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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 19 to October 20, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted according to OECD Guideline 473 with deviations: details of mycoplasma contamination, evaluation criteria and historical data not reported.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
details of mycoplasma contamination, evaluation criteria and historical data not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methoxy-4-propylphenol
EC Number:
220-499-0
EC Name:
2-methoxy-4-propylphenol
Cas Number:
2785-87-7
Molecular formula:
C10H14O2
IUPAC Name:
2-methoxy-4-propylphenol
Test material form:
liquid
Details on test material:
- Physical state: Colourless, clear liquid
- Storage condition of test material: Stored at ambient temperature.

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Source: BIBRA
- Type and identity of media: The cells were routinely grown and subcultured in Hams F12 medium supplemented with 5 % foetal calf serum at 37 °C in a humid atmosphere containing 5 % carbon dioxide in 175 cm2 plastic tissue culture flasks.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 mix: S9-mix from the livers of CD rats of Sprague-Dawley origin treated with Aroclor 1254 at 500 mg/kg bw.
Test concentrations with justification for top dose:
Preliminary toxicity test: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL, with and without S9-mix. All final concentration above 1000 µg/ml caused a precipitate to form in queous tissue culture medium. 1000 µg/ml is referred to as the maximum achievable concentration
Dose levels for metaphase analysis: 7.8, 15.6, 31.3 and 62.5 µg/mL, without S9-mix; 15.6, 31.3, 62.5 and 125 µg/mL, with S9-mix. The choice of concentrations was based on toxicity observed in the preliminary test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Test material preparation: Test material was diluted with DMSO immediately before use. Test material was miscible with the DMSO at a concentration of 166 mg/mL. All final concentration above 1000 µg/ml caused a precipitate to form in queous tissue culture medium. 1000 µg/ml is referred to as the maximum achievable concentration
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9-mix: 0.4 µg/mL of mitomycin C.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9-mix: 20 µg/mL of cyclophosphamide.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 h (without S9-mix); 6 h (with S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.25 µg/mL culture medium)
STAIN (for cytogenetic assays): 10 % Giemsa

NUMBER OF REPLICATIONS: Two cultures/dose for test material, 2 cultures for positive control, 4 cultures for negative control and vehicle control.

NUMBER OF CELLS EVALUATED: Approximately 100 metaphases per culture were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

Other:
- Microscopical examination for mitotic index: Prepared slides were examined at magnification of x160 and the proportion of mitotic cells in each culture was recorded. The concentration of test compound causing a decline in mitotic index to between 20-50 % of the solvent control value (approximating to the EC50 value) was selected and this concentration was used as the highest dose level for metaphase analysis.
- Metaphase analysis: Metaphase spreads were identified using a magnification of x160 and examined at a magnification of x1000 using an oil immersion objective.
- Osmolality measurement: Following the exposure of the cells to the test material, a sample of the supernatants of the two highest doses and the control treatments was removed and a measurement of osmolality made by conventional techniques.
Rationale for test conditions:
The dose levels for metaphase analysis were based on toxicity observed in the preliminary test.
Evaluation criteria:
No data
Statistics:
Statistical significance was confirmed by means of the Fisher’s test (p< 0.001).

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No significant difference from solvent control values.
- Evaporation from medium: No data
- Water solubility: Miscible with DMSO
- Precipitation: Yes; > 1000 µg/mL in culture medium.
- Other confounding effects: None

CYTOTOXICITY:
- Without S9-mix: Cultures treated with test material at 1000 µg/mL killed virtually all the cells; 500 µg/mL yielded no live cells; 250 µg/mL contained no mitotic cells; 125 µg/mL reduced the mitotic index value to approximately 9 % of that of the solvent control and 62.5 µg/mL caused a decline in mitotic index to approximately 53 % of the solvent control value. The five remaining concentrations of the test material did not reduce the mitotic index value to below 70 % of the solvent control. The concentrations selected for the metaphase analysis were 7.8, 15.6, 31.3 and 62.5 µg/mL.
- With S9-mix: Cultures treated with 1000 µg/mL of test material yielded no live cells, and those treated with 500 µg/mL contained no metaphase figures. 250 µg/mL of the test material caused a decline in mitotic index to approximately 6 % of the solvent control value, and 125 and 62.5 µg/mL reduced the mitotic index values to approximately 42 and 46 %, respectively, of that of the solvent control. The five remaining concentrations of the test material did not reduce the mitotic index value to less than 70 % of the solvent control. The concentrations selected for the metaphase analysis were 15.6, 31.3, 62.5 and 125 µg/mL.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the test material is not considered as clastogenic according to the criteria of the Annex VI of the Regulation (EC) No.1272/2008 (CLP) and to the GHS.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No. 473 and in compliance with GLP, Chinese hamster ovary (CHO) cells of K1-BH4 strain were exposed to test material diluted in dimethylsulphoxide (DMSO) at the following concentrations: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL, without S9 mix (24 h exposure without recovery) and with S9-mix (6 h exposure and 18 h recovery). Two hours before the cells were harvested, mitotic activity was arrested by addition of colchicine to each culture at a final concentration of 0.25 µg/mL. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Negative, solvent (DMSO) and positive control groups were also included in the experiment.

In the absence of metabolic activation, cultures treated with test material at 1000 µg/mL killed virtually all the cells; 500 µg/mL yielded no live cells; 250 µg/mL contained no mitotic cells; 125 µg/mL reduced the mitotic index value to approximately 9 % of that of the solvent control and 62.5 µg/mL caused a decline in mitotic index to approximately 53 % of the solvent control value. In the presence of metabolic activation, cultures treated with 1000 µg/mL of test material yielded no live cells, and those treated with 500 µg/mL contained no metaphase figures. 250 µg/mL of the test material caused a decline in mitotic index to approximately 6 % of the solvent control value, and 125 and 62.5 µg/mL reduced the mitotic index values to approximately 42 and 46 %, respectively, of that of the solvent control. The remaining concentrations of the test material did not reduce the mitotic index value to below 70-75 % of the solvent control. Dose levels for metaphase analysis: 7.8, 15.6, 31.3 and 62.5 µg/mL, without S9-mix; 15.6, 31.3, 62.5 and 125 µg/mL, with S9-mix. Test material caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any concentration in either the presence or absence of metabolic activation. Both positive control compounds, mitomycin C (0.4 µg/mL) and cyclophosphamide (20 µg/mL), caused statistically significant increases in the proportion of metaphase figures containing aberrations when compared with the relevant solvent controls. Thus, the sensitivity of the test system and the efficacy of the S-9 mix were validated.

Under the test condition, the test material is not considered as clastogenic according to the criteria of the Annex VI of the Regulation (EC) No.1272/2008 (CLP) and to the GHS.