Tocopherols

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

18 November 1998 - 27 November 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test was conducted according to OECD Test Guideline No. 471, 1997, under GLP Standards and QA, please refer to IUCLID section 13 for read across justification.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

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

IUPAC Name:

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

Details on test material:

- 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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: rfa, ¿uvrB

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: rfa, ¿uvrB; pKM101

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: rfa, ¿uvrB; pKM101

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: rfa, ¿uvrB; pKM101

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

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:

Migrated to IUCLID6: + S-9; 1.0 µg/plate

Controls 2

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: - S-9; 0.4 µg/plate; TA102

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: - S-9; 1.0 µg/plate; TA1535 and TA100

Controls 4

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: - 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

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

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

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

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

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No remarks

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

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). Five Salmonella typhimurium test 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-acetate was 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 with all - rac a tocopheryl-acetate.

Thus it can be concluded that neither all - rac a tocopheryl-acetate per se, nor any of the metabolites formed is mutagenic in the Ames test under the described experimental conditions.