L-Glutamic acid, N-coco acyl derivs., compds. with triethanolamine (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames (Yuasa, 2000)

Under the conditions of this study the test material is not considered to have mutagenic potency.

Ames (Iwahara and Sakamoto, 1980)

Under the conditions of this study the test material is not considered to have mutagenic potency.

Ames (read-across; Hargitai, 2012)

Under the conditions of the study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, therefore the test material is considered to be non-mutagenic.

Chromosome Aberration (read-across; Murata 2011)

Under the conditions of the test, the frequency of structural and numerical chromosome aberrations was less than 5.0 %, determined in test systems exposed under short term (with and without metabolic activation) and continuous exposure (without metabolic activation). Cytotoxicity was observed at concentrations ≥ 1.89 mg/mL under short term exposure, independent of metabolic activation. Under continuous exposure cytotoxicity was also observed at concentrations ≥ 0.945 mg/mL without metabolic activation. Accordingly the test material was considered to be negative for clastogenic potential.

Mouse Lymphoma Assay (read-across; Hargitai, 2013)

No mutagenic effect of the test material was observed either in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

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:

23 February 2000 to 06 March 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The mutagenic potential of the test material was assessed using a reverse mutation assay in which four S. typhimurium strains and one E. coli strain were tested with and without metabolic activation.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The definite amount of test material was weighed at the time of use and water for injection of Japanese Pharmacopoeia was added to this to prepare the test material solution of high concentration. Purity conversion was performed at the time of preparation.
The test material solutions of low concentration were prepared by diluting the test material solution of high concentration with the same medium one after another.

Target gene:

S. typhimurium: Histidine
E. coli: Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The bacterial strains are widely used in mutagenicity studies for their capacity to induce reverse mutations because of high sensitivity to known mutagens.
Inherent genetic characteristics of the respective strains, amino acid requirement, membrane mutation rfa property, UV sensitivity, number of spontaneous revertants, drug resistance factor and sensitivity to know mutagens (relative value to solvent control, relative value to positive control and dose response) were confirmed.
- Methods for maintenance in cell culture if applicable: The microorganisms were dispersed in Nutrient broth No. 2 (Oxoid) overnight to make dispersions. As the seed bacteria, the bacterial dispersions were spread on Nutrient broth No. 2 agar plate and a single colony was dispersed again in Nutrient broth No. 2 and by the early time of the rest period it was incubated under shaking at 37 °C and 0.07 mL DMSO was added to each 0.8 mL of dispersion and stored at -80 °C until use.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Nutrient broth No. 2 was used for pre-incubation of test bacterial strains. The top agar prepared according to the standard defined in Ministry of Labour No. 77 was used (Bacto-Agar, Difco). Minimum glucose agar plate medium, which was prepared according to the same standard, was purchased and stored at room temperature until used.
- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: For confirmation of genetic characteristics and sensitivity to known mutagens, bacterial strains stored by freezing at -80 °C were thawed and 100 µL of each thawed bacteria was inoculated in 30 mL of nutrient broth No. 2 and incubated at 37 °C under shaking for 10 hours.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

-Dose-finding test: 5, 20, 78, 313, 1 250 and 5 000 µg/plate (with and without S9 mix).
-Main study with S9 mix: 156, 313, 625, 1 250, 2 500 and 5 000 µg/plate.
-Main study without S9 mix: 39, 78, 156, 313, 625, 1 250, 2 500 and 5 000 µg/plate.
-Concentrations for the main study were determined as a result of the dose-finding test.

Vehicle / solvent:

- Vehicle: water for injection
- Justification for choice of solvent/vehicle: Water for injection of Japanese Pharmacopoeia was selected as the medium of this test substance because this substance is stable in water and the provided test material is an aqueous solution containing 30 % of the test material.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide and 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

PRE-INCUBATION OF TEST BACTERIUM
The test bacterium strain frozen and stored at -80 °C was thawed, 100 μL of it was inoculated in 30 mL of Nutrient broth No.2, and incubated at 37 °C under shaking for 10 hours. Remaining thawed bacterium strain was all discarded after inoculation. The inoculated Nutrient broth No.2 was stored in a refrigerator before the start of shaking incubation.
After the end of pre-incubation, the optical density (OD value) of bacterial suspension was measured with a photoelectric spectrophotometer, and confirmed that the viable count calculated from the OD value was almost constant (> 10^9 cells/mL).

PREPARATION OF S9 MIX
S9 was thawed. This thawed S9 was added to cofactor solution, which was prepared before use, to make 10 v/v% solution, and agitated gently to prepare the S9 Mix of the following composition. The prepared S9 Mix was stored in a refrigerator until it was used.
Composition in each mL of S9 Mix: S9 0.1 mL, MgCl2 8.0 µmol, KCl 33.0 µmol, Glucose 6 phosphate 5.0 µmol, NADPHA 4.0 µmol, NADH 4.0 µmol and Sodium phosphate buffer (pH 7.4)100.0 µmol.

TEST PROCEDURE
-A 0.1 mL volume of the test material solution was dispensed into a test tube, 0.5 mL of 0.1 M phosphate buffer solution (pH 7.4) if there was no metabolic activation or S9 Mix if there was metabolic activation was added, and then 0.1 mL of respective bacterial suspension was added, and pre-incubated under shaking at 37 °C for 20 minutes. To this, 2.0 mL of top agar was added, and after agitation, it was poured over minimum glucose agar plate medium. Similar operations were performed for the negative (solvent) control and positive control.
-Minimum glucose agar plate medium was prepared 2 plates each for each dose of all of negative (solvent) control, positive control and test substance. In addition, for S9 Mix, 0.1 M phosphate buffer, Nutrient broth, and test substance of high concentration, sterility test was performed, confirming existence or absence of contamination of microorganisms.
-The number of revertant colonies that appeared after incubation at 37 °C for 48 hours was counted, and existence or absence of antibacterial activity was observed by a stereoscopic microscope, and existence or absence of precipitation of test substance was observed visually. The counting of number of revertant colonies was performed by the equipment measurement method using counting loss correction of the equipment.
The results are shown by the actual number and mean value of number of revertant colonies on each plate. In addition, dose-response curve was added for each bacterium strain.

DOSE CONCENTRATIONS
-In order to set the dose levels of each test, a dose finding test was performed, and the number of revertant colonies, existence or absence of antibacterial activity and precipitation were observed.
-From the results of dose finding test, 5 000 μg/plate was set as the high dose and the common ratio was set to be 4, and a total of 6 doses were set.
-In the dose finding test the test material showed antibacterial activity at more than 1 250 μg/plate to Salmonella typhimurium TA100, TA1535, TA98, TA1537 without metabolic activation, and at dose of 5 000 μg/plate with metabolic activation. In both presence and absence of metabolic activation, no precipitation was observed at any dose step, and no increase of more than 2 times of number of revertant colonies relative to the negative (solvent) control was observed in any bacterium strains.
-From the above result, for Salmonella typhimurium TA100, TA1535, TA98, TA1537 in reference to the dose where antibacterial activity was observed, the dose steps of this test were set 1 250 μg/plate for the case of no metabolic activation and 5 000 μg/plate for the case of metabolic activation as the high dose, and setting the common ratio to be 2, dose steps were set 6 doses in total.
-Because no antibacterial activity was observed for Escherichia coli WP2 uvrA regardless of existence or absence of metabolic activation in the dose finding test, 5 000 μg/plate was set as the high dose and the common ratio was set to be 2, with a total of 5 doses set.

