2-(2H-Benzotriazol-2-yl)-6-(1-methyl-1-phenylethyl)-4-(1,1,3,3-tetramethylbutyl)phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial and a mammalian cell mutagenicity as well as a mammalian cell cytogenetic test have been performed. The test item did neither induce a mutagenic nor a clastogenic effect 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:

03-Jan-1997 to 27-Jan-1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD test guideline 471)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on 26-May-1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

CCR Cytotest Cell Research GmbH & Co. KG, 64380 Roftdorf, In den Leppsteinswiesen 19

Type of assay:

bacterial reverse mutation assay

Target gene:

trp, his

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from phenobarbital (i.p.) and beta-naphthoflavone (p.o.) induced male Wistar rats

Test concentrations with justification for top dose:

33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate

Vehicle / solvent:

On the day of the experiment, the test item was dissolved in acetone. The solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

solvent

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Remarks:

all controls were concurrent

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation (experiment I), preincubation (experiment II and IIb)

DURATION
- Preincubation period: 1 hour
- Exposure duration: 48 hours
- Expression time: at least 48 hours

NUMBER OF REPLICATIONS: 3 replicates

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity was assessed as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

POSITIVE CONTROLS
without S9 mix:
sodium azide for strains TA 1535, TA 100 (10 µg/plate)
4-nitro-o-phenylene-diamine for strains TA 98 (10 µg/plate) and TA 1537 (50 µg/plate)
methyl methane sulfonate for strain WP2uvrA (5 µl/plate)
with S9 mix:
2-aminoanthracene for strains TA 1535, TA 1537, TA 98, TA 100 (2.5 µg/plate) and WP2 uvrA (10 µg/plate)

Evaluation criteria:

The generally accepted conditions for the evaluation of the results were:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates
A test item was considered as positive if either a dose related and reproducible increase in the number of revertants or a reproducible increase for at least one test concentration was induced. A test item producing neither a dose related and reproducible increase in the number of revertants nor a reproducible positive response at any one of the test points was considered non-mutagenic in this system.
A test item was considered as mutagenic if in the strains TA 98, TA 100 and WP2 uvrA the number of reversions were at least twice as high and in the strains TA 1535 and TA 1537 were at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants was regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

The data were not evaluated statistically.

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar from 333.3 up to 5000.0 µg/plate. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES:
A pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with three plates each. According to the results of this pre-experiment the concentrations applied in the main experiments were chosen.

Experimental Result

Without S9 Mix

	Revertants/plate (mean from three plates)
	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
Concentration µg/plate	I	II	I	II	I	II	I	II	I	II
Negative control	17	18	11	11	20	17	141	104	44	28
Solvent control	16	15	9	9	18	18	139	102	39	32
Positive control	1167	1109	134	128	430	568	1213	1102	1086	993
33.3	16	13	11	7	21	14	147	103	34	37
100	16	16	8	9	20	16	142	108	42	39
333.3	17	14	12	7	24	13	146	89	39	33
1000	13	14	11	7	26	16	145	106	46	35
2500	17	10	8	8	27	13	134	94	39	35
5000	16	9	9	8	24	25	129	110	45	34

With S9 -Mix

	Revertants/plate (mean from three plates)
	TA 1535		TA 1537		TA 98		TA 100		WP2 uvrA
Concentration µg/plate	I	II	I	II	I	II	I	II	I	II	IIa
Negative control	18	19	17	15	33	33	158	147	44	32	38
Solvent control	16	13	15	12	30	25	154	119	52	28	33
Positive control	275	118	92	70	1119	482	1244	604	236	146	174
33.3	17	16	13	9	24	26	151	131	47	30	32
100	17	18	14	14	30	24	138	119	53	30	31
333.3	17	14	16	12	27	28	149	137	58	41	39
1000	19	14	16	12	31	26	152	132	50	52	35
2500	17	16	16	14	27	32	139	156	57	67	38
5000	17	14	14	16	24	40	156	164	52	50	31

I = Experiment I (standard plate test)

