Bisisopropyl peroxydicarbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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 and in the mouse lymphoma assay, both in the presence and absence of a rat liver metabolizing system. Diisopropyl peroxydicarbonate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

 

OECD 471

The potential of diisopropyl peroxydicarbonate to induce reverse mutations was evaluated in Salmonella typhimurium (Brient 2017). 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 analyzable 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.

 

OECD 490

The objective of this study was to evaluate the potential of bisisopropyl peroxydicarbonate to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells (Brient, 2017). The study was performed according to international guideline (OECD No. 490, adopted on 28 July 2015) and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, the test item, dissolved in dimethylsulfoxide (DMSO), was tested in a single experiment, with or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Cultures of 20 mL at 5 x 105cells/mL (3-hour treatments) or cultures of 50 mL at 2 x 105cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid. Since the test item was found freely soluble but cytotoxic in the preliminary test, the highest dose level selected for the main experiment was based on the level of cytotoxicity, according to the criteria specified in the international guidelines (i.e. decrease in Adj. RTG). Neither precipitate nor emulsion was observed in the culture medium at the end of the treatment periods, at any dose levels.

Experiments without S9 mix

Dose levels selected for treatment:

.            25, 50, 100, 200, 400, 700 and 1000 µg/mL for the 3-hour treatment,

.            6.25, 12.5, 25, 50, 100, 150 and 300 µg/mL for the 24-hour treatment.

Cytotoxicity

Following the 3-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 200 µg/mL, as shown by a 51 to 95% decrease in Adj. RTG.

Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 150 µg/mL, as shown by a 59 to 100% decrease in Adj. RTG.

Mutagenicity

Following the 3-hour treatment, dose-related increases in the mutation frequency were noted. These increases exceeded the GEF at dose levels of 400 and 700 µg/mL, the latest inducing the recommended level of cytotoxicity. These results met the criteria of a positive response.

Following the 24-hour treatment, slight increases in the MF, remaining substantially below the GEF, were observed at dose levels inducing acceptable levels of cytotoxicity (i.e.up to 150 µg/mL), but a dose-response relationship was demonstrated by the linear regression.

None of the tested dose levels induced the recommended level of cytotoxicity (i.e.Adj. RTG between 10 and 20%). Since there was no negative data point between either between 20 and 25% or between 1 and 10% Adj. RTG, these results were considered as equivocal.

Experiment with S9 mix

Dose levels selected for treatment: 12.5, 25, 50, 100, 200, 300 and 400 µg/mL.

Cytotoxicity

A slight to severe cytotoxicity was induced at dose levels = 100 µg/mL, as shown by a 40 to 96% decrease in Adj. RTG.

Mutagenicity

Dose-related increases in the mutation frequency were noted. These increases exceeded the GEF at dose levels of 200 and 300 µg/mL, the latest inducing the recommended level of cytotoxicity. These results met the criteria of a positive response.

Under the experimental conditions of this study, bisisopropyl peroxydicarbonate showed a mutagenic activity in the mouse lymphoma assay, both in the presence and absence of a rat liver metabolizing system.

 

OECD 487

The potential of diisopropyl peroxydicarbonate to induce an increase in the frequency of micronucleated cells was evaluated in the mouse cell line L5178Y TK+/- (Brient, 2017). The study was performed according to the international guideline (OECD No. 487, adopted 26 September 2014) and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, the test item, dissolved in dimethylsulfoxide, was tested in a single experiment with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

- Without S9 mix:3 h treatment + 24 h recovery

- With S9 mix:24 h treatment + 0 h recovery and3 h treatment + 24 h recovery

Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. Then, after the final cell counting, the cells were washed and fixed. Then, cells from three dose levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation (”blind” scoring). For each treatment condition, micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid. Since the test item was found freely soluble but cytotoxic in the preliminary test, the highest dose level selected for the main experiment was based on the level of cytotoxicity, according to the criteria specified in the international regulations. Neither precipitate nor emulsion was observed in the culture medium at the end of the treatment periods, at any dose levels. With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatments were 10, 20, 40, 60, 80, 100, 150, 175 and 200 µg/m L with and without S9 mix.