PREPARATION OF POSITIVE CONTROL SUBSTANCES
Among positive control substances, NaN3 was prepared to the following test concentration using water for injection of Japanese Pharmacopoeia and other positive controls were prepared to their respective test concentrations using DMSO. The prepared positive control substances were frozen at -20 °C and stored and were thawed at time of use. The frozen storage period was one year after the preparation of the positive control substance stock solution that was used after diluting to the test concentrations and the item diluted to the test concentration was 3 months after dispensing. It was confirmed that the frozen positive control substances are stable for more than 1 year.

Evaluation criteria:

When the number of revertant colonies of the treated group with the test material increased more than 2 times relative to the negative (solvent) control and the dose-response relationship and repeatability were evident, it was judged to be positive.

Statistics:

No statistical analysis was performed.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action and at more than 5 000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action and at more than 5 000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action and at more than 5 000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action and at more than 5 000 μg/plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

-The test material did not increase the number of revertant colonies by more than 2 times of negative (solvent) control in any test with bacterium strain regardless of existence or absence of metabolic activation.
-It was concluded that the test material had no mutagenic potency under the conditions of the study.

CONTROL DATA
-There was no existence of intermingled bacteria in Nutrient broth, S9 Mix, 0.1 M phosphate buffer or test material of high concentration used in the test. This was confirmed by a sterility test.
-The number of revertant colonies of the negative (solvent) control and positive control were within the standard values based on background data. In addition, the positive control produced revertant colonies of more than 2 times of the negative (solvent) control in respective bacterium strains, demonstrating that the test was conducted appropriately and was therefore valid.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test material did not produce precipitation at any dose step with or without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY
-The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action to Salmonella typhimurium TA98, TA100, TA1535, TA1537 and at more than 5 000 μg/plate with metabolic activation.

Conclusions:

Under the conditions of this study the test material is not considered to have mutagenic potency.

Executive summary:

The potential of the test material to cause mutagenic potency was investigated using five strains of bacteria: Salmonella typhimurium TA98, TA100, TA1535, and TA 1537 and Escherichia coli WP2 uvrA, both in the presence and absence of metabolic activation in the form of S9 mix.

A 0.1 mL volume of the test material solution was dispensed into a test tube, 0.5 mL of 0.1 M phosphate buffer solution (pH 7.4) if there was no metabolic activation or S9 Mix if there was metabolic activation was added, and then 0.1 mL of respective bacterial suspension was added, and pre-incubated under shaking at 37 °C for 20 minutes. To this, 2.0 mL of top agar was added, and after agitation, it was poured over minimum glucose agar plate medium. Similar operations were performed for the negative (solvent) control and positive control. Duplicate tests were performed for all test groups. The number of revertant colonies that appeared after incubation at 37 °C for 48 hours was counted.

The test material did not increase the number of revertant colonies by more than 2 times the negative (solvent) control in any test with bacterium strain regardless of existence or absence of metabolic activation.

There was no existence of intermingled bacteria in Nutrient broth, S9 Mix, 0.1 M phosphate buffer or test material of high concentration used in the test. This was confirmed by a sterility test. The number of revertant colonies of the negative (solvent) control and positive control were within the standard values based on background data. In addition, the positive control produced revertant colonies of more than 2 times of the negative (solvent) control in respective bacterium strains, demonstrating that the test was conducted appropriately and was therefore valid. The test material did not produce precipitation at any dose step with or without metabolic activation.

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action to Salmonella typhimurium TA98, TA100, TA1535, TA1537 and at more than 5 000 μg/plate with metabolic activation.

Under the conditions of this study the test material is not considered to have mutagenic potency.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2011 to 11 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's MEM supplemented with 10% (v/v) bovine calf serum was used.
- Properly maintained: Yes, cell suspension at a density of 1.5 x 10^6 cells/mL with 10% (v/v) DMSO was stored in liquid nitrogen.
- Periodically checked for Mycoplasma contamination: Yes.
- Periodically checked for karyotype stability: Yes.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1, short term exposure: 1.89, 1.34, 0.945 and 0.668 mg/mL (6 hours exposure, without S9) and 1.89, 0.945, 0.473 and 0.236 mg/mL (with S9).
Experiment 2, continuous exposure: 0.945, 0.668, 0.473 and 0.334 mg/mL (24 hours exposure without S9) and 0.668, 0.473, 0.334 and 0.236 mg/mL (without S9).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle: The test material was soluble and stable in saline (at 100 mM). Therefore, saline was selected as the solvent.

Untreated negative controls:

no

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:

METHOD OF APPLICATION: in medium.

> EXPERIMENT 1: Short-term treatment.
DURATION
- Preincubation period: 3 days, cells seeded at 2 x 10^4 cells per plate were preincubated.
- Exposure duration: 6 hours, at 37 °C and 5 % CO2 concentration with 0.3 mL of the appropriate test solution, and 0.5 mL of S9 as necessary.
- Expression time (cells in growth medium): 18 hours, after being washed with PBS and fresh culture medium was added.

> EXPERIMENT 2: Continuous treatment.
DURATION
- Preincubation period: 3 days, cells seeded at 2 x 10^4 cells per plate were preincubated.
- Exposure duration: 24 or 48 hours, with 0.5 mL of the appropriate test solution.

SELECTION AGENT: Colcemid solution, added to cultures 2 hours prior to the end of incubation at a concentration of 0.2 µg/mL.
SPINDLE INHIBITOR (cytogenetic assays): Trypsin solution 0.25%.
STAIN (for cytogenetic assays): 2% Giemsa solution.

NUMBER OF REPLICATIONS: Duplicate.

NUMBER OF CELLS EVALUATED: 100 cells per replicate.

PRELIMINARY DOSE FINDING TEST
- Two preliminary tests were performed to determine the dosing range for both the short term and continuous treatment definitive tests. Results were based on cell growth inhibition. Tests were performed under the same conditions as described above for the definitive tests.
- Number of replications: Single culture plates per dose.
- Determination of inhibition: After the end of incubation, cell growth conditions were examined under a microscope, and the cell growth rate was determined. The cell growth rate was calculated as a percentage of the negative control, which was set at 100%.
- Dosing range:
> Experiment 1, short term exposure: 0.0591 to 3.78 mg/mL.
> Experiment 2, continuous exposure: 0.0597 to 3.78 mg/mL.
- Dose selection: Based on the cytotoxicity results, the dosing range for the definitive test was set as follows: The lowest dose causing cytotoxicity, under the test conditions, was set as the highest test dose in the definitive study, a further three lower dosing levels were set with a common ratio of 2^½.

