reaction mass of isomers of: C7-9-alkyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 20th, 1989 to October 20th, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with international test guidelines (Japan/EU) and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

with the exception of statistical analysis

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

with the exception of statistical analysis

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

yes

Remarks:

with the exception of statistical analysis

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

yes

Remarks:

with the exception of statistical analysis

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

406-040-9

EC Name:

-

Cas Number:

125643-61-0

Molecular formula:

C24-26 H40-44 O3

IUPAC Name:

C7-9-(branched)-alkyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate

Details on test material:

Appearance: Yellow liquid

Method

Target gene:

Histidine (his- to his+) and tryptophan (tryp- to tryp+) genes in Salmonella typhimurium and Escherichia coli respectively

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from rats induced with Aroclor 1254 and cofactors.

Test concentrations with justification for top dose:

313, 625, 1250, 2500 and 5000 µg/0.1 mLl (with and without microsomal activation)

Vehicle / solvent:

Acetone (p.a., Merck, Darmstadt, Germany)

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

see vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

Acetone alone was used for the negative vehicle control

True negative controls:

no

Controls 2

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

other: Strain TA 98: 2-nitrofluorene, 10 µg/0.1 mL ; Strain TA 100 and TA 1535: sodium azide 2 µg/0.1 mL ; Strain TA 1537: (5)-aminoacridine hydrochloride monohydrate, 150 µg/0.1 mL; Strain E.coli WP2uvrA: 4-nitroquinoline-N-oxide 1 µg/0.1 mL.

Remarks:

2-nitrofluorene, (5)-aminoacridine hydrochloride monohydrate and 4-nitroquinoline-N-oxide were dissolved in DMSO; sodium azide was dissolved in bidistilled water.

Controls 3

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

other: Strain TA 98 and TA 100: 2-aminoanthracene, 2.5 µg/0.1 mL; Strain TA 1535: cyclophosphamide monohydrate, 400 µg/0.1 mL bidistilled water; Strain TA 1537: 2-aminoanthracene, 5 µg/0.1 mL ; Strain E.coli WP2uvrA: 2- aminoanthracene, 50 µg/0.1 mL.

Remarks:

2-aminoanthracene was dissolved in DMSO; cyclophosphamide monohydrate was dissolved in bidistilled water.

Details on test system and experimental conditions:

The tests are carried out in accordance with the method described by Ames et al. (Ames BN et al. (1973) Proc. Natl. Acad. Sci. USA 70, 782-786; Ames BN et al. (1973) Proc. Natl. Acad. Sci. USA 20, 2281-2285; Ames BN et al. (1975) Mutation Res. 31, 347-364).
The test concentrations were selected on the basis of a preliminary toxicity test which had been carried out with concentrations ranging from 20 to 5000 µg/0.1 mL; accordingly, the concentration of 5000 µg/0.1 mL was retained as the highest in the mutagenicity test and the remaining test concentrations were set at 313, 625, 1250 and 2500 µg/mL.
Each Petri dish contained: 1) approx. 20 mL of minimum agar, 2) 0.1 mL of a solution of the test substance or the vehicle and 0.1 mL of a bacterial culture (in 2.5% nutrient broth) in 2.0 mL of soft agar. For the Salmonella strains the soft agar was supplemented with histidine; for E. coli, the soft agar was supplemented with tryptophan.
In the experiments without microsomal activation 0.5 mL of sodium phosphate buffer was added. In the experiments in which the substance is metabolically activated, the buffer is replaced by 0.5 ml of an activation mixture (S9 mix), which, as already indicated, consisted of the S9 fraction of liver from Sprague-Dawley rats induced with Aroclor 1254 and a solution of co-factors.
In the experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). The plates were incubated for about 48 hours at 37 +/-0.5 °C in darkness. At test ending, the number of counted back-mutant colonies in the cultures treated with the various concentrations was compared with controls.

Evaluation criteria:

The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the tester strains TA 98, TA 1535, TA 1537 and E. coli WP2uvrA;
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100. Generally a concentration-related effect should be demonstrable.

Statistics:

The data reported include individual numbers of revertants per plate, mean values and standard deviation for each bacterial strain and each concentration.

Results and discussion

Test results

Species / strain:

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

Remarks:

(at the highest test concentration of 5000 µg/0.1 mL the substance precipitated in soft agar)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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 revealed no marked deviations. No evidence of the induction of point mutations by the substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and Escherichia coli used in these experiments.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

SUMMARY OF RESULTS

Experiment I

	TA 1535		TA 100		TA 1537		TA 98		WP2 uvra
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Control	9	7	137	139	4	7	14	27	15	19
313	9	6	125	133	4	7	17	22	15	16
625	12	9	139	128	6	5	21	35	18	20
1250	8	5	101	120	3	9	19	33	19	19
2500	9	5	112	118	4	10	16	34	19	18
5000	7	6	108	123	3	7	19	28	16	16
positive control	510	234	580	646	446	123	1107	1584	240	480

Experiment II

	TA 1535		TA 100		TA 1537		TA 98		WP2 uvra
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Control	11	18	126	151	6	16	18	30	27	24
313	11	16	109	162	5	20	16	37	23	25
625	13	14	118	151	8	22	20	35	26	22
1250	11	10	127	159	6	16	21	36	34	24
2500	15	14	137	145	6	13	25	36	22	25
5000	7	13	136	168	6	13	20	24	24	23
positive control	201	352	737	944	1198	311	963	1430	397	558

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

No evidence of the induction of point mutations by the test item or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and Escherichia coli used in these experiments.

Executive summary:

The test substance was tested for mutagenic effects on histidine-auxotrophicmutants of Salmonella typhimurium (strain TA 1535, TA 1537, TA 98 and TA 100) and on a tryptophan-auxotrophic strain of E. coli (WP2 uvrA) according to the OECD TG 471 (1983). The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 313, 625, 1250, 2500 and 5000 µg/0.1 mL. In order to confirm the results, the experiments were repeated.

For all tested concentrations, without and with microsomal activation,comparison of the number of back-mutant colonies in the controls and the treated bacteria cultures revealed no marked deviations. No evidence of the induction of point mutations by the substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium and Escherichia coli used in these experiments. No cytotoxicity was observed up to the highest tested concentration of 5000 µg/0.1 mL, which precipitated in soft agar.