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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1988-10-10 to 1988-12-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1983
Deviations:
no
Qualifier:
according to
Guideline:
other: US Toxic Substances Control Act (TSCA) Final Rule, EPA (1985)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent

Method

Target gene:
Chromosome aberration assays detect the induction of chromosome breakage (clastogenesis). Although mutagenic substances produce structural chromosome aberrations by a variety of mechanisms, the endpoint is a discontinuity in the chromosomal DNA which is left unrejoined, or rejoined inaccurately, thus producing a mutated chromosome. Chromosome aberrations are generally evaluated in first post treatment mitoses. The majority of chemical mutagens induce aberration of the chromatid type, but chromosome type aberrations also occur.
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5a culture medium supplemented with 10% FCS (foetal calf serum) 1% L-glutamine, and 1% penicillin and streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fractions obtained from livers of male Sprague-Dawley rats induced with Aroclor 1254 (commercially purchased)
Test concentrations with justification for top dose:
with metabolic activation: 15.0 to 199 µl/ml
without metabolic activation: 3.74 to 74.8 µl/ml
microscopic analyses:
with metabolic activation: 49.9, 99.7, 150 and 199 µl/ml
without metabolic activation: 24.9, 37.4, 49.9 and 74.8 µl/ml
Results from the range finding assay were used to determine the dose range to be used in the chromosomal aberrations assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: 0.250 and 0.500 µg/ml, positive control without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: 25.0 and 50.0 µg/ml, positive control with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Range finding Assays:

In these assays, the cells were cultured for approximately 24 hours prior to treatment by seeding approximately 0.3 x 10exp6 cells per 25 cm² flask into 5 ml of complete McCoy's 5a culture medium. The thymidine analogue, 5-bromo-2'-deoxyuridine (BrUdR), was added at a final concentration of 10 µM approximately two hours after the initial exposure of the cells to the test article.

Range finding Nonactivation Assay:
- The cultures were dosed with the test article for 2 hours when 5-bromo-2'-deoxyuridine (BrUdR) was added at a final concentration of 10 µM. The cultures were then reincubated for approximately 23 hours. Approximately 2.75 hours prior to the harvest of the cells, the test article was washed from the cells with phosphate buffered saline and fresh complete medium with BrUdR (10 µM), and Colcemid® (final concentration 0.1 µg/ml) was added. The cultures were then harvested and were differentially stained for the analysis of cell cycle delay using a modified fluorescence-plus-Giemsa (FPG) technique.

Range finding Assay with Metabolic Activation:
- In this assay, the CHO cells were exposed to the test article for two hours in the presence of a rat liver S9 reaction mixture (S9 15 µl /ml, NADP 1.5 mg/ml, and isocitric acid 2.7 mg/ml). The S9 fraction was derived from the liver of male Sprague-Dawley rats which had been previously treated with Aroclor 1254 to induce the mixed function oxidase enzymes which are capable of metabolizing chemicals to more active forms. The two hour incubation time was used because prolonged exposure to the S9 mixture might be toxic to the cells and the enzyme activity of S9 is lost rapidly at 37°C.
The medium did not have FCS during the exposure period to avoid possible inactivation of short-lived and highly reactive intermediates produced by the S9 enzymes by binding to serum proteins. In this assay, the CHO cells were incubated at 37°C for two hours in the presence of the test article and the S9 reaction mixture in the growth medium without FCS. After the exposure period the cells were washed twice with buffered saline. Complete McCoy's 5a medium with 10 µM BrUdR was added to the cultures which were then incubated for approximately 23 hours. Colcemid® (final concentration of 0.1 µg/ml) was then added for 2.5 hours to collect metaphase cells. The cultures were then harvested, fixed, and slides were prepared and
stained as was described for the nonactivation range finding assay.

Range finding Assay Evaluation:
- The highest three doses with metaphase cells in both trials were analyzed for toxicity (visual observations of monolayer confluence) and cell cycle kinetics. One hundred consecutive metaphase plates, if available, were assessed for the number of cell cycles through which the cells had progressed while in the presence of BrUdR.

