Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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 and is considered relevant and reliable for classification.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 487
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 male or female 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:
* 5 mL of Chromosome complete culture medium and 1% Penicillin/Streptomycin
* After 48 hours the medium was replaced by 4.95 mL of fresh Ham’s F10 medium with FCS and 50 µL of the vehicle control, the 5 concentrations of the test item or the 2 concentrations of the positive control (Mitomycin C, Colchicine) were added to each culture.
* Afterwards the medium was removed and the cultures were washed two times with Ham’s F10 medium. After addition of 5 mL Chromosome medium containing 5 µg/mL Cytochalasin B the cultures were incubated for further 20 hours at 37°C.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary test: 10, 25, 100, 250, 1000, 2500 and 4020 µg test item/mL medium
Main test with S9-mix: 125, 250, 500 or 1000 µg test item/mL medium (at 2500µg test item/mL there were no cells of sufficient quality due to cytotoxicity of the test item)
Main test without S9-mix: 62.5, 125, 250 or 500 µg test item/mL medium (at 1000µg test item/mL there were no cells of sufficient quality due to cytotoxicity of the test item)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the other solvents recommended.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.2 µ/mL, without S9-mix
Positive controls:
yes
Positive control substance:
other: Colchicine
Remarks:
0.02 µg/mL, without S9-mix
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
20 µg/mL, with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Experiment 1
- Exposure duration: 4 hours (with and without S9-mix)
- Selection time (if incubation with a selection agent): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours
Experiment 2:
- Exposure duration: 4 hours (with S9-mix) or 20 hours (without S9-mix)
- Selection time (if incubation with a selection agent): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (with S9-mix) and 40 hours (without S9-mix)

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

NUMBER OF REPLICATIONS: Duplicate cultures were used for each test item concentration and for the vehicle and positive control cultures.

NUMBER OF CELLS EVALUATED:
The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration)

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytokinesis-Biock Proliferation Index, Replicative Index
CBPI = ((No. mononucleate cells)+(2×No. binucleate cells)+(3×No. multinucleate cells)) /(Total number of cells)
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.
1000 binucleated cells per duplicate cell culture were scored with a phase contrast microscope (optical magnification of 600)to assess the frequency of cells with one, two, or more than two micronuclei. Additionally, the cells were classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity.
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 vehicle 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.
The assessment was carried out by a comparison of the samples with the positive and the vehicle control.
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.
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[1]) 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 lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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 and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
pH values: using a digital pH meter type WTW pH 525 (series no. 51039051),
Osmolality: with a semi-micro osmometer.
The pH value of the test item supplied was measured as 5.62.
No relevant changes in pH of the formulations were noted.
- Effects of osmolality:
No relevant changes in osmolality of the formulations were noted.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 4020 µg test item/mL medium were employed. Pronounced to complete cytotoxicity was noted at concentrations of 1000 µg test item/mL and higher in the experiment with metabolic activation. In the experiment without metabolic activation concentrations of 1000 µg test item/mL and higher caused complete cytotoxicity. Hence, 2500 µg/mL was employed as the top concentration for the mutagenicity tests with metabolic activation and 1000 µg test item/mL in the tests without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The micronucleus frequencies of the vehicle controls without and with metabolic activation for the last 8 or 7 studies (most recent background data, not audited by the QAU-department) are given as follows:
Micronucleus frequency per 1000 cells:
Without metabolic activation(4-h or 20-h exposure)
Untreated control (n = 8)
mean: 4.9
SD: 2.0
range: 1-9
Vehicle control (n = 8)
mean: 7.2
SD: 4.6
range: 1-18
Mitomycin C Positive control (n=7)
mean: 95.8
SD: 66.1
range: 24-286
Colchicine Positive control (n = 7)
mean: 25.4
SD: 10.2
range: 7-43

With metabolic activation (4-h exposure)
Vehicle control (n = 8)
mean: 10.8
SD: 6.2
range: 2-25
Cyclophosphamide Positive control (n = 7)
mean: 60.3
SD: 37.8
range: 20-147

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study cytotoxicity was noted starting at concentrations of 500 or 1000 µg/mL in the experiments without and with metabolic activation, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite 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 chromosomal damage in the in vitro micronucleus test.
Executive summary:

Test sample of Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite was 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: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after the end of exposure. The study was conducted in duplicate.
The test item was diluted with aqua ad iniectabilia. A correction factor of 2.49 was used as the supplied test item contains only 40.20% solid matter. Aqua ad iniectabilia served as the vehicle control.
The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item concentrations of 10, 25, 100, 250, 1000, 2500 and 4020 µg test item/mL medium were employed. Pronounced to complete cytotoxicity was noted at concentrations of 1000 µg test item/mL and higher in the experiment with metabolic activation. In the experiment without metabolic activation concentrations of 1000 µg test item/mL and higher caused complete cytotoxicity. Hence, 2500 µg/mL was employed as the top concentration for the mutagenicity tests with metabolic activation and 1000 µg test item/mL in the tests without metabolic activation.
In the main study cytotoxicity was noted starting at concentrations of 500 or 1000 µg/mL in the experiments without and with metabolic activation, respectively.
Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation.
Tests without metabolic activation (4- and 20-hour exposure)
The micronucleus frequencies of cultures treated with the test item at concentrations of 62.5, 125, 250 or 500 µg test item/mL medium (4-h or 20-h exposure) in the absence of metabolic activation ranged from 0.5 to 8.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentrations. The dose level of 1000 µg test item/mL medium led to cytotoxicity, no cells of sufficient quality were available for evaluation.
Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487. In this test the following frequencies were observed: vehicle control: 7.5 or 4.0 micronuclei per 1000 binucleated cells and untreated controls: 8.0 or 4.5 micronuclei per 1000 binucleated cells (4-hour and 20-hour exposure, respectively). Vehicle and untreated control values fell within acceptation ranges.
Test with metabolic activation (4-hour exposure)
The micronucleus frequencies of cultures treated with the test item at concentrations of 125, 250, 500 or 1000 µg test item/mL medium (4-h exposure) in the presence of metabolic activation ranged from 3.0 to 8.5 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. The dose level of 2500 µg test item/mL medium led to cytotoxicity, no cells of sufficient quality were available for evaluation.
Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487. In this test the following frequencies were observed: vehicle control: 5.0 micronuclei per 1000 binucleated cells and untreated controls: 5.0 or 6.0 micronuclei per 1000 binucleated cells. Vehicle and untreated control values fell within acceptation ranges.
Under the present test conditions, Reaction products of ricinoleic acid with 2-aminoethanol and maleic acid and sodium hydrogensulfite 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 chromosomal damage in the in vitro micronucleus test.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.