OO-tert-butyl monoperoxymaleate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential of OO-tert-butyl monoperoxymaleate (target substance) to induce genotoxic effects was tested in an in vitro test battery. The target substance was tested negative in a bacterial reverse gene mutation test conducted according to OECD 471 and in a mammalian cell HPRT mutation assay conducted according to OECD 476. OO-tert-butyl monoperoxymaleate induced structural and/or numerical chromosomal damage in an in vitro micronucleus test in Chinese hamster V79 cells (OECD 487).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-03-24 to 2017-10-26

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:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS & MEDIA USED
V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections were carried out before freezing.
For the experiments thawed cultures were set up in 75 cm² cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 105 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made every 3-4 days.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

microsomal liver enzymes (S9)

Test concentrations with justification for top dose:

Pre-experiment for toxicity:
With and without metabolic activation: 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
Experiment 1:
Without metabolic activation: 20, 30, 40, 50, 55, 60 ,62.5, 65, 67.5, 70, 72.5, 75, 80, 90, 100 µg/mL
With metabolic activation: 100, 125, 150, 160, 170, 175, 180, 185, 190, 195, 200, 210, 225, 250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation, final concentration: 20 µg/mL

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation, final concentration: 2.5 µg/mL

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: colchicine

Remarks:

without metabolic activation, final concentration: 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 50000 cells were seeded per cell culture flask, containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS)
Seeding of the cultures: Three or four-day-old stock cultures (in exponential growth), more than 50% confluent, were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment. Cells subsequently were trypsinised with a solution of 0.2% trypsin in Ca-Mg-free PBS at 37 °C for 5 min. By adding complete culture medium the detachment was stopped and a single cell suspension was prepared.
Exponentially growing V79 cells were seeded into 25 cm2 cell culture flasks (two flasks per test group). Approx. 50000 cells were seeded per cell culture flask, containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS). After an attachment period of approx. 48 h, the complete culture medium was removed and subsequently the test item was added to the treatment medium in appropriate concentrations. The cells were incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 µg/mL cytochalasin B for 20 h at 37 °C.
At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 mL complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. Prior to this an aliquot of each culture was removed to determine the cell count by a cell counter (AL-Systems). After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.
Analysis of Micronuclei:
At least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to criteria of Fenech, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmatic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.
Number of cultures: Duplicate cultures were performed at each concentration level except for the pre-experiment.

DETERMINATION OF CYTOTOXICITY
- Methods: Cytokinesis Block Proliferation Index, % cytostasis

Evaluation criteria:

Acceptability of the Assay:
A mutation assay is considered acceptable if it meets the following criteria:
- The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database.
- Concurrent positive controls should induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
- Cell proliferation criteria in the negative control according to OECD 487 should be fulfilled.
- All experimental conditions are tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analysable.
- Criteria for the selection of top concentration are fulfilled.

Evaluation of Results:
A test item is considered to be clearly positive, if any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
- The increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

The nonparametric Chi-Quadrat Test was performed to verify the results of the experiment.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment (see Table 3 in box “Any other information on results incl. tables”).

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: The pH-value detected with the test item was within the physiological range (pH 7.4).
- Precipitation: No precipitate of the test item was noted in any concentration group evaluated in the experiment.
The cytotoxicity for the tested concentrations did not exceed the limit of 55% ± 5% cytotoxicity according OECD 487.

HISTORICAL CONTROL DATA
In experiment I without metabolic activation the micronucleated cell frequency of the negative control (0.50%) was within the historical control limits of the negative control (0.39% – 1.40%). The mean values of micronucleated cell frequencies found after treatment with the test item were 1.45% (40 µg/mL), 2.15% (60 µg/mL) and 2.90% (67.5 µg/mL). The micronucleus frequencies at concentrations of 40 µg/mL, 60 µg/mL and 67.5 µg/mL showed an increase compared to the concurrent negative control and the corresponding micronuclei frequencies were above the upper limit of the historical control limits of the negative control.
In experiment I with metabolic activation the micronucleated cell frequency of the negative control (1.05%) was within the historical control limits of the negative control (0.37% – 1.68%). The mean values of micronucleated cell frequencies found after treatment with the test item were 1.45% (100 µg/mL), 2.25% (125 µg/mL) and 3.50% (150 µg/mL). The micronuclei frequencies at concentrations of 125 µg/mL and 150 µg/mL were increased compared to the concurrent negative control and the corresponding micronuclei frequencies were above the upper limit of the historical control limits of the negative control.

TEST RESULTS

Cytotoxicity:
In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 40 µg/mL. At a concentration of 60 µg/mL a cytostasis of 39% and at a concentration of 67.5 µg/mL a cytostasis of 54% was noted.
In experiment I with metabolic activation an increase of the cytostasis above 30% was noted up to a concentration of 100 µg/mL. At a concentration of 125 µg/mL a cytostasis of 32% and at a concentration of 150 µg/mL a cytostasis of 50% was noted.

Clastogenicity/Aneugenicity:
In the main experiment I with and without metabolic activation biologically relevant increases of the micronucleus frequency were noted.
In experiment I without metabolic activation the micronucleated cell frequency of the negative control (0.50%) was within the historical control limits of the negative control (0.39% – 1.40%). The mean values of micronucleated cell frequencies found after treatment with the test item were 1.45% (40 µg/mL), 2.15% (60 µg/mL) and 2.90% (67.5 µg/mL). The micronucleus frequencies at concentrations of 40 µg/mL, 60 µg/mL and 67.5 µg/mL showed an increase compared to the concurrent negative control and the corresponding micronuclei frequencies were above the upper limit of the historical control limits of the negative control.
In experiment I with metabolic activation the micronucleated cell frequency of the negative control (1.05%) was within the historical control limits of the negative control (0.37% – 1.68%). The mean values of micronucleated cell frequencies found after treatment with the test item were 1.45% (100 µg/mL), 2.25% (125 µg/mL) and 3.50% (150 µg/mL). The micronuclei frequencies at concentrations of 125 µg/mL and 150 µg/mL were increased compared to the concurrent negative control and the corresponding micronuclei frequencies were above the upper limit of the historical control limits of the negative control.
The nonparametric x² Test was performed to verify the results of the experiment. Statistically significant enhancements (p< 0.05) of cells with micronuclei were noted in the concentration groups 40 µg/mL, 60 µg/mL and 67.5 µg/mL in experiment I without metabolic activation and in the concentration groups 125 µg/mL and 150 µg/mL in experiment I with metabolic activation.
The x² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. Statistically significant increases in the frequency of micronucleated cells under the experimental conditions of the study were observed in experiment I with and without metabolic activation.
MMS (20 µg/mL) and CPA (2.5 µg/mL) were used as clastogenic controls and colchicine as aneugenic control (1.5 µg/mL). They induced distinct and statistically significant increases of the micronucleus frequency. This demonstrates the validity of the assay.

