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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
Study period:
from 2012-08-16 to 2013-04-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted under GLP and according to OECD TG 473

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 487 "In vitro Mammalian Cell Micronucleus Test"
Deviations:
yes
Remarks:
The expression phase and harvest time were slightly modified compared to the OECD TG 487
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Reference substance name:
4-methylacetophenone
IUPAC Name:
4-methylacetophenone
Constituent 2
Chemical structure
Reference substance name:
4'-methylacetophenone
EC Number:
204-514-8
EC Name:
4'-methylacetophenone
Cas Number:
122-00-9
Molecular formula:
C9H10O
IUPAC Name:
1-(4-methylphenyl)ethanone
Test material form:
other: clear liquid, colourless to yellowish
Details on test material:
- Name of test material (as cited in study report): Methyl Acetophenone - Para
- Physical state: clear liquid, colourless to yellowish
- Storage condition of test material: at room temperature

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: 9.1-1400.0 (±S9 mix)

Experiment IIA:
40 h / 20 h: 9.1-1400.0 (-S9 mix)
40 h / 4 h: 85.3-1400.0 (+S9 mix)

Experiment IIB:
40 h / 20 h: 100.0-1000.0 (-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:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
mitomycin C
other: demecolcin
Untreated negative controls:
no
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-mutagenic 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 mutagenic 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 resultsopen allclose all
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Remarks:
at and above 800.0 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000.0 µg/mL
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: precipitation of the test item in the culture medium was observed microscopically in Experiment IIA in the absence of S9 mix at 1400.0 µg/mL.

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Remarks on result:
other: other: Experiment I
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 Methyl Acetophenone - Para 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 Methyl Acetophenone - Para, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. The following study design was performed:

 

Without S9-Mix

With S9-Mix

 

Exp. I

Exp. IIA & IIB

Exp. I and IIA

Exposure period

 4 hrs

20 hrs

 4 hrs

Recovery

16 hrs

-

16 hrs

Cytochalasin B exposure

20 hrs

20 hrs

20 hrs

Preparation interval

40 hrs

40 hrs

40 hrs

Total culture period

88 hrs

88 hrs

88 hrs

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were evaluated for cytogenetic damage. The highest applied concentration in the pre-test on toxicity (1400.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (96.8 %) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. The chosen treatment concentrations were 9.1-1400.0 µg/mL (±S9 mix) for Experiment I, and (-S9 mix) for Experiment IIA, 85.3 -1400.0 µg/mL (+S9 mix) for Experiment IIA and 100.0-1000.0 µg/mL (-S9 mix) for Experiment IIB.

In Experiment I in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix no clear cytotoxicity was observed up to the highest applied concentration. In Experiment IIA in the absence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage and therefore had to be repeated in an independent experiment (Experiment IIB). In Experiment IIB in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration.

In the absence and presence of S9 mix, no increase in the number of micronucleated cells was observed after treatment with the test item. However, one statistically significant increase was observed in Experiment IIA in the presence of S9 mix after treatment with 1400.0 µg/mL (0.75 % micronucleated cells) which was clearly within the range of the historical control data (0.0 – 1.70 %) and therefore not biologically relevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. It can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, Methyl Acetophenone - Para is considered to be non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to cytotoxic, the highest evaluable or required concentrations.