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Toxicological information

Genetic toxicity: in vitro

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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

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2007
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 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 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:
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. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
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:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (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:
- 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
Negative controls:
yes
Remarks:
Minimal essential medium with Hanks salts
Solvent 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 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.