A mixture of: isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-(n)-dodecylphenol; isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-(n)-tetracosylphenol; isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-5,6-didodecyl-phenol. n=5 or 6

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not mutagenic in the Ames test in Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537, tested both in the absence and presence of S9 mix (Ciba-Geigy 851123, 1992). No genotoxicity was observed in a HPRT test (RCC 747797, 2000) and in a cytogenetic test with Chinese Hamster Cells (Biosafety Research Center, Report No. 1007, 1987), both with and without microsomal activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30.09. - 14.01.2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

RCC Cytotest Cell Research GmbH

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimum essential medium) supplemented with 10 % FCS (foetal calf serum).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital and ß-naphthoflavone induced liver of male Wistar rats.

Test concentrations with justification for top dose:

Cytotoxicity test
4hrs, with metabolic activation: 3.5, 7.0, 14.1, 28.1, 56.3, 112.5, 225.0, 450 µg/mL
4hrs, without metabolic activation: 3.5, 7.0, 14.1, 28.1, 56.3, 112.5, 225.0, 450 µg/mL
24hrs, without metabolic activation: 3.5, 7.0, 14.1, 28.1, 56.3, 112.5, 225.0, 450 µg/mL

Mutagenicity test
Experiment I (with and without metabolic activation):
4hrs: 14.1, 28.1, 56.3, 112.5, 225, 450 µg/mL (14.1 µg/ml was not continued)

Experiment II (without metabolic activation):
24 hrs: 14.1, 28.1, 56.3, 112.5, 225, 450 µg/mL (14.1 µg/ml was not continued)

Vehicle / solvent:

- Vehicle/solvent used: ethanol (0.5 % v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: Ethylmethane sulfonate (0.3 mg/mL); with metabolic activation: 7,12-dimethylbenz(a)anthracene (2.5 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hrs (with and without metabolic activation) and 24 hrs (without metabolic activation).
- Expression time (cells in growth medium): approx. 7 days
- Selection time (if incubation with a selection agent): 7-8 days
- Fixation time (start of exposure up to fixation or harvest of cells): after 15-16 days

SELECTION AGENT (mutation assays): 6-Thioguanin (11 µg/mL)

STAIN: 10 % methylene blue in 0.01 % KOH solution

NUMBER OF INDEPENDENT EXPERIMENTS: 2

NUMBER OF REPLICATIONS:
In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (DMSO) control.

EVALUATION: Counting of colonies formed after seeding of 3 - 5x10E5 cells in selective medium

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The test item will be considered to be mutagenic if:
1. A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.

2. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

Assay evaluation criteria
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 10E6 cells found in the negative and/or solvent controls fall within the laboratory historical control data range of 1996 - 1997.
b) the positive control substances must produce a significant increase in mutant colony frequencies.
c) the cloning efficiency (absolute value) of the negative and/or solvent controls must exceed 50 %.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test item formed a fine emulsion in the medium at 56.3 µg/mL and above in the absence and presence of metabolic activation in the first experiment and at 14.1 µg/mL and above in the second experiment. The maximal applicable concentration was 450 µg/mL since higher concentrations led to inhomogeneous emulsions with phase separation in the medium.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects were observed up to the maximal concentration of the test item. The concentration range of the test item was limited by solubility rather than toxicity.