Evaluation criteria:

The frequency (%) of structural aberrations and numerical aberrations was determined for dose levels according to the following criteria:
Negative = <5 %
Equivocal = ≥ 5 % and <10 %
Positive = ≥ 10 %
The final judgment regarding the positive results was that the values of the treatment group showed both obvious increase compared with the negative control values and dose dependency. Additionally, where test series was judged "positive" for a single dose or "equivocal", it was comprehensively judged "positive" if reproducibility of the result could be confirmed.

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 examined

Positive controls validity:

valid

Additional information on results:

DEFINITIVE TESTS
> Experiment 1: The frequency of chromosome aberrations was less than 5.0 % for both structural aberrations and numerical aberrations at any dose in both groups.
> Experiment 2: The frequency of chromosome aberrations was less than 5.0 % for both structural aberrations and numerical aberrations at any dose in both groups.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None observed in either test at initiation or termination.

RANGE-FINDING/SCREENING STUDIES:
> Experiment 1: Cell growth inhibition of ≥ 50 % was observed at doses of ≥ 1.89 mg/mL in both groups.
> Experiment 2: Cell growth inhibition of ≥ 50 % was observed at doses of ≥ 0.945 mg/mL in both groups.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
> Experiment 1: Cell growth inhibition of ≥ 50 % was observed at doses of ≥ 1.34 mg/mL in the absence of a metabolic activation system, and at doses of ≥ 0.945 mg/mL in the presence of a metabolic activation system.
> Experiment 2: Cell growth inhibition of ≥ 50 % was observed at doses of ≥ 0.668 mg/mL in the 24 h treatment, and at doses of ≥ 0.473 mg/mL in the 48 h treatment.

Table 1: Results of Short Term Exposure

Treatment time (h)	S9 mix	Dose (mg/mL)	Number of Cells with Structural Chromosome Aberration							gap	Cell Growth (%)	Number of cells with numerical chromosome aberrations
			Cells observed*	ctb	cte	csb	cse	others	Total (%)			polyploid	others	Total (%)
6 - 18	-	Saline	200	0	0	0	0	0	0 (0.0)	0	100	0	0	0 (0.0)
		0.668	200	1	0	0	0	0	1 (0.5)	0	79	0	0	0 (0.0)
		0.945	200	0	1	0	0	0	1 (0.5)	2	65	0	0	0 (0.0)
		1.34	200	0	0	0	0	0	0 (0.0)	1	42	0	0	0 (0.0)
		1.89	Cytotoxicity								19
		MMC 0.09 µg/mL	200	24	49	1	1	0	67 (33.5)	2	-	0	0	0 (0.0)
6 - 18	+	Saline	200	0	0	0	0	0	0 (0.0)	0	100	0	0	0 (0.0)
		0.236	200	0	0	0	0	0	0 (0.0)	0	85	0	0	0 (0.0)
		0.473	200	0	0	0	0	0	0 (0.0)	0	59	0	0	0 (0.0)
		0.945	200	0	0	0	0	0	0 (0.00	0	43	0	0	0 (0.0)
		1.89	Cytotoxicity								23
		BP 10 µg/mL	200	3	28	0	0	0	31 (15.5)	0	-	0	0	0 (0.0)

 

 

Table 2: Results of Short Term Exposure

Treatment time (h)	S9 mix	Dose (mg/mL)	Number of Cells with Structural Chromosome Aberration							gap	Cell Growth (%)	Number of cells with numerical chromosome aberrations
			Cells observed*	ctb	cte	csb	cse	others	Total (%)			polyploid	others	Total (%)
24 - 0	-	Saline	200	0	0	0	0	0	0 (0.0)	0	100	0	0	0 (0.0)
		0.334	200	0	1	0	0	0	1 (0.5)	0	80	0	0	0 (0.0)
		0.473	200	0	0	0	0	0	0 (0.0)	0	71	0	0	0 (0.0)
		0.668	200	0	0	0	0	0	0 (0.0)	0	49	0	0	0 (0.0)
		0.945	Cytotoxicity								33
		MMC 0.05 µg/mL	200	38	57	0	0	0	86 (43.0)	0		0	0	0 (0.0)
48 - 0	-	Saline	200	0	0	0	0	0	0 (0.0)	0	100	0	0	0 (0.0)
		0.236	200	0	0	0	0	0	0 (0.0)	0	76	0	0	0 (0.0)
		0.334	200	0	0	0	0	0	0 (0.0)	0	60	0	0	0 (0.0)
		0.473	200	0	0	0	0	0	0 (0.0)	0	45	0	0	0 (0.0)
		0.668	Cytotoxicity								20
		MMC 0.03 µg/mL	200	22	45	0	1	0	65 (32.5)	2		0	0	0 (0.0)

ctb = chromatid break; cte = chromatid exchange; csb = chromosome break; cse = chromosome exchange; other = other structural aberration (fragmentation), other numerical aberrations (endoreduplication).

* Cells from replicates 1 and 2; MMC = Mitomycin C (positive control); BP = Benzo[a]pyrene (positive control)

Cytotoxicity, chromosome analysis was terminated due to cell toxicity

Conclusions:

Under the conditions of the test, the frequency of structural and numerical chromosome aberrations was less than 5.0 %, determined in test systems exposed under short term (with and without metabolic activation) and continuous exposure (without metabolic activation). Cytotoxicity was observed at concentrations ≥ 1.89 mg/mL under short term exposure, independent of metabolic activation. Under continuous exposure cytotoxicity was also observed at concentrations ≥ 0.945 mg/mL without metabolic activation. Accordingly the test material was considered to be negative for clastogenic potential.

Executive summary:

The clastogneic potential of the test material was determined in an in vitro mammalian cell chromosome aberration test with Chinese hamster lung fibroblasts (V79). The study was performed under GLP conditions and according to OECD 473. Two experiments were performed during this study. Experiment 1 was conducted with a short term exposure of 6 hours in the presence and absence of metabolic activation system, S9. Experiment 2 was conducted with continuous exposure, for either 24 or 48 hours, in the absence of metabolic activation. Solvent and positive controls were run concurrently for comparison.

Under the conditions of the test, the frequency of structural and numerical chromosome aberrations was less than 5.0 %, determined in test systems exposed under short term (with and without metabolic activation) and continuous exposure (without metabolic activation). Cytotoxicity was observed at concentrations ≥ 1.89 mg/mL under short term exposure, independent of metabolic activation. Under continuous exposure cytotoxicity was also observed at concentrations ≥ 0.945 mg/mL without metabolic activation. Accordingly the test material was considered to be negative for clastogenic potential.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For the justification for read-across, please refer to the read-across assessment framework report that is attached to Section 13.

Reason / purpose for cross-reference:

read-across source

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 examined

Positive controls validity:

valid

Conclusions:

Under the conditions of the test, the frequency of structural and numerical chromosome aberrations was less than 5.0 %, determined in test systems exposed under short term (with and without metabolic activation) and continuous exposure (without metabolic activation). Cytotoxicity was observed at concentrations ≥ 1.89 mg/mL under short term exposure, independent of metabolic activation. Under continuous exposure cytotoxicity was also observed at concentrations ≥ 0.945 mg/mL without metabolic activation. Accordingly the test material was considered to be negative for clastogenic potential.

