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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, comparable to guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
no mitotic index determined but MTD reached; 3 times 50 instead of 2 times 100 cells scored
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Santoflex 14

Method

Target gene:
chromosome aberrations
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix, S9 from Aroclor 1254-induced rat liver homogenate (S9)
Test concentrations with justification for top dose:
pre-test I(+/-S9): 5, 20, 50, 100, 200, 500, 1000, 2000, 5000 µg/ml; pre-test II (+/-S9): 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20 µg/ml,
main experiment I (+/-S9): 0, 7.5, 10, 15 µg/ml, main experiment II (-S9): 7.5, 10 µg/ml;
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: -S9: methylmethanesulfonate, +S9: cyclophosphamide

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
other: solvent aceton induced a relative high number of aberrant cells in main experiment I
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Chinese hamster Ovary (CHO)
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

1. Range-Finding Experiments:

CHO cells were treated with 5 to 5000 µg/ml of SANTOFLEX 14 both in the presence and absence of activation. The cells

were scored for both mitotic index and average cell generation time and compared to the solvent (acetone) control. The average cell generation time for the solvent control was approximately 12 hours for both with and without activation, with a mitotic index of 5

to 8 %. Cytotoxic effects were observed at 20 µg/ml both in the absence and presence of activation as indicated by a sharp reduction in mitotic index and an extended average generation time.

The cytotoxicity of SANTOFLEX 14 was further defined in a second range-finding experiment where concentrations ranged

from 1 to 20 µg/ml with and without activation. The cells were scored for both mitotic index and average cell generation time. Cytotoxic effects were observed at 12.5 µg/ml and higher concentrations in the absence and presence of activation as indicated by an extended average generation time (-S9 20.3 h at 12.5 µg/ml vs. 12.0 control; +S9: at 12.5 µg/ml 15.2 h vs. 12.7 control).

Based on the range-finding experiments, the highest concentrations of SANTOFLEX 14 tested were 15 µg/ml with and without activation. The harvest times were 12 and 24 hours for 1.5, 5, 7.5 and 10 µg/ml with and without activation, 18 and 36 hours for 15 µg/ml with activation and 24 and 48 hours for 15 µg/ml without activation. The harvest times were

chosen to represent approximately 1X and 2X that of the cell generation times.

2. Cytogenetics Studies:

A. Without activation: The SANTOFLEX 14 levels of 7.5, 10 and 15 µg/ml were chosen as the highest three scorable doses.

Statistically significant increases in number of cells with structural aberrations (12 cells with aberrations vs. 3 cells with aberrations solvent control) and average structural aberrations per cell (0.060 vs. 0.015 solvent control) were observed at the 15 ug/ml dose level for the 48 hour harvest time ( % aberrant cells: 6 % vs. 1.5% solvent control) and for average structural aberrations per cell for the 24 hour harvest time (0.120 vs. 0.045 solvent control). A significant dose-response was not observed.

B. With activation: The SANTOFLEX 14 dose levels of 7.5, 10 and 15 µg/ml were selected as the three highest scorable

doses. Statistically significant increases in the number of cells with structural aberrations (at 10 µg/ml 39 vs. 15 solvent control) and average structural aberrations per cell (10 µg/ml: 0.383 vs. 0.090 solvent control) were observed for the 10 µg/ml dose level (% aberrant cells: 19.9 % vs. 7.5% solvent control) and for the number of cells with aberrations at the 7.5 µg/ml dose level for the 12 hour harvest time (at 7.5 µg/ml 29 cells with aberrations vs. 15 cells with aberration solvent control). A dose-related response was not observed.

C. Retest: Because of the relatively high aberration levels (-S9: 10%, +S9: 7.5%) for the 12 hour harvest time in the solvent control, the experiment was repeated using dose levels of 7.5 and 10 µg/ml with and without activation. The 24 and 48 hour harvests were not repeated as the solvent background was low and therefore the statistically significant response of the 15 ug/ml dose level without activation was concluded to be treatment induced. The 10 µg/ml dose level with activation was again statistically significant for the number of cells with aberrations ( 17 cells with aberrations vs. 6 cells solvent control) and for structural aberrations per cell (0.090 vs. 0.030 solvent control) A dose-response relationship was not observed.

Conclusion:

Without activation, a. statistically significant increase in aberration frequency was observed for the 15 µg/ml dose level. In the presence of activation, a statistically significant response was observed at the 10 µg/ml dose level for the 12 hour harvest.

A significant dose-response relationship was not observed. The retest experiment confirmed the statistically significant

response of the 10 µg/ml dose level in the presence of activation. A significant dose-response was not observed.

The authors suggested that the observed effects might be secondary because the effects were observed in the range of cytotoxicity indicated by an increased generation time (10 µg/ml +S9: pre-experiment: 13.7h vs. 12.7 h solvent control, 15 µg/ml -S9: 22.0 h vs. 12.1 h solvent control) and in addition because of a lack of a dose response relationship.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive