that displayed no abnormality throughout the acclimatization and quarantine period were used. These animals were stratified according to body weight on the day of group allocation (administration day), and allocation was performed so that the mean body weights of the groups were as uniform as possible. Allocation was performed using a computer, by a combination of the block placement method and the random extraction method (the required groups were assembled by the block placement method, and the test groups and individual numbers therein were assigned randomly). Animals remaining after group allocation were euthanized under deep ether anesthesia on the study completion day.
- Fasting period before study: not specified
- Housing: individually
- Diet: Solid feed CRF-1, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-22°C
- Humidity: 47-54%
- Air changes: 10-15 times per hour
- Photoperiod: illumination 12 hours a day (07:00-19:00)

IN-LIFE DATES: not specified

Route of administration:

oral: gavage

Vehicle:

- Vehicle 1: Sodium carboxymethyl cellulose (CMC-Na)
- Type and concentration of dispersant aid: 0.1 % Tween® 80
- Justification for choice of solvent/vehicle: Because the test substance is poorly soluble in water, the vehicle selected was aqueous 0.5 w/v% sodium carboxymethyl cellulose solution with 0.1 % Tween® 80 added, in which the present test substance can be obtained as a homogenous suspension.
- Concentration of test material in vehicle: 0.5 w/v% sodium carboxymethyl cellulose solution with 0.1 % Tween® 80
- Amount of vehicle: 20 ml/kg bw
- Lot/batch no.: Lot No.: 4Y01 (CMC-Na) and 7398H (Tween® 80)
- Purity: not specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Treatment groups: For each concentration, the test substance was weighed, and thereto was added aqueous 0.5 w/v% CMC-Na solution with 0.1 % Tween® 80 added; the test substance was suspended therein using a homogenizer (high speed homogenizer Polytron Model K, Kinematica AG, for approx. 30 seconds, at setting 3), and the resulting suspension was made up to the specified volume. Any residual test solution was absorbed using a high-absorbency paper towel, and incinerated.
- Negative control group: The required amount of CMC-Na was weighed out and dissolved in water for injection [Lot No.: 6F74 to obtain 0.5 w/v% aqueous CMC-Na solution. Thereto was added Tween®80, to obtain aqueous 0.5 w/v% CMC-Na solution with 0.1 % Tween® 80 added.
-Positive control group: 0.1 mg/ml aqueous MMC solution was prepared at the time of use. Specifically, the contents of one vial of MMC (2 mg/vial) were dissolved in 5 ml water for injection, then 2 ml was collected and made up to 8 ml by adding 6 mL physiological saline.

For more details please see table 1 in section "Any other information on materials and methods incl. tables"

Storage temperature:
- Treatment groups: Prepared at time of use, and not stored.
- Negative control group: The vehicle was stored in a refrigerator (measured temperature for storage: preliminary study 3-5°C, main study 3-4°C), and used within 4 days after preparation (expiry: 8 days after preparation).
Positive control group: Prepared at time of use, and not stored.

Duration of treatment / exposure:

24 h

Frequency of treatment:

single administration via a flexible gastric tube

Dose / conc.:

2 000 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

6/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C (MMC)
- Justification for choice of positive control: Because MMC is widely used for micronucleus studies and there is a lot of background data; selected according to “toxicity study guidelines”.
- Route of administration: intraperitoneally administered once using a 25G injection needle
- Doses / concentrations: 1 mg/kg (dosage volume of 10 ml/kg bw)

Tissues and cell types examined:

- Sample material: bone marrow cells from the bilateral femoral bones
- For each sample, the number of immature erythrocytes (polychromatic erythrocytes (PCE)), and the number of normochromatic erythrocytes, per 200 total erythrocytes were counted, and at the same time, the number of polychromatic erythrocytes having micronuclei (MNPCE) per 2000 PCE was counted.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In the preliminary study, the maximum dose was set at 2000 mg/kg, which is the maximum dose as stipulated in the guidelines for toxicity tests; then doses of 1000, 500 and 250 mg/kg were set by dividing by a common ratio of 2. There there were no changes in general condition and no deaths. Accordingly, the doses in the main study were set as follows: the preliminary study maximum
dose of 2000 mg/kg was set as the high dose; then doses of 1000 and 500 mg/kg were set by dividing by common ratio of 2, giving 3 doses in total.