Executive summary:

The clastogneic potential of the test material was determined in anin vitromammalian cell chromosome aberration test with Chinese hamster lung fibroblasts (V79). The study was performed under GLP conditions and according to OECD 473. Two experiments were performed during this study. Experiment 1 was conducted with a short term exposure of 6 hours in the presence and absence of metabolic activation system, S9. Experiment 2 was conducted with continuous exposure, for either 24 or 48 hours, in the absence of metabolic activation. Solvent and positive controls were run concurrently for comparison.

Under the conditions of the test, the frequency of structural and numerical chromosome aberrations was less than 5.0 %, determined in test systems exposed under short term (with and without metabolic activation) and continuous exposure (without metabolic activation). Cytotoxicity was observed at concentrations ≥ 1.89 mg/mL under short term exposure, independent of metabolic activation. Under continuous exposure cytotoxicity was also observed at concentrations ≥ 0.945 mg/mL without metabolic activation. Accordingly the test material was considered to be negative for clastogenic potential.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 September 2011 to 01 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

tk+/- (thymidine kinase) locus.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI-1640 medium.
- Properly maintained: Yes, cells were stored as frozen stocks in liquid nitrogen. For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. Subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study).
- Periodically checked for Mycoplasma contamination: Yes.
- Periodically "cleansed" against high spontaneous background: Yes, Each batch of frozen cells was purged of TK-/- mutants.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial fraction (2 % S9 fraction).

Test concentrations with justification for top dose:

- Assay 1: Tested at concentrations of 300, 250, 200, 150, 100, 75, 50, 25, 12.5 and 6.25 μg/mL with S9-mix, and at 1000, 500, 400, 300, 200, 100, 50, 25, 12.5 and 6.25 μg/mL without S9-mix.
- Assay 2: Tested at concentrations of 300, 250, 200, 175, 150, 100, 75, 50, 25 and 12.5 μg/mL with S9-mix, and at 300, 250, 200, 150, 100, 75, 50, 25, 12.5 and 6.25 μg/mL without S9-mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI-1640 medium.
- Justification for choice of solvent/vehicle: Based on the available solubility information, the test material is soluble in distilled water at 200 mg/mL, however, strong foaming was observed during the dissolution process. Therefore, the solubility of the test material was further investigated in RPMI-1640 medium. 250 mg/mL concentration was achievable using this vehicle; therefore, it was selected for vehicle of the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI-1640 medium and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Remarks:

DMSO was used as a solvent for positive control substances.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.
A suitable volume (0.2 mL) of solvent, test material formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or of 150 mM KCl (treatments without metabolic activation) were added to a final volume of 20 mL per culture in each experiment. For the 3-hour treatment, at least 10^7 cells were placed in each of a series of sterile flasks (culturing surface 75 cm²). For the 24-hour treatment, at least 4 x 10^6 cells were placed in each of a series of sterile flasks (culturing surface 25 cm²). The treatment medium contained a reduced serum level of 5% (v/v) (RPMI-5).

INCUBATION CONDITIONS
Cultures were incubated at 37 °C ± 1 °C (approximately 5% CO2 in air). Gentle shaking was used during the 3-hour treatments.

DURATION
- Exposure duration:
> Assay 1: 3 hour exposure with and without metabolic activation (S9-mix)
> Assay 2: 3 hour exposure with metabolic activation and 24 hours without metabolic activation.
- Expression time (cells in growth medium): Approximately 3 days.
- Selection time (if incubation with a selection agent): 2 weeks.

SELECTION AGENT: 5-trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.

NUMBER OF CELLS EVALUATED: 384 wells (in four 96-well microplates) at 2 x 10^3 cells per well. Microplates were incubated at 37 °C ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the mechanism of action of the test material.

DETERMINATION OF CYTOTOXICITY
- Method: Measured as relative survival.

Evaluation criteria:

ASSAY ACCEPTANCE CRITERIA
An assay is considered valid if all of the following criteria are met:
1. The mutant frequency in the negative (solvent) control cultures fall within the normal range (50-170 mutants per 10^6 viable cells).
2. The positive control chemicals induce a statistically significant increase in the mutant frequency.
3. The plating efficiency (PEviability) of the negative controls is within the range of 65 % to 120 % at the end of the expression period.
4. At least four test concentrations are present, where the highest concentration produces 80 – 90 % toxicity (by %RS or RTG), results in precipitation, is 5 mg/mL, or is the highest practical concentration.

EVALUATION CRITERIA
A test material is considered to be mutagenic in this assay if all the following criteria are met:
1. The assay is valid.
2. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding negative control values at one or more concentrations.
3. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. There is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05).
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 10^6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was performed a spreadsheet. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No large changes in pH were observed.
- Effects of osmolality: No large changes in osmolality were observed.
- Water solubility: No insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments of Assay 1 and 2.

RANGE-FINDING/SCREENING STUDIES: The highest concentration tested in the preliminary experiment was 5000 μg/mL.
No insolubility, but cytotoxicity was observed in the preliminary experiment. Therefore, the concentrations selected for the main experiments were expected to cover the concentration range from the maximum cytotoxicity (resulting approximately 10 – 20 % relative survival) to little or no cytotoxicity. The selected highest concentration was 1000 μg/mL. The lower test concentrations were generally separated by factor of two. More closely spaced concentrations were used in the expected cytotoxic concentration range. At least ten test concentrations were selected for the main experiments.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequency of the negative (solvent) control was in the recommended range (50-170 mutants per 10^6 viable cells) in each experiment. The positive controls (cyclophosphamide in the presence of metabolic activation and 4-nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
> Assay 1: In the presence of S9-mix (3-hour treatment), expressive cytotoxicity of the test material was observed at 300, 250 and 200 μg/mL, cells in these samples died during the treatment. An evaluation was made using data of first surviving concentration of 150 μg/mL (relative survival value of 10 %) and next six lower concentrations (a total of seven samples).
In the absence of S9-mix (3-hour treatment), excessive cytotoxicity of the test material was observed at 1000, 500 and 400 μg/mL, cells in these samples died during the treatment. Marked cytotoxicity of the test material was still detected at 300 μg/mL concentration (relative survival values of 1 %), therefore this concentration was excluded from the evaluation. An evaluation was made using data of the next concentration of 200 μg/mL (relative survival value of 39%) and the next five concentrations (a total of six samples).

> Assay 2: In the presence of S9-mix (3-hour treatment), expressive cytotoxicity of the test material was observed at 300 and 250 μg/mL, cells in these samples died during the treatment. Marked cytotoxicity of the test material was still detected at 200 and 175 μg/mL (relative survival values of 2 and 8 %, respectively); therefore these concentrations were excluded from the evaluation. An evaluation was made using data of next concentration of 150 μg/mL (relative survival value of 13 %) and next five lower concentrations (a total of six samples).
In the absence of S9-mix (24-hour treatment), excessive cytotoxicity of the test material was observed at 300, 250 and 200 μg/mL, cells in these samples died during the treatment. An evaluation was made using data of the first surviving concentration of 150 μg/mL (relative survival value of 27%) and the next six concentrations (a total of seven samples).

