3,5-dimethylpyrazole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2002 - 29 March 2002

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.

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:

other: EPA 712-C-98-247

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: CPMP/ICH/141/95

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: CPMP/ICH/174/95

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his G 46 in TA 1535 and TA 100,
his C 3076 in TA 1537,
his D 3052 in TA 1538 and TA 98,
trpE locus in E. coli.

his G 46 is a mis-sense mutation which is reverted to prototrophy by a variety of mutagens that cause base-pair substitutions

his C 3076 contains a frameshift mutation which appears to have added a GC base pair. This mutation is reverted by 9-aminoacridine, ICR-191 and epoxides of polycyclic hydrocarbons.

his D 3052 also contains a frameshift mutation which is reverted by the deletion of 2 base-pairs, CG GC. It is readily reverted by aromatic amines and derivatives.

E.col WP2uvrA contains an ochre mutation at the trpE locus and can be mutated to tryptophan independence either by a base-pair reversion of an A-T base-pair at the tprE locus, or, more likely, by a base-pair substitution within a number of transfer RNA loci elsewhere in the chromome.

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: Oxoid Nutrient Broth No. 2 (25 g per litre).
- Properly maintained: yes
- Samples of each strain were grown by culturing for 16 hrs at 37 ºC in nutrient broth. Cultures were kept for up to 4 days at +4 ºC to allow relevant checks to be performed but fresh cultures were used for the experiment.

Additional strain / cell type characteristics:

other: rfa, uvrB, pKM101

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 mix

Test concentrations with justification for top dose:

- 17, 50, 167, 500, 1667 and 5000 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Two mutation assays were carried out; one with direct plate incorporation and the second with preincubation.

DURATION
- Direct plate method: Plates were prepared by mixing 0.5 mL of S9 mix or 0.05 M phosphate buffer and 0.1 mL of bacteria to 2 mL of soft agar, 0.1 mL aliquots of the solvent (DMSO) or test material were added last. Once mixed, the cooling agar was then poured onto minimal medium plates. These plates contained 25 mL of 1.5% purified agar, in Vogel-Bonner Medium E with 2% glucose. Once set the agar plates were inverted and incubated at 37º C.
- Preincubation period: Preparation began with adding 0.5 mL volumes of either S9 mix or 0.05 M phosphate buffer to sterile tubes, followed by 0.1 mL of bacteria and finally 0.1 mL of either the test solution or solvent (DMSO). Tubes were then sealed and placed in a shaking incubator at 37 ºC for 20 minutes. After which 2 mL of soft agar was added. The cooling contents were then mixed and poured into agar plates, as above. Once set the agar plates were inverted and incubated at 37 °C.
- Exposure duration: Plates were incubated for 2 or 3 days.

NUMBER OF REPLICATIONS: All concentrations were performed with and without S9 mix in triplicate for both tests.

COLONY EVALUATION: Colonies of ≥ 0.1 mm in diameter were counted.

TOXICITY TEST
- Strain: TA 100
- Concentrations: 17, 50, 167, 500, 1667 and 5000 µg per plate.
- Method: One plate was prepared for each concentration in the presence and absence of the S9 mix.

OTHER EXAMINATIONS
Quality Control: Each strain was tested for its resistance to amplicillin (indicating pKM101) and its sensitivity to ultraviolet light and crystal violet (indicating persistence of the uvrB and rfa mutations).

Evaluation criteria:

EVALUATION CRITERIA
A positive response was recorded if the following criteria were met:
1) For S. typhimurium strains TA 1353, TA 1537, and TA 98 and for E. coli at least doubling of the mean concurrent vehicle control values at some concentration of the test material. For S. typhimurium stain TA 100, a 1.5-fold increase over the control value was considered significant. If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases, a minimum count of 20 was required before a significant mutagenic response was registered.
2) A dose related response, although at high dose levels this relationship could be inverted because of, for example (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
3) A reproducible effect in independent tests.

ACCEPTABILITY
The test was considered acceptable if the following criteria were met:
1) The bacteria demonstrated their typical response to crystal violet, ampicillin and U.V. light.
2) At least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and E. coli WP2uvrA 1-60.
3) On at least 2 of the positive control plates, there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5.
4) No toxicity or contamination was observed in at least 4 dose levels.
5) In cases where a mutagenic response was observed, no more than one dose level was discarded before the dose that gave the highest significant mean colony number.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

other: All results were valid with the exception of the counts obtained for TA 100 with 9-aminoacridine. The mutant counts were higher than those reported in the historical data. However this was not thought to have affected the integrity of the study results.

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precippitation was observed at any of the concentrations tested.