DETAILS OF SLIDE PREPARATION:
Samples for observing micronuclei were produced in accordance with the Schmid method (W. Schmid, Mutation Res. 31, 9-15 (1975), W. Schmid, “Chemical Mutagens,” Vol. 4, ed. by A. Hollaender, Plenum Press, N.Y., London, 1976, pp.76-78.). Specifically, at the specified time after administration, the respective mice were euthanized by cervical dislocation, and the bilateral femoral bones were extracted. Then, using a 1 ml disposable syringe and a 23G injection needle, bone marrow cells were washed out into a centrifuge tube using about 0.1-0.2 ml bovine fetal serum. Next, using the same syringe and injection needle,
the bone marrow cells and bovine fetal serum were mixed to loosen the cells, the mixture was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the precipitate was mixed thoroughly in a mixer and then smeared
onto a slide glass (for the right and left femoral bones, one slide was prepared for each side, per animal). The smear samples were air-dried, fixed with methanol for 3 minutes, and then air-dried again. When preparing the smear samples, a corresponding table showing the animal numbers and sample numbers was compiled, and a label stating the respective test number, stage, sample number, type of test and preparation date was attached to each sample.

METHOD OF ANALYSIS:
In order to conduct the observation by a blinded method, one slide with excellent smearing was selected based on sample number. The selected bone marrow smear sample was then stained using acridine orange fluorescent stain and observed in accordance with the method of Hayashi et al. (M. Hayashi, T. Sofuni, M. Jr. Ishidate, Mutat. Res., 120, 241 (1983); Hayashi, M. “Micronucleus tests”, Scientist Press Co., Tokyo, 1991, pp. 44-55.). A small amount of 40 µg/mL aqueous acridine orange solution was dropped onto a cover glass, then the bone marrow smear sample was mounted thereon. The sample was observed at 600x total magnification using a fluorescent microscope equipped with excitation light of wavelength around 490 nm, and an observation filter permeable to ≥515 nm wavelengths.

Statistics:

For each sample, the number of MNPCE per 2000 PCE, the MNPCE frequency (%), the number of PCE per 200 total erythrocytes, and the PCE frequency (%) were calculated. In addition, the mean values and standard deviation for the number of MNPCE, the MNPCE frequency (%), the number of PCE and the PCE frequency (%) were calculated for each group, and the maximum and minimum values for each frequency (%) were also calculated. Furthermore, to assess the significance of the micronuclei frequencies in the main study, it was confirmed that the MNPCE frequencies in the negative control group and in the positive control group were within mean ± 3 S.D. of the background data at the present laboratories (this institute), and then the negative control group and test substance administration groups were compared, and subjected to a Kastenbaum and Bowman test (one-sided p≤0.05) based on binominal distribution, and a Cochran Armitage trend test (two-sided, p≤0.05, 0.01). For the PCE frequencies, homogeneity of variance (one-sided p<0.05) between the negative control group and each test substance administration group was examined using the F-test, and as the distributions were equal, the Student t-test (two-sided, p<0.05, 0.01) was performed.
Moreover, for the positive control group, the MNPCE frequency (%) was compared with the negative control group, and a Kastenbaum and Bowman test (one-sided p≤0.05) based on binominal distribution was performed. For the PCE frequencies, homogeneity of variance (onesided p<0.05) with regard to the negative control group was examined using the F-test, and because the distributions were equal, the Student t-test (two-sided, p<0.05, 0.01) was performed.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: the test substance was administered at 250, 500, 1000 and 2000 mg/kg
- Solubility: Because the test substance is poorly soluble in water, the vehicle selected was aqueous 0.5 w/v% sodium carboxymethyl cellulose solution with 0.1 % Tween® 80 added, in which the present test substance can be obtained as a homogenous suspension.
- Clinical signs of toxicity in test animals: No changes in general condition were observed in any test substance administration group.
- Evidence of cytotoxicity in tissue analysed: No dose-dependent increase in MNPCE frequency in any test substance administration group was observed, at any sampling time. The PCE frequency per 200 total erythrocytes in each test substance administration group revealed a decrease in immature erythrocytes in one male in the 2000 mg/kg group 72 hours after administration, but not other dose-dependent decrease was observed at any sampling time.
- Rationale for exposure: not specified
- Observation time points: 24, 48 and 72 hours after administration

