Bisisopropyl 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:

13 March 2017 - 16 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

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:

Bisisopropyl peroxydicarbonate

EC Number:

203-317-4

EC Name:

Bisisopropyl peroxydicarbonate

Cas Number:

105-64-6

Molecular formula:

C8H14O6

IUPAC Name:

2-[({[(propan-2-yloxy)carbonyl]peroxy}carbonyl)oxy]propane

Constituent 2

Reference substance name:

Ethyl acetate

EC Number:

205-500-4

EC Name:

Ethyl acetate

Cas Number:

141-78-6

Molecular formula:

C4H8O2

IUPAC Name:

ethyl acetate

Test material form:

liquid

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

Since the test item was found freely soluble but toxic 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.
Without S9 mix:
- 156.3, 312.5, 625, 1250, 2500 and 3750 µg/plate for the TA 98 strain in the 1st experiment,
- 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 1535 and TA 1537 strains in the 1st experiment,
- 156.3, 312.5, 625, 1250, 2500, 3750 and 5000 µg/plate for the TA 98 strain in the 2nd experiment,
- 312.5, 625, 1250, 2500, 3750 and 5000 µg/plate for the TA 100 and TA 102 strains in the first experiment, and for the TA 98 strain in the 3rd experiment,
- 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 1535, TA 1537, TA 100 and TA 102 strains in the 2nd experiment.
With S9 mix:
- 312.5, 625, 1250, 2500, 3750 and 5000 µg/plate for the 5 strains in the 1st experiment, for the TA 1535 strain in the third experiment using the direct plate incorporation method, and for the TA 102 strain in the 3rd experiment,
- 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the 5 strains in the 2nd experiment and for the TA 98 strain in the 3rd experiment,
- 312.5, 500, 625, 937.5, 1250 and 2500 µg/plate for the TA 1535 strain in the 3rd experiment using the pre-incubation method,
- 250, 312.5, 500, 625, 1250 and 2500 µg/plate for the TA 100 strain in the 3rd experiment.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide
- Justification for choice: test item was found soluble in the vehicle at a concentration of 100 mg/mL. Using a treatment volume of 50 µL/plate, the highest recommended dose level was avhievable.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Anthramine (2AM) Benzo(a)pyrene (BAP)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);
- The direct plate incorporation method was used for all the experiments without S9 mix and for the first main test with S9 mix, as well as for the third experiment in the TA 1535 strain.
- The pre-incubation method was used for the second experiment with S9 mix, as well as for the third experiment with S9 mix conducted with the TA 1535, TA 98, TA 100 and TA 102 strains.

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

NUMBER OF REPLICATIONS:
Three plates/dose-level.

Evaluation criteria:

In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
- 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 mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose levels,
- nor any evidence of a dose-response relationship is noted.

Statistics:

no

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Pre-incubation method

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

moderate to strong toxicity at the highest dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Pre-incubation method

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

moderate to strong toxicity at the highest dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

moderate to strong toxicity at the highest dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

pre-incubation method

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

moderate to strong toxicity at the highest dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate to strong toxicity at the highest dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate to strong toxicity at the highest dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate to strong toxicity at the highest dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate to strong toxicity at the highest dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 10

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Pre-incubation method

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

moderate to strong toxicity at the highest dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Neither precipitate nor emulsion was observed in the Petri plates when scoring the revertants at any of the tested dose levels, either with or without S9 mix

RANGE-FINDING STUDY: In the absence of S9 mix, a moderate to strong toxicity (decrease in the number of revertants and/or of thinning of the bacterial lawn) was noted at dose levels = 2500 µg/plate in the TA 98 strain, and at the highest tested dose level of 5000 µg/plate in the TA 100 strain.
No noteworthy toxicity was noted in the presence of S9 mix.