QUALITY CONTROL:
All strains were sensitive to crystal violet, whereas only the plasmid-containing stains, TA 98 and TA 100, were resistant to ampicillin. The strains were also tested for sensitivity to U.V light emitted over a period of 5-10 s from a lamp set at 254 nm. Increased sensitivity to U.V. light was demonstrated. These results are consistent with the known properties of these bacteria.

TOXICITY TEST:
No toxicity to bacteria was observed and no precipitation of the test material occurred in either the presence or absence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Direct Plate Method: Mean Number of Revertant Colonies in the Presence and Absence of S9 Mix

Substance	Concentrations ( µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
In the Presence of S9
DMSO	100 µL	22 ± 5	17 ± 6	25 ± 8	117 ± 8	9 ± 0
Test Material	17	21 ± 1	22 ± 1	22± 6	115 ± 15	11 ± 5
	50	22 ± 1	15 ± 3	20 ± 2	116 ± 11	9 ± 3
	167	18 ± 3	16 ± 4	24 ± 3	111 ± 6	14 ± 2
	500	18 ± 5	19 ± 4	21 ± 7	118 ± 3	10 ± 2
	1667	20 ± 2	19 ± 3	26 ± 6	115 ± 9	16 ± 3
	5000	18 ± 6	15 ± 8	25 ± 2	117 ± 8	13 ± 2
In the Absence of S9
DMSO	100 µL	24 ± 8	14 ± 3	11 ± 4	110 ± 6	12 ± 6
Test Material	17	24 ± 11	15 ± 5	19 ± 4	121 ± 14	11 ± 3
	50	20 ± 7	13 ± 3	11 ± 2	114 ± 12	9 ± 3
	167	22 ± 7	13 ± 5	15 ± 4	112 ± 9	10 ± 3
	500	23 ± 10	15 ± 3	16 ± 3	109 ± 6	12 ± 1
	1667	23 ± 8	13 ± 2	14 ± 3	117 ± 5	13 ± 2
	5000	22 ± 10	11 ± 1	13 ± 2	99 ± 13	14 ± 2

 

Table 2. Preincubation Method: Mean Number of Revertant Colonies in the Presence and Absence of S9 Mix

Substance	Concentrations ( µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
In the Presence of S9
DMSO	100 µL	20 ± 3	9 ± 1	40 ± 1	139 ± 14	10 ± 1
Test Material	17	17 ± 5	16 ± 4	39 ± 8	146 ± 17	12 ± 3
	50	21 ± 5	12 ± 2	41 ± 5	144 ± 16	15 ± 5
	167	20 ± 3	9 ± 4	33 ± 6	149 ± 10	13 ± 1
	500	19 ± 7	10 ± 1	38 ± 3	164 ± 7	12 ± 3
	1667	16 ± 3	10 ± 1	37 ± 6	134 ± 8	12 ± 3
	5000	20 ± 3	7 ± 2	41 ± 6	125 ± 12	13 ± 1
In the Absence of S9
DMSO	100 µL	20 ± 5	9 ± 1	26 ± 2	142 ± 11	11 ± 3
Test Material	17	15 ± 3	8 ± 1	29 ± 5	125 ± 11	11 ± 6
	50	18 ± 5	13 ± 3	25 ± 7	124 ± 13	12 ± 3
	167	17 ± 2	8 ± 3	23 ± 3	130 ± 3	9 ± 3
	500	19 ± 2	10 ± 3	27 ± 5	121 ± 9	10 ± 4
	1667	18 ± 6	9 ± 4	31 ± 3	122 ± 9	8 ± 4
	5000	19 ± 2	6 ± 2	20 ± 2	136 ± 5	10 ± 4

 

Table 3. Positive Control Results

Test Method	Presence/Absence of S9 mix	Compound	Strain	Concentration (µg/plate)	Colony Count (mean ± SD)
Direct Plate Method	+	2AAN	TA 1535	2	608 ± 40
			TA 1537	2	705 ± 55
			TA 98	0.5	235 ± 29
			TA 100	0.5	1136 ± 71
			WP2uvrA	20	701 ± 31
	-	NaN₃	TA 1535	1	335 ± 80
		9AA	TA 1537	80	2945 ± 276
		2NF	TA 98	1	468 ± 30
		NaN₃	TA 100	1	938 ± 61
		ENNG	WP2uvrA	2	864 ± 26
Preincubation Method	+	2AAN	TA 1535	2	465 ± 32
			TA 1537	2	441 ± 6
			TA 98	0.5	955 ± 34
			TA 100	0.5	1994 ± 23
			WP2uvrA	20	673 ± 52
	-	NaN₃	TA 1535	1	490 ± 26
		9AA	TA 1537	80	4048 ± 546
		2NF	TA 98	1	723 ± 73
		NaN₃	TA 100	1	1160 ± 24
		ENNG	WP2uvrA	2	902 ± 60