Main test:

- Nonactivation Aberrations Assays:
Cultures were initiated by seeding approximately 1.5 x 10exp6 cells per 75 cm² flask into 10 ml of complete McCoy's 5a medium). One day after culture initiation, the CHO cells to be used in the nonactivation trial were treated with the test article at predetermined doses for 7.25 hours. The cultures were then washed with buffered saline and complete McCoy's 5a medium containing 0.1 µg/ml Colcemid® was placed back onto the cells. Two and one half hours later the cells were harvested and air dried slides were made. The slides were then stained in pH 6.8 buffered 5% Giemsa solution for the analysis of chromosomal aberrations.

- Aberrations Assays with Metabolic Activation:
Cultures were initiated by seeding approximately 1.5 x 10exp cells per 75 cm² flask into 10 ml of complete McCoy's 5a medium. One day after culture initiation. the cultures that were treated under the conditions of metabolic activation were incubated at 37°C for two hours in the presence of the test article and the S9 reaction mixture in McCoy's 5a medium without FCS. After the two hour exposure period the cells were washed twice with buffered saline and the cells were refed with complete McCoy's 5a medium. The cells were incubated for an additional 7.75 hours with 0.1 µg/ml Colcemid®present during the last 2.5 hours of incubation. The metaphase cells were then harvested and prepared for cytogenetic analysis.
NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED:
At least 200 well spread metaphases per concentration and negative/positive controls were scored for cytogenetic damage. The cells scored contained 21 ± 2 centromeres.

HARVEST PROCEDURE AND DETERMINATION OF CYTOTOXICITY
- Prior to the harvest of the cultures visual observations of toxicity were made. These observations included an assessment of the percent confluence of the cell monolayer within the culture flasks. The cultures were also evaluated for the presence of mitotic (large rounded cells) or dead cells floating in the medium. The metaphase cells were collected by mitotic shake-off (Terasima and Tolmach, 1961) and were treated with 0.075 M KCl hypotonic solution. This treatment helps to swell the cells and thus disperse the chromosomes. The cultures were then fixed with an absolute methanol: glacial acetic acid (3:1) fixative and were washed several times before air-dried slides were prepared.

SLIDE PREPARATION AND STAINING
-Slides were prepared by dropping the harvested cultures on clean slides. The slides from the range finding assays were differentially stained using a modified fluorescence-plus-Giemsa (FPG) technique (after Perry and Wolff, 1974; Goto, et al., 1978). The slides were stained in Hoechst 33258 stain, exposed to ultraviolet light, and then stained with Giemsa Azure B stain. The slides prepared from the aberrations assay were stained with pH 6.8
buffered 5% Giemsa solution for the analysis of chromosomal aberrations. All slides were then air-dried and coverslipped using Depex@ mounting medium.

ABERRATION ANALYSIS
- Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 ± 2 (range 19-23) were analyzed. One hundred cells, if possible, from each duplicate culture at four dose levels of the test article and from each of the negative and solvent control cultures were analyzed for the different types of chromosomal aberrations (Evans, 1962). At least 25 cells were analyzed for chromosomal aberrations from one of the positive control culture. For control of bias, all slides except for the positive controls were coded prior to analysis. Cells with aberrations, but not gaps, were recorded on the data sheets by the microscope stage location.

ACCEPTABILITY OF THE ASSAY
An assay will be considered acceptable for evaluation of test results only if all of the following criteria are satisfied. Activation and nonactivation sections of the aberrations assay form independent units and will be repeated independently, as needed, to satisfy the acceptance criteria.
- Unsatisfactory Controls
The assay will be repeated if:
a. The negative or the solvent control is more than twice the upper limit of the range of historical control values (upper limit 5% or more of cells with aberrations).
b. The positive control result is not significantly higher (p<0.01)) than the pooled negative and solvent controls. If the positive control result in the test with S9 is adequate in an assay where activation and nonactivation assays are run concurrently but the positive-control in the nonactivation assay fails, the test need not be repeated as the activation positive control demonstrates . the sensitivity of the cells.