MUTATION FREQUENCY
> Assay 1: In the presence of S9-mix (3-hour treatment), No significant increase in the mutation frequency was observed at the evaluated concentrations. No dose response to the treatment was indicated by the linear trend analysis.
In the absence of S9-mix (3-hour treatment), no statistically significant increase in the mutation frequency was observed at the evaluated concentrations. Furthermore, no significant dose response to the treatment was indicated by the linear trend analysis. This experiment was considered as negative.

> Assay 2: In the presence of S9-mix (3-hour treatment), no significant increase in the mutation frequency was observed at the evaluated concentrations. No dose response to the treatment was indicated by the linear trend analysis.
In the absence of S9-mix (24-hour treatment), no statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis. This experiment confirmed the negative results of the first assay.

Table 1: Mutation Assay 1 Results

S9-mix	Treatment Period (hours)	Test Material Concentration (µg/mL)	Survival		Viability	Mutation Frequency
			Plating Efficiency (PE %)	Harmonised Relative Survival (%) § (%RS)	Plating Efficiency (PE %)
+	3	300	ND	ND	ND	ND
		250	ND	ND	ND	ND
		200	ND	ND	ND	ND
		150	75.4	10	42.7	161.7
		100	80.5	33	41.7	80.7
		75	84.7	55	52.4	131.6
		50	81.0	67	54.7	120.7
		25	71.7	68	46.7	146.3
		12.5	70.1	73	51.3	127.3
		6.25	88.0	85	53.9	96.6
		Solvent Control	90.0	100	53.9	104.6
		Solvent Control CP	72.7	75	48.8	130.8
		Untreated Control	69.2	88	35.7	164.4
		Positive Control (CP)	67.2	66	43.3	334.0*
-	3	1000	ND	ND	ND	ND
		500	ND	ND	ND	ND
		400	ND	ND	ND	ND
		300	14.4	1	37.7	NE
		200	40.5	39	65.3	90.9
		100	59.2	75	59.2	101.6
		50	64.4	83	57.9	83.7
		25	73.7	96	71.2	88.6
		12.5	73.2	93	67.7	89.9
		6.25	70.2	91	77.0	80.9
		Solvent Control	81.6	100	49.1	100.2
		Solvent Control NQO	64.4	82	64.4	108.3
		Untreated Control	68.2	90	60.9	102.4
		Positive Control (NQO)	30.7	34	62.2	648.2*

CP = Cyclophosphamide (4 μg/mL); NQO = 4-Nitroquinoline-N-oxide (0.15 μg/mL)

Solvent control = 1 (v/v) % RPMI-0

Solvent control for CP = 1 (v/v) % DMSO

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

ND = No data (No cells were plated for colony growing due to excessive cytotoxicity observed during the treatment.)

* = Statistically significant

NE = Not evaluated (due to the high level of cytotoxicity)

 

Table 2: Mutation Assay 2 Results

S9-mix	Treatment Period (hours)	Test Material Concentration (µg/mL)	Survival		Viability	Mutation Frequency
			Plating Efficiency (PE %)	Harmonised Relative Survival (%) § (%RS)	Plating Efficiency (PE %)
+	3	300	ND	ND	ND	ND
		250	ND	ND	ND	ND
		200	21.2	2	64.0	NE
		175	42.7	8	56.7	NE
		150	31.2	13	66.3	124.7
		100	72.7	56	70.6	119.2
		75	65.3	53	55.5	99.09
		50	77.0	73	55.9	107.5
		25	81.0	89	66.3	122.4
		12.5	83.5	91	59.6	100.9
		Solvent Control	79.9	100	62.6	85.5
		Solvent Control CP	75.4	89	74.3	85.9
		Untreated Control	81.6	105	55.1	127.9
		Positive Control (CP)	59.6	66	47.4	478.8*
-	24	300	ND	ND	ND	ND
		250	ND	ND	ND	ND
		200	ND	ND	ND	ND
		150	53.9	27	63.5	102.8
		100	63.1	78	70.2	102.6
		75	45.0	65	64.4	162.4
		50	38.0	69	56.3	125.2
		25	43.6	76	65.8	108.3
		12.5	44.3	96	74.3	86.9
		6.25	46.3	88	65.8	109.4
		Solvent Control	47.4	100	65.4	106.8
		Solvent Control NQO	46.7	108	62.6	97.2
		Untreated Control	62.6	129	53.2	115.8
		Positive Control (NQO)	9.9	8	43.3	1152.2*

CP = Cyclophosphamide (4 μg/mL); NQO = 4-Nitroquinoline-N-oxide (0.1 μg/mL)

Solvent control = 1 (v/v) % RPMI-0

Solvent control for CP = 1 (v/v) % DMSO

§ = Relative survival values (%) corrected with the post treatment cell concentrations.

ND = No data (No cells were plated for colony growing due to excessive cytotoxicity observed during the treatment.)

* = Statistically significant

NE = Not evaluated (due to the high level of cytotoxicity)

Conclusions:

No mutagenic effect of the test material was observed either in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Executive summary:

The mutagenic potential of the test material was determined in an in vitro mouse lymphoma assay, conducted under GLP conditions and in line with the standardised guidelines OECD 476 and EU Method B.17.

Cultures of mouse lymphoma L5178Y cells were exposed to the test material at concentrations ranging from 6.25 to 1000 μg/mL, in two assays, both with and without the addition of a rat metabolic activation system (S9 fraction). Assay 1 was performed with two experiments, both with a three hour exposure period; however the first was performed in the presence and the second in the absence of metabolic activation. Assay 2 was performed again with two experiments. The first was performed with metabolic activation and an exposure period of 3 hours, whereas the second experiment was performed without metabolic activation with a 24 hour exposure period. All experiments were conducted with an expression time of 3 days and a selection time of 2 weeks, with trifuorothymidine as the selection agent.

Treatment with the test material did not result in a statistically significant increase in mutation frequencies either in the presence or absence of metabolic activation system in the Mouse Lymphoma Assay. The test material was therefore considered to have no mutagenic potential under the conditions of this test.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

For the justification for read-across, please refer to the read-across assessment framework report that is attached to Section 13.

Reason / purpose for cross-reference:

read-across source

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:

not examined

Positive controls validity:

valid

Conclusions:

No mutagenic effect of the test material was observed either in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Executive summary:

The mutagenic potential of the test material was determined in anin vitromouse lymphoma assay, conducted under GLP conditions and in line with the standardised guidelines OECD 476 and EU Method B.17.

Cultures of mouse lymphoma L5178Y cells were exposed to the test material at concentrations ranging from 6.25 to 1000 μg/mL, in two assays, both with and without the addition of a rat metabolic activation system (S9 fraction). Assay 1 was performed with two experiments, both with a three hour exposure period; however the first was performed in the presence and the second in the absence of metabolic activation. Assay 2 was performed again with two experiments. The first was performed with metabolic activation and an exposure period of 3 hours, whereas the second experiment was performed without metabolic activation with a 24 hour exposure period. All experiments were conducted with an expression time of 3 days and a selection time of 2 weeks, with trifuorothymidine as the selection agent.