2-AAN = 2-Aminoanthracene

NaN₃ = Sodium azide

9AA = 9-Aminoacridine

2NF = 2-Nitrofluorene

ENNG = N-Ethyl-N-nitro-N-nitrosoguanidine

Table 4. Toxicity Test

Strain	Concentration (µg/plate)	S-9 mix	Revertant Colony Count
TA 100	DMSO	-	114
	17	-	112
	50	-	122
	167	-	114
	500	-	119
	1667	-	115
	5000	-	96
	DMSO	+	121
	17	+	121
	50	+	142
	167	+	122
	500	+	132
	1667	+	112
	5000	+	102

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of the test, no mutagenic response was induced in any of the five strains tested in either the presence or absence of metabolic activation.

Executive summary:

In a GLP compliant study performed to standardised guidelines, the mutagenic potential of the test material was assessed in an Ames test. Both S. typhimurium and E. coli were exposed to the test material by both direct plate application and preincubation methods. The following strains were tested, with and without the presence of metabolic activation by S9 -mix: TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA. Positive and solvent controls were run concurrently.

Under the conditions of the test, no mutagenic response was observed in any of the five strains tested in either the presence or absence of metabolic activation up to a maximum concentration of 5000 µg per plate. The results obtained in both mutation assays were similar. There was no toxicity to the bacteria and no precipitation of the test material at any of the concentrations tested. Positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. All results were valid with the exception of the counts obtained for TA 100 with 9-aminoacridine. The mutant counts were higher than those reported in the historical data. However this was not considered to have affected the integrity of the study results.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 May - 23 July 2012

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Human (male)

Details on mammalian cell type (if applicable):

- Type and identity of media: Culture medium consisted of RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and β-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Test concentration: 100, 333 and 961 µg/mL
Preparation of test solution: The test material was dissolved directly in the solvent. All preparations were used within 3 hours. The pH and the osmolarity of the culture medium containing the highest tested concentration were recorded and compared to the solvent control.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Final concentration of solvent in culture medium: 1.0% v/v.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Details of positive controls can be seen in Table 1 in the field "Any other information on materials and methods incl. tables".

Details on test system and experimental conditions:

PRELIMINARY DOSE RANGE FINDING STUDY
- Dosing range: 10, 33, 100, 33 and 9610 µg/mL
- Control: Negative control, DMSO
- Pre-incubation: Cells were incubated for 48 hours before exposure.
- Exposure duration: Cultures were exposed to the test material at each concentration under the same exposure/fixation procedure as in the definitive study.
- Rinsing: Cells in both of the 3 hour exposure groups were rinsed with HBSS to remove the test material according to the following method; First cultures were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 ml culture medium and incubated for another 20-22 hours to allow for a total fixation time of 24 hours. All other cultures were not rinsed and fixed immediately.
- Results: Based on the results of the dose range finding study it was concluded that the highest test concentration did not provoke >50% toxicity or precipitation, thus this was the highest concentration taken forward for the definitive study and the dosing range was set at 100, 333 and 961 µg/mL. Results can be seen in Table 2 in the field “Any other information on results incl. tables”.

DEFINITIVE STUDY: The definitive test was carried out in two independent experiments.

METHOD OF APPLICATION: in medium

CYTOGENETIC ASSAY 1
DURATION
- Pre-incubation period: 48 hours.
- Exposure duration: 3 hours in the presence and absence of S9-mix.
- Rinsing: After the exposure period, cultures were rinsed with HSBB in the same manner as described for the preliminary study.
- Fixation time: 24 hours.
- Controls: All positive and negative controls were run concurrently.

CYTOGENETIC ASSAY 2
DURATION
- Pre-incubation period: 48 hours.
- Exposure duration: 24 and 48 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.
- Fixation time: Cultures were exposed to the test material for either 3, 24 or 48 hours with fixation times of 48, 24 and 48 hours.
- Rinsing: The 3 hour exposure group was rinsed in the same manner as described in the preliminary study.
- Fixation time: 24 hours for the 3 and 24 hour exposure groups, whereas the 48 hours exposure group received a 48 hour fixation time.
- Controls: All positive and negative controls were run concurrently.

SPINDLE INHIBITOR: Colchicine, 0.5 µg/mL in medium.
STAIN: Slides were stained for 10 to 30 minutes with Giemsa solution in tap water 5% v/v.

NUMBER OF REPLICATIONS: Test performed in duplicate.

