4-isopropyl-m-cresol

Genetic toxicity: in vitro

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

Study period:

From March 30 to June 25, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD 476 Guideline without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

(UK GLP Compliance Programme (inspected on February 28, 2000/ signed on April 26, 2000)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-isopropyl-m-cresol

EC Number:

221-761-7

EC Name:

4-isopropyl-m-cresol

Cas Number:

3228-02-2

Molecular formula:

C10H14O

IUPAC Name:

4-isopropyl-m-cresol

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Biosol
- Physical state: White crystalline Solid
- Storage condition of test material: Room temperature in the dark

Method

Target gene:

thymidine kinase (tk) gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source: L5178Y TK +/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr D Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in LN2 at that time.
- Type and identity of media: RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.
- Properly maintained: Yes
- Periodically "cleansed" against high spontaneous background: Yes; The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. For the following 24 hours the cells were cultured in THG medium (i.e.,THG medium without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (10%); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test:
- 23.44, 46.88, 93.75, 187.5, 375, 750 and 1500 µg/mL in the 3-hour exposure with and without S9
- 7.5, 15, 30, 60, 90, 120 and 150 µg/mL in the 24-hour continuous exposure without S9

Mutation tests:
- Experiment 1: 7.5, 15, 30, 45, 60 and 90 µg/mL in the 3-hour exposure without S9
- Experiment 1: 3.75, 7.5, 15, 30, 45 and 60 µg/mL in the 3-hour exposure with S9
- Experiment 2: 4, 8, 16, 32, 48 and 64 µg/mL in the 24-hour continuous exposure without S9
- Experiment 2: 8, 16, 24, 32, 40 and 48 µg/mL in the 3-hour exposure with S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Test material was accurately weighed and dissolved in DMSO before the appropriate dilutions were made. The molecular weight of the test material was 150.22, therefore the maximum dose level was 1500 µg/mL which was equivalent to 10 mM.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix; 800 and 150 µg/mL for Experiment 1 and 2, respectively

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix; 5 and 2.5 µg/mL for Experiment 1 and 2, respectively

Details on test system and experimental conditions:

CELL CULTURE
The stocks of cells are stored in LN2 at -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 Units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM)+, Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5 % CO2 in air. The cells have a generation time of ca. 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.

DURATION
- Exposure duration:
Preliminary toxicity test: 3-hour exposure with and without S9 and 24-hour continuous exposure without S9
Mutagenicity test:
Experiment 1: 3-hour exposure with and without S9
Experiment 2: 24-hour continuous exposure without S9; 3-hour exposure with S9
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and vehicle control
- Main test: Duplicate cultures/dose for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED: 2 and 2000 cells per well plated for viability (%V) and mutant frequency (MF), respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growth (RSG) and Plating efficiency (viability)
Relative suspension growth (RSG):
The cell counts obtained immediately post treatment and over the 2 day expression period were used to calculate the % Relative Suspension Growth.
Suspension Growth (SG) = (24-hour cell count/2) * (48-hour cell count/2)
Day 0 Factor = dose 0-hour cell count / vehicle control 0-hour cell count
% RSG = [(dose SG * dose Day 0 Factor) / vehicle control SG] * 100

Plating efficiency (viability)
- Since the distribution of colony forming units over the wells is described by the Poisson distribution, the plating efficiency (PE) was calculated using the zero term of the Poisson distribution [P(0)] method.
P(0) = No. of negative wells / total wells plated
PE% = [-In P(0) * 100] / no. of cells per well

OTHER:
Calculation of Mutation Frequency (MF)
MF per survivor = [-In P(0) selective medium) / cells per well in selective medium] / surviving fraction in non-selective medium
The experimental data was analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.

Evaluation criteria:

The normal range for mutant frequency per survivor is 25-150 x 10^-6 for the TK +/- locus in L5178Y cells at this laboratory. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10^-6 mutant frequency per survivor are not normally acceptable and will be repeated.

