3-chloroperbenzoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was negative in a reliable Ames test (bacterial reverse mutation assay) employing Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2 uvrA, both in the presence and absence of a metabolic activation system (rat liver S9).

The substance was negative in a reliable chromosome aberration test employing cultured human lymphocytes, either in the presence or absence of a metabolic activation system (rat liver S9).

The substance was negative in a reliable TK mutation assay, both in the presence and absence of a metabolic activation 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:

6 November 2017 - 14 December 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)

Deviations:

yes

Remarks:

An incorrect amount of the positive control was added to the plates. Since all positive control values were within the laboratory historical control data ranges, this deviation has no influence on the results of the study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: A0378021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: 31 October 2019 (retest date)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was soluble in ethanol.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) from animals treated with Aroclor 1254.

Test concentrations with justification for top dose:

Dose-range finding test - eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of the test item used in the first mutation assay was the level at which the test item inhibited bacterial growth.
First Experiment: Direct Plate Assay - 0.18, 0.55, 1.7, 5.4, 17 and 52 μg/plate
Second Experiment: Pre-Incubation Assay - 0.17, 0.55, 1.7, 5.4, 17, 52 and 164 µg/plate using TA100 and WP2uvrA. Based on the results of this test the following concentrations were used for TA1535, TA1537 and TA98:
Absence of S9-mix: 0.028, 0.088, 0.28, 0.86, 2.7 and 8.4 μg/plate.
Presence of S9-mix: 0.28, 0.86, 2.7, 8.4, 26 and 82 μg/plate.

Vehicle / solvent:

Ethanol.

Untreated negative controls:

yes

Remarks:

Ethanol

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Details on test system and experimental conditions:

All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Not applicable.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

In conclusion, the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In the in vitro genotoxicity study (Ames test) the substance was tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and in Escherichia coli strain WP2 uvrA. A dose-range finding test was conducted using eight concentrations of the substance (1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate) were tested in triplicate.  The highest concentration of the substance used in the first mutation assay was the level at which the substance inhibited bacterial growth. This was followed with the first experiment (Direct Plate Assay) employing concentrations of 0.18, 0.55, 1.7, 5.4, 17 and 52 μg/plate. The second experiment (Pre-Incubation Assay) used concentrations of 0.17, 0.55, 1.7, 5.4, 17, 52 and 164 µg/plate with TA100 and WP2uvrA. Based on the results of this test the following concentrations were used for TA1535, TA1537 and TA98:

in the absence of S9-mix: 0.028, 0.088, 0.28, 0.86, 2.7 and 8.4 μg/plate and in the presence of S9-mix: 0.28, 0.86, 2.7, 8.4, 26 and 82 μg/plate. No evidence of mutagenic activity was seen at any concentration of the substance in either mutation test. It was concluded that the substance showed no evidence of mutagenic activity in these bacterial systems under the test conditions employed.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2017 - 18 February 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

yes

Remarks:

In cytogenetic assay 2A not enough cells were scored in one of the duplicates of the dose level of 250 µg/mL. The duplicate culture showed similar results. Therefore this deviation was considered not to have affected the study integrity.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: A0378021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: 31 October 2019 (retest date)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was soluble in ethanol.

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined.

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

The highest tested concentration was determined by the solubility of 3- Chloroperoxybenzoic Acid in the culture medium.
In the dose-range finding study, blood cultures were treated with 31.3, 62.5, 125, 250, 500 and 1000 µg 3- Chloroperoxybenzoic Acid/mL culture medium with and without S9-mix.
Based on the results of the dose-range finding test the following dose levels were selected for the cytogenetic assay:
With and without S9-mix: 25, 50, 100, 150, 200, 250, 300 and 350 µg/mL culture medium (3 h exposure time, 24 h fixation time)
The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 25, 75, 100, 150, 200, 300, 400 and 500 µg/mL culture medium (24 h and 48 h exposure time, 24 h and 48 h fixation time)

Vehicle / solvent:

Ethanol.

