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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 21st July 1997
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinwiesen 19, Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Oxooel N9
- Physical state: colorless clear liquid
- Lot/batch No.: Tank 252 vom 25.01.2008
- Storage condition of test material: At room temperature, under nitrogene

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I: without S9-mix: 0.16 - 2.5 µg/mL; with S9-mix: 39.1 - 1250 µg/mL
Experiment II: without S9-mix: 1.3 - 20 µg/mL; with S9-mix: 40 - 640 µg/mL
Experiment III: without S9.mxi: 2.5 - 30 µg/mL; with S9-mix: 40 -640 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 15 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: two parallel cultures

NUMBER OF CELLS EVALUATED: at least 50 colonies

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.6 – 31.7 mutants per 1000000 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cloning efficiancy of less than 50 % in both parallel cultures occurred in experiment I at 2.5 μg/mL without metabolic activation (4 hours treatment). In experiment II relevant toxic effects as described above occurred at 20 μg/mL in the absence of metabo
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Phase separation: found at 10µg/mL in 4 hour Experiement without S9-mix and at 80 µg/mL in 4 hour experiment with S9-mix

RANGE-FINDING/SCREENING STUDIES: Relevant toxic effect occurred at 10 μg/mL and above without metabolic activation after 4 hours of treatment. In the presence of metabolic activation (4 h treatment) the cell growth was completely inhibited at 1084.8 μg/mL and above. Following 24 hours treatment a clear toxic effect was determined at the maximum concentration of 40 μg/mL.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Phase separation was observed by the unaided eye at 2169.5 and 4339 μg/mL in the presence of metabolic activation (4 hours treatment).


ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects as indicated by a relative cloning efficiency I (CE I) of less than 50 % in both parallel cultures occurred in experiment I at 2.5 μg/mL without metabolic activation (4 hours treatment). In experiment II relevant toxic effects as described above occurred at 20 μg/mL in the absence of metabolic activation and at 320 μg/mL and above with metabolic activation. In experiment III cytotoxic effects were determined at 30 μg/mL and above without and 320 μg/mL and above with metabolic activation. The substantial variability of the cytotoxicity with metabolic activation is based on phase separation phenomena. If a clear separation in two layers occurs the cells adhering to a wall of the culturing flasks do not get into contact with the upper layer at all. However if an emulsion is formed the cells get into contact with both phases and toxic effects are likely to occur. The protein and lipid content of the S9 fraction used in the experimental parts with metabolic activation tends to stabilize emulsions acting like a detergent.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No relevant and reproducible increase in mutant colony numbers/1000000 cells was observed in the main experiments up to the maximal concentration. The induction factor of three times the corresponding solvent control was exceeded in experiment III, culture II with metabolic activation at almost all of the concentrations. However, this effect was not considered biologically relevant since it was not reproduced in the parallel culture performed under identical experimental conditions and was not dose dependent as indicated by the lacking statistical significance. In fact, this increase is based on the low corresponding solvent control of just 5.0 colonies per 1000000 cells. All of the mutation frequency values remained within the historical range of solvent controls. An isolated but substantial increase of the mutation frequency was noted in the second culture of the second experiment at 20 μg/mL. However, no comparable increase occurred in the parallel culture under identical conditions. Still, the increase was statistically relevant so, the experiment was repeated to verify this increase. No increase of the mutation frequency was observed in the repeat experiment though the cytotoxic range of approximately 10-20% relative cloning efficiency I was covered.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in the second culture of the first experiment with metabolic activation. Since the mutation frequency did not exceed the threshold of three times the corresponding solvent control the statistical result was considered as biologically irrelevant. Another significant trend was detected in the second culture of the second experiment without metabolic activation. This trend was relevant since the mutation frequency exceeded the threshold as well and resulted in an additional verifying experiment. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was also detected in the first culture of the first experiment with metabolic activation and in the second culture of the second experiment with metabolic activation. These trends however, were judged as irrelevant since both were reciprocal, going down versus increasing concentrations.

In the main experiments (with and without S9 mix) the range of the solvent controls was from 5.0 up to 31.0 mutants per 1000000 cells; the range of the groups treated with the test item was from 0.0 up to 453.1 mutants per 1000000 cells.

EMS (0.15 mg/mL in experiment I and II, and 0.075 mg/mL in experiment III) and DMBA (1.1 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Applicant's summary and conclusion

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.