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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:
experimental study
Adequacy of study:
key study
Study period:
4.12.- 12.12.1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test was conducted according to OECD Test Guideline No. 471, 1997, and under GLP Standards (US-FDA, 1978, OECD, 1981), 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:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes
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): Tetrahydrolinalool
- Analytical purity: > 97%
- Physical state: liquid
- Storage condition of test material: protected from light, refrigerator

Method

Target gene:
His-gene: Amino acid histidine - GC base pairs, and AT base pairs (TA102)
Species / strain
Species / strain / cell type:
other: TA 1535, TA 97, TA98, TA 100, TA102
Additional strain / cell type characteristics:
not applicable
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:
Standard plate incorporation assay: 10, 33, 100, 333, and 1000 µg/plate
Preincubation assay: 10, 33, 100, 333, and 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
Untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: variable per strain
Remarks:
All strains (± S9): 2-Aminoanthracene; TA1535 and TA100 (-S9): sodium azide; TA98 (-S9): 2-nitrofluorene; TA102 (- S9): mitomycin C; TA97 (-S9): ICR 191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (standard plate incorporation assay); 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 for the test compound and negative control (2 plates for positive controls)

NUMBER OF CELLS EVALUATED: 100 µl of overnight culture (0.3 to 1.9 x 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. Each test substance dose, as well as the appropriate solvent control, was evaluated in duplicate in strain TA100 (± S-9) in the standard plate incorporation version of the assay.

OTHER EXAMINATIONS:
- Other: The solubility of the compound in the solvent, the occurrence of precipitation in the test tube after addition of the soft agar and on the agar plates after the incubation period are noted in the toxicity test with TA100. An examination of precipitation after addition of the phosphate buffer (as in the preincubation test) is included in the prescreen. In this case cells were not added to the mixture.
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
Species / strain:
other: S. typhimurium TA 1535, TA 97, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Strain dependent toxic effects in the selected concentration range
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The formation of a milky suspension was observed starting at 333 µg/plate using the preincubation modification, whereas using the
standard method no precipitation was observable.

RANGE-FINDING/SCREENING STUDIES: Milky suspensions were observed starting at 500 µg/plate. In addition a precipitate was formed using the preincubation method. Toxic effects (absence of revertant colonies) were noticed starting at 1666 µg/plate. Based on the observed toxicity the concentration range 10 to 1000 µg/plate was chosen for the main experiments.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY: Strain dependent toxic effects were observed in the selected concentration range with both methods used. Toxicity was more pronounced using the preincubation method, known to be more sensitive for several classes of compounds. Toxic effects in the standard plate incorporation appeared at 1000 ug/plate (reduced background growth), in the preincubation assay at 100, 333, and
1000 ug/plate (no reduced background growth, no revertant colonies).
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Not relevant

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that neither Tetrahydrolinalool per se, nor any of the metabolites formed under the experimental conditions is mutagenic in the Ames test.
Executive summary:

Tetrahydrolinalool was evaluated for mutagenic activity in the Ames test. A standard plate incorporation and a preincubation modification assay in absence and in presence of an exogenous metabolic activation system (S9) were performed. 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.

Tetrahydrolinalool was dissolved in dimethylsulfoxide (DMSO). Toxic effects were observed in a preliminary toxicity experiment starting at 1666 ug/plate. Upon addition to the aqueous medium increasingly milky suspensions were observed starting at 500 ug/plate. In the preincubation assay in addition an oily precipitation was observed. Based on the observed toxicity the concentration range 10 to 1000 ug/plate was chosen for the main experiments. No precipitation of the compound was apparent using the standard assay, whereas increasingly a milky suspensions was obtained starting at 333 ug/plate by using the preincubation modification. Strain dependent toxic effects were noticed in the evaluated concentration range. Toxicity evident by reduced background growth and / or absence of revertant colonies was more pronounced with the preincubation modification, known to be more sensitive for several classes of compounds.

No significant increase in the number of revertant colonies was apparent in all five test strains (TA1535, TA97, TA98, TA100, and TA102) using both methods, after treatment with Tetrahydrolinalool.

Based on the data presented here it is concluded that neither Tetrahydrolinalool per se, nor any of the formed metabolites is mutagenic in the Ames test under the described experimental conditions.