Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Genetic toxicity in bacteria: The test substance tested with four bacterial strains showed no mutagenicity neither in the presence nor absence of rat liver preparation in the dosage range of 2500 to 4 microgram. An in vitro micronucleus test with Chinese Hamster V79 cells: In the presence of S-9mix (metabolic activation with S-9) p-tert-butylbenzoic acid had to be considered as mutagenic as it had induced micronuclei in V79 cells. Remarks in the EU risk assessment were that the result was weakly positive with metabolic activation and furthermore that therefore no confirmation experiment was performed. An in vivo chromosomal aberration test with rats according to OECD TG 475: In this study a negative result was found for single oral gavage doses of 600 (males) or 300 (females) mg/kg bodyweight.
Link to relevant study records
Reference
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).
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
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.
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.

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.

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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

In respect of mutagenicity of 4-TERT-BUTYLBENZOIC ACID the EU risk assessment report (2009) states (p. 67 and p. VII) that due to the positive in vitro micronucleus test and the fact that clastogenicity and aneugenicity were not distinguished in this test there would be concern for local clastogenic effects and aneugenic effects could not be excluded. Therefore further testing for clarification was recommended, preferably a combination of an in vivo COMET assay (directly exposed tissue and liver) and a bone marrow micronucleus test.

The EU risk assessment also laid out: An in vivo test on chromosomal aberrations in rats is negative for doses which correspond to the MTD. Oral bioavailability can be assumed from the physico-chemical data. This is in line with the fact that toxic effects were observed after acute and subacute oral application of low doses of the substance as well as the weak local effects (reduction of mitotic indices) in the in vivo chromosomal aberration test. There is sufficient evidence to conclude, that a clastogenic potential of ptBBA observed in vitro is unlikely to be expressed in germ cells in vivo.

The presented data in this dossier falls short of the recommendation in the EU risk assessment to generate more in vivo data. However, two out of three studies gave negative results and the prevention of animal testing is also a target of REACH wherever possible. The registrants will therefore await and abide the decision of the ECHA if the recommended studies have to be conducted.


Justification for selection of genetic toxicity endpoint
Treatment with S9 mix induced weak increases in micronucleus.

Justification for classification or non-classification

Regarding all given results there is not enough evidence for a classification as mutagenic at present. However, the substance was found to be genotoxic in one study. Therefore the substance is flagged as "positive" in respect of genetic toxicity in this preliminary evaluation.