Methyl salicylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from 2008/05/14 to 2008/07/21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study satisfying the requirement for OECD 476 for in vitro mutagenicity.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L 1362000, Annex 4E, dated May 19, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Salicylic acid

EC Number:

200-712-3

EC Name:

Salicylic acid

Cas Number:

69-72-7

Molecular formula:

C7H6O3

IUPAC Name:

2-Hydroxybenzoic acid

Method

Target gene:

Thymidine kinase locus (TK+/-)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium supplemented with 15% horse serum, 100 U/100 µg/ml Penicillin/Streptomycin,
220 µg/ml sodium-pyruvate, and 0.5-0.75 U/ml Amphotericin used as antifungal.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 induced by Phenobarbital/beta-Naphthoflavone

Test concentrations with justification for top dose:

For experiment I and II with and without S9 mix: 87.5, 175.0, 350.0, 1400.0 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water. The final concentration of deionised water in the culture medium was 10% (v/v).

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

deionised water

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: without metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no
- Exposure duration: 4 h for the first experiment and 24h for the second experiment without S9 mix.
- Expression time (cells in growth medium): 2 days
- Fixation time (start of exposure up to fixation or harvest of cells): 52 h for the first experiment and 72 h for the second experiment.


SELECTION AGENT (mutation assays):
after expression time:
- seeding of 4x1000 cells/well in selective medium (TFT)
- incubation 10-15 day: determination of mutant colonies

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED:
- Cell density: 10E7 cells/ flask (80 cm2 flasks)
- 4x1000 cells/well for Mutagenicity evaluation.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency:
1- Survival: after treatment time:
- seeding of 2 cells/well in complete culture medium
- incubation 10-15 day: determination of cloning efficiency 1
2- Viability: after expression time:
- seeding of 2 cells/well in medium without TFT
- incubation 10-15 day: determination of cloning efficiency 2

Other:
- The pH and the osmolarity value were determined in culture medium at 1400 µg/ml in the pre-experiment without S9mix:
1- Osmolarity: 265 for slovent control and 286 for SA
2- pH value: 7.35 for slovent control and 7.59 for SA (adjusted with 0.6 ml 2N NaOH).

Evaluation criteria:

- A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10E6 above the corresponding solvent control.
- A relevant increase of the mutation frequency should be dose-dependant.
- A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls and the mutation rates of all negative and vehicle controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, and the cloning efficiency 1 is less than 10% of the vehicle control.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No relevant toxic effects indicated by a relative cloning efficiency 1 or a relative total growth of less than 50% of survival were observed up to the maximum concentration with and without metabolic activation.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
According the results of the pre-test at least four adequate concentrations were chosen for the muation assay experiment: 87.5, 175.0, 350.0,
1400.0 µg/ml. Following the expression phase of 72 hours the cultures at 43.8 µg/ml in both main experiments were not continued.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the second experiment, 24 h treatment without metabolic activation, relevant toxic effects were noted at 700 µg/ml and above. The data at the
maximum concentration of 1400 µg/ml are considered valid even though the relative total growth fell short of the limit of 10%. The corresponding
relative cloning efficiency 1 however, was in a toxic but fully acceptable range. The recommended toxic range of approximately 10-20% of survival
or RTG was covered in experiment II.

Remarks on result:

other: strain/cell type: L5178Y cells

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, salicylic acid pharmaceutical grade did not induce mutations in the mouse lymphoma thymidine kinase locus assay using
the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

A study was performed to investigate the potential of Salicylic acid pharmaceutical grade to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each at 87.5, 175.0, 350.0, 1400.0 µg/ml SA. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours. The highest concentration (1400 µg/ml) was chosen with regard to the molecular weight of the test item corresponding to a molar concentrations of about 10 mM. Relevant cyto-toxic effects were solely noted following 24 h treatment in the second experiment at 700 µg/ml and above. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, salicylic acid pharmaceutical grade is considered to be non-mutagenic in this mouse lymphoma assay.