tert-pentylbenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

other: In vitro mammalian chromosome aberration test.

Test material

Test material information

Constituent 1

Reference substance name:

tert-pentylbenzene

EC Number:

218-076-0

EC Name:

tert-pentylbenzene

Cas Number:

2049-95-8

Molecular formula:

C11H16

IUPAC Name:

(2-methylbutan-2-yl)benzene

Details on test material:

- Substance type: Technical product.
- Analytical purity: 97.4%
- Impurities: Cumene: 0.7%, Heptylbenzene (C13H18): 0.6%, Nonylbenzene (C15H22): 0.6%
- Lot/batch No.: Tank BV1112 du 18 avril 2007.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: no data
- Expiration date of the lot/batch: no data
- Purity: 99.5%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Sealed in a cool, dark place (Measured temperature range: 1°C-8°C)
- Stability under test conditions: stability during experiments was confirmed.
- Solubility and stability of the test substance in the solvent/vehicle: Insoluble in water. Easily soluble in ethanol, ether.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

other: Chinese hamster lungs (CHL/IU)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle MEM culture medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix was prepared from the liver homogenate of rat that was enzyme induced by intraperitoneal administration of phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

- Preliminary test: 5.86, 11.7, 23.4, 46.9, 93.8, 188, 375, 750 and 1500 µg/mL with and without metabolic activation.
- Chromosomal aberration test: 13.1, 16.4, 20.5, 25.6, 32, 40 and 50 µg/mL without metabolic activation (experiment 1 and 2)
- Chromosomal aberration test: 26.2, 32.8, 41, 52.1, 64, 80 and 100 µg/mL with metabolic activation (experiment 1)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (Lot No. SL045, Dojindo Laboratories).
- Justification for choice of solvent/vehicle: no data

Controls

Untreated negative controls:

yes

Remarks:

Vehicle

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Remarks:

With and without S9 mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no
- Exposure duration:
- Experiment 1: 6 hours with and without S9
- Experiment 2: 20 hours without S9
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours (experiment 1 with and without S9 mix)/24 hours (experiment 2 without S9 mix)

SELECTION AGENT (mutation assays): Not applicable

SPINDLE INHIBITOR (cytogenetic assays): colcemid (Lot No. 1335046, GIBCO) (Two hours before).

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: Analysis of 200 metaphases / dose level was made

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A positive result was based on an increase in frequency (%) of total structural aberrations or total numerical aberrations of 10% or higher with this frequency being dose-dependent, or an increase in frequency of 5% or higher when this increase was reproducible by a confirmation test. All other findings gave a negative result.

Statistics:

No statistical methods were used in evaluation.

Results and discussion

Test results

Key result

Species / strain:

other: Chinese hamster lungs (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with and without metabolic activation.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50% or greater cell growth inhibition was observed at doses of 40.0 μg/mL (Experiment 1 without S9), 80.0 μg/mL and above (experiment 1 with metabolic activation), and 32.0 μg/mL and above (experiment 2 without S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: measurements on post-treatment media without and with S-9 mix indicated that
treatment with the test substance had no effect on pH.
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: the test substace is insoluble.
- Precipitation:
At the start of test solution treatment precipitation of the test article was observed in each test system at a dose of 188 μg/mL and above or 375 μg/mL, and at the completion of test solution treatment test article precipitation was observed at a dose of 1,500 μg/mL in the short-term treatment method both without metabolic activation and with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
A preliminary test (cell growth inhibition test: 5.86-1,500 μg/mL) revealed cell growth inhibition occurred in each test system. IC50 values were 35.1 μg/mL with the short-term treatment method without metabolic activation (6 hours of treatment), 67.9 μg/mL with the short-term treatment method with metabolic activation (6 hours of treatment), and 32.7 μg/mL with the continuous treatment method with 24-0 hour treatment without metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Solvent and positive controls are in the range of historical control data.

Applicant's summary and conclusion

Conclusions:

Under the test conditions of this study, tert-pentylbenzene did not induce a significant level of chromosome aberrations with or without metabolic activation. Therefore, tert-pentylbenzene is not considered as clastogenic.

Executive summary:

In an in vitro chromosome aberration test, performed according to OECD guideline N° 473 and in compliance with GLP, Chinese hamster Lungs (CHL/IU) cells were treated with tert-pentylbenzene dissolved in DMSO in two independents experiments, both with and or without a liver metabolizing system (S9-mix), obtained from rats previously treated with phenobarbital and 5,6-benzoflavone.

A preliminary test (cell growth inhibition test: 5.86-1,500 μg/mL) revealed cell growth inhibition occurred in each test system. IC₅₀values were 35.1 μg/mL with the short-term treatment method (6 hours) without metabolic activation, 67.9 μg/mL with the short-term (6 hours) treatment method with metabolic activation, and 32.7 μg/mL with the continuous treatment method with 24-0 hour treatment without metabolic activation. At the start of test solution treatment precipitation of the test article was observed in each test system at a dose of 188 μg/mL and above or 375 μg/mL, and at the completion of test solution treatment test article precipitation was observed at a dose of 1,500 μg/mL in the short-term treatment method both without metabolic activation and with metabolic activation. No test article effect on culture medium pH was observed.

Based on the results of the preliminary test, the present test (chromosomal aberration test) was performed with a maximum dose above the IC₅₀ for each test system. In experiment 1, treatment was continuous for 6 hours at 13.1, 16.4, 20.5, 25.6, 32, 40 and 50 µg/mL in the absence of S-9 and at 26.2, 32.8, 41, 52.1, 64, 80 and 100 µg/mL with metabolic activation. In experiment 2,

treatment was continuous for 24 hours at 13.1, 16.4, 20.5, 25.6, 32, 40 and 50 µg/mL in the absence of S-9.

The incidence of structural chromosomal aberrations and numerical chromosomal aberrations observed in the present test was below 5% with the short-term treatment method without metabolic activation (evaluated doses: 20.5, 25.6, and 32.0 μg/mL), the short-term treatment method with metabolic activation (41.0, 51.2, 64.0, and 80.0 μg/mL), and the continuous treatment method with 24-0 hour treatment (16.4, 20.5, 25.6, and 32.0 μg/mL).The incidence of structural chromosomal aberrations in the positive control group was a clearly positive result for all test systems, which confirmed that these test systems are appropriately sensitive.

Under the test conditions of this study, tert-pentylbenzene did not induce a significant level of chromosome aberrations with or without metabolic activation. Therefore, tert-pentylbenzene is not considered as clastogenic.