Diallyl 2,2'-oxydiethyl dicarbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames (OECD 471, GLP): TA98, TA100, TA102, TA1535 and TA1537, +/- S9: negative

HPRT (OECD 476, GLP): Chinese hamster ovary cells, +/-S9: negative

CA (473, GLP): human perypheral lymphocytes: negative

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

Histidine

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.

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

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

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

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

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

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

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

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

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

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

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

see results in tabular form in the attachment

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 SEPT 2020 - 19 NOV 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hypoxanthineguanine phosphoribosyl transferase enzyme locus (hprt)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO K1

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Sub-line (K1) of Chinese hamster ovary cell line CHO, purchased from ECACC (European Collection of Authenticated Cell Cultures)
- Suitability of cells: one of the most commonly used cells
- Normal cell cycle time: cell doubling time 22 h

For cell lines:
- Absence of Mycoplasma contamination: absence of mycoplasma contamination was tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary; results were fully documented within the raw data file
- Methods for maintenance in cell culture: The cell stocks are kept in liquid nitrogen. Each batch of frozen cells was purged of HPRT mutants and was considered free from mycoplasma infections, tested by Central Agricultural Office National Animal Health Institute Budapest Hungary. For each experiment, cells were thawed rapidly and diluted in Ham's F12 medium containing 10 % (v/v) foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air.
- Cell cycle length, doubling time or proliferation index: cell doubling time 22 h


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The CHO K1 cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the treatments with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium (F12*-SEL) for 6-TG resistant mutants contained 3.4 μg/mL 6-thioguanine (6-TG). Cells were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The S9 was provided by Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
- method of preparation of S9 mix: fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
- concentration or volume of S9 mix and S9 in the final culture medium: 40 μL/mL medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A certificate of analysis was obtained from the supplier and documented with the raw data.

Test concentrations with justification for top dose:

A Pre-test on Toxicity was performed to establish an appropriate concentration range for the main Mutation Assay, both in the absence (125, 250, 500, 1000 and 2000 μg/mL) and in the presence (2, 3.9, 7.8, 15.6, 31.3, 62.5 and 125 μg/mL) of metabolic activation (rodent S9-mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control. In the absence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 2000 μg/mL. In the presence of metabolic activation concentration-dependent toxicity was observed. At the highest applied concentration of 125 μg/mL 100 % cytotoxicity was observed (0 % relative survival). Thus, the highest concentration in the presence of metabolic activation was set to achieve 10-20 % but not less than 10% relative survival in accordance with OCED Guideline 476. The following concentrations were chosen for the Main study:
5-h treatment, without S9: 125, 250, 500, 1000 and 2000 μg/mL
5-h treatment, with S9: 3.9, 7.8, 15.6, 31.3, 62.5 and 93.8 μg/mL

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The test item was dissolved in DMSO and diluted prior to treatment. This solvent is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary solubility test. The suitability of the solvent was confirmed with the available laboratory’s historical database.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 5 replicates of 2 cultures
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5 x10^6 cells in 15 mL medium (or in 14.4 mL medium + 0.6 mL S9 mix) were each placed in 4 sterile dishes per treatment and incubated for approximately 24 hours at 37 °C in a humidified atmosphere of 5 % CO2 before treatment.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5-hour treatment
- Harvest time after the end of treatment (sampling/recovery times): Cells were harvested after 19-hour incubation period


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): After the treatment period, cells were cultured to allow expression of the mutant phenotype. During the expression period, cells were regularly sub-cultured to maintain them in exponential growth until day 8.
- Selection time (if incubation with a selective agent): The selection period was 8 days.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: Cells were cultured in selection medium (F12*-SEL medium) containing 3.4 μg/mL of 6-thioguanine (6-TG, Sigma-Aldrich Chemie GmbH, Germany, Lot: SLBZ6113).
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: At the end of the expression period, cultures from each concentration were adjusted to 2 x10^5 cells/dish in 4 x five dishes per treatment, yielding 4x10^6 cells per treatment in selection medium .
Also for the viability testing, at the end of the expression period cell number in the samples was adjusted to 10^5 cells/mL. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test. A total of 5 mL (200 cells/dish) of the final concentration of each culture was plated into 3 parallel dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for growing colonies. Then, colonies were fixed with methanol, stained with Giemsa and counted.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cloning efficiency; relative survival (RS), other: relative population growth
- Any supplementary information relevant to cytotoxicity:
Determination of "Relative Survival to Treatment" is performed by the following calculation:
Relative Survival (%) = (Average number of colonies per treated culture / Average number of colonies per Solvent (vehicle) control dish) x 100.