Experiments without S9 mix

Following the 3-hour treatment, a marked to severe cytotoxicity was induced at dose levels = 60 µg/mL, as shown by a 68 to 100% decrease in the PD. Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 40 µg/mL, as shown by a 32 to 100% decrease in the PD.

The dose levels selected for micronucleus analysis were 10, 20 and 40 µg/mL, the latter inducing levels of cytotoxicity of 23 and 32% for the 3-hour and 24 -hour treatments, respectively, but higher dose levels being too cytotoxic.No statistically significant increase in the frequency of micronucleated cells was noted either after the 3- or 24-hour treatments. Furthermore, frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed. The recommended level of cytotoxicity was not reached in either condition (3- or 24-hour treatments), however, considering the narrow dose levels spacing used, the available results were considered as suitable to allow a reliable interpretation. In the absence of S9 mix, the results met the criteria of a negative response.

Experiment with S9 mix

A slight to severe cytotoxicity was induced at dose levels = 40 µg/mL, as shown by a 36 to 100% decrease in the PD.

The dose levels selected for micronucleus analysis were 20, 40 and 60 µg/mL, the latter inducing the recommended level of cytotoxicity (i.e.58% decrease in the PD). No statistically significant increase in the frequency of micronucleated cells was noted. Furthermore, frequencies of micronucleated cells remained consistent with vehicle control historical range and no dose-response relationship was evidenced.

Under the experimental conditions of the study, Diisopropyl peroxydicarbonate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

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:

13 March 2017 - 16 May 2017

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:

21 July 1997

Deviations:

no

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

Target gene:

Histidine operon

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.

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

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

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

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

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

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2017- 27 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell mutation assay

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

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:

Experiments without S9 mix
- 25, 50, 100, 200, 400, 700 and 1000 µg/mL for the 3-hour treatment,
- 6.25, 12.5, 25, 50, 100, 150 and 300 µg/mL for the 24-hour treatment.
Experiment with S9 mix
- 12.5, 25, 50, 100, 200, 300 and 400 µg/mL.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide
- Justification for choice: Since the test item was found freely soluble but cytotoxic in the preliminary test, the highest dose level selected for the main experiment was based on the level of cytotoxicity, according to the criteria specified in the international guidelines (i.e. decrease in Adj. RTG).. Using a test item concentration of 400 mg/mL and a treatment volume of 0.5% (v/v) in the culture medium, the highest recommended dose level of 2000 µg/mL was achievable.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.

Evaluation criteria:

In all cases, biological relevance was taken into consideration when evaluating the results.

Evaluation of a positive response:
Based on IWGT recommendations (d, e, f), a test item was considered clearly positive if, in any of the experimental conditions examined:
- at least at one dose level the mutation frequency minus the mutation frequency of the vehicle control (IMF) did equal or exceed the Global Evaluation Factor (GEF) of 126 x 10-6,
- a dose-response relationship was demonstrated by a statistically significant trend test.

Evaluation of a negative response:
A test item was considered clearly negative if, in all experimental conditions, no dose-response relationship was demonstrated or, if there was an increase in MF, it did not exceed the GEF.

Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose levels with Adj. RTG between 10 and 20%, were not considered as positive results.

A test item may be considered as non-mutagenic when there was no culture showing an Adj. RTG value between 10 and 20% if (g):
- there was at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there was no evidence of mutagenicity in a series of data points between 100 and 25% and there was also a negative data point between 10 and 1% Adj. RTG.

Statistics:

To assess the dose-response relationship, a linear regression was performed between dose levels and individual mutation frequencies obtained at dose levels showing a mean Adj. RTG = 10% (see Appendix 4). This statistical analysis was performed using SAS Enterprise Guide software.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: At the end of the treatment periods, neither precipitate nor emulsion was observed in the culture medium at any of the tested dose levels.

