3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-[2-(4-chlorophenoxy)-5-(trifluoromethyl)phenyl]benzamide]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 07, 2012 - May 25, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report Date:

2012

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. certificate)

Remarks:

Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: nanoform

Details on test material:

Expiration Date: October 27, 2019
Physical state, appearance: Solid, yellow
Molecular weight: 916.65 g/mol
Storage conditions: Room temperature
Batch: 4038273

TEM: mostly rods with aspect ratios around 5, diameters from 30 nm to 150 nm
BET (SSA) 57 m2/g

Specific details on test material used for the study:

- Lot/batch No.: 0004038273
- Expiration date of the lot/batch: October 27, 2019
- Storage condition of test material: room temperature

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1 %)
- 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:

Phenobarbital/b-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I: 6.6, 13.1, 26.3, 52.5, 105.0, 210.0 µg/mL
Experiment II: 13.1, 26.3, 52.5, 105.0, 210.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: the test substance was diluted in medium

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

EMS: without S9 mix; DMBA: with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Experiment I: 4 hours without and 4 hours with S9 mix
Experiment II: 24 hours without and 4 hours with S9 mix
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below.
Three or four days after treatment 1500000 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 300000 - 500000 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days.

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
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.
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.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the first experiment precipitation was observed at 105.0 µg/mL and above in the absence and at 26.3 µg/mL and above in the presence of metabolic activation. In the second experiment precipitation was observed at 210.0 µg/mL in the absence and at 52.5 µg/mL and above in the presence of metabolic activation.

Any other information on results incl. tables

Table 1: Summary of results

	conc.	P	S9 mix	releativ cloning efficiancy I	relative cell density	releativ cloning efficiancy II	mutant colonies/10^6cells	induction factor	releativ cloning efficiancy I	relative cell density	releativ cloning efficiancy II	mutant colonies/10^6cells	induction factor
	µg/mL			culture I					culture II
Experiment I / 4 h treatment
solvent control in medium			-	100	100	100	12.9	1	100	100	100	9.7	1
Positive control (EMS)	150		-	96	84.9	91.5	70.2	5.4	93.4	127.8	86.1	50.7	5.2
Test item	6.6		-	96.4	culture was not continued#				98.8	culture was not continued#
Test item	13.1		-	104	96.6	98.7	20.6	1.6	104.5	104.8	98.5	4.7	0.5
Test item	26.3		-	95.1	94.2	90.7	10.7	0.8	99	98.1	97.2	12.1	1.2
Test item	52.5		-	94.8	83.2	94.6	9.8	0.8	105.1	94.9	106.8	6.6	0.7
Test item	105	P	-	97	89.4	91	10.8	0.8	96	131.6	107.3	6.4	0.7
Test item	210	P	-	99	68.2	91	21.1	1.6	98.2	126.2	101.6	29.4	3
solvent control in medium			+	100	100	100	23.9	1	100	100	100	9.2	1
Positive control (DMBA)	1.1		+	50.6	76.3	94	446	18.6	58.2	107.2	86	376.5	41.1
Test item	6.6		+	97.2	96	112.7	12.6	0.5	100.6	113.8	100.8	12.7	1.4
Test item	13.1		+	94.7	95.1	122.7	17	0.7	103.1	117.2	95.4	9.5	1
Test item	26.3	P	+	90.1	97.2	118.1	11.7	0.5	94.3	112.1	96.3	19.6	2.1
Test item	52.5	P	+	94.8	culture was not continued##				94.9	culture was not continued##
Test item	105	P	+	93.3	87.6	110.3	14	0.6	101.6	111.5	109.2	11.8	1.3
Test item	210	P	+	89.7	87.3	123.3	11	0.5	101.5	115.1	92.4	10.9	1.2
Experiment II / 24 h treatment
solvent control in medium			-	100	100	100	6	1	100	100	100	11	1
Positive control (EMS)	150		-	95.8	95.2	92.9	405.8	67.2	78.5	81.6	63.2	524.4	47.8
Test item	6.6		-	98.4	culture was not continued#				93.3	culture was not continued#
Test item	13.1		-	100.3	97.5	100.2	6.1	1	98.6	93	80.2	15.4	1.4
Test item	26.3		-	100	99.5	103.4	15.7	2.6	95.2	73.7	78.1	12.6	1.1
Test item	52.5		-	98.7	108	100.7	11.7	1.9	100	99.9	84.5	7.5	0.7
Test item	105		-	100.1	102	98.8	9.5	1.6	98.1	81.9	72.4	18.1	1.6
Test item	210	P	-	96.8	82.5	90	29.5	4.9	97.8	76.5	57.9	14.9	1.4
Experiment II / 4 h treatment
solvent control in medium			+	100	100	100	8.2	1	100	100	100	6.1	1
Positive control (DMBA)	1.1		+	86.6	112.7	99.5	369.1	44.8	88.2	51	63.4	495.3	81.6
Test item	3.3		+	101.7	culture was not continued#				104.3	culture was not continued#
Test item	6.6		+	100.3	142.2	105.1	15.1	1.8	102.4	90.9	102.3	7.9	1.3
Test item	13.1		+	100.9	204	94.7	13.1	1.6	106.2	121.7	94.2	6.5	1.1
Test item	26.3		+	102	186.1	104.1	19.5	2.4	101.6	75.6	97.1	15.3	2.5
Test item	52.5	P	+	100.3	195	81.2	11	1.3	97.6	71.3	96.2	5.8	1
Test item	105	P	+	99.9	290.9	92	9.9	1.2	91.6	83.2	87.6	7.8	1.3

P = Precipitation

# cutlture was not continued as a minimum of only four concentrations is required

## culture was not continued to avoid analysis of too many precipitating concentrations

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls. An increase of the induction factor reaching or exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the first experiment without metabolic activation at 210 µg/mL and in the first culture of the second experiment without metabolic activation at 210 µg/mL. However, the increases were based on a rather low mutation frequency of the corresponding solvent controls of just 9.7 and 6.0 colonies per 10⁶cells, respectively. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions. Therefore, the increases of the induction factor were judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency. A single significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second experiment at culture I without metabolic activation. However, the trend was judged as biologically irrelevant as all of the values of the mutation frequency remained within the historical range of solvent controls.

Applicant's summary and conclusion

Conclusions:

negative