Reaction mass of 1H-Benzotriazole-1-methanamine, N,N-bis(2-ethylhexyl)-6-methyl- and 2H-Benzotriazole-2-methanamine, N,N-bis(2-ethylhexyl)-5-methyl- and N,N-bis(2-ethylhexyl)-4-methyl-1H-benzotriazole-1-methylamine and 2H-Benzotriazole-2-methanamine, N,N-bis(2-ethylhexyl)-4-methyl- and N,N-bis(2-ethylhexyl)-5-methyl-1H-benzotriazole-1-methylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test compound did not cause mutations in bacteria and in mammalian cell culture. Furthermore, the test compound is considered to have no chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes

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:

August 09, 2012 - November 12, 2012

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: parameters of the experiments remain similar because of the reproducible characteristics of the cells.
- Methods for maintenance: cells are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium.

MEDIA USED
- 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/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

See any other information on materials and methods incl. tables.

Vehicle / solvent:

On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol. The solvent was chosen based on solubility properties and its relative non-toxicity to the cell cultures. The final concentration of ethanol in the culture medium was 0.5 % (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
- Expression/fixation time: Three or four days after treatment 1.5x10^6 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 3 - 5x10^5 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 7-10 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.

SELECTION AGENT: 6-thioguanine

NUMBER OF REPLICATIONS: The study was performed in two independent experiments, using identical experimental procedures.

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
The data of this study comply with the above mentioned criteria.

Evaluation of Results
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.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS GENOTOXICITY:
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A p-value below 0.05 was detected in culture I of the first experiment with metabolic activation and in culture II of the second experiment without metabolic activation. These trends however, were judged as biologically irrelevant as the mutation frequency remained well within the range of the historical solvent controls.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 8.8 up to 25.0 mutants per 10^6 cells; the range of the groups treated with the test item was from 3.8 up to 35.3 mutants per 10^6 cells.

EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 39.1 to 5000 μg/mL according to the current OECD guideline 476 to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects were observed at 78.1 μg/mL and above after 4 hours treatment without metabolic activation and at 156.3 μg/mL with metabolic activation. Following 24 hours treatment the cell growth was completely inhibited down to the lowest concentration.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation was observed at 625 μg/mL and above in the presence of metabolic activation following 4 hours treatment and at 312.5 mg/mL and above in the absence of metabolic activation following 24 hours treatment.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations generally spaced by a factor of 2 was applied. Narrower spacing was used at high concentrations with metabolic activation to cover the onset of cytotoxicity more closely.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I and/or a relative cell density below 50% in both cultures were observed in the first experiment at 78.0 μg/mL with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 10.0 μg/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered without metabolic activation. In the presence of metabolic activation moderate cytotoxic effects were noted in the first experiment with metabolic activation at the highest analysable concentration of 78 μg/mL with metabolic activation. Exceedingly severe cytotoxic effects occurred at the next higher, phase separating concentration of 156 μg/mL. In the second experiment with metabolic activation turbidity observed at the maximum concentration of 120 μg/mL indicated that the limit of solubility was reached.

Experimental Result:

	concentration (µg/ml)	PST	S9 Mix	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor
Experiment I / 4h treatment				culture I					culture II
solvent control (ethanol)			-	100.0	100.0	100.0	8.8	1.0	100.0	100.0	100.0	25.0	1.0
positive control (EMS)	150.0		-	101.7	122.4	92.9	98.5	11.2	85.8	146.3	74.5	157.8	6.3
test item	0.16		-	110.4	culture was not continued#				95.1	culture was not continued#
test item	0.31		-	110.2	culture was not continued#				87.9	culture was not continued#
test item	0.63		-	119.0	117.1	83.0	14.7	1.7	87.9	102.1	128.4	14.4	0.6
test item	1.3		-	111.1	119.7	88.1	7.9	0.9	97.6	112.3	111.7	25.2	1.0
test item	2.5		-	114.4	97.7	77.6	19.5	2.2	98.2	72.9	123.4	10.1	0.4
test item	5.0		-	122.6	100.2	86.2	20.1	2.3	100.0	52.6	124.7	14.6	0.6
test item	10.0		-	96.7	56.5	78.8	19.5	2.2	66.8	45.2	99.2	25.6	1.0
solvent control (ethanol)			+	100.0	100.0	100.0	18.0	1.0	100.0	100.0	100.0	22.7	1.0
positive control (DMBA)	1.1		+	62.8	91.9	41.4	1579.1	87.7	59.3	104.1	73.7	1200.1	52.9
test item	9.8		+	102.2	106.8	84.3	15.5	0.9	98.4	93.5	89.1	22.0	1.0
test item	19.5		+	97.0	123.0	94.6	17.8	1.0	92.2	93.5	101.9	15.7	0.7
test item	39.0		+	99.7	113.1	98.4	19.3	1.1	96.8	85.8	82.0	23.2	1.0
test item	78.0		+	44.1	68.1	70.2	25.7	1.4	54.0	41.5	56.4	35.3	1.6
test item	156.0	PS	+	0.0	culture was not continued##				0.0	culture was not continued##
test item	234.0		+	0.0	culture was not continued##				0.0	culture was not continued##
Experiment II / 24h treatment
solvent control (ethanol)			-	100.0	100.0	100.0	12.5	1.0	100.0	100.0	100.0	14.3	1.0
positive control (EMS)	150.0		-	89.7	113.2	91.1	364.3	29.1	89.7	96.8	72.9	368.9	25.9
test item	0.31		-	98.8	culture was not continued#				101.3	culture was not continued#
test item	0.63		-	100.6	114.7	76.7	21.8	1.7	98.2	88.5	124.6	7.8	0.5
test item	1.3		-	98.9	109.6	178.7	3.8	0.3	98.5	83.5	95.2	7.0	0.5
test item	2.5		-	95.8	118.0	89.8	25.2	2.0	96.1	100.1	130.8	9.4	0.7
test item	5.0		-	77.6	107.9	122.0	17.6	1.4	72.9	75.8	79.1	11.4	0.8
test item	10.0		-	21.1	18.3	87.8	29.2	2.3	23.8	15.2	82.0	26.6	1.9
test item	20.0		-	0.0	culture was not continued##				0.0	culture was not continued##
Experiment II / 4h treatment
solvent control (ethanol)			+	100.0	100.0	100.0	14.5	1.0	100.0	100.0	100.0	14.7	1.0
positive control (DMBA)	1.1		+	83.9	80.9	87.3	544.1	37.6	79.3	78.5	71.8	577.2	39.3
test item	5.0		+	100.4	culture was not continued#				92.6	culture was not continued#
test item	10.0		+	101.1	86.0	103.7	15.0	1.0	89.6	93.6	91.5	18.5	1.3
test item	20.0		+	99.6	88.9	100.8	17.0	1.2	86.3	100.0	88.2	18.8	1.3
test item	40.0		+	93.8	78.5	108.7	15.6	1.1	93.5	108.0	54.8	20.5	1.4
test item	80.0		+	94.9	79.2	101.2	18.1	1.3	94.3	103.4	69.9	32.8	2.2
test item	120.0	T	+	94.9	75.6	113.8	16.6	1.1	88.2	100.0	70.4	24.3	1.7

PS = Phase Separation visible at the end of treatment

T = Turbidity

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

## culture was not continued due to exceedingly severe cytotoxic effects

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Guideline of Japanese Ministry of Labor, dated June 12, 1979

Deviations:

yes

Remarks:

6 strains tested (preincubation method), no confirmatory experiment; no data on test substance purity

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

His- and Trp-operon

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 purchased from Kikkoman Co., Ltd. and S-9 mixture prepared (S9: 0.3 mL, MgCl2 8 µmol, KCl 33 µmol, glucose-6-phosphate 5 µmol, NADPH 4 µmol, NADH 4 µmol Na2HPO4/KH2PO4 buffer (pH 7.4) 100 µmol + distilled water)

Test concentrations with justification for top dose:

0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate method (preincubation method)

DURATION
- Preincubation period: 20 minutes (with shaking)
- Exposure duration: 48 hours
- Incubation temperature: 37°C
- 2 Petri dishes per strain and per concentration

The sterility was tested with sample solution or S9-mix mixed with soft agar solution, displayed over the minimal agar plate without preincubation method.

The strains of S. typhimurium and E. coli were obtained from National Institute of Hygenic Sciences, Tokyo, Japan on April 11, 1980 and preserved at -82°C.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (compared to control), reduced titer, reduced background lawn

Evaluation criteria:

A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

depending on the strain from 10 µg/plate onwards without S9 and from 500 µg/plate onwards with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate onwards with and without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

depending on the strain from 10 µg/plate onwards without S9 and from 500 µg/plate onwards with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

depending on the strain from 10 µg/plate onwards without S9 and from 500 µg/plate onwards with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

depending on the strain from 10 µg/plate onwards without S9 and from 500 µg/plate onwards with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No evidence of the induction of point mutations by the compound or by the metabolites of the substance formed as a result of microsomal activation was detectable in any of the strains of Salmonella typhimurium and Escherichia coli used in this experiment.