Treatment with the test material did not result in a statistically significant increase in mutation frequencies either in the presence or absence of metabolic activation system in the Mouse Lymphoma Assay. The test material was therefore considered to have no mutagenic potential under the conditions of this test.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

A reverse mutation assay was performed using four Salmonella typhimurium strains in a plate incorporation assay with and without metabolic activation.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.5, 1, 2 and 5 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Once dissolved in distilled water, the test material was filter-sterilised with a membrane filter.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

- Seed bacteria were inoculated into nutrient broth with 0.5 % sodium chloride (Difco) in an L-shaped test tube, and cultured on a reciprocal shaker at 37 °C for 16 hours. The resultant cultures were used as inocula for the test.

-2 mL of top agar was mixed well with a 0.1 mL aliquot of inoculum, 0.1 mL of test material solution, and 0.5 mL of phosphate buffer (0.5 mL of a S-9 mixture in the metabolic activation test) in a small test tube.

-The mixture was overlaid on a base agar plate and allowed to solidify. The plate was incubated at 37 °C for 48 hours, and then reverse mutant colonies emerged were calculated.

-Top agar: Bacto agar 0.6 %, NaCl 0.5 %, L-Histidine 0.05 mM and Biotin 0.05 mM.
-S9-Mixture (in 1 mL): S-9 0.1 mL, MgCl2 8 µm, KCl 33 µm, G-6-P 5 µm, G-6-PDH 0.5 U, NADPH 4 µm and 0.2 M Phosphate buffer solution (pH 7.4) 0.5 mL.
-Base agar (per 1L culture medium): MgSO4.7H2O 0.2 g, Citric acid.H2O 2 g, K2HPO4 10 g, NaNH4HPO4 3.5 g, Glucose 20 g, NaCl 5 g and Bacto agar 15 g.
A 30 mL of base agar is added into a petri dish of 90 mm in diameter and allowed to solidify.

Key result

Species / strain:

bacteria, other: Salmonella typhimurium TA98, TA100, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Both with and without metabolic activation no obvious increase in mutant colony was found in the 4 tester strains of Salmonella typhimurium TA-100, TA-98, TA-1535, and TA-1537 at a dose range of 5 to 0.5 mg/plate.

Conclusions:

Under the conditions of this study the test material is not considered to have mutagenic potency.

Executive summary:

The potential of the test material to cause mutagenic potency was investigated using the following strains of bacteria: Salmonella typhimurium TA98, TA100, TA1535, and TA 1537, in the presence and absence of metabolic activation in the form of S9 mix.

The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Seed bacteria were inoculated into nutrient broth with 0.5 % sodium chloride (Difco) in an L-shaped test tube, and cultured on a reciprocal shaker at 37 °C for 16 hours. The resultant cultures were used as inocula for the test. Following the plate incorporation method, 2 mL of top agar was mixed well with a 0.1 mL aliquot of inoculum, 0.1 mL of test material solution, and 0.5 mL of phosphate buffer (0.5 mL of a S-9 mixture in the metabolic activation test) in a small test tube. The mixture was overlaid on a base agar plate and allowed to solidify. The plate was incubated at 37 °C for 48 hours, and then reverse mutant colonies emerged were calculated.

Both with and without metabolic activation no obvious increase in mutant colony was found in the 4 tester strains of Salmonella typhimurium TA-100, TA-98, TA-1535, and TA-1537 at a dose range of 5 to 0.5 mg/plate.

Under the conditions of this study the test material is not considered to have mutagenic potency.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 September 2011 to 20 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan operon.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient Broth No.2, 25.0 g per 1000 mL. Sterilization was performed at 121 °C in an autoclave.
- Properly maintained: Yes, all strains were stored at -80 ± 10 °C. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent. Additional strain characteristics were checked regularly.
- Periodically "cleansed" against high spontaneous background: Yes.

Additional strain / cell type characteristics:

other: rfa, uvrB, uvrA and pKM101

Metabolic activation:

with and without

Metabolic activation system:

Liver extract, S9 fraction.

Test concentrations with justification for top dose:

Main test: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water.
- Justification for choice of solvent/vehicle: The solvent was chosen based on a preliminary solubility test. The solubility of the test material was examined in distilled water, dimethyl sulfoxide (DMSO), and acetone. The test material was soluble in distilled water at 100 mg/mL concentration, but only partial dissolution was observed using DMSO or acetone as solvent at the same concentration. Therefore, distilled water was chosen for solvent of the study. The obtained formulations (50 μL) with the solution of top agar (2 mL) and phosphate buffer (500 µL) were examined in a test tube without test bacterium suspension and found to form a clear solution at 5000 µg/tube.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene; 4-nitro-1,2-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation). Before the overlaying, 50 μL of the test material solution (or solvent or reference control), 100 μL of the overnight culture of the tester strains (cultured in Nutrient Broth No.2) and 0.5 mL of the S9 mix or phosphate buffer (pH 7.4) were added into appropriate tubes to provide direct contact between bacteria and the test material. After the pre-incubation period, 2 mL of molten top agar (kept at 45°C) were added to the tubes; the content was mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activation and an activation test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated.

DURATION
- Preincubation period: 20 mins at 37 °C in a shaking incubator.
- Exposure duration: 48 hours, incubated at 37°C.

NUMBER OF REPLICATIONS: Each concentration was performed in triplicate. A second test was performed at the same dosing range to confirm the results of the first assay.

OBSERVATIONS
The colony numbers on the untreated / negative / positive control and test material treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported.

RANGE FINDING TEST:
- Test concentrations: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
- Tester strains: TA98 and TA100.

Evaluation criteria:

The study was considered valid if:
- The number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests.
- At least five analyzable (non-cytotoxic) concentrations were presented in all strains of the main tests.

Criteria for a positive (mutagenic) response:
- A dose related increase in the number of revertants occurred and/or,
- A reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- In all strains: the number of reversion more than twice higher than the reversion rate of the solvent control

Criteria for a negative (non-mutagenic) response:
The test material was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test material and for the controls using a spreadsheet.

Mutation factor (MF) = mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 & TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MAIN TESTS
In the Initial Mutation Test and Confirmatory Mutation Tests none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed in the main tests.

RANGE-FINDING STUDIES
- Results used for determination of the concentration range for the definitive test: The observed numbers of revertant colonies were mostly in the normal range. Lower colony counts compared to controls were considered to reflect the biological variability of the test system. Inhibition was observed in tester strains TA98 and TA100 at 5000, 2500 and 1000 μg/plate concentrations without metabolic activation; and in S. typhimurium TA100 tester strain at 5000, 2500 and 1000 μg/plate concentrations with metabolic activation. No precipitation was observed in the preliminary experiment.
Based on the observed result of the preliminary experiment, 5000 μg/plate concentration was selected as the highest examined dose in the main test and lower concentrations will be evenly spaced by approximately half log intervals to cover the range from cytotoxicity to no cytotoxicity.