II and IIa = Experiment II and IIa (preincubation method)

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test article is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

A GLP-compliant Ames study following OECD guideline 471 was performed to investigate the test article's potential to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation and an additional experiment (experiment IIa) following the preincubation procedure with the Escherichia coli strain WP2 uvrA in the presence of metabolic activation. Each concentration and the controls were tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in both experiments. No toxic effects occurred in the test groups with and without metabolic activation. No substantial and reproducible increase in revertant colony numbers of any of the five tester strains was observed after treatment with test material at any dose level, neither in the presence nor in the absence of metabolic activation (S9 mix). A slight increase in the number of revertants occurred in the second experiment in strain WP2 uvrA in the presence of metabolic activation exceeding the threshold of twice the number of the corresponding solvent control at 2500 µg/plate. This effect could not be verified under identical conditions in an additional experiment (experiment IIa) in strain WP2 uvrA in the presence of metabolic activation. Therefore, the slight increase observed in the second experiment was judged to be caused by statistical fluctuations of the rather low numbers of revertant colonies and does not represent a mutagenic effect. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

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:

02-Dec-1996 to 28-Feb-1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD test guideline 473)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted on 26-May-1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No target gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from the livers of 8 - 12 weeks old male Wistar rats

Test concentrations with justification for top dose:

Experiment I:
without S9 mix (18 or 28 hour treatment interval):
18 hour preparation interval: (5), 10, 30, 50, (100), 800 µg/mL
28 hour preparation interval: (30), 50, (100), 800 µg/mL

with S9 mix (4 hour treatment interval):
18 hour preparation interval: (3), 5, 10, 30, (100), 800 µg/mL
28 hour preparation interval: (10), 30, (100), 800 µg/mL

Experiment II:
without S9 mix (18 or 28 hour treatment interval):
18 hour preparation interval: (5), 10, 30, 50, (100), 800 µg/mL
28 hour preparation interval: (30), 50, (100), 800 µg/mL

with S9 mix (4 hour treatment interval):
18 hour preparation interval: 5, 10, (20), 30, (100), 800 µg/mL
28 hour preparation interval: (20), 30, (100), 800 µg/mL

Concentrations in parentheses were not evaluated.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone.
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative non-toxcicity for the cells. The final concentration of acetone in the culture medium did not exceed 1 % v/v.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

solvent (acetone)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9 mix: 600 µg/ml ethylmethane sulfonate; with S9 mix: 0.71 µg/ml Cyclophosphamide

Remarks:

with an without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with S9 mix and 18 and 28 hours without S9 mix.
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 h

SPINDLE INHIBITOR: colcemid (0.2 µg/mL culture medium)
STAIN: Giemsa stain

NUMBER OF REPLICATIONS: 2 parallel cultures in each experimental group

NUMBER OF CELLS EVALUATED: 100 well spread metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells was determined

Evaluation criteria:

A test item was classified as mutagenic if it induced reproducibly either a significant concentration related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points. A test item producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any one of the test points was considered non-mutagenic in this system.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test. However, both biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
In the cytogenetic experiments examination of the cultures 4 hours after treatment revealed precipitation of the test item at concentrations of 50 µg/mL and above (without metabolic activation) and 30 µg/mL and above (with metabolic activation).

RANGE-FINDING/SCREENING STUDIES:
The applicable concentration range of the test item for the cytogenetic experiment was determined in a pre-test using the determination of cell numbers 24 hours after treatment start as indicator for cytotoxicity. As the dose selection of the test item was influenced by its solubility, concentrations from 1-800 µg/mL were applied for the treatment of the cell cultures. Since no clear toxicity was observed in the absence and the presence of S9 mix, 800 µg/mL were applied as top concentration for the cytogenetic experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence and the presence of the metabolic activation system, in both experiments no substantial reduction of the mitotic indices occurred at the evaluated experimental points, neither at concentrations with visible precipitation nor at concentrations without precipitation.