The assay parameter "Relative Population Growth" shows the cumulative growth of the treated cells, relative to the control, over the expression period and prior to mutant selection:
Relative Population Growth (%) = (Treated culture population increase over the expression period / Solvent (Vehicle) control culture population increase over the expression period) x 100.

The colony forming ability of cells is measured by "Absolute Cloning Efficiency"(CE) at the time of mutant selection:
Absolute Cloning Efficiency (%) = (Average number of viable colonies per dish / 200) x100.

Rationale for test conditions:

Based on the result of the preliminary cytotoxicity assay (cytotoxicity in the presence of metabolic activation), the concentration levels for the performed Mutation Assay were chosen according to the recommendation in OECD Guideline 476, as well as the number of cells analysed and further test parameter.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (based 95% control limit).

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
- The lower and upper 95% confidence intervals of historical control were calculated with C-chart.
- The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

Species / strain:

Chinese hamster Ovary (CHO)

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 applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH values of test item solutions did not show any significant alterations compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay.
- Data on osmolality: In the Pre-test on Toxicity and Main Mutation Assay the osmolality values of treatment solutions were similar compared to the concurrent control
- Precipitation and time of the determination: Precipitation of the test item in the final culture medium were not observed at the applied concentrations.

RANGE-FINDING/SCREENING STUDIES (if applicable):
A Pre-test on Toxicity was performed to establish an appropriate concentration range for the main Mutation Assay, both in the absence (125, 250, 500, 1000 and 2000 μg/mL) and in the presence (2, 3.9, 7.8, 15.6, 31.3, 62.5 and 125 μg/mL) of metabolic activation (rodent S9-mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control. Results were noted as percentage of cells in relation to the negative control.
In the absence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 2000 μg/mL. In the presence of metabolic activation concentration-dependent toxicity was observed. At the highest applied concentration of 125 μg/mL 100 % cytotoxicity was observed (0 % relative survival) while at 62.5 µg/mL relative survival was approx. 26 %.
Based on the result of the preliminary cytotoxicity assay, the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended for lower- cytotoxic substances in OECD Guideline 476 (updated 2016) and based on the cytotoxicity noted in the preliminary study. The Mutation Assay was performed with and without metabolic activation system at the concentrations and treatment intervals given below:

Mutation Assay 5-hour treatment period without S9-mix:
125, 250, 500, 1000 and 2000 μg/mL*
Mutation Assay 5-hour treatment period with S9-mix:
3.9, 7.8, 15.6, 31.3, 62.5 and 93.8 μg/mL
*: mild opalescence was observed

STUDY RESULTS
- Concurrent vehicle negative and positive control data
without S9-mix [MUTANT FREQ. X 10^-6]:
Solvent control: 7.00, 6.93, 6.93, 7.00
Pos. control (EMS 1.0 µL/mL): 1541.79**, 1477.94**, 1426.09**, 1437.68**
** = p < 0.01 to the concurrent negative (solvent) control and to the historical control

with S9-mix [MUTANT FREQ. X 10^-6]:
Solvent control: 7.00, 7.00, 7.00, 6.93
Solvent control of DMBA: 7.14, 7.07, 7.22, 7.07
Pos. control (DMBA 20 µg/mL): 700.00**, 705.48**, 716.67**, 719.44**
** = p < 0.01 to the concurrent negative (solvent) control and to the historical control

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: In the presence of metabolic activation concentration-dependent toxicity, no concentration-response relationship in mutant freqency was observed
- Statistical analysis: Positive controls showed statistically significant increase in mutant freqencies (p < 0.01), test item showed no statistically significant increase in mutant frequencies

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
On Day 1, in the absence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 2000 μg/mL and there was very clear concentration-dependent toxicity up to the highest tested concentration (93.8 μg/mL) achieving 11-13 % relative survival in the presence of metabolic activation when compared to the negative (solvent) controls, confirming the response seen in the Pre-test on Toxicity (Concentration selection). The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period. The observed cloning efficiency was in the range of 98-101%.

