Dipropyl peroxydicarbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential of LUPEROX 221 to induce reverse mutations was evaluated inSalmonella typhimurium (Brient, 2016). The study was performed according to the international guidelines and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of LUPEROX 221, dissolved in ethanol, to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteriaSalmonella typhimuriumwere used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at, 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.

According to available solubility data, the test item was found to be freely soluble in the vehicle at the concentration of 200 mg/mL. Consequently, using a maximum treatment-volume of 25 µL/plate, the dose-levels used for the preliminary toxicity test were 10, 100, 500, 1000, 2500 and 5000 µg/plate. No precipitate was observed in the Petri plates when scoring the revertants, at any of the tested dose-levels. A strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at dose-levels >= 100 µg/plate in the three strains without S9 mix, and at dose-levels >= 500 µg/plate in the three strains with S9 mix. Since the test item was found to be toxic in the preliminary test, the selection of the highest dose-level to be used in the mutagenicity experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were six analysable dose-levels for each strain and test condition. The study was therefore considered to be valid. No precipitate was observed in the Petri plates when scoring the revertants, at any of the tested dose-levels.

The selected dose-levels were 0.69, 2.06, 6.17, 18.5, 55.6 and 166.7 µg/plate without S9 for the five strains in both mutagenicity experiments. A moderate to strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at 166.7 µg/plate towards the five strains in the first experiment, then at dose-levels >= 55.6 µg/plate in the TA 102 strain and >=18.5 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains in the second experiment. The selected dose-levels were 2.06, 6.17, 18.5, 55.6, 166.7 and 500 µg/plate with S9 for the five strains in both mutagenicity experiments. A strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at the highest tested dose-level of 500 µg/plate towards the five strains used in both mutagenicity experiments. The test item did not induce any noteworthy increase in the number of revertants, in either experiment, in any of the five tested strains.

LUPEROX 221 did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella typhimuriumstrains, either in the presence or absence of a rat liver metabolizing system. 

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 March 2016 - 08 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

The selected dose-levels for the main mutagenicity experiments were the following (based on the toxicity observed in the preliminary test):without S9 mix: 0.69, 2.06, 6.17, 18.5, 55.6 and 166.7 µg/plate for the five strains in both mutagenicity experiments. with S9 mix: 2.06, 6.17, 18.5, 55.6, 166.7 and 500 µg/plate for the five strains in both mutagenicity experiments.

Vehicle / solvent:

- Vehicle: ethanol - Justification for choice of vehicle: test item was found to be freely soluble in the vehicle at the concentration of 200 mg/mL.

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

benzo(a)pyrene

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar.DURATION- Preincubation period: 60 minutes- Incubation time: 48 to 72 hours.DETERMINATION OF TOXICITY- Method: decrease in number of revertant colonies and/or a thinning of the bacterial lawn.

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.

Key result

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

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 to be toxic in the preliminary test, the selection of the highest dose-level to be used in the mutagenicity experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix
The selected dose-levels were 0.69, 2.06, 6.17, 18.5, 55.6 and 166.7 µg/plate for the five strains in both mutagenicity experiments.
No precipitate was observed in the Petri plates when scoring the revertants, at any of the tested dose levels.
A moderate to strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at 166.7 µg/plate towards the five strains in the first experiment, then at dose-levels = 55.6 µg/plate in the TA 102 strain and = 18.5 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains in the second experiment.
The test item did not induce any noteworthy increase in the number of revertants, in either experiment, in any of the five tested strains.

Experiments with S9 mix
The selected dose-levels were 2.06, 6.17, 18.5, 55.6, 166.7 and 500 µg/plate for the five strains in both mutagenicity experiments.
No precipitate was observed in the Petri plates when scoring the revertants, at any of the tested dose levels.
A strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at the highest tested dose-level of 500 µg/plate towards the five strains used in both mutagenicity experiments.
The test item did not induce any noteworthy increase in the number of revertants, in either experiment, in any of the five tested strains.

Conclusions:

LUPEROX 221 did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The potential of LUPEROX 221 to induce reverse mutations was evaluated in Salmonella typhimurium. The study was performed according to the international guidelines and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose-levels of LUPEROX 221, dissolved in ethanol, to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at, 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.

According to available solubility data, the test item was found to be freely soluble in the vehicle at the concentration of 200 mg/mL. Consequently, using a maximum treatment-volume of 25 µL/plate, the dose-levels used for the preliminary toxicity test were 10, 100, 500, 1000, 2500 and 5000 µg/plate. No precipitate was observed in the Petri plates when scoring the revertants, at any of the tested dose-levels. A strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at dose-levels >= 100 µg/plate in the three strains without S9 mix, and at dose-levels >= 500 µg/plate in the three strains with S9 mix. Since the test item was found to be toxic in the preliminary test, the selection of the highest dose-level to be used in the mutagenicity experiments was based on the level of toxicity, according to the criteria specified in the international guidelines. The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were six analysable dose-levels for each strain and test condition. The study was therefore considered to be valid. No precipitate was observed in the Petri plates when scoring the revertants, at any of the tested dose-levels.

The selected dose-levels were 0.69, 2.06, 6.17, 18.5, 55.6 and 166.7 µg/plate without S9 for the five strains in both mutagenicity experiments. A moderate to strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at 166.7 µg/plate towards the five strains in the first experiment, then at dose-levels >= 55.6 µg/plate in the TA 102 strain and >=18.5 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains in the second experiment. The selected dose-levels were 2.06, 6.17, 18.5, 55.6, 166.7 and 500 µg/plate with S9 for the five strains in both mutagenicity experiments. A strong toxicity (thinning of the bacterial lawn and/or decrease in the number of revertants) was noted at the highest tested dose-level of 500 µg/plate towards the five strains used in both mutagenicity experiments. The test item did not induce any noteworthy increase in the number of revertants, in either experiment, in any of the five tested strains.

LUPEROX 221 did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)