Mean number of revertant colonies per plate (average of 2):

	µg/plate	S9 mix	TA 100	TA 1535	WP2uvrA	TA 98	TA 1537	TA 1538
DMSO	0	-	128	22	21	24	8	10
Sample	0.5	-	150	23	19	24	9	21
	1	-	142	16	20	12	8	14
	5	-	142	15	24	13	6	16
	10	-	130	21	18*	6*	6	15
	50	-	96*	14*	10*	5*	3*	11*
	100	-	70*	12*	12*	0*	0*	6*
	500	-	1*	0*	16*	0*	0*	1*
	1000	-	0*	0*	22*	0*	0*	0*
Positive control - S9 mix	Name		ENNG	ENNG	ENNG	NF	AAC	NF
	Concentrations (μg/plate)		2	10	5	2	10	5
	Mean No. of colonies/plate (average of 2)		330	484	986	998	26	877
DMSO	0	+	138	30	16	42	18	40
Sample	0.5	+	140	25	20	43	9	43
	1	+	159	30	17	43	15	40
	5	+	143	27	20	44	7	36
	10	+	163	22	15	41	13	37
	50	+	165	20	22*	47	7	36
	100	+	163	21	29*	38	7	33
	500	+	83*	19	24*	2*	0*	4*
	1000	+	52*	10*	27*	0*	0*	0*
Positive control + S9 mix	Name		BP	2AA	2AA	BP	BP	BP
	Concentrations (μg/plate)		5	1	40	5	5	5
	Mean No. of colonies/plate (average of 2)		892	54	384	456	189	129

*growth inhibition observable

 

ENNG: ethyl-3-nitro-1-nitrosoguanidine

NF: 2-nitrofluorene

AAC: 9-aminoacridine

BP: benzo(a)pyrene

2AA: 2-aminoanthracene

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations in the bacterial strains used.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

N/A

Species / strain / cell type:

lymphocytes: Primary Human Lymphocytes

Details on mammalian cell type (if applicable):

In this test system primary human lymphocytes were analyzed for the occurrence of
micronuclei. Fresh Blood was collected from a single donor for each experiment.
Only healthy, non-smoking donors and not receiving medication were used. In this study, in
the 1st Experiment a 31 year old male donor, in the 2nd Experiment a male (29 years old) donor and in the 3rd Experiment a 30 year old male donor were used.
The lymphocytes of each donor have previously shown to respond well to stimulation of
proliferation with phytohemagglutinin (PHA) and to the used positive control substances. Whole blood cultures were treated with the test substance, the erythrocytes were lysed by hypotonic treatment before preparation of the slides.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

cytokinesis block method using the actin polymerisation inhibitor cytochalasin B (Cyt B)

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction
The S9 fraction was prepared according to Ames et al. (7) at BASF SE in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive.
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH, Sulzfeld, Germany) received 80 mg/kg b.w. phenobarbital i.p. and beta-naphthoflavone orally (both supplied by Sigma-Aldrich, Taufkirchen, Germany) each on three consecutive days.
During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am to 6 pm and dark from 6 pm to 6 am.
Standardized pelleted feed and drinking water from bottles were available ad libitum.
24 hours after the last administration, the rats were sacrificed, and the induced livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution (1 mL ≙ 1 g wet liver), then homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5-mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
The sterility of the S9 fraction was determined by incubating 1 mL S9 fraction on an agar plate at 37°C for 48 hours. The content of protein was measured with an appropriate method (e.g. Bradford). To check the activity of the S9 fraction a bacterial reverse mutation assay according to Ames et al. (7) was performed (results see Appendix 8).

Test concentrations with justification for top dose:

Following the requirements of the current OECD Guideline 487 a test substance with defined composition should be tested up to a maximum concentration of 2 mg/mL, 2 µL/mL or 10 mM, whichever is the lowest. When the test substance is not of defined composition, e.g. substance of unknown or variable composition, complex reaction products or biological materials (socalled UVCBs), or environmental extracts, the top concentration should be higher to increase the concentration of each of the components (e.g. 5 mg/mL). In case of toxicity, the top concentration should produce reduction of the proliferation index (CBPI) to 45 ± 5% (leading to a cytostasis increase of 55 ± 5%) of the concurrent vehicle control. For relatively insoluble
test substances only one concentration should be tested showing turbidity or precipitation in culture medium at the end of exposure period.

