Reaction mass of 3-[(3aS,7aR)-octahydro-1H-4,7-methanoinden-1-yl]propanal and 3-[(2s,3aR,7aS)-octahydro-1H-4,7-methanoinden-2-yl]propanal and 3-[(2r,3aR,7aS)-octahydro-1H-4,7-methanoinden-2-yl]propanal

Genetic toxicity: in vitro

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:

06 January, 2014 - 10 February, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study is conducted according to OECD TG 471, in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 3-[(3aS,7aR)-octahydro-1H-4,7-methanoinden-1-yl]propanal and 3-[(2s,3aR,7aS)-octahydro-1H-4,7-methanoinden-2-yl]propanal and 3-[(2r,3aR,7aS)-octahydro-1H-4,7-methanoinden-2-yl]propanal

EC Number:

942-467-1

Molecular formula:

C13H20O

IUPAC Name:

Reaction mass of 3-[(3aS,7aR)-octahydro-1H-4,7-methanoinden-1-yl]propanal and 3-[(2s,3aR,7aS)-octahydro-1H-4,7-methanoinden-2-yl]propanal and 3-[(2r,3aR,7aS)-octahydro-1H-4,7-methanoinden-2-yl]propanal

Test material form:

other: liquid

Details on test material:

- Name of test material in report: FRET 09-0536
- Physical state: Clear colourless liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

- Dose range finding test:
TA 100 and E.coli WP2uvrA (without and with S9): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate

- Experiment 1:
Due to cytotoxicity, the following dose levels were used:
TA 1535, TA1537 and TA98 (without S9): 0.3, 1, 3.33, 10, 33 and 100 µg/plate
TA 1535, TA1537 and TA98 (with S9): 3, 10, 33, 100, 333 and 1000 µg/plate

- Experiment 2:
The following dose levels were used:
TA 1535, TA1537, TA98 and TA100 (without S9): 0.3, 1, 3, 10 and 100 µg/plate
TA 1535, TA1537, TA98 and TA100 (with S9): 3, 10, 33, 100, 333 and 1000 µg/plate
E.coli WP2uvrA (without S9): 3, 10, 33, 100, 333 and 1000 µg/plate
E.coli WP2uvrA (with S9): 10, 33, 100, 333, 1000 and 3330 µg/plate

- Experiment 3:
Since in tester strain WP2uvrA in the second experiment no dose level with toxicity or precipitate on the plates was observed in the absence of S9-mix, a third mutation experiment was performed with this strain in the absence of S9-mix:
E.coli WP2uvrA (without S9): 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: Ethanol
- Justification for choice of solvent: the test substance was found to be soluble in ethanol up to 5000 µg/plate

Controls

Negative solvent / vehicle controls:

yes

Remarks:

(100 µL/plate Ethanol)

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all 4 experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of FRET 09-0536 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 μg/plate and upwards in tester strains TA100 and WP2uvrA (dose range finding test). In tester strains TA1535, TA1537 and TA98 precipitation of FRET 09-0536 on the plates was only observed at the start of the incubation period at the concentration of 1000 μg/plate (first mutation experiment).
Precipitation of FRET 09-0536 on the plates was observed at the start of the incubation period at the concentration of 1000 μg/plate and upwards. Precipitation of FRET 09-0536 on the plates at the end of the incubation period was observed in tester strains TA100 and WP2uvrA at concentrations of 1000 μg/plate and upwards in the presence of S9-mix (experiment 2). Precipitation of FRET 09-0536 on the plates was observed in tester strain WP2uvrAat the start of the incubation period at concentrations of 1000 μg/plate and upwards and at 3330 and 5000 μg/plate at the end of the incubation period (experiment 3).

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 33 μg/plate and upwards in the absence of S9-mix and 333 μg/plate and upwards in the presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 1000 μg/plate and upwards in the absence of S9-mix and 3330 and 5000 μg/plate in the presence of S9-mix. Results of this dose range finding test were reported as part of the first mutation assay.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative control values were within the laboratory historical control data ranges, except for TA1535 in the absence of S9-mix (first experiment). However, since this value was just outside the limit of the range, the validity of the test was considered to be not affected. The strain-specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the Salmonella strains toxicity was observed at the higher doses (at >= 33 µg/plate), in the absence of S9-mix and at higher doses (at >= 100 µg/plate), in the presence of S9-mix.
- In tester strain WP2uvrA in the second experiment no toxicity was observed in the absence and presence of S9-mix. In the third mutation experiment an extreme reduction of the revertant colonies was observed in strain WP2uvrA at test substance concentrations of 3330 and 5000 μg/plate in the absence of S9-mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay performed according to OECD 471 (1997) and GLP principles.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in all strains. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in strain WP2uvrA , both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.