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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The test substance is considered to be not mutagenic in the Ames test. The test substance is considered to be genotoxic in the chromosomal aberration test and sister chromatid exchange test.
Link to relevant study records
Reference
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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant near-guideline study, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: Chinese Hamster Lung (CHL/IU)
Details on mammalian cell type (if applicable):
- Type and identity of media: Neonatal calf serum (Lot No. NBQ07, Mitsubishi Chemical Corporation) was added to Eagle MEM medium (Lot No. 421001, Nissui Pharmaceutical Co., Ltd.) at 10 v/v%.
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and 5,6-benzoflavone induced SD rats rat liver S9-mix
Test concentrations with justification for top dose:
Chromosomal aberration study: 0, 40, 80, 160 µg/mL in the absence of S9 mix and 0, 70, 140, 280 µg/mL in the presence of S9 mix.
Chromosomal aberration study (confirmatory study): 0, 80, 110, 140 µg/mL in the absence of S9 mix and 0, 140, 200, 260 µg/mL in the presence of S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: This solvent was selected because the test substance was found to be dissolved at 150 mg/mL or more in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the precence of metabolic activation
Details on test system and experimental conditions:
CELL GROWTH INHIBITION TEST: A preliminary toxicity test was performed on cell cultures using a 6-hour exposure period (both with and without metabolic activation) followed by an 18-hour recovery period in treatment-free media. The dose range used was 20 to 360 µg/mL.

CHROMOSOMAL ABERRATION STUDY:
EXPERIMENT 1
- METHOD OF APPLICATION: in medium
- DURATION: A chromosomal aberration study was conducted in the same way as the cell growth inhibition study.
- DOSE LEVELS SELECTED FOR METAPHASE ANALYSIS: Chromosomal aberration study: 0, 40, 80, 160 µg/mL in the absence of S9 mix and 0, 70, 140, 280 µg/mL in the presence of S9 mix; Chromosomal aberration study (confirmatory study): 0, 80, 110, 140 µg/mL in the absence of S9 mix and 0, 140, 200, 260 µg/mL in the presence of S9 mix

SPINDLE INHIBITOR: Colcemid

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: cell growth inhibition study: 50 metaphase cells; chromosomal aberration study: 100 cells per dish.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

DETERMINATION OF CYTOTOXICITY
- Cell growth rate
Evaluation criteria:
The study result was regarded as positive if incidence of cells with structural or numerical aberrations increased 10% or more and increases in the incidence were dose-dependent or if 5% of higher incidence was shown to be reproducible. Otherwise, the result was regarded as negative. If the result was judged to be positive, the D20 value (concentration at which 20% or more cells show abnormality) was determined.
Statistics:
Cell growth rates were calculated as percentages of the cell count in the negative control group, and concentrations at which 50% inhibition is achieve were calculated.
Species / strain:
mammalian cell line, other: Chinese Hamster Lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The 50% inhibition concentration was about 140 µg/mL in the absence of S9 mix and about 250 µg/mL in the presence of S9 mix. Evidence of apparent inhibition of cell growth with cell growth rates lower than 50% was obtained at 160 µg/mL in the absence of S9 mix and 280 µg/mL in the presence of S9 mix, both the highest doses selected in the chromosomal aberration study. In addition, structural aberrations slightly increased in the absence of S9 mix.

CHROMOSOMAL ABERRATION STUDY
In the absence of S9-mix, the cell growth rate was 90.7%, 78.5% and 7.8%, respectively, at 40, 80 and 160 µg/mL. The incidence of structural aberrations was 0.5%, 0.5% and 15.5%, respectively, at 40, 80 and 160 µg/mL compared to 0% in the negative control group and 53.5% in the MMC (positive control) group. Incidence of numerical aberrations was less than 5.0% in all groups. The D20 value was calculated to be 0.23 mg/mL (S=0.016) in terms of structural aberrations. In the presence of S9-mix, the cell growth rate was 101.2%, 93.7% and 16.9%, respectively, at 70, 140 and 280 µg/mL.
The incidence of structural aberrations was 1.0%, 0% and 22.5%, respectively, at 40, 80 and 160 µg/mL compared to 0% in the negative control group and 32.0% in the MMC (positive control) group. The incidence of numerical aberrations was less than 5.0% in all groups. The D20 value was calculated to be 0.30 mg/mL (S=0.021) in terms of structural aberrations.

CONFIRMATORY STUDY
In the absence of S9-mix, the cell growth rate was 76.7%, 71.4% and 12.1%, respectively, at 80, 110 and 140 µg/mL. The incidence of structural aberrations was 1.0%, 0% and 19.0%, respectively, at 80, 110 and 140 µg/mL compared to 1.0% in the negative control group and 56.0% in the MMC (positive control) group. The incidence was less than 5.0% in all groups. The D20 value was calculated to be 0.21 mg/mL (S=0.019) in terms of structural aberrations. In the presence of S9-mix, the cell growth rate was 91.9%, 84.1% and 16.2%,respectively, at 140, 200 and 260 µg/mL. The incidence of structural aberrations was 2.5%, 5.0% and 23.5%, respectively, at 140, 200 and 260 µg/mL compared to 0% in the negative control group and 60.0% in the CPA (positive control) group. Incidence of numerical aberrations was 2.0%, 5.0% and 0%, respectively, at 140, 200 and 260 µg/mL compared to 0.5% in the negative control group. The D20 value was calculated to be 0.28 mg/mL (S=0.021) in terms of structural aberrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
positive

