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Toxicological information

Genetic toxicity: in vitro

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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test was conducted according to OECD Test Guideline No. 471, 1997, under GLP Standards and QA.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 / strainopen allclose all
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 / 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 / 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 / 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 / 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/beta-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.
Controlsopen allclose all
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
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: - S-9; 0.4 µg/plate; TA102
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: - S-9; 1.0 µg/plate; TA1535 and TA100
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: - S-9; 0.5 µg/plate; TA98
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 resultsopen allclose all
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
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
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
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
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.