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: Approximately 5x10^6 cells were placed in 4 sterile dishes per concentration and incubated for 24 hours before treatment, duplicate cultures with approximately 20x10^6 cells / concentration (2 x 10x10^6 cells) were treated.
o Number of cells plated in selective medium: 2x10^5 cells /dish for mutant selection (4x 5 dishes, yielding 4x10^6 cells per treatment)
o Number of resistant colonies in selective medium, and related mutant frequency: MEAN COLONY NUMBER +/- S.D. and MUTANT FREQ. X 10^-6 of solvent control (4x 5 replicates without and with S9):
[-S9] 199.0 ± 1.00 and 7.00, 200.0 ± 1.00 and 6.93, 199.7 ± 0.58 and 6.93, 201.0 ± 0.00 and 7.00, [+S9] 200.7 ± 1.15and 7.00, 201.0 ± 0.00 and 7.00, 200.7 ± 3.21 and 7.00, 201.3 ± 0.58 and 6.93


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
Without S9 mix EMS:
Mean: 1487.41
SD: 27.12
Range: 1357.81 – 1671.46
Lower confidence interval: 1432.61
Upper confidence interval: 1542.21
n: 40

With S9 mix DMBA:
Mean: 735.07
SD: 17.11
Range: 667.11 – 810.29
Lower confidence interval: 700.49
Upper confidence interval: 769.66
n: 40

- Negative (solvent/vehicle) historical control data:
Without S9 mix (Ham's F12 medium)
Mean: 6.58
SD: 0.59
Range: 4.90 – 8.91
Lower confidence interval: 5.36
Upper confidence interval: 7.81
n: 20

With S9 mix (Ham's F12 medium)
Mean: 6.95
SD: 0.84
Range: 4.90 – 11.76
Lower confidence interval: 5.19
Upper confidence interval: 8.71
n: 20

Without S9 mix (DMSO)
Mean: 6.66
SD: 0.58
Range: 4.95 – 10.89
Lower confidence interval: 5.44
Upper confidence interval: 7.89
n: 20

With S9 mix (DMSO)
Mean: 6.85
SD: 0.69
Range: 4.12 – 11.88
Lower confidence interval: 5.41
Upper confidence interval: 8.29
n: 20

see results in tabular form in the attachment

Conclusions:

In conclusion, Diallyl 2,2’-oxydiethyl dicarbonate tested up to the maximum recommended concentration (2000 μg/mL) without metabolic activation and up to cytotoxic concentrations with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control. Thus, it is concluded that the test item, Diallyl 2,2’-oxydiethyl dicarbonate, was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

Executive summary:

The test item, Diallyl 2,2’-oxydiethyl dicarbonate was tested in a Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells in accordance with the most recent Guideline OECD 476 and in compliance with GLP.

The purpose of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthineguanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells.

The test item was dissolved in DMSO and the concentrations of the main test were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver. Based on the result of the preliminary cytotoxicity assay, the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended for lower- cytotoxic substances in OECD Guideline 476 (updated 2016) and based on the cytotoxicity noted in the preliminary study. The Mutation Assay was performed at the concentrations and treatment intervals given below:

Mutation Assay 5-hour treatment period without S9-mix: 125, 250, 500, 1000 and 2000 μg/mL

Mutation Assay 5-hour treatment period with S9-mix: 3.9, 7.8, 15.6, 31.3, 62.5 and 93.8 μg/mL

Following treatment and 19 hours incubation period, phenotypic expression was allowed for 8 days followed by a mutant selection period for another 8 days. In the absence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 2000 μg/mL. In the presence of metabolic activation clear cytotoxicity (survival between 11-13 %) of the test item was observed at the highest concentration applied (93.8 μg/mL). Thus, the requirements for the highest test concentrations with regard to cytotoxicity as given in the guideline were met.

