3-methyl-1,5-pentanediyl diacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April 2013 -- 04 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1,5-pentanediyl diacrylate

EC Number:

264-727-7

EC Name:

3-methyl-1,5-pentanediyl diacrylate

Cas Number:

64194-22-5

Molecular formula:

C12H18O4

IUPAC Name:

3-methyl-5-(prop-2-enoyloxy)pentyl prop-2-enoate

Method

Target gene:

Histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Main test:
- 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains in the first experiment both with and without S9 mix, for the TA 1535, TA 98, TA 100 and TA 102 strains in the second experiment with and without S9 mix, and in TA 1537 strain with S9 mix only in the second experiment,
- 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for TA 1537 strain without S9 mix in the second experiment.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO), batch No. K42474850 145.
- Justification for choice according to solubility assays performed, the highest dose-level of 5000 µg/plate was achievable using a test item solution at 100 mg/mL under a treatment volume of 50 µL/plate.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.

Statistics:

no

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Experiments without S9 mix
In the first experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels superior or equal to 1250 µg/plate (TA 1537 and TA 102 strains) and at dose-levels superior or equal to 2500 µg/plate (TA 1535, TA 98 and TA 100 strains).
 
In the second experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels superior or equal to 1250 µg/plate (TA 98 and TA 100 strains), and at dose-levels superior or equal to
2500 µg/plate (TA 1535, TA 1537 and TA 102 strains).
 
The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.
 
Experiments with S9 mix
In the first experiment, a moderate to strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at 312.5 µg/plate and at dose-levels superior or equal to 1250 µg/plate (TA 1537 strain), at dose-levels superior or equal to 1250 µg/plate (TA 98 strain), and at dose-level of 5000 µg/plate (TA 1535, TA 100 and TA 102 strains).
 
In the second experiment, a strong toxicity (thinning of the bacterial lawn and decrease in the number of revertants) was noted at tested dose-levels
superior or equal to 1250 µg/plate in the five strains.
 
The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or in the absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

This study was conducted in compliance with OECD No. 471 and the principles of Good Laboratory Practices.

Methods

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

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 pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

 

The number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were six analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

 

Since the test item was found to be cytotoxic in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

The treatment-levels were:

- 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains in the first experiment both with and without S9 mix, for the TA 1535, TA 98, TA 100 and TA 102 strains in the second experiment with and without S9 mix, and in TA 1537 strain with S9 mix only in the second experiment,

- 78.13, 156.3, 312.5, 625, 1250 and 2500 µg/plate for TA 1537 strain without S9 mix in the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants whatever the strain and the tested dose-level, either with or without S9 mix.

Experiments without S9 mix

In the first experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels superior or equal to 1250 µg/plate (TA 1537 and TA 102 strains) and at dose-levels superior or equal to 2500 µg/plate (TA 1535, TA 98 and TA 100 strains).

 

In the second experiment, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels superior or equal to 1250 µg/plate (TA 98 and TA 100 strains), and at dose-levels superior or equal to

2500 µg/plate (TA 1535, TA 1537 and TA 102 strains).

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.

 

Experiments with S9 mix

In the first experiment, a moderate to strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at 312.5 µg/plate and at dose-levels superior or equal to 1250 µg/plate (TA 1537 strain), at dose-levels superior or equal to 1250 µg/plate (TA 98 strain), and at dose-level of 5000 µg/plate (TA 1535, TA 100 and TA 102 strains).

 

In the second experiment, a strong toxicity (thinning of the bacterial lawn and decrease in the number of revertants) was noted at tested dose-levels

superior or equal to 1250 µg/plate in the five strains.

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains used.

Conclusion

 

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium either in the presence or in the absence of a rat liver metabolizing system.