NUMBER OF CELLS EVALUATED: 1000 with a maximum deviation of 5%.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index.

Evaluation criteria:

To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was
≥ 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2
Chromosomes were analysed. The number of cells with aberrations and the number of aberrations were calculated.

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.

Statistics:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

If the result is small (p< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.

Key result

Species / strain:

lymphocytes: Human (male)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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 effects were observed on pH in comparison of the 961 µg/mL test solution to the solvent control. Determined pH values were 7.6 and 7.5, respectively.
- Effects of osmolality: No effects were observed on osmolality in comparison of the 961 µg/mL test solution to the solvent control. Determined osmolality values were 437 and 435 mOsm/kg, respectively.

RANGE-FINDING/SCREENING STUDIES: A concentration of 0.01 M (961 μg/ml) showed no precipitation in the culture medium. Therefore, a concentration of 961 μg/ml was used as the highest concentration for the definitive study. The mitotic index's for the range finding study ranged from 62 to 100 as a percentage of the control.

DEFINITIVE STUDY: All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix, 3,5-Dimethylpyrazole did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. Both in the absence and presence of S9-mix, 3,5-Dimethylpyrazole did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 2. Cyctogenetic Assay 1, 3 Hour Exposure in the Absence of S9 -mix

Concentration (µg/mL)		Mitotic Index (%)	No. of Cells with Aberrations (+ gaps/-gaps)	g’	b’	b”	exch.	misc.	Total Aberr (+gaps/-gaps)
DMSO (1.0% v/v)	A	100	1/1		1				1/1
	B		0/0						0/0
	A+B		1/1
100	A	93	0/0						0/0
	B		1/1		1			poly	1/1
	A+B		1/1
333	A	88	1/1		1				1/1
	B		1/0	1					1/0
	A+B		2/1
961	A	76	1/0	1					1/0
	B		0/0					poly	0/0
	A+B		1/0
MMC-C; 0.5	A	65	10/10		5	4	1		10/10
	B		10/10		7	3	1		11/11
	A+B		20/20***

 

Table 3. Cyctogenetic Assay 1, 3 Hour Exposure in the Presence of S9 -mix

Concentration (µg/mL)		Mitotic Index (%)	No. of Cells with Aberrations (+ gaps/-gaps)	b’	b”	exch.	Total Aberr (+gaps/-gaps)
DMSO (1.0% v/v)	A	100	1/1	1			1/1
	B		1/1	1			1/1
	A+B		2/2
100	A	96	0/0				0/0
	B		0/0				0/0
	A+B		0/0
333	A	93	0/0				0/0
	B		1/1	1			1/1
	A+B		1/1
961	A	89	0/0				0/0
	B		1/1	1			1/1
	A+B		1/1
CP; 10	A*	23	25/25	25	4	2	31/31
	B		15/15	15			15/15
	A+B		40/40***
*Only 50 metaphases scored for this culture

 

Table 4. Cyctogenetic Assay 2, 24 Hour Exposure in the Absence of S9 -mix

Concentration (µg/mL)		Mitotic Index (%)	No. of Cells with Aberrations (+ gaps/-gaps)	b’	b”	m”	exch.	misc.	Total Aberr (+gaps/-gaps)
DMSO (1.0% v/v)	A	100	2/2	1	1				2/2
	B		0/0						0/0
	A+B		2/2
100	A	96	0/0						0/0
	B		3/3	2	1				3/3
	A+B		3/3
333	A	76	2/2	2				poly	2/2
	B		2/2	2					2/2
	A+B		4/4
961	A	64	0/0						0/0
	B		1/1		1			2 poly	1/1
	A+B		1/1
MMC-C; 0.2	A	56	7/7	2	6		1		9/9
	B		21/21	10	9	1	2		22/22
	A+B		28/28***
MMC-C; 0.3	A	50	21/21	10	7		4		21/21
	B		29	15	9		5		29/29
	A+B		50***

 

Table 5. Cyctogenetic Assay 2, 48 Hour Exposure in the Absence of S9 -mix

Concentration (µg/mL)		Mitotic Index (%)	No. of Cells with Aberrations (+ gaps/-gaps)	b’	b”	m’	m”	exch.	misc.	Total Aberr (+gaps/-gaps)
DMSO (1.0% v/v)	A	100	1/1		1					1/1
	B		0/0						poly	0/0
	A+B		1/1
100	A	90	1/1	1						1/1
	B		0/0							0/0
	A+B		1/1
333	A	78	3/3	2	1					3/3
	B		1/1	1						1/1
	A+B		4/4
961	A	45	0/4							0/0
	B		1/1	1					3 poly	1/1
	A+B		1/1
MMC-C; 0.1	A	64	11/11	6	2		1	2		11/11
	B		5/5	2	1		1	1		5/5
	A+B		16/16***
MMC-C; 0.15	A	60	22/22	7	13			3		23/23
	B		22/22	11	7	1		3		22/22
	A+B		44/44***