The test substance is considered to be clastogenic under the conditions of this test.
Executive summary:

In a GLP-compliant chromosome aberration test, Chinese Hamster Lung (CHL/IU) cells were exposed to the test substance with and without metabolic activation (S9 -mix).The study consisted of a cell growth inhibition test and two separate experiments. Except in the cell growth inhibition test, duplicate cultures of Chinese Hamster Lung (CHL) cells that had been treated with the test material in either the presence or absence of metabolic activation, were evaluated for induction of chromosome aberrations. Duplicate vehicle and positive controls were included in parallel in both experiments. In both experiments, the exposures were 6(18)-hour both with and without metabolic activation. The dose levels used were selected on the results of the cell growth inhibition test. The dose levels in experiment 1 were 0, 40, 80, 160 µg/mL in the absence of S9 mix and 0, 70, 140, 280 µg/mL in the presence of S9 mix. The dose levels in experiment 2 were 0, 80, 110, 140 µg/mL in the absence of S9 mix and 0, 140, 200, 260 µg/mL in the presence of S9 mix. Experiments were considered to have been conducted properly because there were no significant differences in the incidence of aberrant cells between two dishes used; incidence of aberrant cells was lower than 5% in the negative control group; and incidence of cells with aberrations other than gaps was 20% or higher in the positive control group. In the first experiment, chromosomal aberrations increased at the highest dose in both the presence and absence of S9 mix, but increases were not dose-dependent. In the second experiment, structural aberrations increased in both the presence and absence of S9 mix, showing reproducibility of the finding obtained in the first study. In addition, increases in structural aberrations were dose-dependent in the presence of S9 mix.In conclusion, the test material was considered to be clastogenic to CHL cells in vitro.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

Ames test:

In a GLP compliant bacterial mutagenicity assay (Ames preincubation test), using four Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and one E. Coli WP2uvrA strain, the test substance was evaluated in the presence and absence of rat liver derived metabolic activation system (S9-mix). Two tests were performed. In the dose-finding study, the substance was tested in duplicate at 4.88, 19.5, 78.1, 313, 1250, and 5000 µg/platewith and without S9 mix.The main experiment was performed in duplicate dosing 156, 313, 625, 1256, 2500, 5000 µg/plate with and without S9 mix.The vehicle used was dimethyl sulphoxide. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. The revertant colony count on test substance-treated plates showed less than 2-fold increases compared to the spontaneous revertant colony count observed in the negative control group in the presence (metabolic activation method) and absence of liver S9 mix (non-metabolic activation method), indicating that the test substance has no mutagenic potential.

Chromosomal aberration test:

In a GLP-compliant chromosome aberration test, Chinese Hamster Lung (CHL/IU) cells were exposed to the test substance with and without metabolic activation (S9 -mix). The study consisted of a cell growth inhibition test and two separate experiments. Except in the cell growth inhibition test, duplicate cultures of Chinese Hamster Lung (CHL) cells that had been treated with the test material in either the presence or absence of metabolic activation, were evaluated for induction of chromosome aberrations. Duplicate vehicle and positive controls were included in parallel in both experiments. In both experiments, the exposures were 6(18)-hour both with and without metabolic activation. The dose levels used were selected on the results of the cell growth inhibition test. The dose levels in experiment 1 were 0, 40, 80, 160 µg/mL in the absence of S9 mix and 0, 70, 140, 280 µg/mL in the presence of S9 mix. The dose levels in experiment 2 were 0, 80, 110, 140 µg/mL in the absence of S9 mix and 0, 140, 200, 260 µg/mL in the presence of S9 mix. Experiments were considered to have been conducted properly because there were no significant differences in the incidence of aberrant cells between two dishes used; incidence of aberrant cells was lower than 5% in the negative control group; and incidence of cells with aberrations other than gaps was 20% or higher in the positive control group. In the first experiment, chromosomal aberrations increased at the highest dose in both the presence and absence of S9 mix, but increases were not dose-dependent. In the second experiment, structural aberrations increased in both the presence and absence of S9 mix, showing reproducibility of the finding obtained in the first study. In addition, increases in structural aberrations were dose-dependent in the presence of S9 mix. In conclusion, the test material was considered to be clastogenic to CHL cells in vitro.

Chromatid exchange assay:

2,4,6-Trimethylbenzaldehyde was tested in a sister chromatid exchange assay (Jansson 1988) using lymphocytes, collected from healthy non-smoking donors, mixed with Medium 199 containing Earle's salt. After 24 h incubation at 37°C the test substance was added to the culture. The cells were exposed to several concentrations between 0 and 1.0 mM. Styrene-7,8-oxide was used as a positive control. After 88 h of incubation at 37°C, cell divisions were blocked by colchicine for 2 hours. Subsequently, cells were treated with KCl and fixed in methanol/acetic acid. Chromosome preparations were made on slides and stained. 25 metaphases were scored and analysed per tested concentration. The dose-response data obtained was subjected to linear regression analysis by the method of least squares. It was concluded that the test substance induces sister chromatid exchanges.

Justification for classification or non-classification

There are two reliable studies available investigating the genotoxic potential of the test substance. The test substance is considered to be not mutagenic in the Ames test , but genotoxic in the chromosomal aberration test. In accordance with Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not warrented, because the positive result was observed in vitro and structural analogues such as benzaldehyde (cas no: 100 -52 -7) and 3,4 -dimethylbenzaldehyde (cas no: 5973 -71 -7) are not genotoxic.