Diallyl 2,2'-oxydiethyl dicarbonate

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:

16 MAR 2020 - 03 SEPT 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21. Jul. 1997

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:

Diallyl 2,2'-oxydiethyl dicarbonate

EC Number:

205-528-7

EC Name:

Diallyl 2,2'-oxydiethyl dicarbonate

Cas Number:

142-22-3

Molecular formula:

C12H18O7

IUPAC Name:

3-({[2-(2-{[(prop-2-en-1-yloxy)carbonyl]oxy}ethoxy)ethoxy]carbonyl}oxy)prop-1-ene

Test material form:

liquid

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB- (TA98, TA100, TA1535, TA1537); rfa- (all)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : S9 was obtained by Trinova Biochem GmbH, Gießen (Batch nos. 4183, 4053, 4120, 4063, 4126, produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally)
- method of preparation of S9 mix:
Phosphate buffer 22.5 mL
0.1M NADP-solution 1.0 mL
1M G6P-solution 0.125 mL
Salt solution 0.5 mL
Rat liver S9 1.0 mL

- concentration or volume of S9 mix and S9 in the final culture medium : 500 µL S9-mix / plate
- quality controls of S9: each batch of S9 is characterized with a mutagen that requires metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene).

Test concentrations with justification for top dose:

Plate incorporation:
without metabolic activation: 5, 1.5, 0.5, 0.15 and 0.05 µL/plate, highest as recommended in the guideline, spacing factor of 3.16 with metabolic activation: TA1535, TA1537: 1.5, 0.5, 0.15, 0.05, 0.015, 0.005, 0.0015 µL/plate and TA98, TA100, TA102: 0.5, 0.15, 0.05, 0.015, 0.005, 0.0015, 0.0005 µL/plate, lowest toxic concentration of the initial experiment will be chosen as highest concentration.
Pre-incubation:
without metabolic activation: 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 µL/plate, highest as recommended in the guideline, spacing factor of 2 with metabolic activation: TA98, TA102, TA1535, TA1537: 0.5, 0.25, 0.125, 0.063, 0.031, 0.016, 0.008 µL/plate, TA100: 0.15, 0.075, 0.0375, 0.0188, 0.0094, 0.0047, 0.0023 µL/plate, lowest toxic concentration of the initial experiment will be chosen as highest concentration, spacing factor of 2.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was determined in demineralised water and dimethyl sulfoxide (DMSO). The test item is soluble at a concentration of 50 mL/L in DMSO. Based on the results of the non-GLP pre-test, DMSO will be used as solvent in the experiments.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine, 2-Amino-Anthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 5 (1, 1b, 1c, 2, 2b )

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: in agar (plate incorporation) - Exp. 1; preincubation - Exp. 2

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48h
- Harvest time after the end of treatment (sampling/recovery times): After incubation for 48 hours, the number of revertants is counted and the numbers for each plate are recorded.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

Lowest toxic concentration in cultures with metabolic activation of the initial experiment were chosen as highest concentration, due to OECD guideline 471 requirements at least five non-toxic concentrations were analysed

Evaluation criteria:

A result is considered as positive if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the concentrations occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in the bacteria strains TA 98, TA 100 and TA 102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2)
- if in the bacteria strains TA 1535 and TA 1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3).

A substance is not mutagenic if it does not meet the criteria above. If the criteria listed above are not clearly met, the results are assessed as equivocal and will be discussed.

Statistics:

not performed

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 10

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Plates were checked for precipitation of the test item at the end of the incubation period by visual inspection: In all experiments, no precipitation of the test item Diallyl 2,2'-oxydiethyl dicarbonate was observed at any of the tested concentrations up to 5 µL/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data

Ames test:
- Signs of toxicity : Yes, in the presence of metabolic activation:
Exp. 1:
5 µL/plate: TA98, TA100, TA102, TA1535, TA1537
1.5 µL/plate: TA98, TA100, TA102, TA1535, TA1537
0.5 µL/plate: A98, TA100, TA102, TA1537
Exp. 1c:
1.5 µL/plate: TA1535, TA1537
0.5 µL/plate: TA98, TA100, TA1535, TA1537
0.15 µL/plate: TA102

Exp. 2:
0.5 µL/plate: TA98, TA102, TA1535, TA1537
0.25 µL/plate: TA98, TA102, TA1535

Any other information on results incl. tables

see results in tabular form in the attachment

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Diallyl 2,2'-oxydiethyl dicarbonate is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the absence and presence of metabolic activation under the experimental conditions of the present study.