Remarks on result:

other: Experiment 1

Table 3: Test for cytotoxicity

without metabolic activation
Dose Group	Concentration (µg/mL)	CBPI	Relative Cell Growth [%]	Cytostasis [%]	Precipitate +/-
C	0	1.53	100	0	-
S	0	1.53	100	0	-
1	3.9	1.58	109	0	-
2	7.8	1.54	102	0	-
3	15.6	1.50	94	6	-
4	31.3	1.41	78	22	-
5	62.5	1.09	17	83	-
6	125	1.05	9	91	-
with metabolic activation
Dose Group	Concentration (µg/mL)	CBPI	Relative Cell Growth [%]	Cytostasis [%]	Precipitate +/-
C	0	1.44	100	0	-
S	0	1.44	100	0	-
1	3.9	1.45	102	0	-
2	7.8	1.42	97	3	-
3	15.6	1.41	95	5	-
4	31.3	1.42	98	2	-
5	62.5	1.41	92	8	-
6	125	1.43	53	47	-

C: Negative Control (Culture medium)

CBPI: Cytokinesis Block Proliferation Index, CBPI = ((c₁x 1) + (c₂x 2) + (c_xx 3))/n

Relative Cell Growth:100 x ((CBPI_{Test conc}– 1) / (CBPI_control-1))

c₁:mononucleate cells

c₂:binucleate cells

c_x:multinucleate cells

n:total number of cells

 

Cytostasis [%] = 100- Relative Cell Growth [%]

the cytostasis is defined 0, when the relative cell growth exceeds 100%

Table 4: Summary of Micronuclei Effects: Experiment 1 with and without metabolic activation

without metabolic activation
Dose group	Concentration [µg/mL]	Treatment time	Fixation Interval	Micronucleated Cells Frequency [%]
C	0	4 h	24 h	0.50
3	40	4 h	24 h	1.45
6	60	4 h	24 h	2.15
9	67.5	4 h	24 h	2.90
MMS	20	4 h	24 h	3.00
Colchicine	1.5	4 h	24 h	5.75
with metabolic activation
Dose group	Concentration [µg/mL]	Treatment time	Fixation Interval	Micronucleated Cells Frequency [%]
C	0	4 h	24 h	1.05
1	100	4 h	24 h	1.45
2	125	4 h	24 h	2.25
3	150	4 h	24 h	3.50
CPA	2.5	4 h	24 h	5.30

C: Negative Control (Culture medium)

MMS: Methylmethanesulfonate, Positive Control (without metabolic activation) [20 µg/mL]

Colchicine: Positive Control (without metabolic activation) [1.5 µg/mL]

CPA: Cyclophosphamide, Positive Control (with metabolic activation) [2.5 µg/mL]

Conclusions:

OO-tert-butyl monoperoxymaleate did induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells under the experimental conditions of this in vitro experiment.

Executive summary:

In an in vitro mammalian micronucleus assay (OECD 487), V79 cells cultured in vitro were exposed to OO-tert-butyl monoperoxymaleate in cell culture medium in experiment I (short term exposure, 4 h) at concentrations of 40, 60 and 67.5 µg/mL (without metabolic activation) and at 100, 125 and 150 µg/mL (with metabolic activation). In experiment I (with and without metabolic activation) the micronucleus frequencies showed a statistically significant increase compared to the concurrent negative control and the corresponding micronuclei frequencies were above the upper limit of the historical control limits of the negative control. Since these positive findings were obtained in experiment I with and without metabolic activation the performance of experiment II with long term treatment was omitted. The positive controls did induce distinct and biologically relevant increases of the micronucleus frequency. Based on the results, it can be stated that the target substance did induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells in this study. This study is classified as acceptable and satisfies the requirements for Test Guideline OECD 487.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-04-03 to 2017-05-09

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:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name: OO-tert-butyl monoperoxymaleate
- Product: Peroxan PM-50
- CAS No.: 1931-62-0
- Batch No.: M-160502
- Molecular weight:188.18 g/mol
- Density: 1.04 g/cm³
- Physical state: suspension
- Colour: white
- Active components: 48.8% Peroxide
- Expiry date: not provided by the sponsor
- Storage conditions: =< +30°C
- Safety precautions: the routine hygienic procedures were sufficient to assure personnel health and safety

Target gene:

histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS SOURCES:
TA98, TA1535 and TA102: MOLTOX, INC., NC 28607, USA
TA100 and TA1537: Xenometrix AG, Switzerland

MEDIA USED:
Nutrient medium (per litre):
- 8 g Nutrient Borth
- 5 g NaCl
(solution of 125 µL ampicillin (10 mg/mL) (TA98, TA100, TA102) was added to retain phenotypic characteristics of strain)

Vogel-Bonner-salts (per litre)
- 10 g MgSO4 x 7 H2O
- 100 g citric acid
- 175 g NaNH4HPO4 x 4 H2O
- 500 g K2HPO4

Vogel-Bonner Medium E agar plates (per litre)
- 15 g Agar Agar
- 20 mL Vogel-Bonner salta
- 50 mL glucose-solution (40%)

Overlay agar (per litre)
- 7 g Agar Agar
- 6 g NaCl
- 10.5 mg L-histidine x HCl x H2O
- 12.2 mg biotin

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The test item concentrations in the main experiments were chosen according to the results of the pre-experiment:
Experiment 1: 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
Experiment 2: 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate

Vehicle / solvent:

- Vehicle: Aqua dest.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Aqua dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Aqua dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Aqua dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Aqua dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); pre-incubation

EXPERIMENTAL PERFORMANCE:
For plate incorporation method the following materials were mixed in test tube and poured over the surface of minimal agar plate:
- 100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
- 500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
- 100 µL Bacteria suspension (cf. Preparation of bacteria, pre-culture of the strain),
- 2000 µL Overlay agar.
For pre-incubation method 100 µL of test item preparation was pre-incubated with tester strains (100 µL) and sterile buffer or metabolic activation system (500 µL) for 60 min at 37°C prior to adding overlay agar (2000 µL) and pouring onto surface of minimal agar plate.

DURATION
- Pre-incubation period: 60 minutes at 37 °C
- Exposure duration: at least 48 h in the dark

NUMBER Of REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: diminution of background lawn; reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control

Evaluation criteria:

The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of solvent control.

