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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
other: UK Department of Health Report on Health and Social Subjects No.35 Guidelines for the testing of chemicals for mutagenicity (1989).
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): sodium vinyl solfonate (SVS 25%)
- Substance type: transported intermediair
- Physical state: liquid
- Analytical purity: 25%
- Lot/batch No.: 301S59055
- Storage condition of test material: Room temperature in the dark
- Other: supplier: Clariant GmbH

Method

Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
- Type and identity of media: Human blood was collected aseptically from healthy male donors, pooled and diluted with RPMI 1641 tissue culture medium (Sigma Chemical Co. Ltd.) containing 16.7% foetal calf serum (PAA). Aliquots (0.4 ml blood: 4.5 ml media: 0.1 ml phytohaemagglutin (Wellcome)) of the cell suspension were placed in sterile universal containers and incubated at 37°c in a slanted position, for aproximately 48 hours. The cultures were gently shaken once daily to resuspend the cells.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
After 48 hours, 50 µl aliquots of SVS 25% were added to two sets of duplicate cultures to give final concentrations of 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/ml. Sterile deionised water, the solvent control, in 50 µl aliquots, was added to four cultures and mitomycin C, at final concentrations of 0.2, 0.4 and 0.8 µg/ml, was added to duplicate cultures in both sets.
Aliquots of S-9 mix (1.25 ml) were added to each culture in two further sets, followed by 62.5 µl aliquots of the various dilutions of SVS 25%, giving the same series of final concentrations as above. Sterile deionised water (62.5 µl) was added to four cultures and cyclophosphamide was added to duplicate cultures at final concentrations of 20, 25 and 30 µg/ml in both sets.
Vehicle / solvent:
- Solvent(s) used: Sterile deionised water
- Justification for choice of solvent: prior to commencing testing, the solubility of the test substance was assessed. SVS 25% was found to be miscible in sterile deionised water at 125 mg/ml. When dosed at 1% v/v into tissue culturemedium, to give a final concentration of 1250 µg/ml, it remained miscible. The highest concentration used for subsequent testing was, therefore, 1250 µg/ml. This is the highest concentration used in test systems of this nature due to artefactual increases in chromosomal aberrations caused by solutions of high ionic strength and osmolality (Galloway et al. 1987).
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: In the prescence of S-9 mix
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: In the absence of S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
Three hours after dosing, the cultures containing the S-9 mix were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 or 42 hours. The cultures treated in the absence of S-9 mix were incubated for 21 or 45 hours.


DURATION
- Preincubation period: approximately 48 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): wih S-9 mix: 18 or 42 hours / without S9-mix: 21 or 45 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid ® (Sigma) to each culure at a final concentration of 0.1 µg/ml. After 2 hours incubation, each cell suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with a hypotonic solution (0.05 M KCl prewarmed at 37°c). After a 10 minute period of hypotonic incubation at 37°c, the suspensions were centrifuged at 500g for 5 minutes and the cell pellets fixed several times by addition of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid). The pellets were allowed to fix for at least two hours.


SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS:


NUMBER OF CELLS EVALUATED: A 100 metaphase figures were examined from each culture. Only cells with 44 - 46 chromosomes were analysed


DETERMINATION OF CYTOTOXICITY
- Method: mitotic indexr:


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other: metaphase analysis


OTHER:
Evaluation criteria:
Aberrations were scored according to the classification of the ISCN (1985). Traditionally gaps have been excluded from the quantitation of chromosome aberrations. Some gaps, however, have been shown to be real discontinuities in DNA (Heddle and Bodycote 1970, Satya-Prakash, Hsu and Pathak1981). In this study the total number of cells containing aberrations both with and without gaps has been calculated.

Results and discussion

Test results
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No substantial increases in pH were observed in either the prescence or absence of S9-mix
- Effects of osmolality: No substantial increases in osmolality were observed in either the prescence or absence of S9-mix
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES:


COMPARISON WITH HISTORICAL CONTROL DATA:


ADDITIONAL INFORMATION ON CYTOTOXICITY:

Any other information on results incl. tables

First test - 21 hour harvest:

Toxicity data:

In both the absence and prescence of S-9 mix, SVS 25 % was non-toxic, therefore the dose levels selected for metaphase analysis were 1250, 625 and 312.5 µg/ml. The relative mitotic index at 1250 µg/ml was 104% of the solvent control value in the absence of S-9 mix and 95% in its presence.

The concentration of cyclophosphamide selected for analysis was 20 µg/ml. The cultures treated with mitomycin C, at all three concentrations, were not of suitable quality for analysis. Therefore, the positive control cultures treated with mitomycin C from the 45 hour harvest were analysed. Slides from the 21 and 45 hour harvests, in the absence of S-9 mix, were coded together.

Metaphase analysis:

No statistically significant increases in the proportion of aberrant cells when compared to the solvent control were seen in cultures treated with SVS25% in either the absence or prescence of S-9 mix.

Positive control compound, cyclophosphamide, caused large, statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S-9 mix.

First test - 45 hour harvest

Toxicity data

In both the absence and presence of S-9 mix, SVS 25% at 1250 µg/ml was selected for metaphase analysis as this was the highest concentration analysed at the 21 hour harvest. The relative mitotic index of SVS 25% was 92% of the solvent control value in the absence of S-9 mix and 103% in its presence.

The concentrations of the positive control compound selected for analysis were mitomycin C at 0.4 µg/ml.

Metaphase analysis:

No statistically significant increases in the proportion of aberrant cells when compared to the solvent control were seen in cultures treated with SVS 25% in either the absence or presence of S-9 mix.

Positive control compound, mitomycin C, caused large, statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the sensitivity of the test system

Second test - 21 hour harvest

Toxicity data

In both the absence and presence of S-9 mix, SVS25% was non toxic, therefore the dose levels selected for metaphase analysis were 1250, 625 and 312.5 µg/ml. The relative mitotic index at 1250 µg/ml was 85% of the solvent control value in the absence of S-9 mix and 123% in its presence. The concentrations of the positive control compounds selected for analysis were mitomycin C at 0.8 µg/ml and cyclophosphamide at 20µg/ml.

Metaphase analysis

No statistically significant increases in the proportion of aberrant cells when compared to the solvent control were seen in cultures treated with SVS 25% in either the absence or presence of S-9 mix.

Positive control compound, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P<0.001) in the proportion of aberrant cells.

Second test - 45 hour harvest

Toxicity data

In both the absence and presence of S-9 mix, SVS 25% at 1250 µg/ml was selected for metaphase analysis as this was the highest concentration analysed at the 21 hour harvest. The relative mitotic index of SVS 25% was 110% of the solvent control value in the absence of S-9 mix and 104% in its presence.

Metaphase analysis

No statistically significant increases in the proportion of aberrant cells when compared to the solvent control were seen in cultures treated with SVS 25% in either the absence or presence of S-9 mix.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

SVS 25% has shown no evidence of clastogenic activity in this in vitro cytogenic test system.