2-(4,6-bis(2,4-dimethylphenyl)-1,3,5-triazin-2-yl)-5-(3-((2-ethylhexyl)oxy)-2-hydroxypropoxy)phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test and a chromosome aberration test in V79 cells were performed according to GLP compliant OECD471 and 473 studies to evaluate the mutagenic potential of the test substance. The test item did not induce mutations or chromosome aberrations in vitro. An analogue substance did not induce mutagenicity in mammalian cells (OECD 476). Therefore, the substance is considered as non-mutagenic under the conditions of these tests.

Endpoint Conclusion: No adverse effect observed (negative)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr - Jul 1995

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:

26 May 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Dec 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie und Bundesangelegenheiten

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Z 2651-01
- Expiration date of the lot/batch: February, 1997
- Purity: > 95 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: pure of expiration date
- Solubility and stability of the test substance in the solvent/vehicle: not indicated by the sponsor

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment, the test article was dissolved in acetone. The solvent was chosen because of its solubility properties and its relative nontoxicity for the bacteria.
No precipitation ofthe test article occurred up to the highest investigated dose.

OTHER SPECIFICS:
- Aggregate State at RT: solid
- Colour: yellowish

Target gene:

his, trp

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mixture from aroclor induced 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:

- solvent used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity for the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine / 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: materials were mixed in a test tube and poured onto the minimal agar plates

DURATION
- Preincubation period: 60min (Exp. II, IIA, IIB)
- Exposure duration: 48h, 37°C

SELECTION AGENT (mutation assays): ampicilin

NUMBER OF REPLICATIONS: min. 3 plates per dose and strain

NUMBER OF CELLS EVALUATED: all cells per plate were counted and compared with untreated / vehicle treated cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth;

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
A mutagenic response is described as follows:
A test article is considered mutagenic if in the strains TA 100, WP2, and its uvrA derivative the number of reversions will be at least twice as high and in the strains TA 1535, TA 1537, and TA 98 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

Due to intemational guidelines a statistical evaluation of the results is recommended.
However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Species / strain:

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

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

Species / strain:

E. coli WP2

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

Species / strain:

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:

Pre-experiment:
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test).
Toxicity of the test article results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Table 1: Summary of results, without S9 mix

Concentration µg / plate	Revertants/plate mean from three plates
	TA35		TA1537		TA98		TA100		WP2		WP2 UVRA
	I	II	I	II	I	II	I	II	I	II	I	II
Negative control	14	14	14	8	23	17	102	82	39	36	43	40
Solvent control	23	10	10	7	23	20	106	86	39	41	39	37
Positive control *	611	396	68	112	120	137	368	395	672	329	519	418
33.3	15	6	12	12	19	15	149	72	32	37	43	41
100.0	21	6	12	7	21	13	142	55	30	39	40	32
333.3	14	7	10	11	20	13	144	43	32	38	42	30
1000.0	16	7	11	7	19	13	145	65	25	31	32	28
2500.0	17	10	8	7	26	9	145	68	29	27	44	32
5000.0	20	8	10	8	30	10	138	780	30	37	43	35

* Sodium azide (10.0 pg/plate) strains TA 1535 and TA 100

4-nitro-o-phenylene-diamine (10.0 pg/plate) strains TA 1537 and TA 98

Methyl methane sulfonate (5 pl/plate) strains WP2 uvrA and WP2

Table 2: Summary of results, with S9 mix

Concentration µg / plate	Revertants/plate mean from three plates
	TA1535		TA1537				TA98		TA100		WP2		WP2 UVR
	I	II	I	II	IIa	IIb	I	II	I	II	I	II	I	II
Negative control	27	10	14	9	21	10	25	21	124	116	33	37	37	40
Solvent control	26	10	19	7	22	11	23	21	142	85	36	35	39	41
Positive control **	286	120	80	50	336	68	92	309	358	589	203	229	190	204
33.3	14	6	20	6	15	10	25	11	131	110	38	41	41	33
100.0	45	10	13	6	19	11	19	12	126	96	38	33	49	35
333.3	44	8	15	8	20	10	20	12	143	103	37	37	41	41
1000.0	36	7	17	10	18	14	29	14	145	113	41	36	47	41
2500.0	34	9	17	13	17	11	30	19	150	128	43	41	50	47
5000.0	33	18	18	30	25	12	30	33	154	152	44	55	47	60

** 2-aminoanthracene (2.5 pg/plate) strains TA 1535, TA 1537, TA 98, and TA 100

2-aminoanthracene (10.0 pg/plate) strains WP2 uvrA and WP2

Conclusions:

Interpretation of results (migrated information):
negative

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 article is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test article to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II, experiment IIA, and experiment IIB) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in addition the Escherichia coli strains WP2 and WP2 uvrA. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was 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 metabolic activation in all independent experiments. A slight toxic effect evidenced by a reduction in the number of revertants occurred at 2500.0 µg/plate in strain TA 98 without S9 mix in experiment II.