- Lack of Toxicity
If the aberration results are negative and there is no significant reduction in confluence or delay in cell cycle progression, the test may be repeated at higher doses if higher doses can be achieved (i.e. not limited by solubility or not greater than 5 mg/ml).

- Excessive Toxicity
The assay will be repeated if results are not available for at least three dose levels. Data will not be evaluated from dose levels with excessive toxicity. Any culture flask with less confluence of the monolayer than observed at the time of dosing will be deemed to have excessive toxicity. Those cultures from which 200 cells were not available for analysis from the duplicate flasks will also be considered to have cytotoxic effects.

- Sporadic Increase
If a significant increase is seen at one or more dose levels but not in consecutive doses and if there is no clear evidence for a positive dose response, the assay will be repeated to verify the significance.

If the assay has to be repeated because of equivocal results, only three dose levels will be analyzed, instead of four, in the second assay.
Evaluation criteria:
The following factors were taken into account in the evaluation of the chromosomal aberrations data:
1. The overall chromosomal aberration frequencies.
2. The percentage of cells with any aberrations.
3. The percentage or cells with more than one aberration.
4. Any evidence for increasing amounts of damage with increasing dose, i.e., a positive dose response.
5. The estimated number of breaks involved in the production of the different types of aberrations which were observed, i.e., complex aberrations may have more significance than simple breaks.

Chromatid and isochromatid gaps, if observed, were noted in the raw data and were tabulated. They were not, however, considered in the evaluation of the ability of the test article to induce chromosomal aberrations since they may not represent true chromosomal breaks and may possibly be induced by toxicity.
Statistics:
Statistical analysis employed the Fisher's Exact Test with an adjustment for multiple comparisons (Sokal and Rohlf, 1981) to compare the percentage of cells with aberrations in each treatment group with the results from the pooled solvent and negative controls (the solvent and negative controls were statistically evaluated for similarity prior to the pooled evaluation). Test article significance was established where P

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation: >/= 37.4 µl/ml, with metabolic activation: >/= 99.7 µl/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PREEXPERIMENTS on SOLUBILITY, CELL CYCLE KINETICS, and DOSE DETERMINATION
Solubility was evaluated in water and unpipettable, gelatinous mixtures were obtained at 204, 305, and 500 mg/ml. In an assay that was conducted concurrently, a cloudy white liquid was obtained in water at 52.9 mg/ml. This suspension was considered suitable for use in this assay. Therefore, for this assay a stock concentration of 49.6 mg/ml of the test article was prepared in deionized water. Final concentrations were achieved by a 1:10 dosing of the prepared stock solution or the serial dilutions prepared from this stock. A half-log series of concentrations of 0.165 to 4960 µg/ml was tested in the range finding assays.

- Range finding Assay Without Metabolic Activation
A precipitate was visible at the time of dosing and harvest at 165, 496, 1650, and 4960 µg/ml. Dead cell monolayer with no visible mitotic cells were observed at these concentrations and these cultures were not harvested. An unhealthy cell monolayer, 60% reduction in the monolayer confluence, floating dead cells, and a reduction in visible mitotic cells were observed at 49.6 µg/ml. A slight reduction in visible mitotic cells was still discerned at 16.5 µg/ml. Cell cycle kinetics were evaluated at 4.96, 16.5, and 49.6 µg/ml. No significant cell cycle delay was evident at any of the concentrations analyzed. A 10 hour harvest was selected for testing concentrations of 3.74, 4.99, 12.5, 24.9, 37.4, 49.9, and 74.8 µg/ml in the aberrations assay using a 1:100 dosing volume of the 7.48 mg/ml solution prepared from a 19.9 mg/ml solution of the test article or serial dilutions prepared from the 7.48 mg/ml solution.
- Range finding Assay With Metabolic Activation
A precipitate was visible at the time of dosing and harvest at 496, 1650, and 4960 µg/ml. Floating dead cells, floating debris, no visible mitotic cells, and no visible cell monolayer were observed at these concentrations and the cultures were not harvested. At 165 µg/ml, an unhealthy cell monolayer, slight reduction in visible mitotic cells, and 50% reduction in cell monolayer confluence were discerned. Cell cycle kinetics were evaluated at 16.5, 49.5, and 165 µg/ml. No cell cycle delay was evident at these concentrations. A 10 hour harvest was selected for testing concentrations of 15.0, 19.9, 49.9, 99.7, 150, and 199 µg/ml using a 1:100 dosing volume of the 19.9 mg/ml stock solution or the serial dilutions prepared from the stock solution.