Positive control chemicals should give a significant increase in mutant frequency per survivor over the negative controls of at least a three to five- fold increase.

For a test material to give a significant result then 2 or more of the following criteria should be met:
- a statistically significant increase in mutant frequency
- a greater than three-fold increase in the mutant frequency per survivor over the negative control value
- a dose related increase in the mutant frequency per survivor
- an increase in the absolute number of mutants.

A test material may be reported as equivocal if only one of the above criteria is met. The above criteria may also be used at the discretion of the Study Director to over-rule any spurious significant responses designated by computer program.

Statistics:

No data

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

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 and osmolality: No change in osmolality of the medium of more than 50 mOsm/kg was observed compared with the vehicle control. No change in the pH of the medium compared with the vehicle control.

PRELIMINARY TOXICITY TEST:
- A precipitate of test material was observed at and above 750 μg/mL at the end of the exposure period. In both the absence and presence of metabolic activation, there was a reduction in the plating efficiency of cells treated with the test material when compared to the vehicle controls. In the mutagenicity test, the maximum dose was limited due to toxicity.

MAIN TESTS
Experiment 1: There was clear evidence of dose-related toxicity with the test material in both the presence and absence of metabolic activation as indicated by the %RSG and Day 2 (%V) viabilities this is confirmed by the decrease in relative total growth (RTG) values. The toxicity observed at 90 μg/mL in the absence and 60 μg/mL in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, the doses were excluded from the statistical analysis. A precipitate of test material was not observed at any dose level.
Experiment 2: The 24 hour continuous exposure without S9 treatment, demonstrated that the extended time point had no marked effect on the toxicity of the test material. Similar to Experiment 1, there was clear evidence of a dose-related reduction in % RSG values in cultures dosed with the test material in both the presence and absence of metabolic activation. There was evidence of a reduction in Day 2 (% V) viability and RTG, therefore indicating that residual toxicity occurred, in both the presence and absence of metabolic activation. A precipitate of test material was not observed at any dose level.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical control data.

Remarks on result:

other: strain/cell type: L5178Y mouse lymphoma (3.7.2c) cells

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

See the attached document for summary results

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, Biosol was shown to be non-mutagenic to L5178Y cells with and without metabolic activation.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk^+/-(3.7.2C) mouse lymphoma cells were exposed to Biosol at the following concentrations: 

Preliminary toxicity test:

- 23.44, 46.88, 93.75, 187.5, 375, 750 and 1500 µg/mL in the 3-hour exposure with and without S9

- 7.5, 15, 30, 60, 90, 120 and 150 µg/mL in the 24-hour continuous exposure without S9

 

Mutation tests:

- Experiment 1: 7.5, 15, 30, 45, 60 and 90 µg/mL in the 3-hour exposure without S9

- Experiment 1: 3.75, 7.5, 15, 30, 45 and 60 µg/mL in the 3-hour exposure with S9

- Experiment 2: 4, 8, 16, 32, 48 and 64 µg/mL in the 24-hour continuous exposure without S9

- Experiment 2: 8, 16, 24, 32, 40 and 48 µg/mL in the 3-hour exposure with S9

 

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 10 % (v/v) S9 mix; S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and β-naphthoflavone.

 

In preliminary toxicity test, a precipitate of test material was observed at and above 750 μg/mL at the end of the exposure period. In both the absence and presence of metabolic activation, there was a reduction in the plating efficiency of cells treated with the test material when compared to the vehicle controls. In the mutagenicity test, the maximum dose was limited due to toxicity. In main experiments, no precipitate of test material was observed at any dose level.

 

Biosol did not induce any toxicologically significant or dose-related increase in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. In all tests the concurrent vehicle and positive control were within acceptable ranges. 

 

Under the test conditions, test material was shown to be non-mutagenic to L5178Y cells with and without metabolic activation.