Octahydro-4,7-methano-1H-indenecarbaldehyde

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:

29 June, 2015 - 20 July, 2015

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:

2015

Report date:

2015

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:

Octahydro-4,7-methano-1H-indenecarbaldehyde

EC Number:

250-333-2

EC Name:

Octahydro-4,7-methano-1H-indenecarbaldehyde

Cas Number:

30772-79-3

Molecular formula:

C11H16O

IUPAC Name:

(1R,2S,3S,6R,7S)-tricyclo[5.2.1.0²,⁶]decane-3-carbaldehyde; bis((1R,2S,4R,6R,7S)-tricyclo[5.2.1.0²,⁶]decane-4-carbaldehyde)

Test material form:

other: liquid

Details on test material:

- Name of test material in report: Melozone (multi-constituent)
- Appearance: Clear colourless liquid
- Storage condition of test material: At room temperature container flushed with nitrogen

Method

Target gene:

- S. typhimurium: Histidine gene
- Escherichia 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 Aroclor 1254.

Test concentrations with justification for top dose:

Direct plate:
- Dose range finding test:
TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
- Experiment 1:
Due to cytotoxicity (in TA100), the following dose levels were used:
TA 1535, TA 1537 and TA 98 (without S9): 5.4, 17, 52, 164 and 512 μg/plate
TA 1535, TA 1537 and TA 98 (with S9): 5.4, 17, 52, 164, 512 and 1600 μg/plate

Preincubation:
- Dose range finding test:
TA 100 and WP2uvrA (without S9): 0.55, 1.7, 5.4, 17, 52, 164, 338 and 512 µg/plate
TA 100 and WP2uvrA (with S9): 1.7, 5.4, 17, 52, 164, 512, 1056 and 1600 µg/plate
- Experiment 1:
Due to cytotoxicity (in TA 100), the following dose levels were used:
TA 1535, TA 1537 and TA 98 (with and without S9): 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(100 µL/plate DMSO)

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:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: (independent repeat): preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

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

Evaluation criteria:

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent 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 follow up 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:

Toxicity was observed in all tester strains.

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
- Direct plate assay: In strain TA 100 toxicity was observed at the concentration of 512 µg/plate and above, both in the absence and presence of S9. In tester strain WP2uvrA, toxicity was observed at the concentration of 1600 µg/plate and above, both in the absence and presence of S9.
- Preincubation assay: In strain TA 100 toxicity was observed at the concentration of 164 µg/plate and above, in the presence of S9. In the absence of S9, toxicity was observed at the concentration of 52 µg/plate and above. In tester strain WP2uvrA, toxicity was observed at the concentration of 164 µg/plate and above, both in the absence and presence of S9.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Except the response of the positive control for TA1535 in the second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Direct plate assay: In all strains toxicity was observed at 512 µg/plate and at 1600 µg/plate, in the absence and presence of S9, respectively.
- Preincubation assay: In all strains toxicity was observed at the concentrations of 164 µg/plate, in the absence and presence of S9.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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

Executive summary:

The mutagenic activity of Melozone was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose range finding tests in strain TA 100 (direct plate -/+ S9 >= 512 µg/plate, preincubation +S9 >= 164 µg/plate, preincubation -S9 >=

52 µg/plate). In all strains toxicity was observed. 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 E. coli tester 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 Melozone is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.