 

Table 6. Cyctogenetic Assay 2, 3 Hour Exposure in the Presence of S9 -mix

Concentration (µg/mL)		Mitotic Index (%)	No. of Cells with Aberrations (+ gaps/-gaps)	b’	b”	exch.	Total Aberr (+gaps/-gaps)
DMSO (1.0% v/v)	A	100	1/1	1			1/1
	B		1/1	1			1/1
	A+B		2/2
100	A	100	3/3	2	1		3/3
	B		3/3	1	2		33
	A+B		6/6
333	A	93	1/1	1			1/1
	B		1/1	1			1/1
	A+B		2/2
961	A	85	0/0				0/0
	B		1/1		1		1/1
	A+B		1/1
CP; 10	A	_	29/29	31	7	10	38/38
	B		20/20	13	7	4	24/24
	A+B		49/49***

- 100 methaphases were scored per culture

- *** denotes figures that are significantly different from the control group (Chi-square test) P < 0.001

- MMC-C, mitomycin C

- CP, cyclophosphamide

Definitions of Chromosome Aberrations in Tables 2 to 6

- g': Chromosome gap

- b': Chromatid break

- b": Chromosome break

- m': Minute

- m": Double minutes

- exch.: Exchange figure

- poly: Polyploidy

Mitotic Index Range Finding Study (percent of control)

- 3 hour exposure in the absence of S9 -mix; Due to contamination in the control group it was not possible to calculate the percentage metaphases in the treatment groups.

-24 hour exposure in the absence of S9 –mix;

Control: 100

10 µg/mL: 92

33 µg/mL: 90

100 µg/mL: 87

333 µg/mL: 85

961 µg/mL: 62

-48 hour exposure in the absence of S9 –mix;

Control: 100

10 µg/mL: 98

33 µg/mL: 95

100 µg/mL: 93

333 µg/mL: 76

961 µg/mL: 71

-3 hour exposure in the presence of S9 –mix;

Control: 100

10 µg/mL: 115

33 µg/mL: 129

100 µg/mL: 144

333 µg/mL: 142

961 µg/mL: 158

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the condition of the test, both in the absence and presence of S9-mix the test material did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

Executive summary:

The ability of the test material to cause chromosome aberrations was determined in an in vitro study performed on human lymphocytes. The study was conducted according to GLP and the standardised guidelines OECD 473 and EU Method B.10.

Human peripheral lymphocytes were exposed to the test material in two independent experiments. The first test exposed cells to the test material for a duration of 3 hours in both the presence and absence of metabolic activation system S9-mix. Cultures were rinsed with HBSS following exposure and allowed a fixation time of 24 hours. In the second test cultures were exposed to the test material under various three different exposure/fixation conditions. Cultures were exposed for either 24 or 48 hours with equivalent fixation times in the absence of metabolic activation, or for 3 hours with a 48 hour fixation time in the presence of metabolic activation.

A preliminary dose range finding study was performed and based on the results a dosing range of 100, 333 and 961 µg/mL was set for both independent experiments.

Both positive and solvent controls were run concurrently for comparison with all tests; DMSO mitomycin C and cyclophosphamide.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploidy cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemical both produced statistically significant increases in the frequency of aberrant cells. Thus it was concluded that the test conditions were adequate and that the metabolic activation system functioned properly.

Both in the absence and presence of S9-mix the test material did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments

No effects of the test material were observed on the number of polyploid cells and cells with endoreduplicated chromosomes both in the absence and presence of S9-mix. Therefore it can be concluded that the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25th May 2012 to 07 August 212

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.

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

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine-kinase locus (TK-locus)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
> Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), I mM sodium private and 2 mM L-glutamin.
> Growth medium: Prepared from the basic medium supplemented with 10% (v/v) heat-inactivated horse serum.
> Exposure medium: Prepared from the basic medium supplemented with heat-inactivated horse serum, either 5% (v/v) in the 3 hour exposure group or 10% (v/v) in the 23 hour exposure groups (v/v).
> Selective medium: Prepared from the basic medium supplemented with 20% v/v heat-inactivated horse serum and 5 µg/mL trifluorothymidine.
> None selective medium: Basic medium supplemented with 20% (v/v) heat-inactivated horse serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone)

Test concentrations with justification for top dose:

Main study, experiment 1: 0.3, 1, 3.3, 10, 33, 100, 333 and 961 µg/mL
Main study, experiment 2: 0.3, 1, 3, 10, 33, 100, 333 and 961 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO), the final concentration of the solvent in the exposure medium was 0.8% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.