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In the test substance administration groups, the MNPCE frequency was 0.08 ± 0.06% in the 500 mg/kg group, 0.09 ± 0.04% in the 1000 mg/kg group and 0.13 ± 0.04% in the 2000 mg/kg group. Comparing these values with the negative control group, 0.15 ± 0.06%, revealed no statistically significant increase (p≤0.05) or dose-dependent increase (p≤0.05, 0.01) in any of the administration groups. For all test substance administration groups, there was no statistically significant decrease (p≤0.05, 0.01) in PCE frequency per 200 total erythrocytes when compared to the negative control group. Moreover, the MNPCE frequency in the positive control group was statistically significantly higher than in the negative control group. Furthermore, the MNPCE frequencies in the negative control group and the positive control group were within the range of mean ± 3 S.D. of the background data.
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes
- Other: Because there were no obvious differences between the sexes in terms of the appearance of toxicity, the main study was conducted using males only.

Conclusions:

It was judged from the above results that, under the conditions of this study, the test substance does not induce chromosomal aberration in the bone marrow of Crlj:CD1(ICR) SPF mice.

Executive summary:

The test substance was assessed in a similar to OECD TG 474 study for its potential to induce chromosomal aberration in CrlJ:CD1 (ICR) SPF mice using the micronucleus test method. For this purpose, the test substance, suspended in aqueous 0.5 w/v% sodium carboxymethyl cellulose (CHC-Na) solution with 0.1 % Tween® 80 added, was administered once orally to male animals at dose levels of 500, 1000 and 2000 mg/kg in a volume of 20 ml/kg body weight in each case. The vehicle served as negative control while mitomycin C served as positive controls for clastogenicity and for spindle poison effects, respectively. Bone marrow smear samples were produced approximately 24 hours after administration. The results revealed no statistically significant increase in the frequency of immature erythrocytes with micronuclei in any test substance administration group, compared to the negative control group, and no dose-dependent increase was observed. Moreover, there was no statistically significant decrease in the frequency of immature erythrocytes in 200 whole erythrocytes in any test substance administration group, compared to the negative control group, and therefore the test substance was judged to have no inhibitory effect on the proliferation of bone marrow cells. The frequency of immature erythrocytes with micronuclei in the negative and positive control groups were within the range of mean ± 3S.D. of the background data of the present laboratories (present institute), and so the study was thought to have been conducted appropriately. From the above results, the test substance was judged not to induce chromosomal aberration in the bone marrow of CrlJ:CD1 (ICR) SPF mice under the conditions of the present study.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Reliable in vitro studies of the test substance t-butyl methacrylate (t-BMA) were used to assess the potential of the t-BMA for gene mutations in bacteria and mammalian cells, cytogenicity in mammalian cells. Furthermore, an in vivo micronucleous assay was conducted with the test substance and the analogue substance n-butyl methacrylate.

Overall, based on the results obtained in the in vitro and in vivo studies the test item is not considered to be genotoxic/mutagenic or clastogenic.

In vitro assays

Bacterial gene mutation assay

The test substance was tested similar to OECD TG 471 for its mutagenic potential using the bacterial strains Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli (WP2 uvrA-), in a reverse mutation assay. Based on a preliminary study, concentrations were established for TA100, namely a 313 μg/plate for the maximum concentration, plus concentrations of 156, 78.1, 39.1, 19.5 and 9.77 μg/plate; for the other strains, six concentrations were established with 625 μg/plate as the maximum concentration, plus concentrations of 313, 156, 78.1, 39.1 and 19.5 μg/plate. The test was conducted  both in the presence and absence of metabolic activation and vehicle and positive controls were assessed concurrently with the test substance. The first and the second time the main study was carried out, the number of revertant colonies of test strain induced by the test substance was not two or more times greater than the number seen with the negative (solvent) control, either with or without S9 Mix. Antibacterial properties were observed in all the test strains with and without S9 Mix. From the above results, it was concluded that the test substance is not mutagenic.