RESULTS OF CYTOTOXICITY and GENOTOXICITY:
Without S9 mix
A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose levels = 2500 µg/plate in the TA 1537 strain, = 3750 µg/plate in the TA 98 strain (except for the first experiment), and at 5000 µg/plate in the TA 1535 strain (second experiment only) and TA 100 strain (both experiments).
The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains.

With S9 mix
Using the direct plate incorporation method, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose levels = 2500 µg/plate in the TA 1537 strain and at 5000 µg/plate in the TA 98 strain.
Using the pre-incubation method, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose levels = 937.5 µg/plate in the TA 1535 strain, = 1250 µg/plate in the TA 1537 and TA 100 strains, = 2500 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 102 strain (third experiment only).
Dose-related increases in the number of revertants were noted in the second main experiment when using the pre-incubation method, in all strains (except TA 1537 strain) and exceeded the thresholds of positivity. Since the first main experiment with S9 mix was conducted according to the direct plate incorporation method, the reproducibility of these increases was evaluated in a third independent experiment. In this third assay, the reproducibility of the increases was demonstrated in the same bacterial strains.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached

Applicant's summary and conclusion

Conclusions:

Diisopropyl peroxydicarbonate showed a mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains in the presence of a rat liver metabolizing system. No mutagenic activity was evidenced in the absence of metabolic activation.

Executive summary:

The potential of diisopropyl peroxydicarbonate to induce reverse mutations was evaluated in Salmonella typhimurium. The study was performed according to the OECD guideline No. 471 and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose levels of the test item, dissolved in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in three independent experiments, with or without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. The direct plate incorporation method was used for all the experiments without S9 mix and for the first main test with S9 mix, as well as for the third experiment in the TA 1535 strain.

The pre-incubation method was used for the second experiment with S9 mix, as well as for the third experiment with S9 mix conducted with the TA 1535, TA 98, TA 100 and TA 102 strains. Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five 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 mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose levels for each strain and test condition. The study was therefore considered to be valid. Since the test item was found freely soluble but toxic 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. Neither precipitate nor emulsion was observed in the Petri plates when scoring the revertants at any of the tested dose levels, either with or without S9 mix.

Experiments without S9 mix

The selected dose levels were:

. 156.3, 312.5, 625, 1250, 2500 and 3750 µg/plate for the TA 98 strain in the first experiment,

. 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 1535 and TA 1537 strains in the first experiment,

. 156.3, 312.5, 625, 1250, 2500, 3750 and 5000 µg/plate for the TA 98 strain in the second experiment,

. 312.5, 625, 1250, 2500, 3750 and 5000 µg/plate for the TA 100 and TA 102 strains in the first experiment, and for the TA 98 strain in the third experiment,

. 312.5, 625, 1250, 2500 and 5000 µg/plate for the TA 1535, TA 1537, TA 100 and TA 102 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 in all the tested strains (except for the TA 102 strain) at the highest tested dose levels. 

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

Experiments with S9 mix

The selected dose levels were:

. 312.5, 625, 1250, 2500, 3750 and 5000 µg/plate for the five strains in the first experiment, for the TA 1535 strain in the third experiment using the direct plate incorporation method, and for the TA 102 strain in the third experiment (pre-incubation method),

. 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains in the second experiment and for the TA 98 strain in the third experiment,

. 312.5, 500, 625, 937.5, 1250 and 2500 µg/plate for the TA 1535 strain in the third experiment using the pre-incubation method,

. 250, 312.5, 500, 625, 1250 and 2500 µg/plate for the TA 100 strain in the third experiment.

A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted in all the tested strains at the highest dose levels.

Dose-related and/or reproducible increases in the number of revertants, exceeding the thresholds of positivity, were observed in the TA 1535, TA 98, TA 100 and TA 102 strains when using the pre-incubation method.

These results met the criteria for a positive response.

Diisopropyl peroxydicarbonate showed a mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains in the presence of a rat liver metabolizing system. No mutagenic activity was evidenced in the absence of metabolic activation.