ADDITIONAL INFORMATION ON CYTOTOXICITY
Inhibitory, cytotoxic effects (reduced / slightly reduced background lawn development and / or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) of the test material were observed in both assays in all S. typhimurium strains at concentrations of 5000 and 1581 μg/plate without metabolic activation and at 5000 μg/plate with metabolic activation. Cytotoxiciy was observed on the Initial Mutation Test with E. coli WP2 uvrA strain at 5000 μg/plate without metabolic activation. Furthermore in the Confirmatory Mutation Test, in S. typhimurium TA98, TA1535 and TA1537 at 500 μg/plate without metabolic activation, in S. typhimurium TA100 and TA1537 at 1581 μg/plate with metabolic activation, and in E. coli WP2 uvrA strain at 5000 μg/plate without metabolic activation.

Table 1: Summary of Initial Mutation Test

Concentration (µg/plate)		S. typhimurium Tester Strains								E. Coli
		TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Untreated control	Mean	19.0	32.3	84.7	101.0	10.7	10.3	5.7	9.3	31.0	51.7
	MF	0.98	1.13	1.01	0.96	1.03	0.89	0.74	1.65	0.99	1.22
DMSO control	Mean	19.3	27.0	-	94.3	-	12.3	7.0	9.7	-	44.0
	MF	1.00	0.94	-	0.90	-	1.06	0.91	1.71	-	1.04
Distilled water control	Mean	19.3	28.7	83.7	104.7	10.3	11.7	7.7	5.7	31.3	42.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	0.0*#	10.3 #	0.0*#	39.0*#	0.0*#	5.7*#	1.7*#	1.3 #	14.7 #	34.3
	MF	0.00 #	0.36 #	0.00 #	0.37 #	0.00 #	0.49 #	0.22 #	0.24 #	0.47 #	0.81
1581	Mean	7.7*#	17.0	49.3*#	47.3 #	3.7*#	11.7	2.0*#	6.3	21.3	41.3
	MF	0.40 #	0.59	0.59 #	0.45 #	0.35 #	1.00	0.26 #	1.12	0.68	0.98
500	Mean	13.0	21.3	57.3	69.0	6.7	10.7	2.0	8.0	25.3	40.7
	MF	0.67	0.74	0.69	0.66	0.65	0.91	0.26	1.41	0.81	0.96
158.1	Mean	16.7	26.0	80.0	90.7	7.7	12.0	6.0	5.7	30.3	44.3
	MF	0.86	0.91	0.96	0.87	0.74	1.03	0.78	1.00	0.97	1.05
50	Mean	21.3	26.3	83.7	98.0	8.0	10.7	4.7	7.3	29.7	38.3
	MF	1.10	0.92	1.00	0.94	0.77	0.91	0.61	1.29	0.95	0.91
15.81	Mean	22.7	26.0	90.7	104.7	12.7	14.0	5.0	10.3	31.0	41.7
	MF	1.17	0.91	1.08	1.00	1.23	1.20	0.65	1.82	0.99	0.98
5	Mean	21.3	28.7	81.0	96.3	9.3	10.7	4.3	9.3	28.7	36.3
	MF	1.10	1.00	0.97	0.92	0.90	0.91	0.57	1.65	0.91	0.86
1.581	Mean	21.3	27.7	94.3	89.3	9.3	9.3	6.3	10.3	29.0	35.0
	MF	1.10	0.97	1.13	0.85	0.90	0.80	0.83	1.82	0.93	0.83

* = growth inhibition; # = Cytotoxic concentrations; MF = mutation factor (mean number of revertants on the test material plate divided by the mean number of revertants on the vehicle control plate).

 

Table 2: Summary of Confirmatory Mutation Test

Concentration (µg/plate)		S. typhimurium Tester Strains								E. Coli
		TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Untreated control	Mean	28.7	28.0	91.0	109.3	9.7	8.7	8.3	8.7	23.3	27.7
	MF	1.34	0.79	1.04	1.10	0.91	0.70	1.04	1.04	1.13	0.95
DMSO control	Mean	27.3	29.0	-	111.3	-	9.7	4.0	8.7	-	24.7
	MF	1.28	0.81	-	1.12	-	0.78	0.50	1.04	-	0.85
Distilled water control	Mean	21.3	35.7	87.3	99.0	10.7	12.3	8.0	8.3	20.7	29.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	3.3*#	9.0*#	27.0*#	38.7*#	1.0*#	5.0*#	0.0*#	1.7*#	10.7*#	30.3
	MF	0.16 #	0.25 #	0.31 #	0.39#	0.09 #	0.41 #	0.00 #	0.20 #	0.52 #	1.05
1581	Mean	4.7*#	29.7 #	53.7*#	35.3*#	4.0*#	10.3	0.0*#	3.7 #	22.7	33.0
	MF	0.22#	0.83	0.61 #	0.36 #	0.38 #	0.84	0.00 #	0.44 #	1.10	1.14
500	Mean	15.0*#	35.0	66.3	56.0	6.0*#	9.7 #	2.7*#	8.0	21.3	33.7
	MF	0.70 #	0.98	0.76	0.57	0.56 #	0.78 #	0.33 #	0.96	1.03	1.16
158.1	Mean	24.0	29.7	88.7	71.7	8.7	6.7	8.0	9.0	22.7	40.0
	MF	1.13	0.83	1.02	0.72	0.81	0.54	1.00	1.08	1.10	1.38
50	Mean	26.3	33.3	84.7	85.3	7.7	9.7	3.7	9.7	24.3	38.0
	MF	1.23	0.93	0.97	0.86	0.72	0.78	0.46	1.16	1.18	1.31
15.81	Mean	26.3	31.7	84.7	89.7	7.7	9.7	5.3	8.7	24.0	39.7
	MF	1.23	0.89	0.97	0.91	0.72	0.78	0.67	1.04	1.16	1.37
5	Mean	27.0	39.0	86.0	102.0	8.0	14.0	10.3	10.0	25.3	32.3
	MF	1.27	1.09	0.98	1.03	0.75	1.14	1.29	1.20	1.23	1.11
1.581	Mean	21.3	39.0	92.0	93.0	4.7	8.3	8.7	5.7	26.3	35.7
	MF	1.00	1.09	1.05	0.94	0.44	0.68	1.08	0.68	1.27	1.23

* = growth inhibition; # = Cytotoxic concentrations; MF = mutation factor (mean number of revertants on the test material plate divided by the mean number of revertants on the vehicle control plate).

Conclusions:

Under the conditions of the study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, therefore the test material is considered to be non-mutagenic. Cytotoxicity was observed in both S. typhimurium and E. coli at certain concentrations at or above 500 µg/plate, however, this was dependant on the strain and the inclusion of metabolic activation.

Executive summary:

The mutagenic potential of the test material was assessed in an Ames test conducted under GLP conditions and in line with the standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100. During the study S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA were exposed to the test material at concentrations of 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate using the plate incorporation method. Positive and solvent controls were run concurrently for comparison.

