3,7-dimethylocta-2,6-dienenitrile

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:

The experimental phase of this study was performed between 27 August 2002 and 3 October 2002.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Geranitrile T

IUPAC Name:

Geranitrile T

Constituent 2

Reference substance name:

3,7-dimethylocta-2,6-dienenitrile

EC Number:

225-918-0

EC Name:

3,7-dimethylocta-2,6-dienenitrile

Cas Number:

5146-66-7

Molecular formula:

C10H15N

IUPAC Name:

3,7-dimethylocta-2,6-dienenitrile

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

Sponsor's identification: Geranitrile T
Description: Colourless to pale yellow liquid
Batch number: 9000478447
Purity: 97.6 %
Stability in vehicle: Dimethyl sulfoxide: Not indicated
Expiry date: 21 July 2003
Storage conditions: At room temperature in the dark
Stability under storage conditions: Stable

Method

Target gene:

Histidine for Salmonella typhimurium

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254 induced rat liver, S9-mix

Test concentrations with justification for top dose:

Preliminary Toxicity Test:
Dose range finding test 1: 3,10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Dose range finding test 2: 3, 10, 33, 100, 333, 1000,3330 and 5000 µg/plate

Main Test:
Based on the results of the dose range finding study the following dose range was selected for the mutation assay:

Experiment 1: 10, 33, 100, 333, 1000 and 2000 µg/plate.

Experiment 2: 1000, 2500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
dimethyl sulfoxide

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide: 5 µg/plate

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine: 60 µg/plate

Remarks:

without S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin: 4 µg/plate

Remarks:

without S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO = dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate: 650 µg/plate

Remarks:

Without S9 mix

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO = dimethyl sulfoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO = dimethyl sulfoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2.5 µg/plate

Remarks:

With S9 mix

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO = dimethyl sulfoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO = dimethyl sulfoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA102

Negative solvent / vehicle controls:

yes

Remarks:

DMSO = dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8 dihydroxy anthraquinone: 30 µg/plate

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 30 minutes
- Exposure duration: 48hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

DATA EVALUATION AND STATISTICAL PROCEDURES

No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:

a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.

b) The negative response should be reproducible in at least one repeated experiment.

A test substance is considered positive (mutagenic) in the test if:

a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation.

However, any mean plate count of less than 20 is considered to be not significant.

b) The positive response should be reproducible in at least one repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Standard deviation

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose range finding test 1
GERANITRILE T was tested in tester strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix in the direct plate assay.

Precipitate:
The test substance precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of GERANITRILE T on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate. No precipitate on the plates was observed at the end of the incubation period.

Toxicity:
To determine the toxicity of GERANITRILE T, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

A slight reduction of the bacterial background lawn and an extreme decrease in the number of revertants was observed at the test substance concentration of 1000 µg/plate. An extreme reduction of the bacterial background lawn and an increase in the size of the microcolonies was observed at 3330 µg/plate and 5000 µg/plate.

Mutagenicity
No increase in the number of revertants was observed upon treatment with GERANITRILE T under all conditions tested.

Dose range finding test 2
GERANITRILE T was tested in tester strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix in the preincubation assay.

Precipitate:
The test substance precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of GERANITRILE T on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate. No precipitate on the plates was observed at the end of the incubation period.

Toxicity:
An extreme reduction of the bacterial background lawn and an increase in the size of the microcolonies was observed at test substance concentrations of 333 and 1000 µg/plate. A complete lack of any microcolony background lawn was observed at test substance concentrations of 3330 and 5000 µg/plate.

Mutagenicity
No increase in the number of revertants was observed upon treatment with GERANITRILE T under all conditions tested.

MUTATION ASSAY
Direct plate assay 1
Based on the results of the first dose range finding test the following dose range was selected for the mutation assay with the tester strains TA1535, TA1537, TA98 and TA102: 10, 33, 100, 333, 1000 and 2000 µg/plate.

Precipitate:
GERANITRILE T precipitated in the top agar at concentrations of 1000 and 2000 µg/plate. Precipitation of GERANITRILE T on the plates was observed at the start of the incubation period at the concentration of 2000 µg/plate. No precipitate on the plates was observed at the end of the incubation period.

Toxicity
No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed in the tester strains TA1535 and TA1537.

Mutagenicity
No increase in the number of revertants was observed upon treatment with GERANITRILE T under all conditions tested.

Direct plate assay 2
Since not enough toxicity was observed in the tester strains TA98 and TA102 (absence and presence of S9-mix) in the direct plate assay, a second mutation experiment was performed. The following dose range was selected for this mutation experiment: 1000, 2500 and 5000 µg/plate.

Precipitate:
GERANITRILE T precipitated in the top agar at all dose levels. Precipitation of GERANITRILE T on the plates was observed at the start of the incubation period at concentrations of 2500 and 5000 µg/plate. No precipitqte on the plates was observed at the end of the incubation period.

Mutagenicity
No increase in the number of revertants was observed upon treatment with GERANITRILE T under all conditions tested.

PRE-INCUBATION ASSAY
To obtain more information about the mutagenicity of GERANITRILE T, a preincubation assay was performed with the strains TA1535, TA1537, TA98 and TA102. Based on the results of the second dose range finding study the following dose range was selected for the mutation assay: 1, 3, 10, 33, 100 and 333 µg/plate.

Precipitate:
GERANITRILE T did not precipitate in the top agar. Precipitation of GERANITRILE T on the plates was not observed at the start or at the end of the incubation period.

Mutagenicity:
No increase in the number of revertants was observed upon treatment with GERANITRILE T under all conditions tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that GERANITRILE T is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains resulting in histidine-independent strains.

The study procedures described in this report were based on the following guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (adopted July 21, 1997).

- European Economic Community (EEC). Adapting to technical progress for the twenty-sixth time Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.13/14: "Mutagenicity: "Reverse Mutation Assay using bacteria". EEC Publication Commission Directive (Brussels May 19, 2000).

GERANITRILE T was tested in the Salmonella typhimurium reverse mutation assay with five histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102). The test was performed in two separate experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). To obtain more information about the mutagenicity of GERANITRILE T, an additional experiment was performed with the strains TA98 and TA102 in the absence and presence of S9 -mix.

In the direct plate assay, at first GERANITRILE T was tested in a dose range finding study up to concentrations of 5000 µg/plate in strain TA100. GERANITRILE T did not precipitate on the plates at this dose level. Toxicity was observed at dose levels of 1000 µg/plate and upwards.

Secondly, GERANITRILE T was tested up to concentrations of 2000 µg/plate in the strains TA1535, TA1537, TA98 and TA102. Toxicity was observed in the tester strains TA1535 and TA1537 in the absence and presence of S9-mix and no toxicity was observed in the tester strains TA98 and TA102.

In an additional direct plate assay, GERANITRILE T was tested up to concentrations of 5000 µg/plate in the tester strains TA98 and TA102. Toxicity was observed in both tester strains.

In the preincubation assay, at first GERANITRILE T was tested in a dose range finding study in the strain TA100. GERANITRILE T was tested up to concentrations of 5000 µg/plate. Toxicity was observed at dose levels of 333

µg/plate and upwards.

Subsequently, GERANITRILE T was tested up to concentrations of 333 µg/plate in the strains TA1535, TA1537, TA98 and TA102. Toxicity was observed in all tester strains.

GERANITRILE T did not induce a dose-related increase in the number of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA98, TA100 and TA102) both in the absence and presence of S9-metabolic activation. These results were confirmed in separate experiments.

Based on the results of this study it is concluded that GERANITRILE T is not mutagenic in the Salmonella typhimurium reverse mutation assay.