In this In Vitro Mammalian Cell Gene Mutation Test, the frequency of the cells with mutations did not show biologically and statistically significant increases compared to the concurrent and historical controls when Diallyl 2,2’-oxydiethyl dicarbonate was examined in the absence and in the presence of metabolic activation. All values were within the range of the laboratory historical control data and no dose-response relationships were noted.

At concentration of 2000 μg/mL the treatment solution was slightly opaque. At the other concentrations tested, the treatment solutions were clear. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The mutation frequency found in the solvent controls was well within the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 μL/mL) and 7, 12-Dimethyl benzanthracene (20 μg/mL) caused the expected biologically relevant and statistically significant increase of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, Diallyl 2,2’-oxydiethyl dicarbonate tested up to the maximum recommended concentration (2000 μg/mL) without metabolic activation and up to cytotoxic concentrations with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control. Thus, it is concluded that the test item, Diallyl 2,2’-oxydiethyl dicarbonate, was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

Reference 3

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1979-11-19 to 1980-03-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted under GLP but passed a Quality Assurance audit.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.21 (In Vitro Mammalian Cell Transformation Test)

Deviations:

yes

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.8800 (Morphologic transformation of cells in culture)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Draft OECD Guideline for an "In vitro Syrian Hamster Embryo (SHE) Cell transformation Assay"

Deviations:

no

Principles of method if other than guideline:

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was used as positive control (not listed in the EU Method.21).
The test substance was tested in the absence of metabolic activation. It is not noted whether the cell line possess or not an intrinsic metabolic activity known to be appropriate to the test material being tested.

GLP compliance:

no

Remarks:

The study was performed prior to the adoption of the GLP compliance

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: BALB/3T3 Clone A31 mouse embryo cells (3T3)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's Minimal Essential Medium (EMEM) containing 10% fetal bovine serum, 2% L-glutamine, 2% penicillin-streptomycin and 1% nonessential amino acids (complete medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not reported
- Periodically checked for karyotype stability: not reported
- Periodically "cleansed" against high spontaneous background: not reported

Additional strain / cell type characteristics:

other: see "Any other information on materials and methods incl. tables"

Metabolic activation:

without

Metabolic activation system:

not applicable

Test concentrations with justification for top dose:

0.1, 0.03 and 0.01 µL/ml (The stock concentration of T1562 was 500 µL/mL acetone was diluted with medium to achieve the doses tested);

Vehicle / solvent:

The stock solution of T1562 was prepared in acetone; all working dilutions were made with complete medium. All stock solutions and working dilutions were prepared fresh on the day of use.
Acetone served as solvent (negative) control. The concentration of acetone in the highest treatment dose of test material in medium was used as negative control.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

0.2 µL/mL (µL acetone/mL medium)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N-nitro-N-nitrosoguanidine

Remarks:

positive control concentration 0.5 µg/mL medium

Species / strain:

mammalian cell line, other: BALB/3T3 Clone A31 mouse embryo cells (3T3)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

no significant response

Cytotoxicity / choice of top concentrations:

other: dose depended response

Vehicle controls validity:

valid

Untreated negative controls validity:

other: see vehicle control

Positive controls validity:

valid

Remarks on result:

other: only one cell line was tested

Table 1 shows the cytotoxic effects of a 24 hour treatment of BALB/3T3 Clone A31 cells in the absence of exogenous metabolic activation with test article T1562 or positive or negative controls. Relative to the negative control (acetone), normalized to 100% survival, the colony-forming efficiency (RCE) of 3T3 cells exposed to the various doses of T1562 employed ranged from approximately 60-87%. The positive control (MNNG) reduced the colony-forming efficiency of the 3T3 cells by approximately 52%.

