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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other: GLP compliant study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): 2,4,6-Trimethyl Phenol (TMP, CAS # 527-60-6)
- Test article description: White solid
- Test article purity: 80 - 93%
- Storage conditions: Room temperature, protected from exposure to light and moisture

Method

Target gene:
Thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
- Preliminary toxicity assay: nine concentrations between 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1360 µg/mL
- With metabolic activation: 10, 30, 40, 50, 100 µg/mL
- Without metabolic activation: 50, 75, 100, 125, 150 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was determined to be the solvent of choice based on the solubility of the test article and compatibility with the target cells.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2.5, 5, 10, 20 µg/mL
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Remarks:
2.5, 5, 10, and 20 µg/mL methylmethanesulfonate without metabolic activation; 5 and 7.5 µg/mL 7,12-dimethylbenzanthracene with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without metabolic activation 4 or 24 hours; with metabolic activation 4 hours
- Expression time (cells in growth medium): 48 hours after initial exposure
- Selection time (if incubation with a selection agent): 10 - 14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: suspension growth
Evaluation criteria:
- A result was considered positive if a concentration-related increase in mutant frequency was observed and one or more dose levels with 10% or greater total growth exhibited mutant frequencies of ≥100 mutant per 1E6 clonable cells over the background level.
- A result was considered equivocal in the mutant frequency in treated cultures was between 55 and 99 mutants per 1E6 clonable cells over the background level.
- A result was considered negative if the mutant frequency in treated cultures was fewer than 55 mutants per 1E6 clonable cells over the background level.

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No visible precipitation was present at any dose level.

RANGE-FINDING/SCREENING STUDIES:
Visible precipitation was observed in treatment medium at dose level 1360 µg/mL. No visible precipitate was present at concentration ≤500 µg/mL in treatment medium Suspension growth relative to the solvent controls was 0% at 500 µg/mL without activation with 4- and 24-hour exposures and 0% at 150 µg/mL with S9 activation.

ADDITIONAL INFORMATION ON GENOTOXICITY:
- Without metabolic activation, 4 hour exposure: One cloned culture (125 µg/L) exhibited a mutant frequency of 100 mutants per 1E6 clonable cells over that of the solvent control, and five cultures (75, 100, 125, and 150 µg/L) exhibited mutant frequencies that were between 55 and 99 mutants per 1E6 clonable cells over that of solvent control. There was no dose-response trend.
- With metabolic activation, 4 hour exposure: One cloned culture (40 µg/L) exhibited a mutant frequency that was between 55 and 99 mutants per 1E6 clonable cells over that of solvent control. There was no dose-response trend.
- Without metabolic activation, 24 hour exposure: One cloned culture (100µg/mL) exhibited a mutant frequency of 115 mutants per 1E6 clonable cells over that of the solvent control, and two cloned cultures (75 and 100 µg/mL) exhibited mutant frequencies between 55 and 99 mutants per 1E6 clonable cells over that of solvent control. There was no dose-response trend.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Suspension growth of ≤50% of the solvent control, was observed at ≥75 µg/mL without activation with 4-hour exposure and ≥30 µg/mL with activation with a 4-hour exposure. Toxicity in cloned cultures was observed at doses 150 µg/mL without activation with 24-hour exposure.
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):
ambiguous without metabolic activation
negative with metabolic activation

Under the conditions of this study, the test substance was concluded to be equivocal without activation and negative with S9 activation for mutagenicity in mammalian cells.
Executive summary:

In a GLP compliant in vitro Mouse Lymphoma Assay, L5178Y cells were exposed to the test substance with and without metabolic activation (S9 mix) to investigate the potential of the test substance to induce gene mutations. The cells were treated for 4 and 24 hours in the non-activated test system, and for 4 hours in the S9 activated test system. Different dosages (up to 100 and 150 µg/mL, with and without metabolic activation, respectively) were chosen. Cytotoxicity (total growth ≤50% of the solvent control) was observed at ≥75 and 150 µg/mL without activation with 4-hour and 24 -hour exposure, respectively, and at dose levels ≥30 µg/mL with metabolic activation. In the presence of S9 mix the test substance induced in one culture (40 µg/mL) a mutant frequency between 55 and 99 over solvent control. All other mutant frequencies were below 55 over solvent control and therefore the results were negative in the presence of S9 -mix. In the absence of S9 mix results were equivocal, because in both the 4 -hour and 24 -hour treatment one cultures exhibited a mutant frequency of ≥100 mutants per 1E6 clonable cells over that of the solvent control. However, no dose-response was observed, but as for both exposure times several cultures exhibited mutant frequencies between 55 and 99 mutants per 1E6 clonable cells over that of the solvent control, the results in the absence of a metabolic activation system were considered to be equivocal.