DURATION
- Exposure duration:
> Experiment 1: 3 hours (with and without S9 mix)
> Experiment 2: 23 and 3 hours (with and without S9, respectively)
- Initial cell concentrations:
> 3 hour exposure group: 10^6 cells/ml
> 23 hour exposure group: 1.25 x 10^5 cells/ml
- Expression time (cells in growth medium): Remaining cells were cultured for 2 days after the exposure period. During which cells were subcultured (approximately 4 x 10^4 cells per day) in order to maintain log phase growth.
- Incubation: 3 hour exposure samples were incubated in a shaking incubator at 37.0 ± 1.0 ºC and 145 spm. 24 hour exposure samples were incubated at 37.0 ± 1.0 ºC.

STAIN (for cytogenetic assays): 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS: exposure samples and positive controls were not replicated whereas solvent control: were performed in duplicate.

ENVIRONMENTAL CONDITIONS
- Lighting: Dark
- Humidity: 80 to 100% (actual range 43 to 97%)
- CO2 air content: 5.0 ± 0.5%
- Temperature: 37.0 ± 1.0 ºC (actual range 35.7 to 37.9 ºC)
Deviations in environmental conditions occurred due to the opening and closing of the incubator door. These deviations did not exceed 4 hours and based on historical laboratory data these deviations are not thought to have affected the accuracy and reliability of the study.

DOSE RANGE FINDING STUDY
- Exposure Durations: Cells were exposed to the test material in exposure medium under the following conditions; 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix.
- Cell Concentration: 8 x 10^6 cells (10^6 cells/ml for 3 hours treatment) or 5 x 10^6 cells (1.25 x 10^5 cells/ml for 24 hours treatment).
- Test Concentrations: 10, 33, 100, 333 and 961 µg/mL.
- Control: A solvent control was run concurrently.
- Expression time (cells in growth medium): Surviving cells from the 3 hour exposure group were subcultured and counted at 24 hours and then again at 48 hours. Those surviving from the 24 hour exposure group were subcultured and counted only once at 24 hours.
- Determination of cytotoxicity: Relative cell growth.
- Determination of dose range for the definitive test: The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.

Evaluation criteria:

The test material is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

The test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

The test material is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

CALCUALTION OF THE SURVUAL OR VIABILITY
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. This value obtained is the P(0), the zero term of the Poisson distribution:
P(0) = number of empty wells/total number of wells
The cloning efficiency (CE) is therefore:
CE = -ln P(0)/number of cells plated per well
The relative survival (RS) in each treatment group was determined by comparing cloning efficiencies in treatment and control cultures:
RS = [CE(test)/CE(controls)] x 100
The Relative Total Growth (RTG) was calculated as the product of the cumulative relative suspension growth (RSG) and the relative cloning efficiency for each culture:
RTG = RSG x RSday2/100
Suspension Growth (SG) =
[Day 0 cell count/ (1.25 x 10^5) *] x [Day 1 cell count/(1.25 x 10^5) *] x [Day 2 cell count]
* Or appropriate cell concentration
Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100
RSday2 = CE day2 (test) / CE day2 (controls) x 100
The growth rate, as an indicator of optimally growing cultures, was calculated for the solvent control cultures:
Growth Rate (GR) for the 3 hours treatment=
[Day 1 cell count/(1.25 x 10^5) *] x [Day 2 cell count/(1.25 x 10^5) *]
Growth Rate (GR) for the 24 hours treatment=
[Day 0 cell count/(1.25 x 10^5) ] x [Day 1 cell count/(1.25 x 10^5) *] x [Day 2 cell count/(1.25 x 10^5) *]
* Or appropriate cell concentration
 
CALCULATION OF THE MUTATION FREQUENCY
The mutation frequency was expressed as the number of mutants per 10^6 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 10^6
Small and large colony mutation frequencies were calculated in an identical manner.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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 effects were observed, the pH was recorded to be 7.3 in both the 961 µg/mL exposure group and the solvent control.
- Effects of osmolality: No effects were observed, the osmolality was recorded to be 0.417 Osm/kg in the 961 µg/mL exposure group compared to 0.415 Osm/kg in the solvent control.
- Precipitation: The test material did not precipitate in the exposure medium up to and inclusive of the highest concentration tested 961 µg/mL.

RANGE-FINDING:
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 961 μg/mL compared to the suspension growth of the solvent control. See Tables 1 and 2.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. The growth rate over the two-day expression period for cultures treated with DMSO was between 13 and 24 (3 hours treatment) and 81 and 85 (24 hours treatment.

