Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
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 testing guidelines, therefore it 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)
Version / remarks:
July 22, 2010
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: crystalline
Details on test material:
- Name of test material (as cited in study report): Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts; Disodium C12-18-alkyl sulfosuccinate; Disodium Lauryl Sulfosuccinate
- Physical state: White powder
- Analytical purity: > 95% (correction factor: 1.05)

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:
* Ham’s F10 supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin (culture establishment)
* Ham’s F10 medium with FCS
* Chromosome medium
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
-Preliminary test: 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL medium
In this preliminary experiment without and with metabolic activation cytotoxicity was noted starting at a concentration of 156.3 μg test item/mL. Hence, 156.3 μg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.
-Main test: 9.77, 19.53, 39.1, 78.1 or 156.3 µg/mL medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:aqua ad iniectabilia
- Justification for choice of solvent/vehicle: The test item was completely dissolved in aqua ad iniectabilia.
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 µg/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); 40 hours (without S9-mix)

SPINDLE INHIBITOR (cytogenetic assays): 5 µg/mL Cytochalasin B
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: 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.

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytokinesis-Block Proliferation Index
The Cytokinesis-Block Proliferation Index (CBPI) indicates the average number of cell cycles per cell during the period of exposure to cytoB, and is used to calculate cell proliferation.
((No. mononucleate cells)+(2×No. binucleate cells)+(3×No. multinucleate cells))
CBPI = -------------------------------------------------------------------------------
(Total number of cells)
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

Evaluation criteria:
1000 binucleated cells per duplicate cell culture were scored 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.
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, using a chi-square test corrected for continuity according to YATES as recommended by the UKEMS guidelines.
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.
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 and osmolality:
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 .
No relevant changes in pH or osmolality of the formulations were noted.
- Water solubility:
The test item was completely dissolved in aqua ad iniectabilia.
- Precipitation:
Any possible test item precipitation was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary experiment without and with metabolic activation test item concentrations of 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL medium were employed. Cytotoxicity was noted starting at a concentration of 156.3 µg test item/mL. Hence, 156.3 µg/mL were employed as the top concentration for the mutagenicity tests without and with 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
Standard deviation: 2.0
Range: 1-9
Vehicle control (n = 8):
Mean: 7.2
Standard deviation: 4.6
Range: 1-18
Positive control: Mitomycin C (n = 7):
Mean: 95.8
Standard deviation: 66.1
Range: 24-286
Positive control: Colchicine (n = 7):
Mean: 25.4
Standard deviation: 10.2
Range: 7-43
With metabolic activation (4-h exposure):
Vehicle control (n = 8):
Mean: 10.8
Standard deviation: 6.2
Range: 2-25
Positive control: Cyclophosphamide (n = 7):
Mean: 60.3
Standard deviation: 37.8
Range: 20-147


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 a cytotoxic concentration of 156.3 µg/mL medium, 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.
Executive summary:

Test sample of Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts 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 completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. 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 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL medium were employed. Cytotoxicity was noted starting at a concentration of 156.3 µg test item/mL. Hence, 156.3 µg/mL was employed as the top concentration for the mutagenicity tests without and with metabolic activation.

In the main study cytotoxicity was noted at the top concentration of 156.3 µg/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.

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

The micronucleus frequencies of cultures treated with the test item at concentrations of 9.77, 19.53, 39.1, 78.1 or 156.3 µg/mL medium (4 h and 20-h exposure) in the absence of metabolic activation ranged from 5.5 to 15.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487; (in this test: vehicle control: 6.0 or 12.0 micronuclei per 1000 binucleated cells, untreated controls: 4.0 or 9.0 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 9.77, 19.53, 39.1, 78.1 or 156.3 µg/mL medium (4-h exposure) in the presence of metabolic activation ranged from 6.0 to 18.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487; (in this test: vehicle control: 3.5 or 13.0 micronuclei per 1000 binucleated cells, untreated controls: 4.5 or 8.5 micronuclei per 1000 binucleated cells). Vehicle and untreated control values fell within acceptation ranges.

 Under the present test conditions, the test item tested up to a cytotoxic concentration of 156.3 µg/mL medium, 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.