RANGE-FINDING STUDIES: Using a test item concentration of 400 mg/mL and a treatment volume of 0.5% (v/v) in the culture medium (i.e. 100 µL/20 mL culture medium for the 3-hour treatments and 250 µL/50 mL culture medium for the 24 hour treatment), the highest recommended dose level of 2000 µg/mL was achievable.
Thus, the dose levels selected for the treatment of the preliminary test were 4, 40, 200, 400, 1000 and 2000 µg/mL.

At the highest tested dose level of 2000 µg/mL, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 379 mOsm/kg H2O (377 mOsm/kg H2O for the vehicle control). At the end of the treatment periods, neither precipitate nor emulsion was observed in the culture medium at any of the tested dose levels. Based on these results, none of the tested dose levels was considered to produce extreme culture conditions.

Following the 3-hour treatment without S9 mix, a marked to severe cytotoxicity was induced at dose levels = 400 µg/mL, as shown by a 80 to 100% decrease in Adj. RTG.
Following the 24-hour treatment without S9 mix, a severe cytotoxicity was induced at dose levels = 200 µg/mL, as shown by a 92 to 100% decrease in Adj. RTG.
Following the 3-hour treatment with S9 mix, a marked to severe cytotoxicity was induced at dose levels = 200 µg/mL, as shown by a 82 to 100% decrease in Adj. RTG.

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

RESULTS OF CYTOTOXICITY:
Main experiments without S9 mix
Following the 3-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 200 µg/mL, as shown by a 51 to 95% decrease in Adj. RTG.
Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 150 µg/mL, as shown by a 59 to 100% decrease in Adj. RTG.

Main experiment with S9 mix
A slight to severe cytotoxicity was induced at dose levels = 100 µg/mL, as shown by a 40 to 96% decrease in Adj. RTG.

Conclusions:

Diisopropyl peroxydicarbonate showed a mutagenic activity in the mouse lymphoma assay, both in the presence and absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of diisopropyl peroxydicarbonate to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK^+/-mouse lymphoma cells. The study was performed according to international guideline (OECD No. 490, adopted on 28 July 2015) and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, the test item, dissolved in dimethylsulfoxide (DMSO), was tested in a single experiment, with or without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Cultures of 20 mL at 5 x 10⁵cells/mL (3-hour treatments) or cultures of 50 mL at 2 x 10⁵cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO₂ humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. Cytotoxicity wasmeasured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE₂). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria.In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%. The study was therefore considered to be valid. Since the test item was found freely soluble but cytotoxic in the preliminary test, the highest dose level selected for the main experiment was based on the level of cytotoxicity, according to the criteria specified in the international guidelines (i.e. decrease in Adj. RTG). Neither precipitate nor emulsion was observed in the culture medium at the end of the treatment periods, at any dose levels.

Experiments without S9 mix

Dose levels selected for treatment:

.            25, 50, 100, 200, 400, 700 and 1000 µg/mL for the 3-hour treatment,

.            6.25, 12.5, 25, 50, 100, 150 and 300 µg/mL for the 24-hour treatment.

Cytotoxicity

Following the 3-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 200 µg/mL, as shown by a 51 to 95% decrease in Adj. RTG.

Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 150 µg/mL, as shown by a 59 to 100% decrease in Adj. RTG.

Mutagenicity

Following the 3-hour treatment, dose-related increases in the mutation frequency were noted. These increases exceeded the GEF at dose levels of 400 and 700 µg/mL, the latest inducing the recommended level of cytotoxicity. These results met the criteria of a positive response.

Following the 24-hour treatment, slight increases in the MF, remaining substantially below the GEF, were observed at dose levels inducing acceptable levels of cytotoxicity (i.e.up to 150 µg/mL), but a dose-response relationship was demonstrated by the linear regression.

None of the tested dose levels induced the recommended level of cytotoxicity (i.e. Adj. RTG between 10 and 20%). Since there was no negative data point between either between 20 and 25% or between 1 and 10% Adj. RTG, these results were considered as equivocal.

