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Toxicological information

Genetic toxicity: in vitro

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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1994
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA1535, TA97, TA98, TA100, and TA102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-naphthoflavone induced rat liver S-9 mix.
Test concentrations with justification for top dose:
Justification top dose: Slight toxic effects (weak reduction in the background growth) was observed at the top concentration of 5000 µg/plate in the preliminary test.

Concentrations main study:
Experiment 1: All strains (with and without metabolic activation) 0, 50, 166, 500, 1666, and 5000 µg/plate
Experiment 2: All strains (with and without metabolic activation) 0, 50, 166, 500, 1666, and 5000 µg/plate
Experiment 3: TA1535 / TA98 / TA100 / TA102 (without metabolic activation) 0, 0.5, 1.6, 5, 16.6, and 50 µg/plate and TA98 / TA100 (with metabolic acitvation) 0, 5, 16.6, 50, 166, and 500 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The compound was soluble in dimethylsulfoxide (DMSO).
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene (all strains; 4 µg/plate) / ICR191 (TA97; 1µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 in agar (plate incorporation) and experiment 2 and 3 preincubation.
- Cell density at seeding: 1.0 - 2.7 x 10^8 plated per plate.

DURATION
Plate incorporation (experiment 1):
- Exposure duration: 48 hours

Preincubation experiments (2 and 3):
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates per concentration and two plates for positive controls.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (reduction in background lawns)


Evaluation criteria:
The study director was responsible for the ultimate decision in the evaluation of the results.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA1535, TA98, TA97, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary test was performed using strain TA100 and 0, 1, 3, 10 , 33, 100, 333, 1000, 3333, and 5000 µg/plate test substance. Slight toxic effects (weak reduction in the background growth) was observed at the top concentration of 5000 µg/plate in the preliminary test.

HISTORICAL CONTROL DATA
The mutant frequencies of the controls were in the range of the historical controls and the data published in the literature (Maron and Ames, 1983; Levin et at., 1982a, 1982b). The positive controls induced significant increases in the mutant frequencies verifying the sensitivity of the strains used. For TA100 the spontaneous rates were at the low range. Nevertheless the experiments were considered acceptable, since the positive controls verified the responsiveness of the cultures used. Neither in the standard plate incorporation nor in the preincubation assay any increase in the mutant frequency was observed for the five tester strains after treatment with the test substance

ADDITIONAL INFORMATION ON CYTOTOXICITY:
For the main experiments the concentration range 50 to 5000 µg/plate, the generally recommended highest test concentration for non toxic compounds, was selected. Upon addition to the aqueous medium, increasingly milky suspensions were observed at 166 (standard assay) and 50 µg/plate preincubation assay). In addition an oily precipitate was noticed starting at 1666 and 500 µg/plate for the standard and preincubation assay, respectively. Using the standard plate incorporation assay no toxicity was observed. Using the preincubation modification, known to be more sensitive for several class of compounds, toxic effects were pronounced starting for some strains (-S9) at 50 µg/plate. Therefore a repeat experiment using the preincubation method was performed in the concentration range 0.5 to 50 µg/plate with strains TA1535, TA98, TA100, and TA102 without S9 and in the dose range 5 to 500 µg/plate with strains TA98, and TA100 with S9.
Remarks on result:
other: results for plate incorporation and preincubation method

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, Tetrahydrolinalyl acetate was found to be not mutagenic. Therefore, the substance does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

An Ames test was performed according to OECD 471 and in compliance with GLP. A standard plate incorporation and a preincubation modification assay in absence and in presence of metabolic activation system (S9) were performed. Five Salmonella typhimurium tester strains (TA1535, TA97, TA98, TA100, and TA102) were exposed to concentrations ranging from 50 – 5000 µg/plate. Experiments were performed in triplicate. The activity of the S9-mix and the responsiveness of the tester strains were verified by including appropriate controls into each experiment. It was concluded, that neither the test substance per se, nor any of its metabolites formed under the experimental conditions, induced genetic damage in the Ames test. Therefore Tetrahydrolinalyl acetate was concluded to be not mutagenic. Based on this result, the substance does not need to be classified for mutagenicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).