No substantial increases in revertant colony numbers of any of the six tested strains were observed following treatment with the test substance at any dose level, either in the presence or absence of metabolic activation (S9 mix) in experiment I and II. There was also no relevant tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

May 26, 1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Energie, Jugend, Familie und Gesundheit

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: Z-2759
- Expiration date of the lot/batch: Sep 1997
- Purity: 99.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stable until Sep 1997
- Solubility and stability of the test substance in the solvent/vehicle: In solvent stable for 48 h

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment (immediately before treatment), the test article was dissolved in DMSO (E. MERCK, D-64293 Darmstadt).

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Solution

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cells (supplied by Laboratory for Mutagenicity Testing, LMP,
Technical University Darmstadt, D-64287 Darmstadt)
- Suitability of cells: The V79 cell line has been used successfully for many years in in vitro experiments. Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays with cell cultures as many chemicals only develop a mutagenic potential when they are metabolized by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with liver microsome preparations (S9 mix).

For cell lines:
- Absence of Mycoplasma contamination: yes, each batch was screened for mycoplasm contamination
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37° C in 80 cm plastic flasks (GREINER,D-72632 Frickenhausen). About 5 x 10^ ceUs per flask were seeded into 15 ml of MEM medium
- Proliferation index : high proliferation rate (doubling time of clone V79/T5 in stock cultures:
12 h) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %)
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes, each batch was and checked for karyotype stability

MEDIA USED
- Type and composition of media: MEM (Minimal Essential Medium; SEROMED; D-12247 BerUn) supplemented with 10 % fetal calf serum (FCS; Boehringer Mannheim, D-68261 Mannheim)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:

- Method of preparation of S9 mix :
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanlbm (BRL, CH-4414 Fullinnsdorf; weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg Aroclor 1254 (Antechnika, D-76275 Karlsruhe) per kg body weight in olive oil 5 days previously. The livers of the animals were removed, washed in 150 mM KCl and homogenized. The homogenate, diluted 1+3 with KCl was centrifiiged twice at 9000 g for 10 minutes (4° C). A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 3 ml and stored at -80° C

- concentration or volume of S9 mix and S9 in the final culture medium :
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures.

Test concentrations with justification for top dose:

- In cytogenetic experiment I, test article concentrations within a range from 0.3 - 1000 µg/ml (without and with S9 mix) were applied for the evaluation of the potential to induce cytogenetic damage. In experiment II the applied concentration range was 1 - 1000 µg/ml (with and without S9 mix).

- The highest concentration was limited by the solubility of the test article in applicable solvents (aqua deionized, culture medium, acetone, ethanol or DMSO). Only concentrations of 1000 µg/ml in culture medium, pre-formulated in DMSO (100 mg/ml), could be applied as top concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- On the day of the experiment (immediately before treatment), the test article was dissolved in DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells. The final concentration of DMSO in the culture medium did not exceed 1 % (v/v)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

SPINDLE INHIBITOR (cytogenetic assays): Colcemid; 0.2 µg/ml; 2.5 h treatment

NUMBER OF CELLS EVALUATED: 100/culture

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 2
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x 10^4 - 6x10^4 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS (complete medium).

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: treatment intervals were 4 h with metabolic activation, 18 h and 28 h without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- STAIN (for cytogenetic assays): Giemsa (E. Merck, D-64293 Darmstadt).
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis
- Determination of polyploidy: Yes; the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- mitotic index determination

Evaluation criteria:

A test article is classified mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosome aberrations or a significant and reproducible positive response for at least one of the test points.
A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosome aberrations nor a significant and reproducibly positive response at any one of the test points is 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 should be 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:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Yes; 100 µg/ml and 1000 µg/ ml (fixation time 18 h; with and without metabolic activation)/ 1000 µg/ml (fixation time 28 h; with and without metabolic activation) for both experiments

RANGE-FINDING/SCREENING STUDIES (if applicable): The applicable concentration range of the test article for the cytogenetic experiments was determined in a pre-test using the detemiination of cell numbers 24 h after start of treatment as indicator for toxicity response. The top concentration of the test article (1000 µg/ml) was limited by the solubility of the test article in the solvents

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
Experiment 1: A slight reduction of the mitotic index was observed only in the absence of S9 mix at interval 18 h after treatment with concentrations exhibiting no precipitation (3 µg/ml: 68.9 %; 10 µg/ml 69.4 %).
Experiment 2: In experiment II the applied concentration range 1 - 1000 µg/ml (with and without S9 mix) did not reduce the mitotic index.

- Genotoxicity results (for both cell lines and lymphocytes)
o Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
o In both experiments, at both preparation intervals in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen : In both experiments, no biologically relevant increase in the rate of polyploid metaphases were found after treatment with the test article

HISTORICAL CONTROL DATA
- Range of historical control data: 0.00 % - 4.00 %.