Experiment with S9 mix

Dose levels selected for treatment: 12.5, 25, 50, 100, 200, 300 and 400 µg/mL.

Cytotoxicity

A slight to severe cytotoxicity was induced at dose levels = 100 µg/mL, as shown by a 40 to 96% decrease in Adj. RTG.

Mutagenicity

Dose-related increases in the mutation frequency were noted. These increases exceeded the GEF at dose levels of 200 and 300 µg/mL, the latest inducing the recommended level of cytotoxicity. These results met the criteria of a positive response.

Under the experimental conditions of this study, diisopropyl peroxydicarbonatet showed a mutagenic activity in the mouse lymphoma assay, both in the presence and absence of a rat liver metabolizing system.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2017 - 26 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 September 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable (not a gene mutation assay).

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10% inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
L5178Y TK+/- cells are an established cell line recommended by international regulations for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei. The average cell cycle time is approximately 10-12 hours.
L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the intermediate of Biovalley (Marne-La-Vallée, France).
The cells were stored in a cryoprotective medium (10% horse serum and 10% dimethylsulfoxide (DMSO)) at -80°C and each batch of frozen cells was checked for the absence of mycoplasma.

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:

10, 20, 40, 60, 80, 100, 150, 175 and 200 µg/mL

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide
- Justification for choice: using a test item concentration of 400 mg/mL in the vehicle and a treatment volume of 0.5% (v/v) in culture medium (i.e.10 µL/2 mL culture medium), the highest recommended dose level of 2000 µg/mL was achievable.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine; aneugen without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
Since the test item was found freely soluble but cytotoxic in the preliminary test, the highest dose level selected for the main experiment was based on the level of cytotoxicity, according to the criteria specified in the international regulations.

DURATION:
Preliminary cytotoxicity test
Without S9 mix
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery

With S9 mix
3 h treatment + 24 h recovery

Main cytogenetic experiments
Without S9 mix
3 h treatment + 24 h recovery
24 h treatment + 0 h recovery

With S9 mix
3 h treatment + 24 h recovery

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells per dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item was considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- for at least one dose level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose levels.

Evaluation of a negative response: a test item was considered clearly negative if none of the criteria for a positive response was met.

Statistics:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the Khi2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.

To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 mix 3-h treatment: toxicity at dose levels = 60 µg/mL (68 to 100% PD decrease). 24-h treatment: toxicity at dose levels = 40 µg/mL (32 to 100% PD decrease). With S9 mix Toxicity at dose levels = 40 µg/mL (36 to 100% PD decrease).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: none

RANGE-FINDING STUDIES:
Following the 3-hour treatment without S9 mix, a severe cytotoxicity was observed at dose levels = 200 µg/mL, as shown by a 83 to 100% decrease in the PD.
Following the 24-hour treatment without S9 mix, a slight to severe cytotoxicity was observed at dose levels = 40 µg/mL, as shown by a 32 to 100% decrease in the PD.
Following the 3-hour treatment with S9 mix, a severe cytotoxicity was observed at dose levels = 200 µg/mL, as shown by a 100% decrease in the PD.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see Tables enclosed
- Indication whether binucleate or mononucleate where appropriate: mononucleates cells 'L5178Y cell line).

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

RESULTS OF CYTOTOXICITY:
Experiments without S9 mix
Following the 3-hour treatment, a marked to severe cytotoxicity was induced at dose levels = 60 µg/mL, as shown by a 68 to 100% decrease in the PD.
Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 40 µg/mL, as shown by a 32 to 100% decrease in the PD.

Experiment with S9 mix
A slight to severe cytotoxicity was induced at dose levels = 40 µg/mL, as shown by a 36 to 100% decrease in the PD.

