Benzene, (1-methylethyl)-, oxidized, polyphenyl residues

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 according to OECD guideline with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals, TG 487, adopted 26. Sep. 2014 ”In vitro Mammalian Cell Micronucleus Test“

GLP compliance:

yes (incl. QA statement)

Remarks:

Laus GmbH

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Benzene, (1-methylethyl)-, oxidized, polyphenyl residues

EC Number:

269-798-8

EC Name:

Benzene, (1-methylethyl)-, oxidized, polyphenyl residues

Cas Number:

68333-89-1

Molecular formula:

Cannot be given for UVCB

IUPAC Name:

not applicable

Test material form:

liquid: viscous

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Specification: Human whole blood, treated with anti-coagulant (heparin).
Blood Collection and Delivery:
For this study, blood will be collected from young (approximately 18 – 35 years of age) donors with no known illness or recent exposures to genotoxic agents, to avoid an increase of the background level of micronucleate cells. The blood should be collected either on the evening preceding the start of the expe-riment or in the morning of the day of the experimental start.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254

Test concentrations with justification for top dose:

Pre-Exp. With S9 mix 0.16, 0.08 and 0.04 µL/mL
Exp. I Without S9 mix 0.125, 0.15, 0.075 and 0.013 µL/mL
Exp. I With S9 mix 0.25, 0.2, 0.15 and 0.025 µL/mL
Exp. III Without S9 mix 0.1, 0.05, 0.025 and 0.013 µL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Negative solvent / vehicle controls:

yes

Remarks:

NaCl 0.9% is used as solvent control for the positive controls MMC, CPA and Colchicine. DMSO is used as solvent control for the test item.

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Remarks:

0.9% NaCl was used as solvent control for the positive controls Cyclophosphamide monohydrate (CPA), Mitomycin C (MMC) and Colchicine.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 18-19.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): 22-24 h

STAIN (for cytogenetic assays): 10% solution of Giemsa

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was calculated as reduction in Cytokinesis-block proliferation index (CBPI) compared to the CBPI of the concurrent solvent control.

Evaluation criteria:

Acceptability
The genotoxicity assay is considered acceptable if it meets the following criteria:
- All experimental conditions are tested (short exposure with and without metabolic activation, ex-tended exposure without metabolic activation) unless a positive result is achieved in any experiment.
- In each experiment, an adequate number of cells is analyzable both in the controls and in at least 3 test item concentrations.
- The micronucleus induction of the solvent and positive controls is compatible with the historical labor-atory control data or the literature data.
- The positive control shows a statistically significant increase of binucleate cells with micronuclei com-pared with the concurrent solvent control.
- The criteria for cell proliferation and for the selection of concentrations are fulfilled.

Classification
The test item is considered to have no genotoxic effects if:
- Neither a statistically significant nor a concentration-related increase of the number of micronucleate cells in the evaluated test concentrations is observed.
- The obtained results lie within the range of the historical laboratory control data for solvent controls.
The test item is considered to have genotoxic effects if:
- At least one test concentration shows a statistically significant increase of micronucleate cells com-pared to the concurrent solvent control.
- In at least one experimental condition a dose-related increase of micronucleate cells can be ob-served.
- Any of the results lies outside the range of the historical laboratory control data for solvent controls.

Statistics:

Statistical significance will be confirmed by means of the non-parametric χ2 test or Fisher’s exact test for smaller values in the fourfold table (decision about the statistical method rests with the study director). However, both biological and statistical significance will be considered together. If the above mentioned criteria for the evaluation and interpretation of the results are not clearly met, the classification with re-gard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:In the pre-experiment, concentrations up from 0.16 µL/mL (without metabolic activation) resp. 0.31 µL/mL (with metabolic activation) revealed complete cytotoxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: The velues for sovent control and positive controls were in the renage of the historical controls except for Colchicine with a value slightly above the range (9.53% versus 7.08 % MBNC).

ADDITIONAL INFORMATION ON CYTOTOXICITY: A cytotoxicity of 55 ± 5% in the highest test concentration could not be achieved due to the steep concentration-effect relationship for this endpoint (complete cytotoxicity was observed at one concentration above), but this is without any influ-ence on the result of the test, because even the concentrations below this value showed statistically sig-nificant increased numbers of binucleate cells with micronuclei compared with the concurrent solvent control.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Under the experimental conditions of the the “in Vitro Mammalian Cell Micronucleus Test” following OECD TG 487 and EU B.49, High Boiler, Z-Oil is able to induce the formation of micronu-clei in human lymphocytes in vitro. The test item High Boiler, Z-Oil is considered as “genotoxic under the conditions of the test”.