Tab. 1: Cytotoxicity - with S9 mix

 

Concentration [µg/ml]	Number of cells	% of solvent control
solvent control	936	100.0
0.3	655	69.9
1.0	632	67.5
3.0	803	85.8
10.0	789	84.3
30.0	779	83.2
100.0	624	66.7
300.0	557	59.5
1000	601	64.2

 

Tab. 2: Cytotoxicity - without S9 mix

Concentration [µg/ml]	Number of cells	% of solvent control
solvent control	304	100
0.3	299	98.4
1.0	406	133.6
3.0	496	163.2
10.0	315	103.6
30.0	453	149.0
100.0	407	133.7
300.0	340	111.7
1000.0	447	147.0

Conclusions:

It can be stated that in the study described and under the experimental conditions reported, the test article did not induce reproducible structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, according to our evaluation criteria the test article is considered to be non-mutagenic in this chromosome aberration test.

Executive summary:

The test article, dissolved in DMSO, 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 metaphases 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 0.3 - 1000 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, 1000 µg/ml were applied as top concentration for the cytogenetic experiments.

In cytogenetic experiment I, test article concentrations within a range from 0.3 - 1000 µg/ml (without and with S9 mix) were applied for the evaluation of the potential to induce cytogenetic damage. In experiment II the applied concentration range was 1 - 1000 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 100 µg/ml and above. 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, in the absence and the presence of S9 mix, no biologically relevant increases in cells carrying structural chromosome aberrations were observed. In the presence of S9 mix, at both fixation intervals and both experiments, the test article did induce unreproduced statistically significant increases in cells carrying structural chromosome aberrations. These increases must be regarded as biologically not relevant. 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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the attached read-across-justification

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

mammalian cell line

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

An Ames test according to OECD guideline 471 was performed to investigate the potential of the test article to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II, experiment IIA, and experiment IIB) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in addition the Escherichia coli strains WP2 and WP2 uvrA. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicates. The test article was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate. Plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in all independent experiments. A slight toxic effect evidenced by a reduction in the number of revertants occurred at 2500 µg/plate in strain TA 98 without S9 mix in experiment II. No substantial increases in revertant colony numbers of any of the six tester strains were observed following treatment with the test substance at any dose level, neither in the presence nor in the absence of metabolic activation (S9 mix) in experiment I and II. There was also no relevant tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. In summary, treatment of different bacteria strains with the test substance in presence and absence of S9-mix did not induce increases in revertant colonies.

Chromosome aberration test:

The test substance, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in a GLP-compliant experiment according to OECD guideline 473. The chromosomes were prepared 18 h and 28 h after start of treatment with the test substance. The treatment intervals were 4 h with metabolic activation and 18 h as well as 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosome aberrations. The applicable concentration range of the test substance 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. The dose selection of the test substance was influenced by the solubility of the test substance concentrations from 0.3 – 1000 µg/mL. Since no clear toxicity was observed in the absence and the presence of the S9 mix, 1000 µg/mL of the test substance were applied as top concentration for the cytogenetic experiments. In cytogenetic experiment I, the test substance concentrations within a range from 0.3 – 1000 µg/mL (without and with S9 mix) were applied for the evaluation of the potential to induce cytogenetic damage. In experiment II the applied concentration range was 1 – 1000 µg/mL (with and without S9 mix). Examination of the cultures 4 h after treatment revealed precipitation of the target substance at concentrations of 100 µg/mL and above. 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, in the absence and the presence of S9 mix, no treatment related increases in cells carrying structural chromosome aberrations were observed. Moreover, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test substance as compared to the frequencies of the controls. In summary, the test substance did not induce reproducible structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

HPRT test:

There are no data on the mutagenic activity (HPRT test) of the test substance available. However, another structural analogue source substance was tested for this endpoint and the result can be transferred to the test substance in order to fill this data gap.

In detail, a GLP-compliant study according to OECD 476 was performed with another analogue substance. Based on preliminary toxicity test four concentrations were selected for the original mutagenicity experiment ranging from 18.52 to 500 µg/ml in the presence and absence of metabolic activation. 500 µg/ml was the highest attainable concentration due to the solubility limit inthe vehicle.The mutagenic activity was measured in the presence and absence of metabolic activation. Although the original experiment revealed statistically significant differences at some of the concentrations, both in the absence and in the presence of metabolic activation, the criteria for a positive response were not fullfilled. The increase was not concentration dependent in both parts and the number of mutant colonies differed from the respective number of the negative control by much less than the required 20. Furthermore, the effects were not reproducible. The positive controls induced a clear increase in mutant frequency. Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the substance and its metabolites did not show any mutagenic activity in this forward mutation system.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP) and in the in-vitro chromosome aberration assay (OECD 473, GLP). An analogue substance did not induce mutagenicity in the HPRT test in mammalian cells (OECD 476,GLP). The same result is assumed for the test substance.As a result, the test substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No 2018/1480.