Thiodiethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

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:

1984-07-17 to 1984-07-27

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

fifth strain is missing in order to detect cross-linking mutagens

GLP compliance:

no

Remarks:

but QAU statement included

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Thiodiethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

EC Number:

255-392-8

EC Name:

Thiodiethylene bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

Cas Number:

41484-35-9

Molecular formula:

C38H58O6S

IUPAC Name:

2-[(2-{[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoyl]oxy}ethyl)sulfanyl]ethyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate

Details on test material:

- Physical state: solid
- Analytical purity: > 99.5% (CIBA-GEIGY Ltd, Analyse Number: 5659, 25.10.1984)

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

20, 80, 320, 1280, and 5120 µg/0.1 mL with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see in "Any other informations on materials and methods"

Details on test system and experimental conditions:

METHOD OF APPLICATION: agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

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

Statistics:

Arithmetic mean values and standard deviation were calculated.

Results and discussion

Test results

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 320 µg/0.1 ml and above the substance precipitated in soft agar.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was carried out with concentrations ranging from 0.1 to 5000 µg/0.1 ml. Thereupon, the concentration of 5120 µg/0.1 ml was used as the highest in the mutagenicity test.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Plate incorportation assay - First experiment:

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		Frameshift type
		TA 100	TA1535	TA98	TA1537
–	0	121 ± 10	14 ± 10	32 ± 10	6 ± 1
–	20	103 ± 18	8 ± 3	32 ± 6	7 ± 1
–	80	128 ± 4	19 ± 2	23 ± 3	7 ± 3
–	320	118 ± 28	13 ± 5	24 ± 4	8 ± 5
–	1280	128 ± 23	11 ± 5	19 ± 2	6 ± 2
–	5120	107 ± 8	17 ± 1	17 ± 8	7 ± 2
Positive controls, –S9	Name	4NQO	SA	DR	9AA
	Concentrations (μg/plate)	0.125	2.5	5	50
		0.25	5	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	851 ± 81	1426 ± 105	366 ± 82	100 ± 22
		1456 ± 141	1965 ± 181	841 ± 79	1531 ± 801
+	0	121 ± 6	11 ± 6	45 ± 3	17 ± 3
+	50	108 ± 10	11 ± 6	54 ± 10	16 ± 2
+	150	116 ± 6	13 ± 4	50 ± 6	18 ± 4
+	500	94 ± 9	14 ± 4	45 ± 3	15 ± 5
+	1500	101 ± 10	14 ± 7	53 ± 1	17 ± 3
+	5000	89 ± 13	13 ± 7	47 ± 5	17 ± 2
Positive controls, –S9	Name		CP
	Concentrations (μg/plate)		250
	Mean No. of colonies/plate (average of 3 ± SD)		547 ± 103

4NQO: 4-nitroquinoline-N-oxide

SA: sodium azide

DR: daunorubicin

9AA: 9(5)aminoacridine hydrochloride monohydrate

CP: cyclophosphamide

Table 2: Plate incorportation assay - Second experiment

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		Frameshift type
		TA 100	TA1535	TA98	TA1537
–	0	139 ± 10	12 ± 4	27 ± 6	5 ± 3
–	20	120 ± 23	16 ± 6	23 ± 5	10 ± 6
–	80	148 ± 19	14 ± 6	27 ± 6	9 ± 4
–	320	136 ± 14	15 ± 4	26 ± 7	12 ± 5
–	1280	126 ± 10	12 ± 7	23 ± 3	9 ± 4
–	5120	127 ± 9	12 ± 3	23 ± 2	11 ± 4
Positive controls, –S9	Name	4NQO	SA	DR	9AA
	Concentrations (μg/plate)	0.125	2.5	5	50
		0.25	5	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	992 ± 26	865 ± 65	806 ± 69	66 ± 21
		1617 ± 139	1343 ± 61	1186 ± 188	973 ± 349
+	0	127 ± 17	15 ± 1	47 ± 9	10 ± 4
+	50	130 ± 10	13 ± 7	39 ± 4	10 ± 6
+	150	123 ± 24	17 ± 2	43 ± 5	13 ± 5
+	500	129 ± 9	12 ± 4	46 ± 15	9 ± 4
+	1500	114 ± 5	15 ± 2	33 ± 6	13 ± 5
+	5000	118 ± 20	12 ± 4	35 ± 8	6 ± 3
Positive controls, –S9	Name		CP
	Concentrations (μg/plate)		250
	Mean No. of colonies/plate (average of 3 ± SD)		369 ± 48

4NQO: 4-nitroquinoline-N-oxide

SA: sodium azide

DR: daunorubicin

9AA: 9(5)aminoacridine hydrochloride monohydrate

CP: cyclophosphamide

The slight increase in the number of back-mutant colonies observed in the second experiment without microsomal activation on strain TA 1537 at the concentrations of 20, 320 and 5120 µg/0.1 mL is attributed to fluctuations in the rate of spontaneously occurring back-mutants.

Applicant's summary and conclusion

Conclusions:

No evidence of the induction of point mutations by the test article or by the metabolites of the substance was detectable in the strains of S. typhimurium used in these experiments.

Executive summary:

In an Ames test similar to OECD guideline 471, the test substance was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium, using the plate incorporation method. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 80, 320, 1280 and 5120 µg/0.1 mL. In order to confirm the results the experiments were repeated. In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of the test substance revealed no marked deviations. No evidence of the induction of point mutations by the test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.