Statistics:

not applicable

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: at 2.5 µL/plate and higher (with and without metabolic activation). Experiment II: at 0.316 µL/plate and higher /without metabolic activation) and at 1.0 µL/plate (with metabolic activation)

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

Remarks:

Experiment I: at 2.5 µL/plate and higher (with and without metabolic activation). Experiment II: at 0.316 µL/plate and higher (with and without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: at 2.5 µL/plate and higher (without metabolic activation) and at 5.0 µl/plate (with metabolic activation). Experiment II: at 0.316 µL/plate and higher (with and without metabolic activation)

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

Remarks:

Experiment I: at 2.5 µL/plate and higher (without metabolic activation) and at 5.0 µl/plate (with metabolic activation). Experiment II: at 0.316 µL/plate and higher (with and without metabolic activation).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: at 2.5 µL/plate and higher (with and without metabolic activation). Experiment II: at 0.316 µL/plate and higher (with and without metabolic activation).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

Remarks on result:

other:

Remarks:

Experiment 1

Pre-experiment for Toxicity & Main Experiments 1 and 2:

Result tables: see box "Attached background material"

Conclusions:

The test item is not genotoxic in the bacterial reverse gene mutation assay in the presence and absence of mammalian metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471) strains of S. typhimurium (TA1537, TA1535, TA102, TA100 and TA98) were exposed to OO-tert-butyl monoperoxymaleate in Aqua dest. at concentrations of 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µl/plate (experiment I) and for experiment II at concentrations of 0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 µL/plate in the presence and absence of mammalian metabolic activation. The positive controls did induce the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-03-24 to 2017-09-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Baed on a prior solubility test, the test item was dissolved in cell culture medium (MEM + 0% FBS (4 h treatment); MEM + 10% FBS (20 h treatment)) and diluted prior to treatment

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus of V79 cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins BioPharma Product Testing Munich GmbH stock cultures
- Suitability of cells: V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate


MEDIA USED
Type and identity of media:
Short-term exposure: MEM medium, 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Long-term exposure: MEM Medium with 10 % fetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

microsomal liver enzymes (S9)

Test concentrations with justification for top dose:

Pre-test for toxicity: 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 1.5, 2 mg/mL without/with S9 mix
Main test:
Experiment I: 0.05, 0.1, 0.13, 0.16, 0.18, 0.2, 0.22, 0.25, 0.3, 0.4 mg / mL without S9 mix
Experiment II: 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 mg / mL with S9 mix

The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to OECD guideline 476.

Vehicle / solvent:

- vehicle/solvent used: MEM + 0% FBS (4 h treatment) and MEM + 10% FBS (20 h treatment)
- justification for choice of solvent: The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

Treatment medium (MEM Medium)

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation, final concentration 300 µg/ml

Untreated negative controls:

yes

Remarks:

Treatment medium (MEM Medium) plus S9 mix

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation, final concentration 1.0 and 1.5 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (short time exposure, Experiment I with and without metabolic activation)
20 hours (long time exposure, Experiment II with and without metabolic activation)
- Expression time (cells in growth medium): 6 - 7 days
- Selection time (if incubation with a selection agent): 7 - 9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 - 12 days

SELECTION AGENT (mutation assays): Methanol


DETERMINATION OF CYTOTOXICITY
- Methods: Relative Survival, based on the cloning efficiency

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
- one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

Statistical significance át the 5% level (p < 0.05) was evaluated by means of the non - parametric Mann - Whitney test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The selection of the concentrations used in the main experiments was based on data from the pre-experiments according to the OECD guideline 476. 0.3 mg/mL (without metabolic activation) and 0.5 mg/mL (with metabolic activation) were selected as the highest concentrations. The experiment with and without metabolic activation was performed as a 4 h short-term exposure assay.

POSITIVE CONTROL:
- Positive control: With metabolic activation: The positive controls, DMBA (1.0 and 1.5 µg/mL) and EMS (300 µg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.
Without metabolic activation: The positive control EMS induced a distinct increase in mutant frequency with 272.1 mutants/10^6 cells.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Negative (solvent/vehicle) historical control data: In the experiment with metabolic activation, the mutant values of the negative controls were slightly increased and therefore outside the historic control range. Nevertheless, the negative control values were considered acceptable for inclusion in the historical control data set as they were only slightly increased and no technical reason or human failure was determined. In the experiment without metabolic activation, the mutant values of the negative controls and all mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 7.8 – 39.7 mutants per 10^6 cells).

For individual results, see Tables 2-5 in box "Any other information on results incl. tables".

Table 2: Experiment I – Toxicity, without metabolic activation

Dose Group	Concen-tration	Number of cells at the		Number of colonies per flask			CE[%]	Adjusted CE [%]	Relative Survival (RS) [%]
	[mg/mL]	beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	12801000	188	149	169	84	108	100
NC2		10000000	11679000	171	182	177	88	103
1	0.05	20000000	26010000	188	183	186	93	121	114
2	0.1	20000000	25534000	135	197	166	83	106	100
3	0.13	20000000	23528000	204	167	186	93	109	103
4	0.16	20000000	24582000	158	155	157	78	96	91
5	0.18	20000000	23834000	118	140	129	65	77	73
6	0.2	20000000	23868000	103	150	127	63	75	72
7	0.22	20000000	23732000	101	114	108	54	64	60
8	0.25	20000000	20264000	102	125	114	57	57	55
9	0.3	20000000	15334000	47	46	47	23	18	17
10	0.4	20000000	12682000	9	5	7	4	2	2
EMS	300 µg/ml	10000000	12053000	153	135	144	72	87	82

Table 3: Experiment I – Mutagenicity, without metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration	Number of colonies per flask			CE [%]	Number of colonies per flask							CE [%]	Mutant Frequency per 10^6cells
	[mg/mL]	I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	142	171	157	78	12	11	11	12	10	11.2	0.7	0.0028	35.8
NC2		143	152	148	74	5	8	13	15	6	9.4	3.9	0.0024	31.9
2	0.10	157	142	150	75	4	10	12	10	9	9.0	2.7	0.0023	30.1
4	0.16	147	140	144	72	5	8	9	5	8	7.0	1.7	0.0018	24.4
5	0.18	153	146	150	75	3	5	4	9	2	4.6	2.4	0.0012	15.4
7	0.22	165	130	148	74	7	14	13	8	9	10.2	2.8	0.0026	34.6
8	0.25	126	148	137	69	7	9	5	8	10	7.8	1.7	0.0020	28.5
9	0.30	118	153	136	68	3	5	6	10	5	5.8	2.3	0.0015	21.4
EMS	300 µg/mL	110	137	124	62	50	69	72	75	70	67.2	8.8	0.0168	272.1

