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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented stury report equivalent or similar to OECD guideline 487: GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: F-12K Medium supplemented with 10% fetal bovine serum
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of Aroclor induced rat liver
Test concentrations with justification for top dose:
Rangefinder cytotoxicity:
(+S9): (0.006, 0.02, 0.06, 0.2, 0.6, 2 ul/mL)
(-S9): (0.006, 0.02, 0.06, 0.2, 0.6, 2 ul/mL)
Micronucleus Test:
Short term exposure (0.004, 0.006, 0.008, 0.02, and 0.04 uL/ml)
Long term exposure (0.004, 0.006, 0.008, 0.02, and 0.04 uL/ml)
Vehicle / solvent:
DMSO
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO alone
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: cytosine B-D-arabinofuranoside and digitonin
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Cells were plated at a seeding density of 1.25x104 cells/well in 48-well plates for both cytotoxicity and test runs, allowed to grow for 24 hours post-seeding, and then exposed to the test article and controls. Test article was added to the wells with or without S9 fraction.
DURATION
Short Exposure:
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 21 hours
Long Exposure (absence of S9):
- Exposure duration: 21 hours
- Expression time (cells in growth medium): 21 hours
NUMBER OF REPLICATIONS:
- 3 replicate wells
STAIN
- Microflow Kit, using EMA (nucleic acid dye A solution), PeakFlow Green Flow Cytometry Reference Beads
DETERMINATION OF CYTOTOXICITY
- Method: assessment of relative increase in cell counts (RICC)
Evaluation criteria:
The data from the flow cytometer was collected and compared to the negative control. The sample in each well was analyzed for % of events that are EMA-positive total counts of nucleated events, total counts of hypodiploidy, total bead counts and total micronuclei counts. The data was assessed as follows:
-The percent hypodiploid counts in each well, percent micronuclei counts in each well, and the nuclei to bead ratio in each well was determined
-The fold change in hypodiploidy in each well, fold change in micronuclei in each well, and the fold change in %EMA positive cells in each well was determined
-The mean fold change in hypoploidy EMA positive cell and micronuclei was determined
-The cell viability was determined by dividing the mean nuclei-bead ratio of the test article by the mean nuclei-bead ratio of the negative control, and multiplying by 100
A test article is considered clearly positive if, in any of the experimental conditions examined: at least one of the test concentrations exhibits a statistically significant increase of 2-fold compared with the concurrent negative control, which is considered a biologically relevant induction in this system; if the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test; or if any of the results are outside the distribution of the historical negative control data.
A test article is considered clearly negative if, in all experimental conditions examined: none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control; or if there is no concentration-related increase when evaluated with an appropriate trend test.
Statistics:
For cytotoxicity the mean % viability and standard deviation were calculated. The mean of fold change vs vehicle control (negative) control was determined. A Student’s paired T-test was performed to assess statistical significance (p≤0.01) between each tested concentration and the negative (vehicle) control.

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:
Short term exposure (0.04ul/mL)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Short term exposure - The test substance did not induce a statistically significant increase in micronuclei formation at any of the non-cytotoxic dose levels tested, with or without metabolic activation.
Long term exposure - The test substance did not induce a statistically significant increase in micronuclei formation at any of the dose levels tested without metabolic activation.

Applicant's summary and conclusion

Conclusions:
The mammalian cell micronucleus test to assess the genotoxicity of Exxal 13 was negative.
Executive summary:

Exxal 13 was examined for its potential to induce the formation of micronuclei in Chinese Hamster Ovary (CHO) cells, at both short (with and without S9 metabolic activation) and long (without S9 metabolic activation) exposures. The doses were: short term exposure (0.004, 0.006, 0.008, 0.02, and 0.04 uL/ml) and long term exposure (0.004, 0.006, 0.008, 0.02, and 0.04 uL/ml).

Exxal 13 did not induce a statistically significant increase in the number of cells with micronuclei at any of the non-cytotoxic doses chosen with or without metabolic activation; significant cytotoxicity was observed at 0.02 ul/mL and above in the absence of S9, and at concentrations at and above 0.006ul/mL in the presence of S9. Under the conditions in this study, Exxal 13 was cytotoxic but negative for micronuclei formation in all three tested conditions (short term exposure with and without metabolic activation; long exposure without metabolic activation) for CHO cells.

The mammalian cell micronucleus test to assess the genotoxicity of Exxal 13 was negative.