Untreated negative controls:

yes

Remarks:

Ethanol

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

mammalian cell line, other: lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

Conclusions:

Both in the absence and presence of S9-mix the substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

Executive summary:

The potential for the substance to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix) was evaluated in an in vitro study. The possible clastogenicity of the test item was tested in two independent experiments. In the first cytogenetic assay, the substance was tested up to 300 and 200 µg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, the substance was tested up to 200 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 100 µg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. The substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. In conclusion, the substance is not clastogenic in human lymphocytes under the experimental conditions employed in this study. 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 March 2018 - 14 May 2018

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:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Batch: A0378021
Purity/Composition: 98.8% of 3- Chloroperoxybenzoic acid (72%) balanced with 3- chlorobenzoic acid (8.8%) and water (18%)
Test item storage: In refrigerator (2-8°C)
Stable under storage conditions until: 31 October 2019 (retest date)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/--3.7.2C mouse lymphoma cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 1000 μg/mL (range 63 to 1000 µg/mL) exposure medium.
First Mutagenicity Test: Without and with S9-mix: 1, 5, 10, 50, 100, 300, 600, 700, 800, 900 and 1000 μg/mL exposure medium.
Too many dose levels showed severe cytotoxicity, therefore this experiment was repeated (experiment 1A): the following dose-ranges were selected:
Without S9-mix: 10, 20, 40, 50, 100, 150, 200, 250, 300, 600, 700, 800, 900 and 1000 μg/mL exposure medium.
With S9-mix: 10, 20, 30, 40, 50, 75, 100, 200, 300, 600, 700, 800, 900 and 1000 μg/mL exposure medium.
Second Mutagenicity Test: 3.1, 6.3, 12.5, 25, 50, 100, 200, 300, 400, 450 and 500 µg/mL exposure medium (based on the results of the dose-range finding test and experiment 1)

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Remarks:

Ethanol

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Rationale for test conditions:

Recommended test conditions in international guidelines (e.g. OECD).

Evaluation criteria:

Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A substance is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

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:

In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a substance concentration range of 63 to 1000 µg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.
3 hours of treatment: In the absence and presence of S9-mix, the relative suspension growth was 2 and 8% at the substance concentration of 1000 μg/mL, respectively, compared to the relative suspension growth of the solvent control.
24 hours of treatment: The relative suspension growth was 8% at the substance concentration of 500 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the substance concentration of 1000 μg/mL.

Conclusions:

The substance is not mutagenic in the TK mutation test system under the experimental conditions described in this study.

Executive summary:

The mutagenic potential of the substance was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix).  The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period based on the most recent OECD guideline. In the first experiment, the substance was tested up to the concentration of 1000 µg/mL in the absence and presence S9-mix. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 21 and 30% in the absence and presence of S9-mix, respectively. The substance precipitated in the culture medium at this dose level. In the second experiment, the substance was tested up to the concentration of 400 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 11%. The substance did not precipitate in the culture medium at this dose level. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. In the absence of S9-mix, the substance did not induce a biologically relevant increase in the mutation frequency in the first experiment, i.e. none of the tested concentrations reached a mutation frequency of MF_(controls)+ 126. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the substance did not induce a biologically relevant increase in the mutation frequency, i.e. none of the tested concentrations reached a mutation frequency of MF_(controls)+ 126. the study concluded that the substance is not mutagenic in the TK mutation test system under the experimental conditions described in this study.

  

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 March 2018 - 19 June 2018

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:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test concentrations with justification for top dose:

First Mutagenicity Test:
Without S9-mix: 10, 20, 40, 50, 100, 150, 200, 250, 300, 600, 700, 800, 900 and 1000 μg/mL exposure medium.
With S9-mix: 10, 20, 40, 50, 100, 300, 800 and 1000 μg/mL exposure medium.

Second Mutagenicity Test
3.1, 6.3, 12.5, 25, 50, 100, 200, 300, 400, 450 and 500 µg/mL exposure medium.

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The substance is not mutagenic in the TK mutation test system

Executive summary:

The mutagenic potential of the substance was evaluated by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The vehicle of the test item was ethanol. In the first experiment, the substance was tested up to the concentration of 1000 µg/mL in the absence and presence S9-mix. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 21 and 6% in the absence and presence of S9-mix, respectively. The substance precipitated in the culture medium at this dose level. In the second experiment, the substance was tested up to the concentration of 400 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 11%. The substance did not precipitate in the culture medium at this dose level. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, the substance did not induce a biologically relevant increase in the mutation frequency in the first experiment, i.e. none of the tested concentrations reached a mutation frequency of MF_(controls)+ 126. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the substance did not induce a biologically relevant increase in the mutation frequency, i.e. none of the tested concentrations reached a mutation frequency of MF_(controls)+ 126. In conclusion, the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the findings of reliable in vitro genotoxicity studies conducted on the substance, classification of the substance is not justified.