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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP and valid methods, therefore the study is considered relevant, adequate and reliable for classification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
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:
other: liquid

Method

Species / strain
Species / strain / cell type:
lymphocytes: Human peripheral blood was obtained by venipuncture from young (approximately 18 – 35 years of age), healthy, non-smoking individuals with no known recent exposures to genotoxic chemicals or radiation, and collected in heparinised vessels.
Details on mammalian cell type (if applicable):
- Type and identity of media:
* Ham’s F10 supplemented with 10% fetal calf serum (FCS) and 1% Penicillin/Streptomycin
* fresh Ham’s F10 medium
* chromosome medium with 5 µg/mL Cytochalasin B
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary experiment: 10, 25, 100, 250, 1000, 2500 and 4358 µg active ingredient/mL medium
First And Second MainTest (4 h and 20-h exposure) without S9: 7.81, 15.63, 31.3, 62.5 or 125 µg/mL medium
First and Second Main Test (4h exposure) with S9: 7.81, 15.63, 31.3, 62.5 or 125 µg/mL medium
Third Main Test (4 h exposure) without S9: 31.3, 62.5, 125, 250 or 500 µg /mL
Third Main Test (4h exposure) with S9: 31.3, 62.5, 125, 250 or 500 µg /mL
The concentrations employed were chosen based on the results of a preliminary cytotoxicity study. Cytotoxicity was noted starting at a concentration of 100 µg active ingredient/mL in the preliminary experiment without and with metabolic activation.
Hence, 125 µg active ingredient/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure).
In a third experiment without and with metabolic activation (4-hour exposure) 500 µg active ingredient/mL were employed as the top concentration for the mutagenicity tests. A third experiment with two higher concentrations was added as it was thought that a concentration of 125 µg active ingredient/mL had not resulted in sufficient clear-cut cytotoxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiol. saline (aqua ad iniectabilia)
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated medium
Negative solvent / vehicle controls:
yes
Remarks:
aqua ad iniectabilia
True negative controls:
yes
Positive controls:
yes
Remarks:
clastogen
Positive control substance:
mitomycin C
Remarks:
0.2 µg/mL without S9
Positive controls:
yes
Remarks:
clastogen
Positive control substance:
cyclophosphamide
Remarks:
20 µg/mL with S9
Positive controls:
yes
Remarks:
aneugen
Positive control substance:
other: Colchicine
Remarks:
0.02 µg/mL without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4h (with and without S9)
Experiment 2: 4h (with S9) and 20 h (without S9)
Experiment 3: 4h (with and without S9)
- Fixation time (start of exposure up to fixation or harvest of cells):
28 h (Experiment 1with and without S9; Experiment 2 with S9 and Experiment 3 with and without S9)
44h (Eperiment 2 without S9)

SPINDLE INHIBITOR (cytogenetic assays): 5µg/mL Cytochalasin B
STAIN (for cytogenetic assays): 10% Giemsa.

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 2000/concentration

DETERMINATION OF CYTOTOXICITY
- Method: other: Treatment of cultures with cytoB, and measurement of the relative frequencies of mononucleate, binucleate, and multi-nucleate cells in the culture, provides an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment and ensures that only cells that divided during or after treatment are scored.

Evaluation criteria:
Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction. Concurrent measures of cytotoxicity and/or cytostasis for all treated and solvent control cultures were determined. Individual culture data were provided.
If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing micronuclei, it is classified as a positive result.
Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response.
A positive result from the in vitro micronucleus test indicates that the test item induces chromosome damage or damage to the cell division apparatus.
Negative results indicate that, under the test conditions used, the test substance does not induce chromosome breaks and/or gain or loss in cultured mammalian cells.
There is no requirement for verification by additional testing of a clear positive or negative response.
Equivocal results may be clarified by analysis of another 1000 cells from all the cultures to avoid loss of blinding. If this approach does not resolve the result, further testing would be necessary. Modification of study parameters over an extended or narrowed range of conditions, as appropriate, would be considered in follow-up experiments. Study parameters that might be modified include the test concentration spacing, the timing of treatment and cell harvest, and/or the metabolic activation conditions.
Although most experiments give clearly positive or negative results, in some cases the data set would preclude making a definite judgement about the activity of the test item. These equivocal or questionable responses may occur regardless of the number of times the experiment is repeated.
Statistics:
The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971[3]) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity data).

