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, other: TA98, TA100, TA102, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA98, TA100 & TA102 at 2.5 µL/plate and higher (with and without metabolic activation); For TA1535 & TA1537 at 2.5 µL/plate and higher (without metabolic activation) and at 5.0 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, other: TA98, TA100, TA102, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

For TA98 at 0.316 µL/plate and higher /without metabolic activation) and at 1.0 µL/plate (with metabolic activation). For TA100, TA1535, TA1537 and TA102 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, conducting an in vivo micronucleus test (OECD 474) is proposed in line with ECHAs recommendation for the assessment of genotoxic effects.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:

- Name of the substance on which testing is proposed to be carried out: OO-tert-butyl monoperoxymaleate (CAS 1931-62-0)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:

- Available GLP studies:
The potential of OO-tert-butyl monoperoxymaleate to induce genotoxic effects was tested in a suitable in vitro test battery, conducted under GLP. 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 an in vitro micronucleus test in Chinese hamster V79 cells (OECD 487), OO-tert-butyl monoperoxymaleate did induce structural and/or numerical chromosomal damage.

- Available non-GLP studies:
There are no non-GLP studies available to address this potential mode of action.

- Historical human data:
There is no historical human data available to address this potential mode of action.

- (Q)SAR:
The use of QSARs models for the prediction of genotoxicity is a possibility to address this endpoint. However, the outcome of a QSAR assessment will not overrule the positive results of an in vitro micronucleus test.

- In vitro methods:
Further in vitro testing is not considered sufficient to provide information to conclude on this endpoint.

- Weight of evidence:
There is no additional relevant information available that can be used in a WoE to cover this endpoint for registration and classification purposes. Experiments with other members of the chemical group of peroxyester indicate that this functionality might lead to false positive results in in vitro tests. However, false positive results do not occur in all members of the group, and this mode of action would not account for the non-peroxide part of the molecule and/or degradation products.

- Grouping and read-across:
No further experimental data on potential analogues are available.

- Substance-tailored exposure driven testing [if applicable]:
The uses of this substance do not allow to use substance tailored exposure driven arguments to avoid the requirement for this study.

- Approaches in addition to above:
Not applicable

- Other reasons [if applicable]:
No data is available that would allow a general adaptation possibility as identified in Annex XI of the REACH Regulation. More specific, no in vivo data is available which would allow to draw a conclusion on the classification of the substance.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:

- In accordance to ANNEX IX of the REACH Regulation 1907/2006, column 2 of standard information requirement 8.4: "If there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant". Based on the positive results from the in vitro micronucleus test (OECD 487, GLP, IUCLID section 7.6.1, Donath (2017)) and as no additional in vivo data is available, it is necessary to propose an appropriate in vivo somatic cell genotoxicity test (OECD 474).

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:

- Details on study design / methodology proposed [if relevant]: To assess if the induction of micronuclei on V79 cells in vitro is also prominent in vivo, it is proposed to conduct an in vivo micronucleus test in accordance to OECD TG 474 (Mammalian Erythrocyte Micronucleus Test). This study will be contracted and conducted at a GLP certified laboratory.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

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

The chemical group of peroxyester shows a consistent result pattern of in vitro and in vivo genotoxicity tests. This is demonstrated by the table below, listing the respective study results for ten peroxyesters, for which an in vivo chromosomal aberration study is available. These ten substances were, with one exception, positive in the Ames test (OECD 471). Also, the two available chromosomal aberration (OECD 473) test were positive. Results of in vitro gene mutation test (OECD 476) were primarily negative. As at least one positive result was obtained with two or three in vitro tests for each substance, an in vivo chromosomal aberration assay was conducted for each of these peroxyesters. Regardless of the route of administration (oral or intraperitoneal), all in vivo studies were negative. Also for tert-butyl monoperoxy-maleate (CAS 1931-62-0) one in vitro study, i.e. an in vitro micronucleus study, was positive. Following the ECHA recommendation for assessing ambiguous results in the genotoxicity in vitro battery, an in vivo micronucleus test according to OECD 474 is proposed. Based on the negative in vivo result for the other ten peroxyester, it can be anticipated that the in vivo test with tert-butyl monoperoxy-maleate will also be negative. Therefore, no classification for mutagenicity is considered justified as an interim measure until the results of the proposed in vivo mammalian erythrocyte micronucleus test will be available.    

When the results from the in vivo test are available the classification of OO-tert-butyl monoperoxymaleate will be re-assessed.

 

peroxyester name	CAS no.	In vitro gene mutation study in bacteria (OECD 471)	In vitro chromosomal aberrations (OECD473)	In vitro gene mutation in mammalian cells (OECD 476)	In vivo chromosomal aberration assay (micronucleus)
tert-butyl peroxybenzoate	614-45-9	Positive	Positive	Positive	Negative
tert-butyl peroxy-3,5,5-trimethylhexanoate	13122-18-4	Negative	Positive	Negative	Negative (oral)
tert-butyl peroxy-2-ethylhexanoate	3006-82-4	Positive	na	Positive	Negative (oral)
tert-amyl peroxy-2-ethylhexanoate	686-31-7	Positive	na	Negative	Negative (oral)
tert-butyl peroxyneodecanoate	26748-41-4	Positive	na	Negative	Negative (ip)
tert-butyl peroxypivalate	927-07-1	Positive	na	Negative	Negative (ip)
tert-amyl peroxypivalate	29240-17-3	Positive	na	Negative	Negative
2,5-dimethyl-2,5-di(2-ethylhexanoyl peroxy)hexane	13052-09-0	Positive	na	Negative	Negative (ip)
tert-butyl peroxyisobutyrate	109-13-7	Positive	na	Positive	Negative (oral)
tert-butyl peroxyacetate	107-71-1	Positive	na	na	Negative (oral)
tert-butyl monoperoxy-maleate	1931-62-0	Negative	positive (OECD 487)	Negative	testing proposal

           na: not available; ip: intraperitoneal

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). Without additional information, it is not possible to assess the relevance of this finding for humans. Therefore, an in vivo test according to OECD 474 is proposed.