Executive summary:

This study was performed in order to evaluate the mutagenic potential of Diallyl 2,2'-oxydiethyl dicarbonatein the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537), based on the most recent Guideline OECD 471 and EU Method B.13/14 in compliance with GLP.

Five experiments (1, 1b, 1c, 2 and 2b) were performed in the presence and absence of an exogenous metabolic activation (±S9). The experiments 1, 1b and 1c were performed by using the plate incorporation method while for the experiments 2 and 2b the pre-incubation method was applied. Experiment 1 showed cytotoxicity in the strains TA98, TA100, TA102, TA1535 and TA1537 with metabolic activation and was invalid for TA102 (-S9), experiment 1b was invalid for all bacterial strains due to contamination of the bacterial culture and experiment 2 was invalid for TA102 (-S9).

In the first experiment, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations. Cytotoxicity of the test item could be observed in the presence of metabolic activation, however, in the lower concentrations, no signs of toxicity could be observed.

The results of this experiment showed that none of the tested non-toxic concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. For TA102 (-S9) experiment 1 was invalid as the positive control did not meet the acceptance criteria. To achieve the required number of analyzable non-toxic concentrations for the strains TA98, TA100, TA102, TA1535 and TA1537 with metabolic activation and a valid experiment for TA102 without metabolic activation, experiment 1 was repeated.

The repetition was performed with additional test item concentrations for the strains to which the test item showed a toxic effect and exactly as experiment 1 for TA102 without metabolic activation. Experiment 1b was invalid due to a contamination of the bacterial culture and the experiment 1c was performed with the following concentrations:

- TA102 (-S9): 5, 1.5, 0.5, 0.15, 0.05 µL/plate

- TA1535, TA1537 (+S9):  1.5, 0.5, 0.15, 0.05, 0.015, 0.005, 0.0015 µL/plate

- TA98, TA100, TA102 (+S9): 0.5, 0.15, 0.05, 0.015, 0.005, 0.0015, 0.0005 µL/plate

The test item showed no precipitates on the plates and the bacterial background lawn was not affected at any of the test item concentrations. Some cytotoxicity was observed in the presence of metabolic activation and in the lower concentrations, no signs of toxicity could be observed.

Therefore, the number of analyzable non-toxic concentrations was sufficient. The positive control data met the acceptance criteria for all tested bacterial strains. No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed and the experiment 1c was assessed to be valid.

Based on the results of the first experiments (1 and 1c), the test item was tested with the following concentrations in experiment 2:

- TA98, TA100, TA102, TA1535, TA1537 (-S9): 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 µL/plate

- TA98, TA102, TA1535, TA1537 (+S9): 0.5, 0.25, 0.125, 0.063, 0.031, 0.016, 0.008 µL/plate

- TA100 (+S9): 0.15, 0.075, 0.0375, 0.0188, 0.0094, 0.0047, 0.0023 µL/plate

Experiment 2 was performed by using the pre-incubation method. The test item showed no precipitates on the plates at any of the test item concentrations. Some cytotoxicity could be observed in the presence of metabolic activation, however, the results of this experiments showed that the test item caused no increase in the number of revertants in all bacterial strains compared to the solvent control, in both the presence and absence of metabolic activation.

For TA102 without metabolic activation experiment 2 was invalid as the positive control did not meet the acceptance criteria. To achieve a valid experiment for TA102 in the absence of metabolic activationexperiment 2 was repeated under the same conditions (experiment 2b). No signs of cytotoxicity could be observed. The positive control met the acceptance criteria and thus, results obtained with the test item are valid for TA102 in the absence of metabolic activation.

The results of this experiments showed that the test item caused no increase in the number of revertants in strain TA102 in the absence of metabolic activation compared to the solvent control DMSO.

Based on the results of this study it is concluded that Diallyl 2,2'-oxydiethyl dicarbonate is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions of this study.