EXPERIMENTAL RESULT

	concentration (µg/ml)	S9 Mix	relative cloning efficiency (%)*	relative cell density (% of control)	viability factor**	mutant colonies / 10^6cells	relative cloning efficiency (%)*	relative cell density (% of control)	viability factor**	mutant colonies / 10^6cells
Experiment I, 4h treatment			culture I				culture II
Negative control		-	100	100	0.82	7.8	100	100	0.75	8.9
Solvent control with Ethanol		-	100	100	0.79	3.9	100	100	0.73	6.3
Positive control EMS	300	-	97.1	67.4	0.8	109.2	89.8	84.6	0.94	119.9
Test item	14.1	-	95.2	92.8	culture was not continued		87.5	97.6	culture was not continued
Test item	28.1	-	99.5	96.6	0.57	8.8	95	82.6	0.66	10.5
Test item	56.3	-	84.9	90.5	0.63	2.3	80.2	79.5	0.78	4.3
Test item	112.5	-	86.8	109.5	1.03	5.1	87.6	75.9	0.69	12.7
Test item	225	-	91.1	81	0.64	9.8	76.1	75.2	0.77	5.6
Test item	450	-	90.4	111	0.8	19.3	81.9	71.2	0.7	7
Negative control		+	100	100	0.67	6.4	100	100	0.47	9
Solvent control with DMSO		+	100	100	0.84	6.7	100	100	0.76	8.8
Solvent control with Ethanol		+	100	100	0.79	1.3	100	100	0.65	4
Positive control DMBA	2.5	+	52	86.4	0.85	453.6	50.6	62.9	0.63	638.3
Test item	14.1	+	112.8	120.3	culture was not continued		123.4	69.3	culture was not continued
Test item	28.1	+	110	122.4	0.75	10.7	107.5	103.6	0.76	3.4
Test item	56.3	+	107.3	100.9	0.7	1.5	115.9	113.5	0.69	14.4
Test item	112.5	+	93.5	117.4	0.65	4	110.7	87.7	0.67	6.5
Test item	225	+	109.8	147.5	0.93	1.6	119.2	91.7	0.68	5.8
Test item	450		110.8	137.4	0.8	3.2	113.4	148.4	0.64	4.9
Experiment II, 24 h treatment			culture I				culture II
Negative control		-	100	100	0.72	7.5	100	100	0.68	5.2
Solvent control with Ethanol		-	100	100	0.9	0.6	100	100	0.6	6.9
Positive control EMS	300	-	35.5	41.8	0.52	578	31.1	83.5	0.26	606.9
Test item	14.1	-	98.8	101.4	culture was not continued		99.5	101.1	culture was not continued
Test item	28.1	-	82.3	83	0.67	7	82.6	135.5	0.6	7.7
Test item	56.3	-	93.2	57.2	0.65	1.7	73.3	113.1	0.57	8.3
Test item	112.5	-	79	85.8	0.93	3.2	77.2	104.8	0.79	7.9
Test item	225	-	93.8	81.1	0.97	3.4	85.1	104	0.68	0
Test item	450	-	87	74.8	0.87	7.3	84.4	112.2	0.57	14.3

* mean number of cells per flask divided by mean number of cells per flask of the negative control multiplied with 100 (from cultures seeded with 500 cells each before treatment with the test substance)

** mean number of cells per flask divided by number of cells seeded (from cultures seeded with 500 cells each after expression)

Up to the highest investigated concentrations no relevant increase in mutant colony numbers was observed in both independent experiments. An isolated increase (19.3 colonies per 10⁶cells) exceeding the threshold of three times the corresponding solvent control and the historical range of our negative and solvent controls was observed at 450.0 µg/ml in experiment I, culture I without metabolic activation. This increase was judged as biologically irrelevant since it was not reproduced in the parallel culture (culture II) under identical conditions. This effect is probably induced by inhomogeneity of the emulsion of the test item in medium at the maximal concentration. The factor of three times the corresponding solvent control was exceeded at almost all concentrations in the first culture of the second experiment. This effect however, is based upon the very low solvent control (0.6 colonies per 10⁶cells) and represents statistical fluctuations at such low absolute numbers. The absolute numbers of colonies remained well within our range of historical negative and solvent controls and are below the corresponding negative control.

Conclusions:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Executive summary:

In an HPRT study according to OECD test guideline 476 and in compliance with GLP principles, the test article’s potential to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was investigated. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. In order to determine the concentration range for the mutagenicity, a pre-test was performed with eight concentrations ranging from 3.5 to 450.0 µg/ml without metabolic activation (4 h and 24 h treatment) and with metabolic activation (4 h treatment). The maximal concentration was limited by the solubility of the test item. Concentrations above 450 µg/ml formed unstable emulsions leading to phase separation. No relevant toxic effects occurred in the pre-experiment up to the maximal concentration. Therefore, the following concentrations were evaluated in the main study: 28.1; 56.3; 112.5; 225.0; and 450.0 µg/ml. In the man test, the test item formed a fine emulsion in the medium at 56.3 µg/ml and above in the absence and presence of metabolic activation in the first experiment and at 14.1 µg/ml and above in the second experiment. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies. Up to the highest investigated concentration no relevant increase in mutant colony numbers was obtained in both independent experiments. An isolated increase exceeding the threshold of three times the corresponding solvent control and the historical range of our negative and solvent controls was observed at 450.0 µg/ml in experiment I, culture I without metabolic activation. This increase was judged as biologically irrelevant since it was not reproduced in the parallel culture (culture II) under identical conditions and based on a very low solvent control value. In conclusion, the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.05. - 17.08.1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Negative result in 6h exposure +S9 was not confirmed and positive contol was erroneus in 6h exposure -S9 (Experiment II).