Dose selection of the 1st Experiment
Without S9 mix 4 hours exposure
1.4 µg/mL - 83.3 µg/mL

With S9 mix
4 hours exposure
2.5 µg/mL - 150.0 µg/mL

Dose selection of the 2nd Experiment
Without S9 mix
20-hours exposure
0.5 µg/mL - 30.0 µg/mL

Dose selection of the 3rd Experiment
Without S9 mix 4 hours exposure
4.0 µg/mL - 25.0 µg/mL

With S9 mix 4 hours exposure
3.5 µg/mL - 60.0 µg/mL

In this study the concentrations are given as rounded values by using a dilution factor of 1.8, except for a dilution factor of 1.5 in the 3rd Experiment with S9mix and a dilution factor of 1.3 in the 3rd Experiment without S9mix.
At least three concentrations were evaluated to detect a possible dose-response relationship.
At least 2 cultures were prepared per test group (referred to as A and B), and at least 1000 cells per culture were evaluated for the occurrence of micronucleated cells.

Vehicle / solvent:

Due to the limited solubility of the test substance in water, ethanol was selected as the vehicle, which has been demonstrated to be suitable in the in vitro micronucleus test and for which historical control data are available.
The final concentration of the vehicle ethanol in culture medium was 1% (v/v).

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

see "any other information on materials and methods"

Rationale for test conditions:

see "any other information on materials and methods"

Evaluation criteria:

Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells in
the control groups (vehicle/positive) and in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our
laboratory’s historical negative control data (95% control limit). Weak outliers can be judged
acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix induced a distinct, statistically significant
increase in the number of micronucleated cells in the expected range.

Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the concurrent vehicle control value and
the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing
micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent
vehicle control value and within the range of our laboratory’s historical negative control data
(95% control limit).

Statistics:

An appropriate statistical analysis was performed. The proportion of cells containing
micronuclei was calculated for each test group. A comparison of the micronucleus rates of each
test group with the concurrent vehicle control group was carried out for the hypothesis of equal
proportions (i.e. one-sided Fisher's exact test, BASF SE).
If the results of this test were statistically significant compared with the respective vehicle
control (p ≤ 0.05), labels (s
) were printed in the tables.
In addition, a statistical trend test (SAS procedure REG (16)) was performed to assess a
possible dose-related increase of micronucleated cells. The used model is one of the proposed
models of the International Workshop on Genotoxicity Test procedures Workgroup Report (17).
The dependent variable was the number of micronucleated cells and the independent variable
was the concentration. The trend was judged as statistically significant whenever the one-sided
p-value (probability value) was below 0.05.
However, both, biological and statistical significance were considered together.

Species / strain:

lymphocytes: Primary Human Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TREATMENT CONDITIONS

Osmolality and pH values were not relevantly influenced by test substance treatment.
In the 1st Experiment, in the absence and the presence of S9 mix, test substance precipitation
was observed macroscopically in culture medium at the end of treatment at 14.3 µg/mL and
above.
After 20 hours continuous treatment test substance precipitation was observed at 16.7 µg/mL
and above (microscopically).
In the 3rd Experiment in the absence of S9 mix, test substance precipitation was observed
macroscopically in culture medium at the end of treatment at 25.0 µg/mL. In the presence of
metabolic activation precipitation in culture medium was observed microscopically at
40.0 µg/mL and above.

CYTOTOXICITY

In this study, no relevantly reduced proliferative activity was observed after 4 hours exposure
interval in the absence and presence of S9 mix in the test groups scored for cytogenetic
damage. In the 1st Experiment at 14.3 µg/mL in the absence of S9 mix a cytostasis of 68.5%
was observed. In the 3rd Experiment without S9 mix cytostasis levels of 62.4% and 84.0%
were observed after treatment with 19.2 µg/mL and 25.0 µg/mL, respectively. These groups
were not evaluated for cytogenetic damage due to the enhanced cytotoxicity levels. After
20 hours continuous test substance treatment without S9 mix in the test group (9.3 µg/mL)
scored for cytogenetic damage, reduced proliferation (cytostasis: 46.8%) was obtained.

