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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: suspension

Method

Species / strain
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
Controlsopen allclose all
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.

Results and discussion

Test results
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

Any other information on results incl. tables

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]

Applicant's summary and conclusion

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.