Summary of results

						Aberrant cells
Exp	Preparation Interval	concentration (µg/ml)	S9 Mix	Polyploid cells (%)	Mitotic indices in % of control	incl. Gaps	excl. Gaps*	exchanges
I	18 h	solvent control	-	3.5	100	2	1.5	0
	18 h	pos. control^1	-	3.5	89.4	32	30.5^S	20.5
	18 h	10	-	3.5	89.4	1	0.5	0
	18 h	30	-	3	88.2	1	1	0
	18 h	50^P	-	4.5	86.8	3.5	2	0
	18 h	800^P	-	4.5	100.3	1.5	1.5	0
II	18 h	solvent control	-	3	100	3	2	0
	18 h	pos. control^1	-	2	33.5	22	21.0^S	12.5
	18 h	10	-	3.5	81.2	4	2	0.5
	18 h	30	-	1.5	70	1.5	0	0
	18 h	50	-	1.5	110.4	4	0.5	0
	18 h	800^P	-	3.5	79.1	2.5	2.5	0.5
I	28 h	solvent control	-	5	100	1.5	0.5	0
	28 h	50	-	3.5	107.2	2	1.5	0
	28 h	800^P	-	3	91	1	0.5	0
II	28 h	solvent control	-	2.5	100	2	1	0
	28 h	50	-	2	112.3	4.5	2.5	1
	28 h	800^P	-	2	97	0.5	0	0
I	18 h	solvent control	+	3	100	0.5	0.5	0
	18 h	pos. control^2	+	2	89.8	28	27.0^S	8.5
	18 h	5	+	1	105.5	1	1	0.5
	18 h	10	+	2.5	93.2	2	1	0
	18 h	30^P	+	3	105.2	1	1	0
	18 h	800^P	+	2	104.5	2	1	0.5
II	18 h	solvent control	+	4	100	2	1	0.5
	18 h	pos. control^2	+	0	58.5	27.5	27.0^S	13
	18 h	5	+	3	105.9	2	1.5	0.5
	18 h	10	+	3	93.4	2.5	1	0.5
	18 h	30^P	+	3.5	97.1	2	1	0
	18 h	800^P	+	3.5	94.9	1	0.5	0
I	28 h	solvent control	+	2.5	100	3	1.5	0
	28 h	30	+	4.5	86.5	2	1.5	0.5
	28 h	800^P	+	3	85.6	0	0	0
II	28 h	solvent control	+	4	100	1	0.5	0
	28 h	30^P	+	2.5	106.9	1	1	0
	28 h	800^P	+	4	117.3	1.5	1	0

* inclusive cells caryying exchanges

^P precipitation occurred

¹ ethylmethane sulfonate 600 µg/ml

² cyclophasphamide 0.71 µg/ml

^S Aberration frequency statistically significant higher than corresponding slovent values

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in vitro in V79 cells (Chinese hamster cell line) and is therefore considered to be non-mutagenic in this chromosome aberration test.

Executive summary:

The test article dissolved in acetone was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment intervals were 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The applicable concentration range of the test article for the cytogenetic experiment was determined in a pre-test using the determination of cell numbers 24 h after start of treatment as indicator for toxicity response. As dose selection of the test article was influenced by the solubility of the test article concentrations from 1 - 800 µg/ml were applied for the treatment of the cultures. Since no clear toxicity was observed in the absence and the presence of S9 mix, 800 µg/ml were applied as top concentration for the cytogenetic experiments. In experiment I, test article concentrations within a range of 5 - 800 µg/ml (without S9 mix) and 3 - 800 µg/ml (with S9 mix) were applied for the investigation of the potential to induce cytogenetic damage. In experiment II the applied concentration range was 5 - 800 µg/ml (with and without S9 mix). In the cytogenetic experiments examination of the cultures 4 h after treatment revealed precipitation of the test article at concentrations of 50 µg/ml and above (without S9 mix) and 30 µg/ml and above (with S9 mix). In the absence and the presence of S9 mix, in both experiments no substantial reduction of the mitotic indices occurred at the evaluated experimental points, neither at concentrations with visible precipitation nor at concentrations without precipitation. Therefore, cultures after treatment with the highest applicable concentration (800 µg/ml) and one concentration at the threshold to precipitation (50 µg/ml or 30 µg/ml, respectively) were evaluated for cytogenetic damage. In both experiments, at both intervals, in the absence and the presence of S9 mix, the test article did not increase the frequency of cells carrying structural chromosome aberrations. The aberration frequencies exclusive gaps after treatment with the test article (exp. I: 0.0 % - 2.0 %; exp. II: 0.0 % - 2.5 %) were near to the range of the solvent control values (exp. I: 0.5 % - 1.5 %; exp, II: 0.5 % - 2.0 %) and within the range of our historical control data: 0.0 % - 4.0 %. In both experiments, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in vitro in V79 cells (Chinese hamster cell line). Therefore, the test article is considered to be non-mutagenic in this chromosome aberration 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:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guideline conform study with well characterized test material

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (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:

hprt

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Population doubling time 8-10 hours.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 from Arochlor-1254 treated rats

Test concentrations with justification for top dose:

Experiment 1: 20, 40, 80, 120, 160, 200, 250, 300 mg/L
Experiment 2: 25, 50, 75, 100, 120, 140, 160, 200, 250 mg/L

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: none
- Exposure duration: 3h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 12-13 days
- Fixation time (start of exposure up to fixation or harvest of cells):19-20 days

SELECTION AGENT (mutation assays): 6-thioguanine


NUMBER OF REPLICATIONS: 18 (during expression time), 32 (during selection time)

NUMBER OF CELLS EVALUATED: Clones generated from treatment of 10exp7 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The assay was considered valid if the following criteria were met:
1.The mutant frequencies in the negative (vehicle) control cultures fell within the normal range (not more than three times the historical mean value)
2. At least one concentration of each of the positive control chemicals induced a clear, unequivocal increase in mutant frequency.

Statistics:

Robinson W D, Green M H L, Cole J, Garner R C, Healy M J R and Gatehouse D (1990). Statistical evaluation of bacterial/mammalian fluctuation tests. In Statistical Evaluation of Mutagenicity Test Data (Ed D J Kirkland) Cambridge University Press, pp 102 - 140

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No marked changes in osmolality or pH were observed in the range-finder experiment at the highest concentration tested (250 mg/L), compared to the concurrent vehicle controls.

Experiment 1 (3 hour treatment in the absence and presence of S-9)

Treatment		-S-9					Treatment		+S-9
(µg/mL)							(µg/mL)
		% RS	Individual mutant frequency §		Mean mutant frequency §				% RS	Individual MF§		Mean MF§
1		100	1.15, 0.94		1.05		0		100	0.82, 1.23		1.01
UTC		111	2.74, 0.96		1.93	NS	UTC		100	0.00, 0.00		0	#
20		98	2.05, 3.24		2.7	NS	20		113	0.53, 3.44		1.79	NS
40		104	0.70, 1.23		0.96	NS	40		100	3.07, 1.89		2.44	NS
80		108	2.93, 1.79		2.54	NS	80		109	1.41, 2.51		1.94	NS
120	P	114	1.95, 3.47		2.65	NS	120	P	116	1.21, 1.78		1.49	NS
160	P,PP	99	1.37, 1.11		1.24	NS	160	P,PP	93	1.78, 1.80		1.81	NS
Linear trend				NS			Linear trend				NS
NQO							B[a]P
0.15		59	26.07				2		61	21.44
0.2		53	38.77				3		33	30.79

UTC = Untreated control cells

P = Indicates precipitation observed at the beginning of treatment

PP= Indicates precipitation observed following treatment incubation period

RS = Relative survival

NQO = 4-nitroquinoline 1‑oxide

B[a]P = Benzo[a]pyrene

 § = 6TG resistant mutants/10⁶viable cells 7 days after treatment

Experiment 2 (3 hour treatment in the absence and presence of S-9)

