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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: According to OECD Guideline 476 (adopted 4 April 1984).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
1) The OECD Principles of Good Laboratory Practice, 1981; and 2) Chemicals Act of the Federal Republic of Germany (Annex 1), 14 March 1990.
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,6-xylenol; 2,6-dimethylphenol
- Physical state: Colourless solid
- Analytical purity: 99.8 + %
- Purity test date: No data
- Stability under test conditions: No data
- Storage condition of test material: Was stored at 4 degrees C

Method

Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
Experiment I: Without S9 mix: 10.0, 30.0, 100.0, 300.0 µg/mL; With S9 mix: 30.0, 200.0, 300.0, 450.0 µg/mL
Experiment II: Without S9 mix: 30.0, 300.0, 350.0, 400.0 µg/mL; With S9 mix: 30.0, 100.0, 200.0, 300.0, 600.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity for the cells. The final concentration of ethanol in the culture medium did not exceed 1% v/v.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
>98% purity; 0.6 mg/mL (4.8 mM); dissolved in nutrient medium

Migrated to IUCLID6: without metabolic activation
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
approx. 95% purity; 3.85 ug/mL (15 uM); dissolved in DMSO

Migrated to IUCLID6: with metabolic activation
Details on test system and experimental conditions:
PRE-TEST ON TOXICITY: A pre-test was performed to determine the concentration range for the mutagenicity experiments. The general culturing and experimental conditions in the pre-test were the same as those described below for the mutagenicity experiments. In this pre-test, the colony forming ability of approximately 500 single cells (duplicate cultures per concentration) after treatment with 2,6-xylenol was observed and compared to the controls. Toxicity of 2,6-xylenol was evidenced by a reduction in cloning efficiency. In the pre-test for toxicity (colony forming ability) the cloning efficiency of the V79 cells was reduced after treatment with 10.0 ug/mL -S9 mix and with 300.0 ug/mL +S9 mix.

DOSE SELECTION: Experiment I was performed with six concentrations ranging from 3.0 ug/mL -S9 mix and 30.0 ug/mL +S9 mix both up to 600.00 ug/mL. Experiment II was performed with six concentrations ranging from 30.0 ug/mL up to 400 ug/mL -S9 mix and up to 600.0 ug/mL +S9 mix. During the course of the main experiments, four concentrations (five in Experiment II +S9 mix) were selected to be evaluated at the end of the experiment.

MUTAGENICITY EXPERIMENTS
SELECTION AGENT: 6-thioguanine
SEEDING: The cell suspension was seeded into plastic culture flasks. Approximately 1.5 x 10^6 (single culture) and 5 x 10^2 cells (in duplicate) were seeded in MEM with 10% FCS (complete medium) for the determination of mutation rate and toxicity, respectively.
TREATMENT: After 24 h the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 50 uL/mL S9 mix. After 4 h this medium was replaced with complete medium following two washing steps with saline G.
INCUBATION: Cultures were incubated at 37 degrees C in a humidified atmosphere with 4.5% CO2.
FIXATION AND STAINING: Cloning efficiency flasks in the main experiments were processed for fixation and staining on Day 16, while mutant selection flasks were processed for fixation and staining on Day 17. Fixation and staining for the Pre-test on Toxicity flasks occurred on either Day 8 or Day 9. The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted.
Evaluation criteria:
A test article was classified as positive if it induced either a concentration-related increase of the mutant frequency or a reproducible and positive response for one of the test points. A test article producing neither a concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points was considered non-mutagenic in this system. A significant response was described as follows: The test article was classified as mutagenic if it induced reproducibly with one of the concentrations a mutation frequency that was three times higher than the spontaneous mutation frequency in the experiment. The test article was classified as mutagenic if there was a reproducible concentration-related increase of the mutation frequency. Such evaluation was also considered in the case that a three-fold increase of the mutant frequency was not observed. However, in a case by case evaluation this decision depended on the level of the corresponding negative control data. If there was by chance a low spontaneous mutation rate in the range normally found (0 - 45 mutants per 10^6 cells), a concentration-related increase of the mutations within this range was discussed.
Statistics:
Not performed.

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
Vehicle controls validity:
other: Absolute cloning efficiency was low for the+S9 vehicle control in Exp. II only. However, this did not affect the validity of the study.
Untreated negative controls validity:
other: Absolute cloning efficiencies were low for both the -S9 and +S9 neg. controls in Exp. II only. However, this did not affect the validity of the study.
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A pre-test on toxicity was performed. The cloning efficiency of the V79 cells was reduced after treatment with 10.0 ug/mL -S9 mix and with 300.0 ug/mL +S9 mix. These results were used to select concentrations for the main mutagenicity experiments.

COMPARISON WITH HISTORICAL CONTROL DATA: Performed.

Any other information on results incl. tables

In Experiment I a concentration-dependent toxic effect evidenced by a reduction in cloning efficiency (CE% relative) was observed in the -S9 cultures beginning at 100.0 ug/mL up to the highest investigated concentration. Also, the cell density at the first subcultivation (-S9 mix) was reduced at 300.0 ug/mL. In the +S9 cultures, toxicity evidenced by a reduction in the cloning efficiency as well as in the cell density at first subcultivation was obtained with 450.0 ug/mL. At the highest concentration (600.0 ug/mL) both with and without metabolic activation, the cultures could not be subcultured for mutant selection due to the high toxicity of 2,6 -xylenol.

In Expermiment II the toxicity of 2,6 -xylenol was less distinctive. In the -S9 cultures, 2,6 -xylenol showed toxic effects beginning at the concentration of 300.0 ug/mL in the cloning efficiency; the cell density at the first subcultivation was slightly reduced at 400.0 ug/mL (highest concentration tested). In the +S9 cultures strong toxic effects in the cloning efficiency as well as in the cell density at first subcultivation were observed at 600.0 ug/mL (the highest concentration). However, this concentration was subcultivated for the selection of mutants; cell density at first subcultivation was 19.2%, which compared to the corresponding solvent control, but was slightly under the recommended 20% limit.

The mutation rates found in the groups treated with 2,6 -xylenol were comparable to those found in the negative and solvent controls; thus, no relevant increase of gene mutations was observed. 2,6 -xylenol did not induce a reproducible concentration-related increase in mutant colony numbers. The mutant values of the groups treated with 2.6 -xylenol were in the range of the negative controls.

The slightly increased value of 36.1 mutants per 10^6 cells in Experiment I at 450 ug/mL +S9, which resulted in a mutation factor of 2.95 was regarded not to be biologically relevant. This effect could not be reproduced in the independent experiment. Additionallyy, the value of 36.1 mutants per 10^6 cells was within the laboratory's historical control range (0 - 45 mutants per 10^6).

In this study in both experiments (-S9 and +S9) the range of the negative controls was from 4.5 up to 18.4 mutants per 10^6 cells; the range of the groups treated with 2.6 -xylenol was from 0.7 up to 36.1 mutants per 10^6 cells.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

As the mutation rates found in the groups treated with 2,6-xylenol were comparable to the negative and solvent controls, it was concluded that no relevant increase of gene mutations was observed. In conclusion,under the experimental conditions reported, 2,6-xylenol did not induce gene mutations at the HPRT locus in V79 cells. Therefore, 2.6-xylenol was considered non-mutagenic in this HPRT assay.