3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl nicotinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

other: published parts of study report

Title:

Unnamed

Year:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Remarks:

Statement of Compliance

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-benzopyran-6-yl acetate

EC Number:

231-710-0

EC Name:

3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-benzopyran-6-yl acetate

Cas Number:

7695-91-2

Molecular formula:

C31H52O3

IUPAC Name:

2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-dihydro-2H-1-benzopyran-6-yl acetate

Test material form:

liquid: viscous

Specific details on test material used for the study:

- Name of test material (as cited in study report): all - rac a tocopheryl-acetate
- Physical state: viscous liquid
- Stability under test conditions:
Retest date: 05.11.99; Detailed data regarding stability during storage and under test conditions are not available. It is, however, to be expected
that no gross degradation is occurring under the specified storage conditions for a period of a few months or when dissolved in the solvent for the test duration (= 6 hours).
- Storage condition of test material: Refrigerated, protected from light, under inert gas (nitrogen), protected against moisture

Method

Target gene:

His-gene: Amino acid histidine - GC base pairs, and AT base pairs (TA102)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: rfa, uvrB

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

other: rfa, uvrB; pKM101

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: rfa, uvrB; pKM101

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: rfa, uvrB; pKM101

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: rfa; pKM101, pAQ1

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 (from phenobarbital/ß-naphthoflavone treated male albino rats)

Test concentrations with justification for top dose:

0, 50, 158, 500, 1580 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: ethanol
- Justification for choice of solvent: The test compound was soluble in ethanol.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

+ S-9; 1.0 µg/plate

Controls 2

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

- S-9; 0.4 µg/plate; TA102

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

- S-9; 1.0 µg/plate; TA1535 and TA100

Controls 4

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

- S-9; 0.5 µg/plate; TA98

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

± S-9; all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation modification assay

DURATION
- Preincubation period: 30 minutes
- Selection time (if incubation with a selection agent): 2 days

SELECTION AGENT (mutation assays): overlay agar containing Histidine

NUMBER OF REPLICATIONS: 3 plates per concentration (2 plates for positive controls)

NUMBER OF CELLS EVALUATED per culture: 100 µl of overnight culture (ca 10*8 cells) plated per plate

DETERMINATION OF CYTOTOXICITY
- Method: in a preliminary toxicity assay determination of reduction in the revertant colony number and/or observation of thinning or absence of the background lawn.

OTHER EXAMINATIONS:
- Other: A range finder assay with strain TA100 (standard plate incorporation version, ± S-9) was performed.

Evaluation criteria:

A positive result is defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA1535 and TA98. For strains TA97, TA100 and TA102 a 1.5 - fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Biological relevance should always be taken into account. A negative result is defined as the absence of a reproducible increase in the number of his+ revertant colonies.

Statistics:

Mean values and standard deviation (SD).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weak reduction of background growth at = 500 µg/plate (preincubation test, - S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium 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 applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: The mutant frequencies of the controls were in the range of the historical control values.

Applicant's summary and conclusion

Conclusions:

The test compound did not induce any relevant increase of the number of revertant colonies/plate in any of the five tester strains.
Thus it can be concluded that all - rac a tocopheryl-acetate is not mutagenic in the Ames test under the described experimental conditions.

Executive summary:

all - rac a tocopheryl-acetate was evaluated for mutagenic activity in the Ames test. A standard plate incorporation and a preincubation modification assay were performed in absence and in presence of an exogenous metabolic activation system (S9). FiveSalmonella typhimuriumtest strains (TA1535, TA97, TA98, TA100, and TA102) were employed. The activity of the S9-mix and the responsiveness of the test strains were verified by including appropriate controls into each experiment.

all - rac a tocopheryl-acetatewas dissolved in ethanol. Upon addition of aliquots to the aqueous medium formation of milky suspensions was apparent already at concentrations (=50 µg/plate) and precipitation in the form of droplets occurred at=500 µg/plate. Since toxic effects were generally not observed with the exception of a weak reduction of background growth in strain TA98 (preincubation test, - S9) the concentration range 50 to 5000 µg/plate, the generally recommended highest test concentration for non toxic compounds, could be evaluated.

No increase in the number of revertant colonies was apparent for any of the five test strains after treatment withall - rac a tocopheryl-acetate.

Thus it can be concluded that neitherall - rac a tocopheryl-acetateper se, nor any of the metabolites formed is mutagenic in the Ames test under the described experimental conditions.