NC:     negative control

P:        precipitation at the end of treatment

CE:     cloning efficiency

EMS:  Ethylmethanesulfonate

 Table 4: Experiment I – Toxicity,with metabolic activation

Dose Group	Concen-tration	Number of cells at the		Number of colonies per flask			CE[%]	Adjusted CE [%]	Relative Survival (RS) [%]
	[mg/mL]	beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	12274000	150	153	152	76	93	100
NC2		10000000	12189000	129	166	148	74	90
1	0.05	20000000	25228000	171	156	164	82	103	113
2	0.1	20000000	25058000	159	174	167	83	104	114
3	0.15	20000000	24004000	160	185	173	86	104	113
4	0.2	20000000	25874000	143	121	132	66	85	93
5	0.25	20000000	26622000	162	138	150	75	100	109
6	0.3	20000000	23868000	143	152	148	74	88	96
7	0.35	20000000	24548000	87	103	95	48	58	64
8	0.4	20000000	22440000	57	70	64	32	36	39
9	0.45	20000000	21012000	34	23	29	14	15	16
10	0.5	20000000	22678000	29	16	23	11	13	14
DMBA1	1.0 µg/mL	10000000	12988000	141	149	145	73	94	103
DMBA2	1.5 µg/mL	10000000	13923000	84	97	91	45	63	69

Table 5: Experiment I – Mutagenicity, with metabolic activation

		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration	Number of colonies per flask			CE [%]	Number of colonies per flask							CE [%]	Mutant Frequency per 10^6cells
	[mg/mL]	I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	169	179	174	87	12	14	16	21	11	14.8	3.5	0.0037	42.5
NC2		155	176	166	83	18	13	15	12	23	16.2	4.0	0.0041	48.9
5	0.25	174	173	174	87	27	24	15	19	25	22.0	4.4	0.0055	63.4
6	0.30	172	171	172	86	14	13	16	12	14	13.8	1.3	0.0035	40.2
7	0.35	177	197	187	94	13	15	11	18	16	14.6	2.4	0.0037	39.0
8	0.40	180	179	180	90	10	18	22	16	16	16.4	3.9	0.0041	45.7
9	0.45	184	194	189	95	8	5	6	4	9	6.4	1.9	0.0016	16.9
10	0.50	174	189	182	91	12	12	12	8	12	11.2	1.6	0.0028	30.9
DMBA1	1.0 µg/mL	155	156	156	78	205	169	214	193	166	189.4	19.1	0.0474	609.0
DMBA2	1.5 µg/mL	148	156	152	76	249	238	236	234	241	239.6	5.2	0.0599	788.2

NC:        negative control

S:           solvent control (DMSO)

P:           precipitation at the end of treatment

CE:        cloning efficiency

DMBA:  7,12-dimethylbenz(a)anthracene

Conclusions:

Under the experimental conditions reported, the test item OO-tert-butyl monoperoxymaleate is considered to be non-mutagenic in the HPRT gene mutation assay using V79 chinese hamster cells.

Executive summary:

In a mammalian cell HPRT gene mutation assay, V79 cells cultured in vitro were exposed to OO-tert-butyl monoperoxymaleate (48.8% Peroxide) at concentrations of 0.25, 0.3, 0.35, 0.4, 0.45 and 0.5 mg/mL in the presence of metabolic activation and at concentrations of 0.1, 0.16, 0.18, 0.22, 0.25 and 0.3 mg/mL in the absence of mammalian metabolic activation (experiment I). The results of the first experiment were found to be negative and thus, a second experiment with long-term treatment was not considered necessary. For all tested treatment groups no dose-response relationship could be observed. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Because of the positive result in an in vitro micronucleus test according to OECD 487, an in vivo mammalian alkaline comet assay was requested by ECHA (decision TPE-D-2114557072-54-01/F from 01 June 2021).

The test item O,O-tert-butyl monoperoxymaleate did not induce biologically relevant increases in DNA-strand breaks in any of the tissues evaluated (liver, glandular stomach, duodenum) after oral (gavage) administration up to the maximum tolerated dose (600 mg/kg bw/day) in male rats. Therefore, the test item is considered to be non-DNA damaging under the experimental conditions of the in vivo mammalian Alkaline Comet Assay (OECD 489).

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:

2022-04-06

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:

adopted July 29, 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Pergan GmbH, batch no.: 1330431-01
- Purity of active component: 37% (w/w); contains phlegmatizer Triacetin at concentrations ≤60% (w/w)
- Expiry Date: 25 November 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8°C, protected from light.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was diluted in PEG400. The test item formulations were prepared freshly on each administration day. The prepared formulations were transferred to BSL Munich protected from light together with the control formulations under consideration of stability.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rats have been used for many years as standard experimental animals in genotoxicity investigations. In addition, they have been used as experimental animal in the toxicological safety characterization of the test item. Data from such experiments are useful for the design of the alkaline Comet Assay and interpretation of its findings.
According to OECD TG 489, healthy young adult rodents are recommended and rats being routinely used in this test.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 7-9 weeks old
- Weight at study initiation: interval within 20% of the mean weight
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: The animals are kept in groups of 2-3 animals/sex/group/cage in IVCs (type III H, polysulphone cages) on Altromin saw fibre bedding
- Diet: ad libitum, Altromin 1324 maintenance diet for rats and mice
- Water: ad libitum, tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: adequate acclimatisation period (at least five days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

:

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Test item (OO-tert-butyl monoperoxymaleate) and additional control (Triacetin) were dissolved in polyethylene glycol (PEG 400).
The positive control ethyl methanesulfonate was dissolved in physiol. saline (0.9% NaCl);
- Justification for choice of solvent/vehicle: The vehicle was chosen according to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 12/30/60 mg/ml for the 120/300/600 mg/kg bw/day dose groups, respectively (administration volume 10 ml/kg bw).
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw.
- Lot/batch no.: PEG 400: Sigma-Aldrich BCCG1486 (pre-experiment), BCCG9382 (Main Study); physiological saline: B.Braun; Batch: 13QLP251

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item was diluted in PEG400. The vehicle was chosen according to its relative non-toxicity for the animals. On sponsors request no correction factor for purity was used in dose calculations. All animals received a single volume orally of 10 mL/kg bw.

- As the test item contained the phlegmatizer Triacetin, Triacetin was additionally used as control item. The final concentration for Triacetin was determined by the sponsor after the dose range finding with 360 mg/kg bw and was based on the concentration of the highest dose group (600 mg/kg bw) of the test material. Triacetin was weighed into a tared plastic vial on a precision balance and the vehicle was added.

The test item formulations were prepared freshly on each administration day. The prepared formulations were transferred to BSL Munich protected from light together with the control formulations under consideration of stability.