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Guideline on toxicity study data for application of new chemical standards" (a notice 237 of Kanpogyo, a notice 306 of Yaku and a notice 303 of 62 Kikyoku; March 31, 1987).

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Fibroblast cell line derived from the lung of Chinese hamsters (CHL/IU)

Details on mammalian cell type (if applicable):

- Type and identity of media: EAGLE-MEM
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from the liver of phenobarbital and 5,6-benzoflavone i.p. treated male SD-rats.

Test concentrations with justification for top dose:

Direct method (experiment I, without metabolic activation)
24h exposure: 0, 1250, 2500, 5000 µg/mL
48h exposure: 0, 1250, 2500, 5000 µg/mL

Activation method (experiment II, with and without metabolic activation)
6h exposure: 0, 1250, 2500, 5000 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: 1% carboxymethyl cellulose

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

carboxymethyl cellulose (1 %)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9-mix: N-methyl-N-nitro-N-nitrosoguanidine (2 µg/mL) in experiment I; with and without S9-mix: 1,2-benzopyrene (30 µg/mL) in experiment II

Details on test system and experimental conditions:

METHOD OF APPLICATION: test item suspension in 1 % CMS added into petri dishes containing cells and medium

DURATION
- Culture period before exposure: 3 days (Experiment I and II)
- Exposure duration: 24 or 48 h (Experiment I, -S9 mix); 6h (Experiment II, +/-S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48 h (Experiment I), 24 h (Experiment II)

SPINDLE INHIBITOR (cytogenetic assays): colcemid solution added 2 h before harvest (0.2 µg/mL culture medium)

STAIN (for cytogenetic assays): Giemsa (1.2 %)

NUMBER OF REPLICATIONS: 2 petri dishes were used per dose
NUMBER OF CELLS EVALUATED: 200 metaphases/dose-level (100 metaphases/culture-petri dish)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: Not specified

Evaluation criteria:

Analysis of data was performed on the frequency of aberrant metaphases, both including and excluding gap type aberrations.
In final judgement, gap type aberration were included as aberrant cells. Where more than two types of aberration occurred in a cell, each type was counted as one aberrant cell. The incidences of polyploid cells and cells with structural aberration in each group were judged according to the following criteria:
- Less than 5 %: Negative (-)
- Greater than 5 %, but less than 10% Suspicious (+)
- Greater than 10 % Positive (+)

Species / strain:

mammalian cell line, other: Fibroblast cell line derived from the lung of Chinese hamsters (CHL/IU) was used.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility/precipitation: the liquid test item was not soluble in the culture medium; at all test concentrations (direct and activation method) the test item floated on the surface of the medium.

RANGE-FINDING/SCREENING STUDIES:
- Growth inhibition test: Cells were exposed to test item concentrations ranging from 313 to 5000 µg/mL for 48 h (-S9) or 6 h (+S9). Cytotoxicity was determined as relative survival after 48 h (exp. wihtout -S9) or 24 h (exp. with S9). No remarkable cytotoxicity was noted both direct and activation method at the highest dose of 5000 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: not indicated

ADDITIONAL INFORMATION ON RESULTS: In the test item treated groups (1,250-5,000 µg/mL), the incidence of structural aberration cells and pollploid cells were less than 2%.