Conclusions:

Diisopropyl peroxydicarbonate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The potential of Diisopropyl peroxydicarbonate to induce an increase in the frequency of micronucleated cells was evaluated in the mouse cell line L5178Y TK^+/-. The study was performed according to the international guideline (OECD No. 487, adopted 26 September 2014) and in compliance with the principles of Good Laboratory Practice. After a preliminary cytotoxicity test, the test item, dissolved in dimethylsulfoxide, was tested in a single experiment with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

- Without S9 mix: 3 h treatment + 24 h recovery

- With S9 mix: 24 h treatment + 0 h recovery and 3 h treatment + 24 h recovery

Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells. Then, after the final cell counting, the cells were washed and fixed. Then, cells from three dose levels of the test item treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each treatment condition, micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid. Since the test item was found freely soluble but cytotoxic in the preliminary test, the highest dose level selected for the main experiment was based on the level of cytotoxicity, according to the criteria specified in the international regulations. Neither precipitate nor emulsion was observed in the culture medium at the end of the treatment periods, at any dose levels. With a treatment volume of 0.5% (v/v) in culture medium, the dose levels selected for the treatments were 10, 20, 40, 60, 80, 100, 150, 175 and 200 µg/m L with and without S9 mix.

Experiments without S9 mix

Following the 3-hour treatment, a marked to severe cytotoxicity was induced at dose levels = 60 µg/mL, as shown by a 68 to 100% decrease in the PD. Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose levels = 40 µg/mL, as shown by a 32 to 100% decrease in the PD.

The dose levels selected for micronucleus analysis were 10, 20 and 40 µg/mL, the latter inducing levels of cytotoxicity of 23 and 32% for the 3-hour and 24 -hour treatments, respectively, but higher dose levels being too cytotoxic. No statistically significant increase in the frequency of micronucleated cells was noted either after the 3- or 24-hour treatments. Furthermore, frequencies of micronucleated cells remained consistent with vehicle control historical ranges and no dose-response relationship was observed. The recommended level of cytotoxicity was not reached in either condition (3- or 24-hour treatments), however, considering the narrow dose levels spacing used, the available results were considered as suitable to allow a reliable interpretation. In the absence of S9 mix, the results met the criteria of a negative response.

Experiment with S9 mix

A slight to severe cytotoxicity was induced at dose levels = 40 µg/mL, as shown by a 36 to 100% decrease in the PD.

The dose levels selected for micronucleus analysis were 20, 40 and 60 µg/mL, the latter inducing the recommended level of cytotoxicity (i.e. 58% decrease in the PD). No statistically significant increase in the frequency of micronucleated cells was noted. Furthermore, frequencies of micronucleated cells remained consistent with vehicle control historical range and no dose-response relationship was evidenced.

Under the experimental conditions of the study, Diisopropyl peroxydicarbonate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK^+/-mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

OECD 489

The search for potential genotoxic activity of Bisisopropyl peroxydicarbonate (CAS 105-64-6) was assessed using the in vivo comet assay under alkaline conditions (SCGE) in the liver, glandular stomach and duodenum, on CD Sprague-Dawley rats, according to OECD Guidelines (No. 489, 2016).
The treatment was carried out by oral route (gavage), using 1 daily treatment for 2 consecutive days with the OCDE guideline 489 (2016) at the dose-levels of 1000 – 500 – 250 mg/kg b.w./day base on the results of preliminary assay up to 2000 mg/kg b.w./day.

No increase in the percentage of DNA in tail was observed in liver and duodenum.

Investigations on the glandular stomach are in progress.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2022 - 19 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat is the species recommended by OECD guidelline for this test.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE
- Age at study initiation: 8 weeks old
- Weight at study initiation: 201 g to 230 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in polypropylene cages
- Diet : A04C-10 from SAFE (batch 19330), ad libitum
- Water : ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %,
- Air changes (per hr): not precised, but ventilated system
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

Dose volume : 10 mL/kg b.w.
emulsion

In the main genotoxicity assay, three preparations of the test item at the concentrations of 100 - 50 and 25 mg/mL were prepared, giving final doses of 1000 - 500 and 250 mg/kg b.w./day (x2), respectively when administered at 10 mL/kg b.w..
Preparations for treatment were performed on the day of the 1st treatment and the formulations were used within 24 hours for the 2nd treatment.

