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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).

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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:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
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) + (cxx 3))/n

Relative Cell Growth:100 x ((CBPITest conc– 1) / (CBPIcontrol-1))

c1:mononucleate cells

c2:binucleate cells

cx: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.

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.

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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 106cells

[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 106cells

[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:
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.