CLASTOGENICITY

- Chromosomal Aberrations Assay Without Metabolic Activation
No precipitate was visible after dosing at any of the concentrations. A precipitate was visible at the time of harvest at 49.9 and 74.8 µg/ml, the two highest concentrations evaluated in this test. An unhealthy cell monolayer, reductions in the cell monolayer confluence (30% at 49.9 and 40% at 74.8 µg/ml), and a severe reduction in visible mitotic cells were observed at 49.9 and 74.8 µg/ml. A slightly unhealthy cell monolayer, 30% reduction in the cell monolayer confluency, and reduction in visible mitotic cells were observed at 37.4 µg/ml. Results were evaluated at 24.9, 37.4, 49.9, and 74.8 µg/ml.
A significant increase in chromosomally aberrant cells was not observed at the concentrations analyzed.
The test article is considered negative for inducing chromosomal aberrations under nonactivation conditions.

- Chromosomal Aberrations Assay With Metabolic Activation
No precipitate was visible after dosing at any of the concentrations. A precipitate was visible at the time of harvest at 49.9, 99.7, 150, and 199 µg/ml. Floating dead cells, floating debris, unhealthy cell monolayer, severe reduction in visible mitotic cells, and reductions in the cell monolayer confluency (30% at 99.7 and 150 µg/ml, and 40% at 199 µg/ml) were observed at 99.7, 150, and 199 µg/ml. A slightly unhealthy cell monolayer, floating debris, and a slight reduction in visible mitotic cells were observed at 49.9 µg/ml. Results were analyzed at 49.9, 99.7, 150, and 199 µg/ml.
There was not a significant increase in chromosomally aberrant cells at the doses analyzed.
The successful activation of the metabolic system is illustrated by the increased incidence of chromosomally aberrant cells in the cultures induced with cyclophosphamide, the positive control agent.
The test article is considered negative for inducing chromosomal aberrations under conditions of metabolic activation.

POSITIVE CONTROLS
Mitomycin C for the nonactivation series and cyclophosphamide in the metabolic activation series were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberration.
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

It can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item partially unsaturated IQAC, DMS quaternised (75 %) did not induce structural chromosomal aberrations in the CHO Chinese hamster cell line.

Therefore, the test item Varisoft 475 (methyl-1-tallow amido ethyl-2-tallow imidazolium-methyl sulfate, 75 %) is considered to be non-clastogenic.
Executive summary:

In a mammalian cell cytogenetics assay according to OECD Guideline 473, 1983 and US EPA-TSCA Guidelines, CHO cell cultures were exposed to the patially unsaturated IQAC, DMS quaternised (75 %) at concentrations of 15.0 to 199 µl/ml with metabolic activation and at 3.74 to 74.8 µl/ml without metabolic activation. Evaluation for aberrant cells was performed at for aberrant cells at 49.9, 99.7, 150 and 199 µl/ml with metabolic activation and at 24.9, 37.4, 49.9 and 74.8 µl/ml without metabolic activation.

Cytotoxic effects of the test item were observed with and without metabolic activation; at the highest evaluated concentrations reduction in the cell monolayer confluency was 40 % in both experiments, without and with metabolic activation.

Neither without metabolic activation nor with metabolic activation a significant increase in chromosomally aberrant cells was observed at the concentrations analyzed.

As positive controls reference mutagens Mitomycin C and cyclophosphamide were tested in parallel to the test item. They induced distinct and biologically relevant increases in cells with structural chromosomal aberration.

There was no evidence of chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement of the mentioned guidelines for in vitro mammalian

cytogenetics assays.