Bis-sec-butyl peroxydicarbonate

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:

17 January 2018 - 07 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis-sec-butyl peroxydicarbonate

EC Number:

243-424-3

EC Name:

Bis-sec-butyl peroxydicarbonate

Cas Number:

19910-65-7

Molecular formula:

C10H18O6

IUPAC Name:

2-[({[(butan-2-yloxy)carbonyl]peroxy}carbonyl)oxy]butane

Test material form:

not specified

Details on test material:

- Storage conditions: frozen at -60°C to -80°C, protected from light

Specific details on test material used for the study:

- Density: 1.06 g/mL at 5°C
- No correction factor was applied

Method

Target gene:

Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor™ 1254-induced rat liver S9 fraction.

Test concentrations with justification for top dose:

Dose range finding experiment (all strains, with and without S9): 25, 50, 100, 250, 500, 1000, 2500, and 5000 μg/plate
First experiment (TA1537, TA98, TA100 and TA1535 without S9): 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 25, 50, and 100 μg/plate
First experiment (TA1537, TA 98, TA100 and TA1535 with S9 and WP2uvrA with and without S9): 0.5, 1.0, 2.5, 5.0, 10, 25, 50, 100, 250, and 500 μg/plate
Second experiment (TA1537, TA98, TA100 and TA1535 without S9): 0.5, 1.0, 2.5, 5.0, 10, 25, 50, and 100 μg/plate
Second experiment (TA1537, TA 98, TA100 and TA1535 with S9 and WP2uvrA with and without S9): 2.5, 5.0, 10, 25, 50, 100, 250, and 500 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: according to OECD test guideline

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: ICR-191; 2-aminoanthracene

Remarks:

For more details on the positive control substances, see table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

PERFORMANCE OF THE ASSAY: Sterile 12 × 75 mm test tubes were placed in heating blocks at 45°C to 47°C and the following items were added in a stepwise manner for each concentration of test or control substance: 2.00 mL of top agar, supplemented with 10% of a 0.5 mM histidine/biotin/tryptophan solution, 0.10 mL of indicator organisms (overnight culture), 0.10 mL of vehicle control or test substance, or 0.05 mL of positive control substance and 0.50 mL of metabolic activation mixture or PBS, for tests with or without S9, respectively. The tube contents were mixed gently and then poured onto minimal glucose plates. The top agar was allowed to set and the plates were incubated at 36°C to 38°C for 2 days.

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn

COLONY COUNTING:
The number of revertant colonies were counted by hand or with an automatic colony counter and then recorded. Plates that had a cytotoxic reduction in background lawn growth (> 50% reduction in the background lawn) were counted.

Evaluation criteria:

POSITIVE RESPONSE:
The test substance was considered positive for mutagenicity if it induced an increase of revertants per plate with increasing concentration. The increases should be at least 2 times the vehicle control background frequency for strains with high spontaneous levels (i.e., TA100) and three times for those with low spontaneous levels (TA1537, TA98, TA1535, and WP2 uvrA).
These increases should be seen in at least 2 or more successive concentrations or the response should be repeatable at a single concentration.

NEGATIVE RESPONSE:
The test substance was considered to be negative for inducing mutagenicity if it did not induce a response which fulfills the criteria for a positive response.

EQUIVOCAL RESPONSE:
Cases which did not clearly fit into the positive or negative criteria may be judged equivocal. In these cases the Study Director, based on sound scientific judgment, may take additional factors into consideration in evaluating the test results.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 250 μg/plate and ≥ 50 μg/plate (DRF study, with and without S9, resp.); ≥ 100 μg/plate and ≥ 50 μg/plate (first experiment, with and without S9, resp.); ≥ 250 μg/plate and ≥ 100 μg/plate (second experiment, with and without S9, resp.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 100 μg/plate and ≥ 50 μg/plate (DRF study, with and without S9, resp.); ≥ 100 μg/plate and ≥ 25 μg/plate (first experiment, with and without S9, resp.); ≥ 100 μg/plate and ≥ 50 μg/plate (second experiment, with and without S9, resp.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 250 μg/plate and ≥ 50 μg/plate (DRF study, with and without S9, resp.); ≥100 μg/plate (first experiment, with and without S9); ≥ 100 μg/plate (second experiment, with and without S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 250 μg/plate and ≥ 25 μg/plate (DRF study, with and without S9, resp.); ≥ 100 μg/plate and ≥ 25 μg/plate (first experiment, with and without S9, resp.); ≥ 100 μg/plate and ≥ 25 μg/plate (second experiment, with and without S9, resp.)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 250 μg/plate and ≥ 500 μg/plate (DRF study, with and without S9, resp.); ≥ 250 μg/plate (first and second experiment, with and without S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- No precipitation was observed in any of the experiments.
- There were no increases in the number of revertant colonies indicating a positive repsonse for inducing mutagenicity. Therefore, the test item is considered to be not mutagenic.
- The means of all positive control data were at least 3-fold greater than the means of the vehicle control data and comparable to the historical data.
- In both the first and second assay, criteria for a negative response were met for all tester strains with and without metabolic activation.

Applicant's summary and conclusion

Conclusions:

Based on the results of an Ames test, performed according to OECD guideline 471 and GLP principles, Di-sec-butyl peroxydicarbonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.