Duration of treatment / exposure:

Test item, control (PEG 400) and additional control (Triacetin): Two consecutive days
Positive control (ethyl methanesulfonate): Once

Frequency of treatment:

daily

Post exposure period:

4 hours after last treatment, animals were sacrificed

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control (PEG 400)

Dose / conc.:

120 mg/kg bw/day

Remarks:

Test item (37% (w/w) active component)

Dose / conc.:

300 mg/kg bw/day

Remarks:

Test item (37% (w/w) active component)

Dose / conc.:

600 mg/kg bw/day

Remarks:

Test item (37% (w/w) active component)

No. of animals per sex per dose:

5 males per dose

Control animals:

yes, concurrent vehicle

other: Phlegmatizer Triacetin at 360 mg/kg bw/day

Positive control(s):

ethylmethanesulfonate
- Justification for choice of positive control(s): Ethyl methanesulfonate (EMS) as an appropriate DNA damaging agent (250 mg/kg bw) was used as positive control.
- Route of administration: oral
- Doses / concentrations: 250 mg/kg bw

Tissues and cell types examined:

Four hours after the last administration of the test item, samples of liver, glandular stomach, duodenum and gonads were taken. The liver, glandular stomach, and duodenum were analyzed for DNA strand breaks, while comet slides for male gonadal calls were prepared for later analysis for DNA strand breaks in case of positive results observed on somatic cells. All samples were kept in ice-cold mincing buffer and directly prepared. One part of the liver, glandular stomach and duodenum were preserved in 10% neutral-buffered formalin for histopathological evaluation.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Due to the severity of clinical signs (reduced spontaneous activity, hunched posture, piloerection and half eyes closed) and histopathological effects observed in a dose range finding study, a dose of 600 mg/kg bw was identified as the maximum tolerated dose (MTD) and selected as the highest dose (HD). Additional details on the results of the range-finding study are provided in section "Results and discussion" under "Additional information on results".
The dose level of the phlegmatizer Triacetin which was used as an additional control, was based on the high dose level of test material (600 mg/kg bw/day) and maximum content of Triacetin (60% (w/w)) in test item formulation.

DETAILS OF SLIDE PREPARATION:
- Preparation of single cells:
A portion of the liver was minced with a pair of scissors to isolate the cells. The cell suspension was kept for not more than 15 seconds until bigger fragments of the liver settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL low-melting agarose (LMA) 0.6% (w/v) in PBS solution.
The duodenum was flushed with a syringe filled with cold mincing buffer to wash out the food. The duodenum was cut open into two halves. One half of the duodenum was minced with a pair of scissors, the other one was kept for histopathology. The cell suspension was kept for not more than 15 seconds until bigger fragments settled on the bottom of the tube. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.
The stomach was cut open and washed free of food using cold water. A portion of the glandular stomach was minced with a pair of scissors. The pieces were further crushed with a pestle to release single cells. The suspension was kept for less than 15 seconds to allow large clumps to settle. A volume of 30 µL of the supernatant was pipetted into a tube and mixed with 270 µL LMA solution.
The end of the epithelial capsule of the gonad was punctured to squeeze out the seminiferous tubules. Without pressure the tubules were flattened with a tissue roller in D-PBS until it became cloudy with released cells. The cell suspension was kept on ice for 1-2 minutes until accidentally collected tubules settled on the bottom of the tube. The cell suspension was transferred to a new tube leaving the settled tubules behind and was centrifuged at 11,000 x g for 30 sec at 4°C. The supernatant was discarded carefully without disturbing the pellet, which was diluted in Mincing buffer. A volume of 30 μL of the supernatant was pipetted into a tube and mixed with 270 μL LMA solution.

The cell pellets (150 cells/animal/tissue) were shortly put on ice and were directly used for preparing comet slides.

The slides used were pre-coated with normal-melting agarose (NMA) and coded with a random number. A volume of 75 µL of cell suspension embedded in low-melting temperature agarose was placed on slides, which were covered with a cover slip and cooled for 10 min on ice (3 slides per animal and tissue).
Cover slips were carefully removed and the slides incubated overnight in chilled lysing solution at 2 - 8 °C in the fridge to lyse cellular and nuclear membranes and allow the release of coiled DNA loops during electrophoresis. After completion of lysis, the slides were rinsed in purified water to remove residual detergent and salts.
Prior to electrophoresis, the slides were incubated in alkaline (pH > 13) electrophoresis solution for 20 min at 4°C. After alkali unwinding, the single-stranded DNA was electrophoresed under alkaline conditions to enable the formation of DNA tails. The electrophoretic conditions were 0.7 V/cm and approximately 300 mA, with the DNA being electrophoresed for 30 min at 4°C. The slides were placed in a horizontal gel electrophoresis chamber, positioned close to the anode and covered with electrophoresis solution. Slides were placed in the electrophoresis chamber in a random order.
After electrophoresis, the slides were neutralised by rinsing with neutralisation buffer three times for 5 min each. The slides were incubated for approximately 10 – 20 min in ice-cold ethanol and air-dried afterwards.
Following dehydration, the cells were stained by applying 75 µL gel red staining solution on top of the slides and covering with a cover slip.

METHOD OF ANALYSIS:
Comet slides were analysed for potential DNA damage using a fluorescence microscope with magnification (200x) coupled to a camera and the Comet Software ‘Comet Assay IV’ (Perceptive Instrument, software version 2.1.2). The slides were coded so that the evaluator was not aware of which dose group was evaluated.
Cells were classified into three potential categories scorable, non-scorable and “hedgehog” (cells that exhibit a microscopic image consisting of a small or non-existent head and a large diffuse tail are considered to be heavily damaged cells). To avoid artefacts only scorable cells and at least 150 cells per sample were scored, if available. The %-tail intensity is the parameter for evaluation and interpretation of DNA damage, and was determined by the DNA staining intensity present in the tail region expressed as a percentage of the cell's total staining intensity including the nucleus [5]. The hedgehogs were manually classified by the evaluator.

Evaluation criteria:

Increases in DNA damage in the presence of a clear evidence for cytotoxicity during e.g. clinical observations should be interpreted with caution. A positive response should minimally yield a statistically significant increase in the %-tail DNA in at least one dose group at a single sampling time in comparison with the negative control value.
Providing all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
- at least one of the test doses exhibits a statistically significant increase in tail intensity compared with the concurrent negative control, and
- this increase is dose-related when evaluated with an appropriate trend test,
- any of these results are outside the distribution of the historical negative control data

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
- none of the test concentrations exhibits a statistically significant increase in tail intensity compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data,
- direct or indirect evidence supports exposure of, or toxicity to, the target tissue(s).