Treatment		-S-9					Treatment		+S-9
(µg/mL)							(µg/mL)
		% RS	Individual MF§		Mean MF				% RS	Individual MF§		Mean MF
1		100	1.27, 0.58		0.93		0		100	2.48, 2.42		2.45
UTC		101	1.10, 2.30		1.63	NS	UTC		105	1.40, 2.49		1.94	NS
25		91	0.73, 1.66		1.15	NS	25		92	2.17, 1.59		1.91	NS
50		88	1.09, 2.73		1.87	NS	50		79	1.65, 0.75		1.15	NS
75		100	2.90, 2.02		2.53	NS	75		94	0.66, 2.59		1.73	NS
100	P	98	1.39, 1.70		1.54	NS	100	P	88	1.35, 3.03		2.16	NS
120	P	109	3.51, 0.25		1.71	NS	120	P	87	1.46, 0.14		0.83	NS
140	P, PP	98	0.90, 0.49		0.7	NS	140	P, PP	95	1.27, 2.77		2.09	NS
Linear trend				NS			Linear trend				NS
NQO							B[a]P
0.15		59	17.06				2		85	10.98
0.2		51	19.25				3		59	21.34

UTC = Untreated control cells

P =Indicates precipitation observed at the beginning of treatment

PP=Indicates precipitation observed following treatment incubation period

RS = Relative survival

NQO =4-nitroquinoline 1‑oxide

B[a]P =Benzo[a]pyrene

 § =6TG resistant mutants/10⁶viable cells 7 days after treatment

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Mutagenicity

In a GLP-compliant guideline test (OECD 471) the test article was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiments II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA (CCR, 1997). The assay was performed in two independent experiments both with and without liver microsomal activation. The test article was tested in triplicates at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in both experiments. No toxic effects occurred in the test groups with and without metabolic activation. No substantial and reproducible increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor in the absence of metabolic activation. A slight increase in the number of revertants occurred in the second experiment in strain WP2 uvrA in the presence of metabolic activation exceeding the threshold of twice the number of the corresponding solvent control at 2500 µg/plate. However, this effect was not reproducible as shown in an additional repeat experiment (experiment IIa). Therefore, the slight increase observed in the second experiment was judged to be caused by statistical fluctuation and does not represent a mutagenic effect. The positive controls showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

This finding is supported by a second Ames test performed previously (Pharmakon, 1984). In this assay, the results for test article were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166, 500, 1666 and 5000 µg/plate.

Chromosomal aberration

In a GLP-compliant in vitro assay following OECD guideline 473 the test article was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in two independent experiments. The chromosomes were prepared 18 h and 28 h after start of treatment with the test article. The treatment intervals were 4 h with metabolic activation, 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. In the cytogenetic experiments examination of the cultures 4 h after treatment revealed precipitation of the test article at concentrations of 50 µg/ml and above (without S9 mix) and 30 µg/ml and above (with S9 mix). In the absence and the presence of S9 mix, in both experiments, no substantial reduction of the mitotic indices occurred at the evaluated experimental points, neither at concentrations with visible precipitation nor at concentrations without precipitation. In both experiments, at both intervals, in the absence and the presence of S9 mix, the test article did not increase the frequency of cells carrying structural chromosome aberrations. In both experiments, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Therefore, neither at concentrations with visible precipitation nor at concentrations without precipitation a clastogenic potential of the test article was apparent. Therefore, the test article is considered to be non-mutagenic in this chromosome aberration test.

Gene mutation in mammalian cells in vitro

In a GLP-compliant in vitro assay following OECD guideline 476 the test article was assessed for its potential to induce gene mutations in mouse lymphoma L5178Y cells in two independent experiments. The highest concentrations that could be tested were determined by precipitation of the test substance in the culture medium. Stock solutions were prepared in acetone and the two highest concentrations evaluated showed precipitates of the test substance. No increase in mutant frequency was observed. Negative and positive control incubations, both with and without metabolic activation, confirmed the validity of the assay.

Justification for classification or non-classification

There are conclusive data for classification of the test item with regard to genotoxicity. No genotoxicity was observed in three in-vitro studies.

The test item is not classified for this endpoint in accordance to Directive 67/548/EEC or the CLP Regulation (EC) No 1272/2008.