(3Z)-hex-3-en-1-yl 2-methylprop-2-en-1-yl ether

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test is available for Vivaldie. The test substance was tested in two independent experiments, with and without a metabolic activation system. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535 , TA 1537, TA 98, TA 100 and TA 102 and one strain of Escherichia coli: WP2 uvrA were used. Each strain was exposed to five dose-levels of the test substance ( three plates/dose-level). Experiments without S9 mix: 31.25 to 2500 µg/plate. A slight to marked toxicity was noted in all tester strains, generally at dose-levels ≥ 125 µg/plate. Experiments with S9 mix: 31.25 to 5000 µg/plate. A slight to marked toxicity was noted, depending on the tester strain.The test substance did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the six strains. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Under the experimental conditions of this test, the test substance Vivaldie does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

Link to relevant study records

Reference

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:

between 19 October 2000 and 29 November 2000.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with OECD guidelines and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes

Remarks:

No certificate in report

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

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

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Test Concentrations:
Preliminary Toxicity Test: 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Experiments without S9 mix:
The selected treatment-levels were as follows:
- 31.25, 62.5, 125,250 and 500 µg/plate: for the TA 1535, TA 100 and TA 102 strains in both experiments as well as for the TA 1537 strain in the second experiment,
- 78.125,156.25,312.5,625 and 1250 µg/plate: for the TA 98 strain in the second experiment,
- 156.25, 312.5, 625, 1250 and 2500 µg/plate: for the WP2 uvrA strain in both experiments as well as for the TA 1537 and TA 98 strains in the first experiment.

Experiments with S9 mix:
The selected treatment-levels were as follows:
- 31.25,62.5, 125,250 and 500 I!g/plate: for the TA 1535, TA 100 and TA 102 strains in both experiments as well as for the TA 1537 strain in the second experiment,
- 312.5,625, 1250,2500 and 5000 I!g/plate: for the remaining tester strains in both experiments as well as for the TA 1537 strain in the first experiment.

Vehicle / solvent:

Vehicle: The vehicle was dimethylsulfoxide (DMSO), batch No.K27073650003 (Merck CJevenot, 77500 Chelles, France).
Justification for choice of solvent/vehicle:
The test substance was freely soluble in the vehicle at 50 mg/ml.
Formulation procedure
The test substance was dissolved in the vehicle at a concentration: of 50 mg/ml for the preliminary toxicity test and the first mutagenicity experiment, of 50 and 5 mg/ml for the second mutagenicity experiment. The preparations were made immediately before use.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA and TA102

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2 nitrofluorine 0.5 µg/plate

Remarks:

without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine 50µg/plate

Remarks:

without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100 and TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide 1 µg/plate

Remarks:

without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA102

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: mitomycin C 0.5 µg/plate

Remarks:

Without S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide 2µg/plate

Remarks:

Without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) - Experiment 1

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- 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.
METHODS OF APPLICATION: in agar (pre-incubation) – Experiment 2
- Pre-incubation period for bacterial strains: 10hrs
- Exposure duration: 48-72hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (in incubation with a selective agent): 60 minutes at 37 degrees C

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.

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:

Acceptance criteria
This study is considered valid if the following criteria are fully met: the number of revertants in the vehicle controls is consistent with historical data. the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.
Evaluation Criteria
The following evaluation criteria were used:
A reproducible 2-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship is considered as a positive result. Reference to historical data, or other considerations of biological relevance may be taken into account in the evaluation of the data obtained. Results are considered generally as clearly negative in the absence of a greater than 1.5 times increase versus the solvent or vehicle control. In the case that clear negative or clear positive results are not obtained at the end of the two mutagenicity experiments, the following criteria are followed:
Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S9 fraction in the S9 mix. Should an increase in revertant colony numbers be observed which satisfies the above mentioned paragraph, the test substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
If no clear "positive" response is obtained the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually analysis of variance followed by Dunnett's test.