Table 1
Cytotoxic Effects of T1562 to BALB/3T3 Cl. A31 Cells Exposed in the Absence of Exogenous Metabolic Activation^a
Treatment	Concentration	RPE^b(%)	RCE^C(%)
Negative Control
Acetone	0.2 µl/ml	43.7	100.0
Positive Control
MNNG	0.5 µg/ml	20.9	47.9
Compound
T1562	0.10 µl/ml	26.0	59.5
	0.0 3 µl/ml	36.9	84.5
	0.01 µl/ml	37.9	86.6

Table 2 shows the ability of the various treatment conditions to induce morphological transformation among 3T3 cells exposed for 24 hours in the absence of exogenous metabolic activation. Of the doses of T1562 employed, 0.1 ul/ml and 0.03 ul/ml failed to induce both Type II and Type III morphologically transformed foci. At a dose of 0.01 ul/ml, however, one Type III transformed focus was observed among 5.685 x 10⁴ cells at risk, giving a transformation frequency (TF) of 1.8 x 10^-5 (which was not statistically significant); no Type II foci were observed under these conditions. No spontaneous transformants were observed in the solvent-treated (negative) controls. MNNG (positive control) induced the formation of one morphologically transformed Type II focus and five morphologically transformed Type III foci per 2.926 x 10⁴ cells at risk, giving a transformation frequency for Type III foci of 1.71 x 10⁴.

a Cells .were seeded at 250 cells/dish and incubated at 37 ± 2°C in the presence or absence of test article for 24 hr. Following treatment, cells were washed and incubated in complete growth medium for 7-10 days. Cells were fixed, stained with Giemsa, and scored for colony formation.

b RPE = relative plating efficiency, i.e. the number of surviving colonies relative to the number of cells seeded per condition.

c RCE = relative colony-forming efficiency, i.e. the number of colonies forming per condition relative (normalized) to 100% survival in the negative control.

Table 2
Transformation Potential of T1562 to BALB/3T3 CI. A31 Cells Exposed in the Absence of Exogenous Metabolic Activation^a
Treatment	Concentration	#CAR^b(x 10^3)	#Type II Foci	#Type III Foci	TF^C(x 10^-4)
Negative Control
Acetone	0.2 ul/ml	61.18	0	0	< 0.16
Positive Control
MNNG	0.5 ug/ml	29.26	1	5	1.71
Compound
T1562	0.10 ul/ml	39.00	0	0	< 0.26
	0.0 3 ul/ml	55.35	0	0	< 0.18
	0.01 ul/ml	56.85	0	1	0.18

a Cells were seeded at 1 x 10⁴ cells/dish and incubated for 24 hr at 37 ± 2°C in the presence or absence of test article. Cells were washed and cultured for 4-6 weeks with scheduled medium changes. Cells were fixed, stained with Giemsa, and scored for morphologically transformed foci.

b #CAR = number of cells at risk for each treatment group.

c TF = transformation frequency for the number of Type III foci per number of cells at risk.

Conclusions:

Interpretation of results: negative not significantly different from the negative control

Under the conditions employed in the assay described in this report, the data suggest that the test article T1562 induces a level of transforming activity in 3T3 cells which is not significantly different (P>0,05) from zero (i.e. the negative control).

Executive summary:

The in vitro Mammalian Cell Transformation Assay was used to detect the ability of test material to produce cellular alterations which result in mutant or neoplastic phenotypes.

The purpose of this study was to employ the BALB/3T3 Clone A31 mouse cell line in the absence of an exogenous metabolic activation system in order to investigate the in vitro morphological transforming potential of a test compound designated in this report as T1562, otherwise known as diallyl diglycol carbonate.

Target cells were plated and treated for 20-24 hours at 37 ± 2°C with different concentrations of test article as well as positive (N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)) and negative controls (acetone), after which they were analyzed for the cytotoxic effects of treatment, and the induction of phenotypic transformation. The transforming potential of a test article is determined by its ability to induce a significant increase in the number of morphologically transformed foci when compared to the negative control.