EXPERIMENTAL RESULT

Experiment I, 24 hours				% aberrant cells
article	concentration (µg/ml)	number of cells analyzed	S9-Mix	incl. Gaps	excl. Gaps	Polyploid cells
solvent control		200	-	1	0.5	0
test article	1250	200	-	1	0.5	0
test article	2500	200	-	1	1	0.5
test article	5000	200	-	0.5	0	0.5
MNNG	2	200	-	75	73	0.5
Experiment I, 48 hours
solvent control		200	-	1	0.5	0
test article	1250	200	-	1	0	0.5
test article	2500	200	-	0.5	0.5	0
test article	5000	200	-	0	0	0
MNNG	2	200	-	45	42.5	3
Experiment II, 6 hours
solvent control		200	+	0	0	0
test article	1250	200	+	0	0	1
test article	2500	200	+	0.5	0	0
test article	5000	200	+	1	0	1.5
B(a)P	30	200	+	30	27.5	0
Experiment II, 6 hours
solvent control		200	-	1	0	0
test article	1250	200	-	0	0	0
test article	2500	200	-	0.5	0	0.5
test article	5000	200	-	1.5	0.5	0
B(a)P	30	200	-	0	0	0

Conclusions:

The frequency of chromosomal aberration in treated cells without or with metabolic activation was comparable to that in the solvent-control. It is therefore concluded that the rest item did not induced chromosomal aberration in the in vitro test system under the reported conditions.

Executive summary:

A GLP-compliant chromosome aberration test was conducted in vitro with Chinese hamster lung fibroblasts cells (CHL) to assess the test article’s potential to induce chromosome aberrations. Dose levels were based on the results of a growth inhibition test and were determined to be as follows: 1250, 2500 and 5000 µg/ml. No cytotoxicity was evident. Both positive controls, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in experiment I (without microsomal activation, 24 and 48 hour treatment) and 1,2-benzopyrene (B(a)P) in experiment II (with microsomal activation, 6 hour treatment) induced chromosomal aberration at a high incidence. The frequency of chromosomal aberration cells treated with the test article without or with metabolic activation was comparable to that in the solvent control. It is therefore concluded that the test substance did not induced chromosomal aberration in the in vitro test system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.11.1985 - 10.02.1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1983

Deviations:

no

GLP compliance:

no

Remarks:

but perforemd under GLP-like quality assurance program

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix prepared from the liver of Tif:RAIf(SPF) rats after induction with Aroclor 1254.

Test concentrations with justification for top dose:

Range finding test: 0.08 - 5000 µg/plate
Mutagenicity test: 0, 20, 78, 313, 1250, 5000 µg/plate; with and without S9-mix tested with all tester strains.

Vehicle / solvent:

- Vehicle used: acetone (0.1 mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9-mix: Daunorubicin-HCl (TA 98), 4-nitroquinoline-N-oxide (TA 100), mitomycin-C (TA 102), sodium azide (TA 1535), 9(5) aminoacridine-HCl (TA 1537). With S9-mix: 2-aminoanthracene (TA 98, TA 100, TA 102, TA 1537), cyclophosphamide (TA 1535).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 48h at 37 °C +/- 1.5 °C

NUMBER OF INDEPENDENT EXPERIMENTS: 2

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: colony counting and comparison with vehicle control

Evaluation criteria:

The test item is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Species / strain:

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

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentration of 5000 µg/plate the test item precipitated in soft agar.

RANGE-FINDING/SCREENING STUDY
A preliminary toxicity test was carried out with the concentrations ranging from 0.08 to 5000 µg/0.1 mL.

EXPERIMENT I

	TA 98		TA 100		TA 102		TA 1535		TA 1537
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	20	43	151	135	324	388	15	17	6	14
20	23	35	159	130	326	439	11	12	8	8
78	25	43	144	143	310	384	15	13	7	16
313	21	27	147	136	294	426	16	11	9	11
1250	20	31	158	124	280	410	15	12	4	8
5000	19	43	140	133	303	308	11	10	4	13
positive control A	210	1241	539	845	1394	946	105	342	182	102
positive control B	496		1206		1954		275		643