Results and discussion

Test results
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
125 µg/mL were employed as the top concentration for the first and second experiment, 500 µg/mL were employed in the third experiment. It was thought that a concentration of 125 µg/mL had not resulted in sufficient clear-cut cytotoxicity.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
The pH of the negative control and the test item formulations in the medium were determined employing a digital pH meter type WTW pH 525 (series no. 51039051). No changes in the pH values were noted (pH range: 7.66 – 7.73).

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, the test item tested up to cytotoxic concentrations, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of any chromosomal damage in the in vitro micronucleus test.
In the same test, Mitomycin C and cyclophosphamide induced significant damage.


Executive summary:

Test samples of the test item were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: two experiments with an exposure time of 4 hours and two different concentration ranges and one experiment with an exposure time of 20 hours. The experiment with S9 mix was carried out threefold with one exposure time of 4 hours employing two different concentration ranges. The harvesting time was 24 hours after the end of exposure. Each treatment was conducted in duplicate.

The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 2.41 was used in order to correct for a content of the solid material of 41.5% only. The vehicle aqua ad iniectabilia served as the vehicle control.

Preliminary experiment

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 10, 25, 100, 250, 1000, 2500 and 4358 µg active ingredient/mL medium were employed. Cytotoxicity was noted starting at a concentration of 100 µg test item/mL in the experiment without and with metabolic activation.

Hence, 125 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation in two independent experiments, each (4-hour and 20-hour exposure). In a third experiment without and with metabolic activation (4-hour exposure) 500 µg/mL were employed as the top concentration for the mutagenicity tests. A third experiment with two higher concentrations was added as it was thought that a concentration of 125 µg/mL had not resulted in sufficient clear-cut cytotoxicity.

Main study

In the main study cytotoxicity was noted starting at a concentration of 125 µg active ingredient/mL in the experiments without and with metabolic activation.

Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation. Positive controls induced significant increases in micronuclei in both experiments with/without metabolic activation.

 

Tests without metabolic activation (4- and 20-hour exposure)

The micronucleus frequencies of cultures treated with the test item at concentrations of 7.81, 15.63, 31.3, 62.5 or 125 µg active ingredient/mL medium in the first and second experiment (4 h and 20-h exposure) or 31.3, 62.5, 125, 250 or 500 µg active ingredient/mL in the third experiment (4-hour exposure) in the absence of metabolic activation ranged from 3.0 to 8.0 micronuclei per 1000 binucleated cells. There was no increase in micronuclei up to the cytotoxic concentration when compared to control (in this test: vehicle control: 8.0, 6.0 or 6.5 micronuclei per 1000 binucleated cells, untreated controls: 5.5, 4.5 or 5.5 micronuclei per 1000 binucleated cells (4-hour and 20-hour exposure, respectively)).

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the test item at concentrations of 7.81, 15.63, 31.3, 62.5 or 125 µg active ingredient/mL medium in the first and second experiment or 31.3, 62.5, 125, 250 or 500 µg active ingredient/mL in the third experiment in the presence of metabolic activation ranged from 3.0 to 9.5 micronuclei per 1000 binucleated cells. There was no increase in micronuclei up to the cytotoxic concentration when compared to control (in this test: vehicle control: 4.5 or 9.0 micronuclei per 1000 binucleated cells, untreated controls: 5.0, 6.0 or 4.5 micronuclei per 1000 binucleated cells).

Under the present test conditions, the test item tested up to cytotoxic concentrations, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of any chromosomal damage in the in vitro micronucleus test.

In the same test, Mitomycin C and cyclophosphamide induced significant damage.