Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-10-20 to 2013-
Reliability:
1 (reliable without restriction)
Justification for data waiving:
other:

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 487
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
4-phenylbutan-2-one
EC Number:
219-847-4
EC Name:
4-phenylbutan-2-one
Cas Number:
2550-26-7
Molecular formula:
C10H12O
IUPAC Name:
4-phenylbutan-2-one
Test material form:
other: colorless to pale yellow, clear liquid
Details on test material:
- Name of test material (as cited in study report): Benzyl acetone
- Physical state: colorless to pale yellow, clear liquid

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/Ham's F12 medium, mixture 1:1) supplemented with 200 mM GlutaMax
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 mix
Test concentrations with justification for top dose:
Experiment I:
40 h / 4 h: 9.6-1482.0 (±S9 mix)

Experiment IIA:
40 h / 20 h: 9.6-1482.0 (-S9 mix)
40 h / 4 h: 9.6-1482.0 (+S9 mix)

Experiment IIB:
40 h / 20 h: 200.0-1482.0 (-S9 mix)
40 h / 4 h: 200.0-1482.0 (+S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO; final concentration of DMSO in the culture medium was 0.5 % (v/v)
- Justification for choice of solvent/vehicle: the solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: mitomycin C other:demecolcin
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 h with and without S9 mix (experiment I), 4 h with and 20 h without S9 mix (experiment IIA) and 20 h without S9 mix (experiment IIB)
- Expression time (cells in growth medium): cells exposed for 4 h have 16 h recovery period before fixation, no recovery period for 20 h exposure cells
- Selection time (if incubation with a selection agent): 20 h with Cytochalasin B (4 µg/mL)
- Cells were prepared 40 hrs after start of the exposure

SELECTION AGENT (mutation assays): Cytochalasin B (4 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
cytotoxic effect the CBPI: ca 500 cells per culture and cytotoxicity is expressed as % cytostasis
micronuclei effects: at least 1000 binucleate cells per culture. The frequency of micronucleated cells was reported as % micronucleated cells

DETERMINATION OF CYTOTOXICITY
- percentages of reduction in the CBPI (cytokinesis-block proliferating index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate
Evaluation criteria:
cytotoxic effect: percentages of reduction in the CBPI (cytokinesis-block proliferating index) in comparison with the controls (% cytostasis)

micronuclei effects: 1000 binucleate cells per culture. The frequency of micronucleated cells was reported as % micronucleated cells

criteria for the evaluation of micronuclei:
The micronuclei were counted in cells showing a clearly visible cytoplasm area. The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

A test item can be classified as non-clastogenic and non-aneugenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

A test item can be classified as clastogenic and aneugenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.
Statistics:
Statistical significance was confirmed by means of the Chi square test.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in Experiment IIA with and without S9 and Experiment IIB without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no effects
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitation of the test item in the culture medium was observed

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes
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):
negative

Under the experimental conditions, the test item Benzyl Acetone (4-phenylbutan-2-one) did not induce micronuclei in human lymphocytes in vitro, when tested up to cytotoxic, the highest evaluable or required concentrations.
Executive summary:

The test item was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Three independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment IIA and IIB, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis.

The highest treatment concentration in this study, 1482.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

No precipitation of the test item in the culture medium was observed. Phase separation was observed microscopically in the absence of S9 mix in Experiment I at 483.9 µg/mL and above and in Experiment IIA at 1482.0 µg/mL at the end of treatment. In Experiment IIB in the absence of S9 mix no phase separation was observed up to the highest applied concentration. In the presence of S9 mix phase separation was observed microscopically in Experiment I and IIA at 846.9 µg/mL and above at the end of treatment.

No relevant influence on osmolarity or pH value was observed.

In Experiment I in the absence and presence of S9 mix and in Experiment IIB in the presence of S9 mix no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In Experiment IIB in the absence of S9 mix cytotoxicity was observed at the highestevaluated concentration.

In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.10 - 1.45 % micronucleated cells) were above the range of the solvent control values (0.40 - 0.70 % micronucleated cells) and within the range of the laboratory historical control data. In Experiment IIB in the presence of S9 mix statistically significant increases were observed after treatment with 1200.0, 1300.0 and 1482.0 µg/mL (1.45, 1.25 and 1.38 % micronucleated cells). The values were in the range of the historical control data (0.20 - 1.70 % micronucleated cells) and are therefore regarded as biologically irrelevant.

Either Demecolcin (75.0 or 125.0 ng/mL), MMC (3.0 µg/mL) or CPA (12.5 or 17.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item Benzyl Acetone did not induce micronuclei in human lymphocytes in vitro, when tested up to cytotoxic or the highest required or evaluable concentrations.