To assess the biological relevance of a positive or equivocal result, information on cytotoxicity of the target tissue can be required. Where positive or equivocal findings are observed solely in the presence of a clear evidence for cytotoxicity, the study should be concluded as equivocal for genotoxicity unless there is enough information supporting a more definitive conclusion.

Statistics:

All slides, including those of positive and vehicle controls were independently coded before microscopic analysis and subsequently scored blinded. The median %-tail DNA for each slide was determined and the mean of the median values was calculated for each of the tissue types from each animal.
For each tissue type, the mean of the individual animal means was then determined to give a group mean. Normality was tested according to Kolmogorov-Smirnov-test. For the determination of statistical significances, the mean values of each animal per dose group were evaluated with one-way ANOVA (Dunnett’s test) at the 5 % level (p<0.05). The p value was used as a limit in judging for significance levels in comparison with the corresponding vehicle control (PEG400).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Dose-related clinical effects in MD (reduced spontaneous activity) and HD group (reduced spontaneous activity, piloerection, half eyes closure, hunched posture) and histopathological effects in HD group (stomach erosion/necrosis, hepatocyte hypertrophy).

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-600 mg/kg bw
- Solubility: The test item was diluted in PEG400, after the use of an aqueous solution/suspension was not possible.
- Clinical signs of toxicity in test animals:
In the pre-experiment, one male and one female rat received a single dose of 500 mg/kg bw orally by gavage on two consecutive days.
The male rat showed reduced spontaneous activity and prone position 30 minutes after the first administration of the test item. The symptoms lasted until 2 h and were gone after 4 h. After the second administration, piloerection and hunched posture were visible after 2 h, but vanished afterwards. The female animal showed reduced spontaneous activity 1 h after the first administration of the test item and reduced spontaneous activity and prone position after 2 h. No clinical signs were observed after 4 h. Two hours after the second administration, reduced spontaneous activity, hunched posture and piloerection were visible but were not present after 4 h.
Based on these observations, the amount of the test item was enhanced to 600 mg/kg bw/d. Three male and three female rats received a single dose of 600 mg/kg bw orally on two consecutive days.
The first male rat showed reduced spontaneous activity and hunched posture 30 minutes after the first administration of the test item, reduced spontaneous activity, prone position and piloerection after 1 h and reduced spontaneous activity and piloerection after 2 h. At 4 h of the observation period, piloerection was still present. After the second administration, reduced spontaneous activity, hunched posture and piloerection were observed after 1 h. These symptoms in addition with half eyes closed were still visible after 4 h of the last administration. The second male rat showed reduced spontaneous activity 30 minutes after the first administration of the test item, while reduced spontaneous activity and prone position was present in the third male rat. Reduced spontaneous activity, prone position and piloerection was observed in both male rats after 1 h and reduced spontaneous activity and piloerection after 2 h till 4 h of the observation period. Thirty minutes after the second administration, the two male rats showed reduced spontaneous activity, hunched posture, piloerection and half eyes closed were observed. Piloerection was still visible after 4 h.
The first female animal showed reduced spontaneous activity and hunched posture 30 minutes after the first administration of the test item, reduced spontaneous activity, prone position and piloerection after 1 h and reduced spontaneous activity and piloerection after 2 h. After 4 h piloerection was still present. One hour after the second administration, reduced spontaneous activity and piloerection were observed. Piloerection, hunched posture and half eyes closed were present after 4 h of the observation period. The other two female rats showed reduced spontaneous activity, prone position and piloerection 1 h after the first administration of the test item and reduced spontaneous activity, hunched posture and piloerection after 2 h. After 4 h of the observation period, piloerection and reduced spontaneous activity were still present. One hour after the second administration, reduced spontaneous activity, hunched posture, piloerection and half eyes closed were observed. Piloerection was still visible after 4 h.
- Evidence of cytotoxicity in tissue analysed: In the glandular stomach, mucosal erosion (4/6 animals), necrosis/apoptosis of epithelial cells at the tips of the gastric pits (6/6 animals), and mixed cell infiltrates (1/6 animals) were observed in different animals that received 600 mg/kg bw test item. Minimal centrilobular hypertrophy was observed in one animal that received 500 mg/kg bw test item and in one animal that received 600 mg/kg bw test item. In addition, minimal, single hepatocytes necrosis/apoptosis were observed in one animal that received 600 mg/kg bw test item.
- Rationale for exposure: Based on the results obtained in the dose-range finding study, the main study was conducted with male rats, as no gender specific differences were determined, and 600 mg/kg bw was chosen as highest dose in the main experiment. Due to the observed severity of clinical signs and the incidence and severity of histopathological effects (for more information, please refer to Table 5 and Table 6 in section "Any other information on results incl. tables"), this dose represents the MTD.

RESULTS OF DEFINITIVE STUDY (Detailed tabular results for each group of animals treated with the test substance, vehicle and positive controls and each tissue are provided in Tables 1 and 2 in section "Any other information on results incl. tables")
- Appropriateness of dose levels and route: Based on the clinical signs observed in the HD and MD groups in the main experiment (All animals treated with the highest dose (HD) showed slight to moderate toxic effects such as reduction of spontaneous activity, piloerection, half eyes closure and hunched posture, after the first administration of the test item, which were also present after the second administration. On both days of treatment, all animals treated with 300 mg/kg bw (MD) showed reduced spontaneous activity 4 h after administration of the test item, while rats treated with 120 mg/kg bw (LD) showed no clinical signs, for more information, please refer to Table 7 in section "Any other information on results incl. tables") and the histopathological evaluation (for more information, please refer to Table 8 in section "Any other information on results incl. tables"), dose groups were selected to range from the MTD which is represented by the HD group (600 mg/kg bw/day) to a dose without any evidence of adverse effects in the LD group (120 mg/kg bw/day).
- Statistical evaluation: A statistically significant increase in induced DNA strand breaks was determined for the positive control group of each organ. No significances compared to the vehicle control (PEG400) were determined for the incidence of induced DNA-strand breaks in any of the dose groups of each organ. A concentration-related increase was noted in male liver cells as the tail intensity value for the highest concentration was increased compared to the lower concentrations tested. However, this positive trend test was not regarded as biologically relevant as the concentration was not significantly increased compared to the vehicle control groups and the tail intensities of all groups were in the historical control range (for more information on historical control data, please refer to Table 4 in section "Any other information on results incl. tables") of the test facility. No concentration-related increase was noted in glandular stomach and duodenum cells of male rats.