GENOTOXICITY - MICRONUCLEUS ANALYSIS

In the presence of metabolic activation, in the 1
st Experiment the obtained values (0.3 – 0.7%
micronucleated cells) were within the range of the 95% control limit of the historical negative
control data (0.1 - 1.1% micronucleated cells; see Appendix 6). In the 3rd Experiment the
values of test substance treated groups varied between 0.3 – 0.4% micronucleated cells. These
values were all within the historical negative control data. In none of the two experiments with
S9 mix a statistically significant increase compared to the respective vehicle control value (0.3
and 0.5% micronucleated cells, 1st and 3rd Experiment, respectively) was observed.
In the 1st Experiment after 4 hours treatment in the absence of S9 mix, the values (0.3 – 0.5%
micronucleated cells) were within the range of the 95% control limit of the historical negative
control data (0.2 – 0.9% micronucleated cells; see Appendix 6). In the 3rd Experiment the
values of test substance treated groups varied between 0.2 – 0.6%. These values were all
within the historical negative control data. In none of the two experiments without S9 mix at 4h
treatment a statistically significant increase compared to the respective vehicle control value
(0.4 and 0.2% micronucleated cells, first and third Experiment, respectively) was observed.
After 20 hours continuous treatment the values (0.4 – 0.8% micronucleated cells) were within
the range of the 95% control limit of the historical negative control data (0.2 – 0.9%
micronucleated cells; see Appendix 6). A statistically significant increase compared to the
vehicle control value was not obseved (0.6% micronucleated cells).
Non of the experimental parts described above showed a positive dose response as assessed
by a trend analysis.
The positive control substances MMC (without S9 mix 0.04 and 0.31 µg/mL), Colchicine
(without S9 mix: 0.05 µg/mL) and CPA (with S9 mix; 2.50 and 3.50 µg/mL) induced statistically
significantly increased micronucleus frequencies in all independently performed experiments.
In this study, in the absence and presence of metabolic activation the frequencies of
micronucleated cells (4 h without S9 mix: 9.1 and 7.8% micronucleated cells; 20 h without S9
mix: 4.4% and 6.7% micronucleated cells (MMC and Col, respectively); 1
st experiment with S9
mix 3.6% and 6.0% micronucleated cells (2.50 and 3.50 µg/mL CPA, respectively) and 3rd
experiment with S9 mix 2.9% and 3.9% micronucleated cells (2.50 and 3.50 µg/mL CPA,
respectively) were compatible to the historical positive control data range (Appendix 7).

Conclusions:

Thus, under the experimental conditions chosen here, the conclusion is drawn that Irgamet 39 has no potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Genetic toxicity in bacterial cells

In a reverse gene mutation assay performed according to a Japanese guideline similar and equivalent to the OECD Test guideline 471, Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 and E. coli WP2 uvrA were exposed to the test substance (no data on purity) in DMSO at concentrations of 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate in the presence and absence of mammalian metabolic activation in a plate incorporation test. The test substance was tested up to precipitating and cytotoxic concentrations. Only one experiment with duplicates was performed. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in this test.

Genetic toxicity in mammalian cells

A GLP-compliant mammalian cell mutagenicity test according to OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The test item was dissolved in ethanol. The concentration range of the main experiments was limited by cytotoxic effects and the solubility of the test item in aqueous medium. The tested concentrations ranged from 0.63 to 120 µg/ml. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. The mutant frequency remained well within the historical range of solvent controls. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item 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 vitro MNT in primary human lymphocytes

The test substance was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Three independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital-and beta-naphthoflavone induced rats (exogenous metabolic activation). According to a solubility test for the determination of the experimental concentrations and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were tested.

Test groups printed in bold type were evaluated for the occurrence of micronuclei:

1st Experiment

4 hours exposure, without S9 mix 0; 1.4; 2.5; 4.4; 7.9; 14.3; 25.7; 46.3; 83.3 µg/mL

4 hours exposure, with S9 mix 0; 2.5; 4.4; 7.9; 14.3; 25.7; 46.3; 83.3; 150.0 µg/mL

2nd Experiment

20 hours exposure, without S9 mix 0; 0.5; 0.9; 1.6; 2.9; 5.1; 9.3; 16.7; 30.0 µg/mL

3rd Experiment

4 hours exposure, without S9 mix 0; 4.0; 5.2; 6.7; 8.8; 11.4; 14.8; 19.2; 25.0 µg/mL

4 hours exposure, with S9 mix 0; 3.5; 5.3; 7.9; 11.9; 17.8; 26.7; 40.0; 60.0 µg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. In this study, ethanol was selected as vehicle. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicine and Cyclophosphamide (CPA), led to the expected increase in the number of cells containing micronuclei. In this study, cytotoxicity indicated by clearly reduced proliferation index (CBPI) was observed at least at the highest applied test substance concentration in all experimental parts without metabolic activation of this study. In the presence of S9 mix no cytotoxicity indicated by reduced proliferation index (CBPI) was observed. On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, the test substance is considered to have no chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Three in vitro studies on genetic toxicity were negative. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.