Duration of treatment / exposure:

2 daily treatments at 24-hour intervals

Frequency of treatment:

2 daily treatments at 24-hour intervals

Post exposure period:

2-6 hours after the second treatment

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

5 males/group, except for the high dose group : 7

Control animals:

yes, concurrent vehicle

Positive control(s):

A total of 5 animals were treated orally twice with Methylmethane sulfonate (Aldrich, batch MKCL6261) in sterile water (Fresenius, Batch 13RPD081), 100 mg/kg b.w./day, gavage, 2 daily treatments at 24-hour intervals and sacrified 2 to 6 hours after the last treatment.

Interpretation of the results
For a test item to be considered positive in the comet assay, it must be observed:
- At least one of the treatment groups exhibits a statistically significant increase in the mean of medians of percentage of DNA in tail compared with the concurrent vehicle control,
- This increase is dose-related when evaluated with an appropriate trend test, and
- Any of these results are outside the distribution of the historical vehicle control data and/or the intervals of historical data for individual values.
When all of these criteria are met, the test chemical is then considered able to induce DNA strand breakage in the tissues studied in this test system.

A test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control,
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical vehicle control data and/or the intervals of historical data for individual values
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.
The test chemical is then considered unable to induce DNA strand breakage in the tissues studied in this test system.

Tissues and cell types examined:

Two to six hours after the second treatment, the rats are killed and cells from the selected target organs (i.e. liver, glandular stomach and duodenum) are isolated for the comet assay.
Five males of each group were assigned for cell isolation and assessed for DNA fragmentation.
Individual animals were anaesthetized with isoflurane and exsanguined.

Single cell preparations were done within one hour after animal sacrifice.
A 'V' shaped incision was made from the centre of the lower abdomen to the rib cage. The skin and muscles were removed to reveal the abdominal cavity.
A portion of the liver, the glandular stomach and the duodenum was removed and washed in the cold mincing buffer until as much blood as possible has been removed. The portion was minced or scrapped with a pair of fine scissors to release the cells. The cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a hemocytometer, and sufficient cells to obtain 25± 5 x 103 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.

Number of cells observed per animal: 150
Number of cells observed per dose: 750

Details of tissue and slide preparation:

After isolation, single cells are embedded in agarose on microscope slides and the obtained microgels are successively submitted to lysis, unwinding and electrophoresis in alkaline conditions and under dimmed light to prevent any additional DNA damage. After neutralization, slides are dried and could then be stained with a fluorescent dye (e.g. propidium iodide) before analysis and scoring. The method used for quantifying DNA migration involves a computerized image analysis system in order to collect comet data; then, the dedicated software allows the calculation of metrics for DNA migration.

Determination of the cytotoxicity of the test item
Statistically significant increases in the percentage of hedgehogs were observed in the 1000 and 250 mg/kg b.w./day (x2) treated groups when compared to the vehicle group (See Table 12) with values of 22.68 and 16.21%, respectively, vs. 12.79% in the relative vehicle control. The values were however clearly below 50%.

Evaluation criteria:

Expression of the results
- the median per slide of the percentage of DNA in tail for at least 50 cells
- the mean of medians of the percentage of DNA in tail per animal (i.e. 3 slides, 150 cells)
- the mean of medians per concentration (i.e. 5 animals, 750 cells)
In addition, each slide was also examined for presence of hedgehogs (possible indicator of toxicity and or apoptosis). Hedgehogs were excluded from image analysis data collection. However, determining their frequency might be useful for data interpretation. Therefore, the percentage of hedgehogs was recorded for each slide per animal and per organ. The hedgehogs, also known as clouds or ghost cells, are morphological indicative of highly damaged cells often associated with severe genotoxicity, necrosis and apoptosis. A hedgehog results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum.
A study is accepted if the following criteria are fulfilled:
- Concurrent vehicle controls should be within the control limits of the distribution of the laboratory’s historical vehicle control database and in the intervals of historical data for individual values.
- The concurrent positive controls should induce responses that are comparable to the historical positive control data and produce a statistically significant increase compared with the concurrent vehicle control.
- The appropriate number of doses and cells must be analysed.

