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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 14, 2016 to March 27, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Requested by ECHA: In vitro cytogenicity study in mammalian cells (Annex VIII, Section 8.4.2., test method: OECD 473).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
OECD Council: July 29, 2016
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzyl Salicylate
- Physical state: colourless to pale yellow liquid
- Analytical purity: 99.9%
- Lot/batch No.: AS00196733
- Expiration date of the lot/batch: 08 January 2016
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 84OJN
- Purity test date: 99.9%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In cool and dark place (actual temperature: 4.1 to 6.0°C from October 4, 2016 to February 13, 2017), in well-closed containers
- Stability under test conditions: stable (confirmed after the end of the experiment)


Method

Species / strain
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Chinese hamster lung fibroblast (CHL/IU) from the National Institute of Biomedical Innovation, JCRB Cell Bank
- Suitability of cells: according the guideline
- Normal cell cycle time (negative control): DMSO, used as the vehicle, was used as the negative control article.

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: 30. The passage number of the cells at the time of use was 18 in the cell-growth inhibition test, and 28 in the chromosomal aberration test.
- Methods for maintenance in cell culture: The cells were cultured in a carbon dioxide gas incubator under conditions of 5% CO2 at 37°C and at high humidity. Subcultivation was carried out every 1 to 4 days.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: carbon dioxide gas incubator under conditions of 5% CO2 at 37°C and at high humidity
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- The S9 and cofactor (S9/cofactor C set, Lot numbers: C160902061 and C161118091) were mixed to prepare S9 mix.
Test concentrations with justification for top dose:
9 dose concentrations, 17.0, 14.0, 12.0, 11.0, 10.0, 8.00, 6.00 and 4.00 mg/mL.
Vehicle / solvent:
DMSO (lot DSH0997 and DSR0111)
DMSO was selected as the vehicle since the test article was not dissolved in water but dissolved in DMSO at 200 mg/mL in the examination for the vehicle.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Evaluation criteria:
Judgment was done using the number of cells with structural / numerical aberrations in chromosomes. The total incidence of cells with structural aberrations was calculated in 2 ways, one including gaps (TAG) and the other excluding gaps (TA), and the latter was used for the final evaluation based on statistical analysis.
Statistics:
For statistical significance of the difference in the incidence of cells with abnormalities, the numbers of the cells with chromosome structural aberrations and numerical aberrations between the negative control group and the test article treatment group were analyzed by Fisher’s exact test (level of significance: 5%, one-tailed) 2) and Cochran-Armitage trend test (level of significance: 5%, one-tailed) 3), while the numbers of the cells with chromosome structural aberrations between the negative control group and the positive control group were analyzed by Fisher’s exact test (level of significance: 5%, one-tailed) 2).

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
It was concluded that benzyl salicylate had neither chromosome structural aberration inducibility nor chromosome numerical aberration inducibility under the conditions of this study.
Executive summary:

In order to evaluate the clastogenic potential of Benzyl Salicylate, a chromosomal aberration test using cultured Chinese hamster (CHL/IU) cells was conducted.

 

As a preliminary study to select dose levels for the chromosomal aberration test, a cell-growth inhibition test was conducted setting the highest dose level at 2000 μg/mL, and this dose diluted using a common ratio of 2 to prepare a total of 8 concentrations. In the results, cell growth inhibition effects of more than 50% were recorded at the dose levels of 125 μg/mL and above for the short-term treatment method without metabolic activation and for the continuous treatment method and at the dose levels of 250 μg/mL and above for the short-term treatment method with metabolic activation, and thus the 50% cell growth inhibition concentration (approximate value) was calculated to be 92 μg/mL for the short-term treatment method without metabolic activation, 178 μg/mL for the short-term treatment method with metabolic activation, and  97 μg/mL for the continuous treatment method. Based on these results, chromosome aberration study was conducted setting the maximum dose concentration at 120 μg/mL and using 5 dose concentrations with common difference of 20 μg/mL for the short-term treatment method without metabolic activation and for the continuous treatment method, and setting the maximum dose concentration at 200 μg/mL and using 5 dose concentrations with common difference of 30 μg/mL for the short-term treatment method with metabolic activation.

 

In the results of the chromosome aberration study, the incidence of the occurrence of cells with chromosomal aberrations not containing gaps, an index for the chromosome structural aberration (TA value), and the incidence of the occurrence of cells with polyploidy (poly value) were not higher than those in the negative control group with statistical significance for any treatment method, and the values for the negative control group within the range of 95% probability distribution of the negative control values in the historical background data of the test facility. Therefore, the test article was judged to be negative for chromosome aberration effects.

 

For all the treatment methods, the incidence of the occurrence of cells with chromosome structural aberrations and the incidence of the occurrence of polyploidy in the negative control group were within the range of 95% probability distribution of the historical background data of the test facility. In the positive control group, a statistically significant increase in the incidence of cells with chromosome structural aberration was recorded in comparison with that of the negative control group. Therefore, it was judged that the study was conducted appropriately.