Table 1: Summary of Mean Tail Intensities [%] in Liver, Glandular Stomach and Duodenum

Dose	Mean Tail Intensity [%]			Clinical Signs
Group	Liver	Glandular Stomach	Duodenum
PC	13.73*	7.70*	6.94*	None
VC1	0.96	2.89	2.72	None
VC2	0.66	2.97	1.24	None
LD	0.79	1.87	1.3	None
MD	1.25	2.16	2.16	Reduced spontaneous activity
HD	1.4	1.7	3.45	Reduced spontaneous activity, piloerection, half eyelid closure and hunched posture
Statistically significant trend	Yes°	No	No	/
Historic Control Range	Negative Control: 0.07% – 3.82% Positive Control: 2.81% – 28.08%	Negative Control: 1.30% - 5.46% Positive Control: 5.99% – 34.62%	Negative Control: 0.89% - 4.06% Positive Control: 1.77% – 30.75%

*      significantly increased (One-way ANOVA with Dunnett’s test after normality test by Kolmogorov-Smirnov)

°      a trend test (One-way ANOVA) revealed a statistically significant increase, which was not considered as biologically relevant.

VC1:	Vehicle Control (PEG400)	PC:	Positive Control (Ethyl methanesulfonate: 250 mg/kg bw)
LD:	Low Dose	MD:	Mid Dose
HD:	High Dose	VC2:	Vehicle Control (Triacetin)

Table 2: Detailed Results for Tail Intensity in Liver, Glandular Stomach and Duodenum cells

Tail intensity in Liver Cells
Animal per Group	Mean of Medians
	PC	VC1	VC2	LD	MD	HD
I	14.19	0.8	0.67	1.17	0.69	1.45
II	14.81	1.11	0.47	0.57	1.57	1.3
III	19.93	0.7	0.52	0.93	1.49	1.11
IV	9.45	1.14	0.53	0.64	1.54	1.14
V	10.24	1.03	1.09	0.65	0.98	2
Group Mean:	13.73*	0.96	0.66	0.79	1.25	1.4
± SD:	4.19	0.19	0.25	0.25	0.4	0.36
Tail intensity in Glandular Stomach Cells
Animal per Group	Mean of Medians
	PC	VC1	VC2	LD	MD	HD
I	9	3.54	3.7	2.91	1.84	3.01
II	10.7	3.5	1.46	2.5	2.41	2.7
III	0.65	2.38	2.37	1.25	3.6	0.81
IV	11.59	2.72	5.75	2	1.95	1.06
V	6.56	2.31	1.59	0.7	1.01	0.9
Group Mean:	7.70*	2.89	2.97	1.87	2.16	1.7
± SD:	4.38	0.6	1.79	0.9	0.95	1.07
Tail intensity in Duodenum Cells
Animal per Group	Mean of Medians
	PC	VC1	VC2	LD	MD	HD
I	3.8	5.53	1.56	1.08	2.85	4.66
II	4.14	2.89	1.57	0.65	1.14	5.62
III	11.27	2.34	0.79	0.89	1.77	1.04
IV	9.85	1.93	0.71	0.89	1.96	0.58
V	5.65	0.93	1.55	3.01	3.07	5.37
Group Mean:	6.94*	2.72	1.24	1.3	2.16	3.45
± SD:	3.41	1.73	0.45	0.97	0.8	2.45
LD:	Low Dose
MD:	Mid Dose
HD:	High Dose
PC:	Positive Control (Ethyl methanesulfonate: 250 mg/kg bw)
SD:	Standard Deviation
VC1:	Vehicle Control (PEG400)
VC2:	Additional Control (Triactin)
* significantly increased (One-way ANOVA with Dunnett’s test after normality test by Kolmogorov-Smirnov)

Table 3: Linear Trend test - Statistical significance of test item treatment at the 5 % level (p < 0.05)

	Liver Cells	Glandular Stomach Cells	Duodenum Cells
Significance	+*	-	-
P Value	0.0159	0.7802	0.0532
+:	statistically significant trend test
+*:	statistically significant concentration-dependency was noted but considered as not biological relevant.
-:	statistically not significant trend test

Table 4: Laboratory Historical Control Data for tail intensity (%) (2017 – 2020)

Negative and Vehicle Controls
	tail intensity [%]
	liver	glandular stomach	duodenum
mean [%]	1.95	3.38	2.48
SD [%]	0.94	1.04	0.79
RSD [%]	48.11	30.72	31.96
min [%]	0.65	1.61	1.11
max [%]	3.97	4.95	3.81
LCL [%]	0.07	1.3	0.89
UCL [%]	3.82	5.46	4.06
n	20	12	14
Median	1.81	3.67	2.38
Positive Controls
	tail intensity [%]
	liver	glandular stomach	duodenum
mean [%]	15.45	20.31	16.26
SD [%]	6.32	7.16	7.24
RSD [%]	40.91	35.25	44.55
min [%]	5.66	10.72	8.21
max [%]	30.94	42.79	37.52
LCL [%]	2.81	5.99	1.77
UCL [%]	28.08	34.62	30.75
n	32	22	27
Median	15.23	17.34	14.94
mean:	Group mean tail intensity [%]
min.:	minimum tail intensity [%]
LCL:	lower control limit [%]
n:	number of assays
SD:	Standard Deviation [%]
max.:	maximum tail intensity [%]
UCL:	upper control limit [%]

Table 5: Clinical Signs observed in the Pre-Experiment at 600 mg/kg bw

Clinical Signs	Time post-administration / Sex
	(3 male and 3 female rats)
	0 h		0.5 h		1 h		2 h		4 h		24 h		24.5 h		25 h		28 h
	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f
Dose level	600 mg/kg bw
Reduction of spontaneous activity	0	0	3	1	3	3	3	3	2	2	0	0	2	0	1	3	1	0
Piloerection	0	0	0	0	3	3	3	3	3	3	0	0	2	0	1	3	3	3
Half eyelid closure	0	0	0	0	0	0	0	0	0	0	0	0	2	0	0	0	1	1
Prone Position	0	0	1	0	3	3	0	0	0	0	0	0	0	0	0	0	0	0
Hunched posture	0	0	1	1	0	0	0	2	0	0	0	0	2	0	1	2	1	1

Table 6: Incidence and severity of relevant microscopic changes in the dose range finding experiment

Findings / Groups	500 mg/kg bw	600 mg/kg bw
Animals Affected / Mean Grade	2	6
Glandular stomach
Erosion	-	4/1.8
Necrosis / Apoptosis	-	6/1.0
Mixed cell infiltrates	-	1/2.0
Liver
Centrilobular hypertrophy	1/1.0	1/1.0
Mononuclear cell infiltrates	2/1.0	5/1.0