POSITIVE CONTROLS:
Mutation frequencies in cultures treated with positive control chemicals were increased 9.4- and 9.1-fold for MMS in the absence of S9-mix, and 15- and 12-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Dose Range Finding Study: Cytotoxicity of the Test Material (3 hour exposure, in the presence and absence of metabolic activation)

Dose (µg/L)	Cell Count (cells/mL x 10⁵)			SG (x10⁵cells/mL)	RSG (%)
	3 Hour Exposure of Treatment	24 Hour Exposure of Subculture	48 Hour Exposure of Subculture
Without Metabolic Activation
SC	6.5	5.0	6.3	131	100
10	7.6	3.5	8.3	142	108
33	7.1	4.1	7.2	134	102
100	6.6	4.4	7.6	141	107
333	7.3	4.0	7.3	136	104
961	7.3	3.3	8.6	132	101
With Metabolic Activation
SC	5.8	4.8	6.6	118	100
10	6.4	4.6	7.3	138	118
33	5.9	4.2	7.7	121	103
100	6.4	3.8	7.1	110	94
333	6.9	3.5	7.2	112	95
961	6.4	3.5	7.0	101	86

Table 2.Dose Range Finding Study: Cytotoxicity of the Test Material (24 hour exposure, in the absence of metabolic activation)

Dose (µg/L)	Cell Count (cells/mL x 10⁵)		SG (x10⁵cells/mL)	RSG (%)
	24 Hour Exposure of Treatment	24 Hours Exposure of Subculture
SC	8.2	5.1	34	100
10	8.8	6.3	45	132
33	9.0	7.9	56	166
100	9.0	5.3	38	111
333	8.7	5.0	34	101
961	6.5	4.5	24	70

Table 3. Cytotoxic and Mutagenic Response of the Test Material in the Mouse Lymphoma L5178Y Test System, Experiments 1 and 2

Treatment Group	Dose (µg/mL)	RSG (%)	CE day2 (%)	RS day2 (%)	RTG (%)	Mutation frequency per 10⁶ survivors
						total	small	large
3 Hour exposure group, without S9	SC1	100	125	100	100	80	42	34
	SC2		107			74	27	44
	0.3	106	95	82	87	102	63	35
	1	98	110	95	93	63	31	29
	3.3	100	99	86	86	64	29	32
	10	110	89	77	84	77	40	34
	33	116	116	100	117	48	25	22
	100	118	118	102	120	68	25	40
	333	104	97	83	87	56	36	19
	961	84	141	121	101	65	43	19
	MMS	72	72	63	45	726	384	252
3 Hour exposure group, with 8% (v/v) S9 mix	SC1	100	95	100	100	61	17	43
	SC2		94			79	33	43
	0.3	100	99	105	105	79	32	44
	1	101	86	91	92	82	35	44
	3.3	103	94	99	102	45	26	18
	10	94	93	98	92	59	29	29
	33	94	95	101	94	70	30	37
	100	82	101	107	88	61	30	30
	333	61	121	128	78	84	34	46
	961	45	111	118	53	135	62	63
	CP	38	72	77	29	1021	515	328
24 Hour exposure group, without S9	SC1	100	121	100	100	56	22	32
	SC2		94			59	21	36
	0.3	109	95	89	97	52	27	23
	1	108	115	106	115	50	25	23
	3	130	86	80	105	54	36	17
	10	114	93	86	98	64	26	36
	33	142	88	81	116	61	29	31
	100	102	93	86	87	62	24	36
	333	101	94	87	88	43	21	20
	961	68	101	94	64	63	27	34
	MMS	89	75	69	61	526	248	228
3 Hour exposure group, with 12% (v/v) S9 mix	SC1	100	94	100	100	59	19	38
	SC2		97			62	21	39
	0.3	137	98	103	141	68	26	40
	1	163	107	112	182	53	24	27
	3	160	98	103	165	68	21	46
	10	112	105	110	123	62	20	40
	33	96	88	92	88	51	17	33
	100	205	99	104	214	62	24	37
	333	162	88	92	148	82	32	47
	961	152	95	100	152	87	44	39
	CP	157	83	87	136	703	287	315

Key for all Tables

Note: all calculations were made without rounding off

SG: Suspension growth,

RSG: Relative suspension growth,

CE: Cloning efficiency,

RS: Relative Survival,

RTG: Relative total growth,

SC: Solvent control (DMSO),

MMS: Methylmethanesulfonate,

CP: Cyclophosphamide.

Conclusions:

Interpretation of results (migrated information):
negative

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with an extended exposure period.

