4-tert-butylbenzoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

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: Study performed according to OECD guideline, detailed study report, GLP conformity. The study was also referred to in the EU risk assessment report of the substance (2009).

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2007

Reference 2

Reference Type:

publication

Title:

Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test.

Author:

Ames et al.

Year:

1975

Bibliographic source:

Ames, B.N., McCann, J. and Yamasaki, E. (1975), Mutation Res., 31, 347-363

Reference 3

Reference Type:

publication

Title:

The production of micronuclei from chromosome aberrations in irridiated cultures of human lymphocytes.

Author:

Countryman and Heddle

Year:

1976

Bibliographic source:

Countryman, P.I. and Heddle, J.A. (1976), Mutation Res., 41, 321-332

Reference 4

Reference Type:

other: EU risk assessment report

Title:

No information

Author:

Federal Institute for Occupational Safety and Health, Division for Chemicals and Biocides Regulation, Germany

Year:

2009

Bibliographic source:

European Union Risk Assessment Report, 4-tert-butylbenzoic acid, CAS No: 98-73-7, EINECS No: 202-696-3, 4.1.2.7 Mutagenicity, p.66-67, Final Approved Version, July 2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD GUIDELINE FOR THE TESTING OF CHEMICALS DRAFT PROPOSAL FOR A NEW GUIDELINE 487: In Vitro Mammalian Cell Micronucleus Test (MNvit), 2nd Version

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butylbenzoic acid

EC Number:

202-696-3

EC Name:

4-tert-butylbenzoic acid

Cas Number:

98-73-7

Molecular formula:

C11H14O2

IUPAC Name:

4-tert-butylbenzoic acid

Details on test material:

- Name of test material (as cited in study report): p-tert-butylbenzoic acid
- Analytical purity: 99.5 area % (HPLC)

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cell line: obtained from LMP, Technical University of Darmstadt, Germany; stored under liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH; before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability - for the reason of quality assurance.


Cell cultures of V79:
Thawed stock cultures are propagated at 37 °C in 80 square centimetre plastic flasks. About 5*10^5 cells per flask are seeded in 15 ml of MEM (minimal essential medium from Seromed, 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (from PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells are subcultured twice weekly. The cell cultures are incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix: Phenobarbital/ß-naphtoflavone induced rat liver S-9, homogenised and diluted with a KCl solution (1:3), then centrifugation at 9000 g, then aliquots stored deep frozen until S-9 mix prepared according to Ames et al. (1975)

Test concentrations with justification for top dose:

2.9, 5.9, 11.7, 23.4, 46.9, 93.8, 187.5, 375.0, 750.0 and 1500.0 microgram/ml; precipitation occurred at the highest concentration with the S-9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
On the day of the experiment immediately before treatment, the test item was dissolved in DMSO (purity 99.5 %). Final concentration of DMSO in the culture medium was 0.5 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity in the cell cultures.

Controls

Untreated negative controls:

yes

Remarks:

Minimal essential medium with Hanks salts

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 0.5 % (v/v) DMSO

Positive controls:

yes

Positive control substance:

other: without metabolic activation: Colcemid, MMC (Mitomycin C); with metabolic activation: CPA (cyclophosphamide)

Details on test system and experimental conditions:

exposure period: 4 h
recovery: 20 h
preparation interval: 24 h

Statistics:

Statistical significance of the results was confirmed by means of the Chi square test.

Results and discussion

Any other information on results incl. tables

In the absence and presence of S-9 mix, no clear cytotoxicity was observed up to the highest applied concentration.

In the absence of metabolic activation, a statistically significant increase in the percentage of micronucleated cells was observed after 4 h treatment with 375 microgram/ml. Although the increase of 2.15 % was statistically significantly higher than the corresponding control (0.80 % micronucleated cells), the value did not exceed the laboratory's historical control range (0.0 -2.2 % micronucleated cells) and no dose-dependency was observed. Therefore, this observation was regarded as biologically irrelevant.

In contrast, in the presence of S-9 mix, the test item induced an increase in the percentage of micronucleated cells in a dose-related manner. At the two highest scored concentrations (750 and 1500 microgram/ml), the values of 3.2 % and 4.95 %, respectively, were statistically significantly higher than the corresponding control (1.60 % micronucleated cells) and exceeded the laboratory's historical control range (0.0 - 2.5 % micronucleated cells). The observation was therefore regarded as biologically relevant.

Either colcemid (7.5 or 10 microgram/ml), Mitomycin C (0.03 or 0.1 microgram/ml), or CPA (10 or 25 microgram/ml) were scored as positive controls and showed a distinct increase in the percentage of micronucleated cells.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
ambiguous

The result from the in vitro micronucleus test is ambiguous since treatment with S9 mix induced weak increases in micronucleus (positive result) and without S9 mix was negative.