EXPERIMENT II

	TA 98		TA 100		TA 102		TA 1535		TA 1537
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	24	40	123	129	284	412	13	12	13	22
20	29	35	130	123	264	326	18	15	16	20
78	19	38	138	133	305	316	16	15	17	27
313	20	30	146	129	287	310	15	20	19	25
1250	24	23	122	136	271	317	18	14	12	20
5000	29	30	143	131	305	330	15	11	18	16
positive control A	251	525	377	822	841	1023	1093	284	1906	238
positive control B	480		790		1056		1469		3174

CONTROLS

Without S9 mix:

TA 98: daunorubicin-HCl, A: 5 and B: 10 µg/0.1 mL phosphate buffer

TA 100: 4-nitroquinoline-N-oxide, A: 0.125 and B: 0.25 µg/0.1 mL DMSO

TA 102: mitomycin-C, A: 0.5 and B: 1.0 µg/0.1 mL phosphate buffer

TA 1535: sodium azide, A: 2.5 and B: 5.0 µg/0.1 mL phosphate buffer

TA 1537: 9(5)- aminoacridine hydrochloride monohydrate, A: 50 and B: 100 µg/0.1 mL DMSO

With S9-Mix:

TA 98, TA 100, TA 1537: 2-aminoanthracene, 5 µg/0.1 mL DMSO

TA 102: 2-aminoanthracene, 20 µg/0.1 mL DMSO

TA 1535: cyclophosphamide, 250 µg/0.1 mL phosphate buffer

Conclusions:

Based on the presented results and under the conditions employed, the test article did not induce point mutations in presence or absence of a metabolic activation system and is therefore regarded as not mutagenic in the Ames test.

Executive summary:

In order to investigate the test article's potential to cause point mutation in bacteria, an AMES test according to the OECD guideline No. 471 and in compliance with GLP principles was carried out with the tester strains Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test article was applied by the plate incorporation method at concentrations of 20, 78, 313, 1250 and 5000 µg/0.1 ml either with or without a metabolic activation system (rat liver S9 mix). The experiment was performed in triplicates and repeated once for confirmation. Positive controls were performed in parallel to check the tester strains sensitivity. In none of the experiments did treatment with the test substance lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the controls. No growth-inhibiting effect was observed. At the highest concentration of 5000 µg/0.l ml the substance precipitated in soft agar. In conclusion, no evidence of the induction of point mutations by the test substance or by its metabolites formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the substance is considered as not mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo micronucleus assay performed in Chinese hamsters (Ciba-Geigy 851224, 1986), no clastogenicity was observed either with or without a metabolic activation system.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13.05. - 29.07.1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

no

Remarks:

but performed under GLP-like quality control with QAU statement included.

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Strain:

other: random outbred strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln
- Age at study initiation: females: 6-10 weeks; males: 4-9 weeks
- Weight at study initiation: females 22-35 g, males 23-35 g (tolerability test), females 28-34 g, males 28-33 g (mutagenicity test)
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: not specified
- Diet: standard diet NAFAG No.924, ad libitum
- Water: tap water, ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 52-56
- Air changes: no data
- Photoperiod: 12 hrs dark /12 hrs light

IN-LIFE DATES: from 24.02. - 13.05.1986

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: arachis oil
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in arachis oil.

Duration of treatment / exposure:

Single treatment.

Frequency of treatment:

The animals received the test item, the vehicle or the positive control substance once. The dosing volume was 10 mL/kg bw.

Post exposure period:

16 h, 24 h or 48 h

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 at 5000 mg/kg and for negative control (8 per sampling time).
8 for positive control.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): as recommended by the guideline
- Route of administration: oral gavage
- Doses / concentrations: 64 mg/kg bw in 10 mL/kg bw arachis oil

Tissues and cell types examined:

Bone marrow smears from femur.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary test was performed to determine the highest dosage of the test item to be applied in the mutagenicity assay. In this experiment the dose of 5000 mg/kg was determined as the highest applicable in the mutagenicity assay.

TREATMENT AND SAMPLING TIMES
The preparation was administered orally to groups of 24 female and 24 male animals each in the negative and in the 5000 mg/kg bw dose group. The positive control group consisted of 8 female and 8 male animals. Treatment consisted of a single application. 16, 24 and 48h after application 8 female and 8 male animals per sampling time were sacrificed. Bone marrow was harvested from the shafts of both femurs.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 µl rat serum the bone marrow was drawn up. In order to receive a homogenous suspension the content of pipette was aspirated gently about three times. Small drops of the homogenous bone marrow suspension in rat serum were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Grünwald solution for 2 min then in May-Grünwald solution/water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 sec and washed off twice in water, they were left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted in Eukitt.

