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Administrative data

in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1996-09-13 to 1996-09-18
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study performed under GLP without deviations

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): Ro 02-2438
- Physical state: clear, yellow liquid
- Analytical purity: 91.2% (Isomer 3+4)
- Lot/batch No.: 05076
- Stability under test conditions: Data regarding stability under test conditions are not available. It is, however, to be expected that no gross degration is occuring when dissolved in the solvent for the test duration (< 6 hours).
- Storage condition of test material: Room temperature


Target gene:
Histidine for Salmonella
Species / strain
Species / strain / cell type:
other: TA1535, TA97, TA98, TA100, TA102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I: 5, 16.6, 50, 166, 500 µg/plate
Experiment II: 5, 16.6, 50, 166, 500 µg/plate
Experiment III: 0.5, 1.6, 5, 16.6, 50 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (Experiment I+II), K22294850 (experiment III)
- Justification for choice of solvent/vehicle: The compound was soluble in dimethylsulfoxide (DMSO).
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
other: Without S9: sodium azide (TA1535, TA100), ICR 191 (TA97), 2-nitrofluorene (TA98), MMC (TA102). With and without S9: 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
Experiment I: in agar (plate incorporation)
Experiment II+III: preincubation

- Preincubation period: 30 min (Experiment II)
- Exposure duration: 48 h


- Method: determination of the growth of the background lawn and/or frequency of spontaneous revertants
Evaluation criteria:
There is as yet no generally accepted statistical treatment of Ames test data. In most tests, the results are either clearly positive or clearly negative. 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. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation and 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. Since it is impossible to define criteria that would apply to every configuration of data generated by the mutation assay, the study director is responsible for the ultimate decision in the evaluation of the results.

Results and discussion

Test results
Species / strain:
other: TA1535, TA97, TA98, TA100, TA102
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Positive controls validity:
Additional information on results:
- Water solubility: The compound was soluble in dimethylsulfoxide (DMSO).
- Precipitation: Upon addition to the aqueous medium, the formation of a milky suspension was observed at 500 µg/plate in the standard plate incorporation assay. Using the preincubation method starting at 166 µg/plate a slight milky suspension with oily precipitation was observable.

RANGE-FINDING/SCREENING STUDIES: For determination of the top dose a preliminary toxicity experiment was performed using strain TA100. Concentrations from 1.6 to 5000 µg/plate were tested. Toxic effects (reduction in the background growth and reduction in the number of revertant colonies) were observed starting at 500 µg/plate.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY: Strain dependent toxicity was observed with both methods used. Using the preincubation modification, known to be more sensitive for several classes of compounds, toxicity was more 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, and TA102 without S9.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Any other information on results incl. tables

Spontaneous rates

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.

Applicant's summary and conclusion

Interpretation of results (migrated information):

Pseudoionone, nor any of the metabolites formed is mutagenic in the Ames test under the used experimental conditions.
Executive summary:

Pseudoionone was evaluated for mutagenic activity in the Ames test in accordance with OECD TG 471. 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 tester strains (TA1535, TA97, TA98, TA100, and TA102) were employed. The activity of the S9-mix and the responsiveness of the tester strains were verified by including appropriate controls into each experiment.

Pseudoionone was dissolved in dimethylsulfoxide (DMSO). Toxic effects were observed in a preliminary toxicity experiment, starting at 500 µg/plate (reduction in the number of revertants and reduced background growth). Therefore concentrations ranging from 5 to 500 µg/plate were evaluated in the main experiments. Upon addition to the aqueous medium, a milky suspension was observed at 500 µg/plate (standard assay) and a slight, milky suspension with oily precipitation starting at 166 µg/plate using the preincubation method. Strain dependent toxicity (reduction in the background growth and/or reduction in the number of revertant colonies) was observed in the selected concentration range. The toxic effects were more pronounced in absence of an exogenous metabolic activation system and in the experiment using the preincubation method, known to be more sensitive for several class of compounds. In order to provide data on a sufficient number of test concentrations, a repeat experiment was performed with strains TA1535, and TA102 in absence of S9 using the preincubation method (concentration range: 0.5 to 50 µg/plate).

Pseudoionone did not cause a mutagenic effect in any of the five investigated strains. Thus it can be concluded that neither Pseudoionone per se, nor any of its metabolites formed under the experimental conditions, induced genetic damage in the Ames test.