Chromosomal aberration assay in mammalian cells

The test substance was tested in an in vitro chromosomal aberration test in Chinese hamster lung fibroblasts cells (CHL/IU) according to the OECD 473 guideline and GLP. The test was conducted with and without metabolic activation using the following test concentrations and exposure times:

without S9 mix (24 h treatment): 0, 100, 200, 400 μg/mL

without S9 mix (48 h treatment): 0, 50, 100, 200 μg/mL

without S9 mix (6 h pulse treatment): 0, 175, 350, 700 μg/mL

with S9 mix (6 h pulse treatment): 0, 188, 375, 750 μg/mL

The test item caused a weak positive response (clastogeniciy) in a mammalian chromosome aberration assay only at the highest test concentration tested with a 24 h exposure in the absence of metabolic activation (400 µg/mL). The remaining test concentrations, whether with or without metabolic activation, did not induce a positive response. Because only a weak increase in clastogenicity was observed for just one concentration and this result was not reproduced with any other test item concentration under similar conditions, the result of this test was regarded as negative. In addition a negative in vivo test is available ( see below).

HPRT assay

This GLP compliant study (according to OECD TG 476) was performed to investigate the potential of the test substance, dissolved in DMSO, to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in one experiment using two parallel cultures. The main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The highest applied concentration in the pre-test on toxicity (1422 µg/mL) was chosen corresponding to 10 mM, with respect to the OECD guideline 476 (2016). Based on the results of the pre-experiment the following concentrations were applied in the main experiment: 11.1, 22.2, 44.4, 88.9, 177.8, 355.5, 711, and 1422 µg/ml.

In the main experiment in the absence and presence of S9 mix, no relevant cytotoxicity was observed up to the highest applied concentration, which showed phase separation.

In the main experiment in the absence and presence of S9 mix, no relevant increases in the numbers of mutant colonies were observed after treatment with the test substance.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

 

In vivo assays

The test substance was assessed in a similar to OECD TG 474 study for its potential to induce chromosomal aberration in CrlJ:CD1 (ICR) SPF mice using the micronucleus test method. For this purpose, the test substance, suspended in aqueous 0.5 w/v% sodium carboxymethyl cellulose (CHC-Na) solution with 0.1 % Tween® 80 added, was administered once orally to male animals at dose levels of 500, 1000 and 2000 mg/kg in a volume of 20 ml/kg body weight in each case. The vehicle served as negative control while mitomycin C served as positive controls for clastogenicity and for spindle poison effects, respectively. Bone marrow smear samples were produced approximately 24 hours after administration. The results revealed no statistically significant increase in the frequency of immature erythrocytes with micronuclei in any test substance administration group, compared to the negative control group, and no dose-dependent increase was observed. Moreover, there was no statistically significant decrease in the frequency of immature erythrocytes in 200 whole erythrocytes in any test substance administration group, compared to the negative control group, and therefore the test substance was judged to have no inhibitory effect on the proliferation of bone marrow cells. The frequency of immature erythrocytes with micronuclei in the negative and positive control groups were within the range of mean ± 3S.D. of the background data of the present laboratories (present institute), and so the study was thought to have been conducted appropriately. From the above results, the test substance was judged not to induce chromosomal aberration in the bone marrow of CrlJ:CD1 (ICR) SPF mice under the conditions of the present study.

The ability of the read-across substance n-butyl methacrylate to cause chromosomal damage in vivo was investigated in a micronucleus OECD 474 test. Dose-levels for treatment were selected on the basis of a preliminary toxicity test. Male and female Swiss CD-1 mice were dosed once intraperitoneally with vehicle only, corn oil, 2000, 1000 and 500 mg/kg bw n-butyl methacrylate and the positive control Mitomycin-C. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time. Following treatment with n-butyl methacrylate, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose-level. Slight increases in the ratio of mature to polychromatic erythrocytes, compared to the vehicle control, were seen at the 48 hour sampling time for both male and female animals from the high-dose group, indicating that the test substance exerted a mild toxic effect on the bone marrow cells. Following treatment with the positive control Mitomycin-C, statistically significant increases in the incidence of micronucleated PCE's over the control values were seen in the positive control group indicating the correct functioning of the test system. It is concluded that n-butyl methacrylate administered intraperitoneally at dose-levels of 2000, 1000 and 500 mg/kg bodyweight to both male and female animals, does not induce micronuclei in the polychromatic erythrocytes of treated mice, under the reported experimental conditions.

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity were observed in in vitro or in vivo studies. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.