METHOD OF ANALYSIS:
The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post-treatment were examined. The slides of five female and five male animals of the positive control group were scored at 24 hours post-treatment only. 1000 polychromatic erythrocytes each were scored for the incidence of micronuclei per animal. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.

Evaluation criteria:

Not specified.

Statistics:

The significance of difference was assessed by X² -test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Three groups of four Chinese hamsters (2 females and 2 males) were treated with three different doses, one receiving the maximum dose of 5000 mg/kg bw, and the other two doses of 1/5 and 1/25 of that amount respectively.
- Solubility: no data
- Mortality: no mortality within the observation period of 24, 48 or 72 hours.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.
- Ratio of PCE/NCE (for Micronucleus assay): After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.
- Appropriateness of dose levels and route: yes
- Statistical evaluation: No statistically significant differences in the frequency of erythrocytes containing micronuclei between the solvent control and the dose groups were observed.

Conclusions:

Under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.

Executive summary:

In a micronucleus test performed under GLP-like quality control, the test article in arachis oil was given by gavage to three groups of 8 male and 8 female Chinese hamsters. The animals were treated once with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made. The negative control animals received the vehicle alone. The bone marrow smears from animals treated with the test substance showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes compared to the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 2.9% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.06%) treated with the vehicle (arachis oil) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the limit dose of 5000 mg/kg bw of the test article.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test

The mutagenic potential of the test substance was investigated in a GLP-compliant Ames test according to OECD 471, performed with Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of a metabolic activation system (S9 mix). None of the tested concentrations (20 to 5000 µg/0.1 ml) led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control either with or without metabolic activation. This result was confirmed in a second independent experiment. Therefore, based on the results of these experiments, and on standard evaluation criteria, it is concluded that the test article and its metabolites did not induce gene mutations in the strains of S. typhimurium used.

in vitro chromosome aberration

In a chromosome aberration test similar to OECD 473 and in compliance with GLP, the substance was investigated for clastogenic effects on Chinese hamster lung fibroblasts in vitro. Experiments were performed with dose levels of 1,250, 2,500 and 5,000 µg/ml either without metabolic activation (direct method, 24 hours or 48 hours incubation) or with metabolic activation (activation method, 6 hours incubation). In all experiments performed, no biologically significant increase in the number of specific chromosome aberrations was observed. Therefore, it is concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster lung fibroblasts in vitro.

HPRT test

In a mammalian gene mutation test according to OECD guideline 476 and in compliance with GLP, the test article was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 h. In all experiments performed comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies. An isolated increase (19.3 colonies per 10⁶ cells) exceeding the threshold of three times the corresponding solvent control and the historical range of our negative and solvent controls was observed at 450.0 µg/ml in experiment I, culture I without metabolic activation. This increase was judged as biologically irrelevant since it was not reproduced in the parallel culture (culture II) under identical conditions. This effect is probably induced by inhomogeneity of the emulsion of the test item in medium at the maximal concentration. The factor of three times the corresponding solvent control was exceeded at almost all concentrations in the first culture of the second experiment. This effect, however, is based upon the very low solvent control (0.6 colonies per 10⁶ cells) and represents statistical fluctuations at such low absolute numbers. The absolute numbers of colonies remained well within our range of historical negative and solvent controls and are below the corresponding negative control. Based on this result and under the given experimental conditions, it is concluded that the test article and its metabolites did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

in vivo micronucleus test

In an in vivo micronucleus test performed similarly to OECD test guideline 474 and under GLP-like quality control, the test article was investigated for mutagenic effects in Chinese hamsters. The animals were treated once by gavage with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made. No statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes compared to the negative control animals at all three sampling times were observed. The respective positive control experiments with cyclophosphamide (64 mg/kg) yielded an average of 2.9% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.06%) treated with the vehicle (arachis oil) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters and the test substance is therefore considered to be non clastogenic.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.