Moreover, in the vehicle group, an eventual increase in the percentage of hedgehogs, must not be >50%.

Statistics:

In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software GraphPad Version 3.10.
As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups.
The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control. This test was also used to compare vehicle control and positive control to determine acceptable criteria of a valid test.

Sex:

male

Genotoxicity:

negative

Remarks:

on liver cells

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

on duodenum cells

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A slight decrease in spontaneous motor activity was noted at 1000 mg/kg b.w. 15 minutes and between 2 to 4 hours after the 2nd treatment.

Liver cells
Determination of the cytotoxicity of the test item
A statistically increase in the percentage of hedgehogs was observed in the 1000 mg/kg b.w./day (x2) treated groups when compared to the vehicle group with 4.12% of ghost cells vs. 2.31% in the relative control group.

Determination of the genotoxicity of the test item
No statistically significant increase in the mean of medians for the percentage of DNA in tail was induced at the three analysed doses of 1000, 500 and 250 mg/kg b.w./day (x2), vs. the vehicle control. Indeed, the means of medians of the percentage of tail DNA ranged from 0.13 to 0.68% vs. 0.09% in the respective vehicle control.
No assessment of systemic exposure was done, however clinical signs such as decrease in spontaneous motor activity were observed at the top dose tested and a slight increase in the percentage of ghost cells was noted.
The test item Bisisopropyl peroxydicarbonate was thus not genotoxic in rat liver. Indeed, no primary DNA damage was detected.

Duodenum cells
Determination of the cytotoxicity of the test item
No statistically significant increase in the percentage of hedgehogs was noted at the doses tested of 1000, 500 or 250 mg/kg b.w./day (x2) treated groups when compared to the vehicle group.

Determination of the genotoxicity of the test item
No statistically significant increases in the mean of medians for the percentage of DNA in tail was induced at the three tested doses of 1000, 500 and 250mg/kg b.w./day (x2), vs. the vehicle control. Indeed, the means of medians of the percentage of tail DNA ranged from 1.23 to 2.29% vs. 2.79% in the respective vehicle control.
The test item Bisisopropyl peroxydicarbonate was thus not genotoxic in rat duodenum. Indeed, no primary DNA damage was detected.

Glandular stomach cells
In progress.

Analytical results:
The control of concentrations of Bisisopropyl peroxydicarbonate in treatment preparations was performed in a GLP compliant laboratory following a validated method. The resuslts are reliable; concentrations were determined to be within the range 85-115% of the treatment with percent recovery between the theoretical and actual concentrations of -1 or -0.7% at the concentrations ranging from 25 to 100 mg/mL (report in progress).

Conclusions:

Under our experimental conditions, the test item does not present DNA strand breaks and/or alkali-labile sites inducer activities toward the liver and the duodenum from CD Sprague-Dawley male rats.
Investigations on the glandular stomach are in progress.

Executive summary:

The search for potential genotoxic activity of Bisisopropyl peroxydicarbonate (CAS 105-64-6) was assessed using the in vivo comet assay in the liver, glandular stomach and duodenum in the rat.
The treatment was carried out by oral route (gavage), using 1 daily treatment for 2 consecutive days with the OCDE guideline 489 (2016) at the dose-levels of 1000 – 500 – 250 mg/kg b.w./day base on the results of preliminary assay up to 2000 mg/kg b.w./day.

No increase in the percentage of DNA in tail was observed in liver and duodenum.

Investigations on the glandular stomach are in progress.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No increase in the percentage of DNA in tail was observed in liver and duodenum. Investigations on the glandular stomach are in progress.

Justification for classification or non-classification

The available data warranted no classification according to CLP/GHS criteria. Investigations on the glandular stomach are in progress.