Table 7: Clinical Signs observed in the Main Experiment

Clinical Signs	Time post-administration / Sex
	(5 male rats)
	0 h	0.5 h	1 h	2 h	3 h	4 h	24 h	24.5 h	25 h	26 h	27 h	28 h
	m	m	m	m	m	m	m	m	m	m	m	m
Dose level	120 mg/kg bw
No clinical signs observed	-	-	-	-	-	-	-	-	-	-	-	-
Dose level	300 mg/kg bw
Reduction of spontaneous activity	0	0	0	0	0	0	5	0	0	0	0	4
Dose level	600 mg/kg bw
Reduction of spontaneous activity	0	0	5	5	5	5	0	5	5	5	5	5
Piloerection	0	0	2	3	4	5	1	0	0	0	0	2
Half eyelid closure	0	0	0	0	0	2	0	0	0	0	0	1
Hunched posture	0	0	0	5	5	5	0	5	5	5	5	5

Table 8: Incidence and severity of relevant microscopic changes in the main experiment

Findings / Groups	Vehicle Control	Additional control group	LD	MD	HD	Positive control
	PEG400	360 mg/kg bw Triacetin	120 mg/kg bw	300 mg/kg bw	600 mg/kg bw	250 mg/kg bw EMS
Animal per Sex Affected / Mean Grade	5(M)	5(M)	5(M)	5(M)	7(M)	5(M)
Glandular stomach
Erosion	-	-	-	-	1/2.0	-
Necrosis / Apoptosis	-	-	-	-	3/1.0	-
Mixed cell infiltrates	1/1.0	2/2.0	1/2.0	-	1/2.0	-
Liver
Centrilobular hypertrophy	2/1.0	3/1.0	4/1.0	4/1.0	6/1.0	4/1.0

Conclusions:

The test item O,O-tert-butyl monoperoxymaleate did not induce biologically relevant increases in DNA-strand breaks in any of the tissues evaluated (liver, glandular stomach, duodenum) after oral (gavage) administration up to the maximum tolerated dose (600 mg/kg bw/day) in male rats.
Therefore, the test item is considered to be non-DNA damaging under the experimental conditions of the in vivo mammalian Alkaline Comet Assay.

Executive summary:

In an in vivo mammalian Alkaline Comet Assay conducted according to OECD 489, the test material O,O-tert-Butyl monoperoxymaleate (purity: 37%) was administered to 5 male Crl(WI):Han rats/dose by oral gavage at dose levels of 120, 300 and 600 mg/kg bw/day in PEG 400 for two consecutive days.

No correction factor for purity was used in dose calculations. The phlegmatizer Triacetin was used as additional control item at a dose of 360 mg/kg bw/day (via oral gavage), based on the selected high dose level of the test material (600 mg/kg bw/day) and the maximum content of Triacetin (60% (w/w)) in the test item formulation.

There were no signs of toxicity in animals treated with 120 mg/kg bw/day. Animals in the 300 mg/kg bw/day group showed reduction of spontaneous activity, while animals of the high dose showed more severe clinical signs such as reduction of spontaneous activity, piloerection, half eyelid closure and hunched posture. No specific biologically-relevant effect on body weight was observed in any of the dose groups. Specific treatment-related histopathological effects were observed in the 600 mg/kg bw/day group, which included erosion and necrosis in the stomach and increased incidences of centrilobular hepatocyte hypertrophy in the liver. Based on these results, systemic availability of the substance can be reasonably assumed and the high dose level can be considered to represent the maximum tolerated dose (MTD).

Cells from the liver, glandular stomach and duodenum were isolated, embedded in agarose, lysed and DNA allowed to migrate under electrophoresis conditions. 150 cells per animal tissue were evaluated.

The tail intensities of the vehicle control group consisting of PEG 400 was within the historical control limits and therefore accepted for addition to the laboratory control data set. The additional control group (Triacetin) was within historical control limits. The positive control ethyl methanesulfonate (250 mg/kg bw by oral gavage) induced a statistically significant increase in DNA damage for all evaluated organs within historical control limits of the positive control of the testing facility, thereby demonstrating the validity of the assay.

No biologically relevant increase of tail intensity was found after treatment with the test item in any of the dose groups and organs evaluated compared to the vehicle controls. Mean tail intensity values for the test item dose groups were within the range of the concurrent vehicle controls and within the historical control limits. A slight dose-related increase was observed for liver cells but considered not biologically relevant as the tail intensity values were not statistically significant on a single dose level and were within historical control range of the testing facility.

Based on these observations, the test item is considered to be non-DNA damaging under the experimental conditions of the in vivo mammalian Alkaline Comet Assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 489 for in vivo mammalian alkaline comet assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The potential of OO-tert-butyl monoperoxymaleate to induce genotoxic effects was tested in a suitable in vitro test battery. The target substance was tested negative in a bacterial reverse gene mutation test conducted according to OECD 471 and in a mammalian cell HPRT mutation assay according to OECD 476.

In contrast, in an in vitro micronucleus test in Chinese hamster V79 cells (OECD 487), OO-tert-butyl monoperoxymaleate did induce structural and/or numerical chromosomal damage.

In experiment I (with and without metabolic activation) the micronucleus frequencies showed a statistically significant increase compared to the concurrent negative control and the corresponding micronuclei frequencies were above the upper limit of the historical control limits of the negative control. Since these positive findings were obtained in experiment I with and without metabolic activation, the performance of experiment II with long term treatment was omitted. The positive controls did induce distinct and biologically relevant increases of the micronucleus frequency.

Based on these results, ECHA requested an in vivo mammalian alkaline comet assay in its decision on testing proposal TPE-D-2114557072-54-01/F. The mammalian alkaline comet assay was conducted in rats as a suitable follow-up for the positive in vitro micronucleus results with OO-tert-butyl monoperoxymaleate.

There were no biologically relevant increases in DNA-strand breaks in any of the tissues evaluated (liver, glandular stomach, duodenum) after oral (gavage) administration up to the maximum tolerated dose (600 mg/kg bw/day) in male rats. Therefore, the test item is considered to be non-DNA damaging under the experimental conditions of the in vivo mammalian alkaline Comet Assay (OECD 489).

The results of the mammalian alkaline Comet Assay demonstrate that the positive results for chromosomal aberrations in vitro were not expressed in vivo and therefore no biologically relevant genetic toxicity is expected in mammals.

Justification for classification or non-classification

OO-tert-butyl monoperoxymaleate induced structural and/or numerical chromosomal damage in an in vitro micronucleus test in Chinese hamster V79 cells (OECD 487). The positive in vitro results were followed up in an in vivo mammalian alkaline comet assay according to OECD 489, in which no biologically relevant increases in DNA-strand breaks were observed in any of the tissues evaluated (liver, glandular stomach, duodenum).

Therefore, the test item is considered to be non-DNA damaging in vivo and classification according to the criteria set out in Regulation (EC) No 1272/2008 is not warranted.