Statistics:

Standard deviation

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A slight to marked toxicity was noted, depending on the tester strain, the dose-levels and the experimental conditions.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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:

cytotoxicity

Remarks:

A slight to marked toxicity was noted, depending on the tester strain, the dose-levels and the experimental conditions.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECFIC CONFOUNDING FACTORS:
A slight emulsion was noted in the plates at 5000 µg/plate.
The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test substance was toxic, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

PRELIMINARY TOXICITY TEST

The test substance was freely soluble in the vehicle (DMSO) at 50 mg/ml. Consequently, with a treatment volume of 100 µl/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Without S9 mix, a slight to severe toxicity was induced generally at dose-levels <': 500 µg/plate. With S9 mix, no toxicity was noted in the WP2 uvrA strain. In the remaining tester strains, a slight and/or strong toxicity was induced at dose-levels <': 500 µg/plate (TA 100 and TA 102 strains) or <'2500 µg/plate (TA 98 strain).

 

MUTAGENICITY EXPERIMENTS

Experiments without S9 mix:

The selected treatment-levels were as follows:

a) 31.25, 62.5, 125,250 and 500 µg/plate: for the TA 1535, TA 100 and TA 102 strains in both experiments as well as for the TA 1537 strain in the second experiment,

b) 78.125, 156.25, 312.5, 625 and 1250 µg/plate: for the TA 98 strain in the second experiment

c) 156.25, 312.5, 625, 1250 and 2500 µg/plate: for the WP2 uvrA strain in both experiments as well as for the TA 1537 and TA 98 strains in the first experiment. A slight to marked toxicity was noted in all tester strains, generally at dose-levels <'125 µg/plate.

No noteworthy increase in the number of revertants was induced in all tester strains, in both experiments.

 

Experiments with S9 mix:

The selected treatment-levels were as follows:

a) 31.25,62.5, 125,250 and 500 µg/plate: for the TA 1535, TA 100 and TA 102 strains in both experiments as well as for the TA 1537 strain in the second experiment,

b) · 312.5,625, 1250, 2500 and 5000 µg/plate: for the remaining tester strains in both experiments as well as for the TA 1537 strain in the first experiment. In the Salmonella typhimurium strains, a slight to strong toxicity was noted, mainly at dose levels ≥ 250 µg/plate, depending on the tester strain, the dose-levels and the experimental conditions. In the WP2 uvrA strain, no toxicity was induced in the first experiment. Therefore, in a first treatment (data archived but not reported) using the preincubation method, the same range of concentrations was used. However, following this treatment a slight to strong toxicity was noted at dose‑levels ≥ 625 µg/plate. Therefore this experiment was repeated using a lower range of dose-levels. For all tester strains, the test substance induced more toxicity when the preincubation method was used. The test substance did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the six strains.

 

FOR TABLES OF RESULTS FOR MUTATION TEST: Please see attached in overall remarks, attachments

 

REFERENCES:

Ames, B. N.; Durston, W. E.; Yamasaki, E. and Lee, F. D.: Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Nat. Acad. Sci. U.S.A., 70,2281-2285 (1973).

Ames, B. N.; Mc Cann, D. and Yamasaki, E.: Methods for detecting carcinogens and mutagens with the Salmonella Mammalian-microsome mutagenicity test. Mutation Research, n, 347-364 (1975).

Maron, D. M. and Ames B. N.: Revised methods for the Salmonella mutagenicity test. Mutation

Research, 113, 173-215 (1983).

Conclusions:

Interpretation of results (migrated information):
negative

Under our experimental conditions, the test substance Vivaldie does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

Executive summary:

The objective of this study was to evaluate the potential of the test substance Vivaldie to induce reverse mutation in Salmonella typhimurium and Escherichia coli. The test substance was then tested in two independent experiments, with and without a metabolic activation system. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535 , TA 1537, TA 98, TA 100 and TA 102 and one strain of Escherichia coli: WP2 uvrA were used. Each strain was exposed to five dose-levels of the test substance ( three plates/dose-level). Experiments without S9 mix: 31.25 to 2500 µg/plate. A slight to marked toxicity was noted in all tester strains, generally at dose-levels ≥ 125 µg/plate.

Experiments with S9 mix: 31.25 to 5000 µg/plate. A slight to marked toxicity was noted, depending on the tester strain. The test substance did not induce any noteworthy increase in the number of revertants, in both experiments, in any of the six strains. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Under the experimental conditions of this test, the test substance Vivaldie does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
Only an Ames study according to OECD guidelines and in compliance with GLP is available.

Justification for classification or non-classification

Based on the only mutagenicity study available, the Ames test, Vivaldie does not need to be classified for this endpoint according to the DSD 67/548/EC and the CLP Regulation EC 1272/2008.