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Toxicological information

Genetic toxicity: in vitro

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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:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L 1362000, Annex 4E, dated May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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).
Controlsopen allclose all
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
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 resultsopen allclose all
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
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.