In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

Under the conditions of the test, the test material is not considered to be mutagenic in the TK mutation test system.

Executive summary:

 The mutagenic potential of the test material was assessed in an in vitro mammalian cell gene mutation test using L5178Y mouse lymphoma cells. The study was conducted according to GLP and in line with the standardised guidelines OECD 476 and EU Method B.17.

The cells were exposed to the test material in two independent tests at concentrations ranging from 0.3 to 961 µg/mL. Cells were exposed to the test material for 3 hours in both the presence and absence of the metabolic activation system S9 mix (8%, v/v), during experiment 1. The test was repeated in Experiment 2, where cells were exposed to the test material for 24 hours without metabolic activation and for 3 hours with increased metabolic activation (12%, v/v).

Data obtained from positive and solvent controls fell within the limits of historical data and within the acceptability criteria set out in this study.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with an extended exposure period.

In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

Under the conditions of the test, the test material is not considered to be mutagenic in the TK mutation test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

IN VITRO GENE MUATION STUDY IN BACTERIA

In Stevenson (2002), the following strains were tested, with and without the presence of metabolic activation by S9 -mix: TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA. Positive and solvent controls were run concurrently. No mutagenic response was observed in any of the five strains tested in either the presence or absence of metabolic activation up to a maximum concentration of 5000 µg per plate. The results obtained in both mutation assays were similar. There was no toxicity to the bacteria and no precipitation of the test material at any of the concentrations tested. Positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. All results were valid with the exception of the counts obtained for TA 100 with 9-aminoacridine. The mutant counts were higher than those reported in the historical data. However this was not considered to have affected the integrity of the study results.

In Jones (1994), the following strains were tested, with and without the presence of metabolic activation by S9 -mix: TA 1535, TA 1537, TA 1538, TA 98 and TA 100. Both the concurrent positive and negative controls were valid, demonstrating the sensitivity of the assay and the metabolising activity of the S9-mix. Under the conditions of the test no substantial increase in revertant colony numbers of any of the tester strains, and the test material is considered to have given a negative response.

Neither of the studies available for this endpoint showed any evidence of genetic toxicity.

IN VITRO CYTOGENICITY

Both in the absence and presence of S9-mix the test material did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments using human peripheral lymphocytes.

No effects of the test material were observed on the number of polyploid cells and cells with endoreduplicated chromosomes both in the absence and presence of S9-mix. Therefore it can be concluded that the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions of the test.

IN VITRO GENE MUTATON IN MAMMALIAN CELLS

The mutagenic potential of the test material was assessed in an in vitro mammalian cell gene mutation test using L5178Y mouse lymphoma cells.

In the absence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with an extended exposure period.

In the presence of S9-mix, the test material did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.

Justification for selection of genetic toxicity endpoint

No single study could be selected as representative of the overall conclusion for this endpoint, as all three key studies assess different aspects of genotoxicity. Stevenson (2002) was selected as the key study assessing in vitro gene mutation in bacteria, as an appropriate strain that detects cross-linkage was included, which was not considered in the supporting in vitro gene mutation in bacteria study. Verbaan (2012) assessed in vitro cytogenicity in mammalian cells and Veerpeek-Rip (2012) assessed in vitro gene mutation in mammalian cells. All studies were performed to a good standard with a sufficient level of detail to assess the quality of the data submitted. Accordingly they have been assigned a reliability score of 1, in accordance with Klimisch (1997) and considered suitable to fulfil these data requirements.

Short description of key information:

IN VITRO GENE MUATION STUDY IN BACTERIA

Key: Stevenson (2002), Ames test performed to GLP, OECD 471, EU Method B.13/14, EPA 712-C-98-247, CPMP/ICH/141/95 and CPMP/ICH/174/95, Klimisch 1, negative without and with metabolic activation.

Supporting: Jones (1994), Ames test performed to GLP, OECD 471, EU Method B13/14 and US Environmental Protection Agency, Method: The S. typhimurium reverse mutation assay, 1984, Klimisch 1, negative without and with metabolic activation.

IN VITRO CYTOGENICITY IN MAMMALIAN CELLS

Key: Verbann (2012), Chromosome aberration test performed to GLP and EU Method B.10, Klimisch 1, negative without and with metabolic activation.

IN VITRO GENE MUTATON IN MAMMALIAN CELLS

Key: Verspeek-Rip (2012), Mammalian cell mutation test performed to GLP, OECD 476 and EU Method B.17, Klimisch 1,negative without and with metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

With respect to the information available, the test material does not meet the criteria for classification as a mutagenic substance in accordance with Regulation (EC) No. 1272/2008.