2,4,6-trimethylphenol

Genetic toxicity: in vitro

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other: GLP compliant study, available as unpublished report, no restrictions, fully adequate for assessment.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

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-trimethylphenol

EC Number:

208-419-2

EC Name:

2,4,6-trimethylphenol

Cas Number:

527-60-6

Molecular formula:

C9H12O

IUPAC Name:

2,4,6-trimethylphenol

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 lot No.: 7/31/01
- Test article description: White solid
- Storage conditions: Room temperature, protected from exposure to light and moisture

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

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 0.136, 0.408, 1.36, 4.08, 13.6, 40.8, 136, 408, 1360 µg/mL
- With metabolic activation, 4 h exposure: 12.5, 25, 50, 100, 150, 200 µg/mL
- Without metabolic activation, 4 h exposure: 25, 50, 100, 150, 200, 300 µg/mL
- Without metabolic activation, 20 h exposure: 25, 50, 100, 150, 200, 300, 400 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

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

0.1 and 0.2 µg/mL mitomycin without metabolic activation; 10 and 20 µg/mL cyclophosphamide with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
- Exposure duration: without metabolic activation 4 and 20 hours, with metabolic activation 4 hours
- Expression time (cells in growth medium): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Two hours prior to harverst Colcemid was added to duplicate flasks for each treatment condition.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: The percentage of cells in mitosis per 500 cells scored (mitotic index) was determined for each treatment group. Metaphase cells with 20 ± 2 centromeres were examined under oil immersion without prior knowledge of treatment groups. A minimum of 200 metaphase spreads were examined and scored for chromate-type and chromosome-type aberrations. Chromatid-type aberrations include chromatid and isochromatid breaks and exchanges figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations includes chromosome breaks and exchanges figures such as dicentrics and rings.

DETERMINATION OF CYTOTOXICITY:
- Method: relative total growth and mitotix index
- Tryptan blue dye exclusion was used to determine viability

STAINING: Slides were stained with 5% Giemsa

Evaluation criteria:

- Selection of doses for microscopic analysis was based on toxicity (the lowest dose with at least 50% reduction in cell growth relative to the solvent control and two lower doses) in the non-activated 4 hour exposure group. Selection of doses for microscopic analysis was based on mitotic inhibition (the lowest dose with at least 50% reduction in mitotic index relative to the solvent control and two lower doses) in the S9 activated 4 hour exposure group and in the non-activated 20 hour exposure group.
- Validity criteria: The frequency of cells with structural chromosome aberrations in the solvent must be within the range of the historical solvent control. The percentage of cells with chromosome aberrations in the positive control must be statistically increased (p≤0.05, Fischer's exact test) relative to solvent control.
- The test article was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant (p≤0.05). However, values that are statistically significant but do not exceed the range of historic solvent controls may be judged as not biologically significant. Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative.

Statistics:

Fisher's exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

structural aberrations

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
- Effects of pH: the pH of the highest concentration of test article in treatment medium was approximately 6.5.
- Effects of osmolality: The osmolality in treatment medium of the highest concentration tested, 400 µg/mL, was 386 mmol/kg. The osmolality of the solvent in treatment medium was 426 mmol/kg.
- Precipitation: No precipitation was observed in the main assay.

RANGE-FINDING/SCREENING STUDIES:
Visible precipitate was observed in treatment medium at dose level 1360 µg/mL. Dose levels ≤408 µg/mL were soluble in treatment medium. The osmolality in treatment medium of the highest soulble concentration, 408 µg/mL, was 384 mmol/kg. The osmolality of the solvent in treatment medium was 424 mmol/kg. The pH of the highest concentration of test article in treatment medium was approximately 6.5. Substantial toxicity (i.e. 50% of growth inhibition) was observed at dose levels ≥ 408 µg/mL in 4 hour exposure group without metabolic activation, at dose levels ≥ 136 µg/mL in the 4 hour exposure group with metabolic activation and in the 20 hour exposure group without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxicity of the test substance in CHO cells when treated for 4 hours in the absence of S9 activation was 63% at 300 µg/mL, the highest test concentration evaluated. The mitotic index at the highest dose level evaluated for chromosome aberrations, 300µg/mL, was 40% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 50, 150, and 300 µg/mL. Toxicity of the test substance in CHO cells when treated for 4 hours in the presence of S9 activation was 35% at 200 µg/mL, the highest test concentration evaluated. The mitotic index at the highest dose level evaluated for chromosome aberrations, 200µg/mL, was 61% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 50, 100, and 200 µg/mL. Toxicity of the test substance was 40% at 100 µg/mL, the highest test concentration evaluated in the non-activated 20 hour continuous exposure group, the highest test concentration evaluated. The mitotic index at the highest dose level evaluated for chromosome aberrations, 100 µg/mL, was 56% reduced relative to the solvent control. The dose levels selected for microscopic analysis were 25, 50, and 100 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The percentage of cells with structural aberrations in CHO cells when treated for 4 hours in the absence of S9 activation was significantly increased above that of the solvent control at dose level 300 µg/mL (p≤0.05, Fischer's test). The Cochran-Armitage test was also positive for a dose-response (p<0.05). However, the percentage of cells with structural aberrations in the treated group (3.0%) was within the historical solvent control range of 0 - 5.5%. Therefore, it is not considered to be biologically significant.

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):
positive with metabolic activation
negative without metabolic activation

The test substance is considered to be mutagenic in the presence of metabolic activation in this chromosomal aberration test.

Executive summary:

In a GLP-compliant chromosome aberration test Chinese hamster ovary (CHO) cells were exposed to the test substance in DMSO with and without metabolic activation (S9 -mix). The cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9 activated test system and all cells were harvested at 20 hours after treatment initiation. Different dosages (up to 200, 300 and 400 µg/mL, for +S9 mix 4 h, -S9 mix 4 h and -S9 mix 20 h, respectively) were chosen in the three groups. Cytotoxicity was observed in all three groups at the highest tested dose. The percentage of cells with structural aberrations in the test article-treated groups was significantly increased above that of the solvent control at dose levels 100 and 200 µg/mL in the S9 activated 4 hour exposure group. The precentage of numerical aberrations was not significantly increased. In the non-activated 4 -hour exposure group, the percentage of cells with structural aberrations was significantly above that of the solvent control at dose level 300 µg/mL. However, this percentage of 3.0 % was within the historical solvent control and therefore not considered to be biologically relevant. In the absence of the S9 mix in the 20 -hour exposure group, the test article did not increase the frequency of cells with aberrations. Based on these results the test substance is considered to be mutagenic in the presence of metabolic activation.