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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

419-740-4

EC Name:

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

Cas Number:

137658-79-8

Molecular formula:

C36 H45 N3 O4

IUPAC Name:

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

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

Method

Target gene:

his, trp

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2

Species / strain 3

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.