The negative control and the positive control fulfilled the requirements for determination of a valid test. The criterion established for the surviving fraction of cells treated with the test article was also satisfied. The treated 3T3 cells failed to exhibit the morphologically transformed phenotypes designated at Type II and Type III foci. Under the same test conditions, the cells exposed to a dose of 0.01 µl/ml of test article developed one focus exhibiting the morphologically transformed Type III phenotype. However, relative to the negative control, the induced transformation frequency (TF = 1.8 x 10^-5) was not statistically significant (P>0.05).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro tests

In 2020 an Ames test was conducted 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. 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.

 

Also, in 2020 the registered substance was tested in a Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells in accordance with the most recent Guideline OECD 476 and in compliance with GLP. The test item was tested up to the maximum recommended concentration (2000 μg/mL) without metabolic activation and up to cytotoxic concentrations with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control. Thus, it is concluded that the test item, Diallyl 2,2’-oxydiethyl dicarbonate, was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

 

An Ames test performed earlier prior to the adoption of the OECD Guideline 471, but the design of the test method is equivalent to the guideline requirements. In this test, no increasing in revertants was noted in strains TA 1535, TA 1537, TA 1538 and TA 100 with and without metabolic activation. Increasing in revertants was noted only the TA98 strain at three lower dose tested with metabolic activation. The positive result in TA98 is questionable, due to following factors: 1) the number of revertants induced was just at the level chosen for significance (3-fold increase), 2) a 10-fold increase in concentration of test material did not cause a dose-dependent increase in the number or revertants , and 3) higher concentrations of test material that were not toxic (0.1 and 0.3%) did not increase the number of revertants as compared to controls.  On the contrary, the positive control induced a significantly greater number of revertants than the test material. Although the authors of the study concluded that the test material was positive in the assay, the fact that mutagenicity was not noted in other strains with frame shift mutation (TA1537 and TA 1538) along with the issues raised about the positive result with TA strain suggest that this substance does not have the potential to be mutagenic or genotoxic.

In the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes (HPBL), diallyl diglycol carbonate was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9 activated test systems. The positive and solvent controls fulfilled the requirements for a valid test.

In the Primary Rat Hepatocyte UDS Assay, primary rat hepatocytes obtained from liver of male Fisher 344 rats were exposed to the test substance at concentrations of 0.3313 – 10 nL/mL without metabolic activation for 1 hour and then washed. The test material, CR-39, did not induce detectable UDS in primary rat hepatocytes over an applied concentration range of 10 nL/mL to 0.313 nL/mL. This concentration range caused toxicity ranging from high to nondetectable 20 hours after treatment. Therefore, the test material is considered to be inactive in this assay.

Diallyl diglycol carbonate was tested in the in vitro Mammalian Cell Transformation Assay (Microbiological Associates, 1980b) to investigate the in vitro morphological transforming potential of the test substance in the BALB/3T3 Clone A31 mouse cell line in the absence of an exogenous metabolic activation system. The negative control and the positive control fulfilled the requirements for determination of a valid test. The criterion established for the surviving fraction of cells treated with the test article was also satisfied. The treated 3T3 cells failed to exhibit the morphologically transformed phenotypes designated at Type II and Type III foci at 0.1 µL/mL and 0.03 µL/mL. Under the same test conditions, the cells exposed to a dose of 0.01 µl/ml of test article developed one focus exhibiting the morphologically transformed Type III phenotype among 5.685 x 10E4 cells at risk giving a transformation frequency (TF) of 1.8 x 10E-5. However, relative to the negative control, the induced transformation frequency (TF = 1.8 x 10E-5) was not statistically significant (P>0.05). 

Justification for classification or non-classification

Based on the information above and especially the most recent guideline studies, the in vitro gene mutation test in bacterial cells (Ames, 2020) and in mammalian cells (HPRT, 2020) conducted with CR 39 (Diallyl diglycol carbonate), it has been concluded that the registered substance does not show the potential to be mutagenic or genotoxic.

Hence, a classification according to the criteria of Regulation (EC) No 1272/2008 is not warranted.