Under the conditions of the study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, therefore the test material is considered to be non-mutagenic. Cytotoxicity was observed in both S. typhimurium and E. coli at certain concentrations at or above 500 µg/plate, however this was dependant on the strain and the presence or absence of metabolic activation.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

For the justification for read-across, please refer to the read-across assessment framework report that is attached to Section 13.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 & TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the conditions of the study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, therefore the test material is considered to be non-mutagenic. Cytotoxicity was observed in both S. typhimurium and E. coli at certain concentrations at or above 500 µg/plate, however, this was dependant on the strain and the inclusion of metabolic activation.

Executive summary:

The mutagenic potential of the test material was assessed in an Ames test conducted under GLP conditions and in line with the standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100. During the studyS. typhimuriumTA 1535, TA 1537, TA 98, TA 100 andE. coliWP2uvrAwere exposed to the test material at concentrations of 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate using the plate incorporation method. Positive and solvent controls were run concurrently for comparison.

Under the conditions of the study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, therefore the test material is considered to be non-mutagenic. Cytotoxicity was observed in bothS. typhimuriumandE. coliat certain concentrations at or above 500 µg/plate, however this was dependant on the strain and the presence or absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames (Yuasa, 2000)

The potential of the test material to cause mutagenic potency was investigated using five strains of bacteria: Salmonella typhimurium TA98,TA100,TA1535, and TA 1537 and Escherichia coli WP2 uvrA,both in the presence and absence of metabolic activation in the form of S9 mix. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

A 0.1 mL volume of the test material solution was dispensed into a test tube, 0.5 mL of 0.1 M phosphate buffer solution (pH 7.4) if there was no metabolic activation or S9 Mix if there was metabolic activation was added, and then 0.1 mL of respective bacterial suspension was added, and pre-incubated under shaking at 37 °C for 20 minutes. To this, 2.0 mL of top agar was added, and after agitation, it was poured over minimum glucose agar plate medium. Similar operations were performed for the negative (solvent) control and positive control. Duplicate tests were performed for all test groups. The number of revertant colonies that appeared after incubation at 37 °C for 48 hours was counted.

The test material did not increase the number of revertant colonies by more than 2 times the negative (solvent) control in any test with bacterium strain regardless of existence or absence of metabolic activation.

There was no existence of intermingled bacteria in Nutrient broth, S9 Mix, 0.1 M phosphate buffer or test material of high concentration used in the test. This was confirmed by a sterility test. The number of revertant colonies of the negative (solvent) control and positive control were within the standard values based on background data. In addition, the positive control produced revertant colonies of more than 2 times of the negative (solvent) control in respective bacterium strains, demonstrating that the test was conducted appropriately and was therefore valid. The test material did not produce precipitation at any dose step with or without metabolic activation.

The test material showed antibacterial activity at more than 1 250 μg/plate without metabolic action to Salmonella typhimurium TA98, TA100, TA1535, TA1537 and at more than 5 000 μg/plate with metabolic activation.

Under the conditions of this study the test material is not considered to have mutagenic potency.

Ames (Iwahara and Sakamoto, 1980)

The potential of the test material to cause mutagenic potency was investigated using the following strains of bacteria; Salmonella typhimurium TA98, TA100, TA1535, and TA 1537, in the presence and absence of metabolic activation in the form of S9 mix.

The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Seed bacteria were inoculated into nutrient broth with 0.5 % sodium chloride (Difco) in an L-shaped test tube, and cultured on a reciprocal shaker at 37 °C for 16 hours. The resultant cultures were used as inocula for the test. Following the plate incorporation method, 2 mL of top agar was mixed well with a 0.1 mL aliquot of inoculum, 0.1 mL of test material solution, and 0.5 mL of phosphate buffer (0.5 mL of a S-9 mixture in the metabolic activation test) in a small test tube. The mixture was overlaid on a base agar plate and allowed to solidify. The plate was incubated at 37 °C for 48 hours, and then reverse mutant colonies emerged were calculated.

Both with and without metabolic activation no obvious increase in mutant colony was found in the 4 tester strains of Salmonella typhimurium TA-100, TA-98, TA-1535, and TA-1537 at a dose range of 5 to 0.5 mg/plate.

Under the conditions of this study the test material is not considered to have mutagenic potency.

Ames (read-across; Hargitai, 2012)

In the key study (Hargitai, 2012) the mutagenic potential of the read-across material was assessed in an Ames test conducted under GLP conditions and in line with the standardised guidelines OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100. The study has been assigned a reliability score of 1 in line with Klimisch et al. (1997).

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA were exposed to the test material at concentrations of 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate using the plate incorporation method. Positive and solvent controls were run concurrently for comparison.

Under the conditions of the test, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used, therefore the test material is considered to be non-mutagenic. Cytotoxicity was observed in both S.typhimurium and E. coli at certain concentrations at or above 500 µg/plate, however this was dependant on the strain and the presence or absence of metabolic activation.

 

Chromosome Aberration (read-across; Murata 2011)

In the key study (Murata, 2011) the clastogneic potential of the read-across material was determined in an in vitro mammalian chromosome aberration test with Chinese hamster lung fibroblasts (V79). The study was performed under GLP conditions and according to OECD 473. The study has been assigned a reliability score of 1 in line with Klimisch et al. (1997).

Two experiments were performed during this study. Experiment 1 was conducted with a short term exposure of 6 hours in the presence and absence of metabolic activation system S9. Experiment 2 was conducted with continuous exposure for either 24 or 48 hours in the absence of metabolic activation. Solvent and positive controls were run concurrently for comparison.

Under the conditions of the test, the frequency of structural and numerical chromosome aberrations was less than 5.0%, determined in test systems exposed under short term (with and without metabolic activation) and continuous exposure (without metabolic activation). Cytotoxicity was observed at concentrations ≥ 1.89 mg/mL under short term exposure, independent of metabolic activation. Under continuous exposure cytotoxicity was also observed at concentrations ≥ 0.945 mg/mL without metabolic activation. Accordingly the test material was considered to be negative for clastogenic potential.

 

Mouse Lymphoma Assay (read-across; Hargitai, 2013)

In the key study (Hargitai, 2013) the mutagenic potential of the read-across material was determined in an in vitro mouse lymphoma assay, conducted under GLP conditions and in line with the standardised guidelines OECD 476 and EU Method B.17. The study has been assigned a reliability score of 1 in line with Klimisch et al. (1997).

Cultures of mouse lymphoma L5178Y cells were exposed to the test material at concentrations ranging from 6.25 to 1000 μg/mL, in two assays, both with and without the addition of a rat metabolic activation system (S9 fraction). Assay 1 was performed with two experiments, both with a three hour exposure period; however the first was performed in the presence and the second in the absence of metabolic activation. Assay 2 was performed again with two experiments. The first was performed with metabolic activation and an exposure period of 3 hours, whereas the second experiment was performed without metabolic activation with a 24 hour exposure period. All experiments were conducted with an expression time of 3 days and a selection time of 2 weeks, with trifuorothymidine as the selection agent.Treatment with the test material did not result in a statistically significant increase in mutation frequencies either in the presence or absence of metabolic activation system in the Mouse Lymphoma Assay. The test material was therefore considered to have no mutagenic potential under the conditions of this test.

Justification for classification or non-classification

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