Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 August - 30 September 1999
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 10% (v/v) horse serum, L-glutamine (2mM) and penicillin/streptomycin (50 U/mL and 50 micro g/mL respectively).
Selective - Medium consisted of F10 complete culture medium, supplemented with 10% (v/v) horse serum and 5 micro g/mL TFT (sigma).
Non-selective - Medium consisted of F10 complete culture medium, supplemented with 10% horse serum.
- 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
Metabolic activation:
with and without
Metabolic activation system:
S9-Rat liver
Test concentrations with justification for top dose:
Without S9- 10, 25, 50, 100, 125, 250, 500, 625, 750 and 1000 micro g/mL; and
With S9- 10, 25, 50, 100, 125, 250, 500, 625, 750 and 1000 micro g/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulphoxide.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: dimethylnitrosamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: In exposition medium in the presence and absence of S-9 mix.

DURATION
- Preincubation period: 4 days
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 3 days

SELECTION AGENT (mutation assays): Selective medium consisted of F10 complete culture medium, supplemented with 10% (v/v) horse serum and 5 micro g/mL TFT (Sigma).
STAIN (for cytogenetic assays): 0.5 mb/mL MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma)

NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED: 9.6 10E05 cells/concentration, containing 2500 cells

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency (CE)

OTHER EXAMINATIONS:
- Other: Mutation frequency
Evaluation criteria:
The test substance was considered positive if:
- It induced at least a 3-fold increase in mutation frequency compared to the solvent control in a dose-dependent manner; and
- The results were reproducible in an independently repeated test.

The test substance was considered negative if:
- None of the tested concentrations showed mutant frequency of at least 3-fold compared to the solvent control.
- The results were confirmed in an independently repeated test.

A mutation assay was considered acceptable if:
- The absolute cloning efficiency of the solvent controls was >/= 50%.
- In at least seven of the eight doses of the test substance, an acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
- The spontaneous mutant frequency in the untreated or solvent control was < 10 per 10E5 clonable cells.
- The positive controls induced significant (at least 3-fold) increases in the mutant frequencies.
Statistics:
Calculations of the CE was determined by dividing the number of empty wells by the total number of wells. This value was called P(0), the zero term of the Poisson distribution.
P(0) = number of empty wells/total number of wells;
CE = -ln P(0)/number of cells plated per well.

The calculations for mutation frequency (MF) was determined, as follows:
MF = {-ln P(0)/number of cells plated per well}/CE3
Mutation frequency was reported as the number of mutants per 10E5 surviving cells.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix after 3 hours of treatment, the toxicity in the suspension growth was 47% at the test substance concentration of 333 micro g/mL compared to the suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 1000 micro g/mL (3%).

In the absence of S9-mix after 3 hours of treatment, toxicity in the suspension growth was 31% at the test substance concentration of 333 micro g/mL compared to the suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 1000 micro g/mL (6%).

In the absence of S9-mix after 24 hours of treatment, the toxicity in the suspension growth was 79% at the test substance concentration of 33 micro g/mL compared to the suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 100, 333, and 1000 micro g/mL after 24 hours of treatment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under experimental conditions the test substance is not mutagenic in the TK mutation test system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Two study reports are available for this evaluating the genetic toxicity of Pymorolac, an in vitro Mouse Lymphoma study and an in vitro Ames study with S. typhimurium and E. coli strains tested. The Mouse Lymphona study conducted according to OECD Guideline 476 and EU Method B.17 concluded that Pymorolac was not mutagenic. The Ames study conducted according to OECD Guideline 471 and EU Method B.17 concluded that Pymorolac was positive for mutagenicity with and without S9 metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100; although Pymorolac was negative for mutagenicity with and without S9 activation in E. coli strain WP2uvrA.


Justification for selection of genetic toxicity endpoint
Under experimental conditions the test substance is not mutagenic in the TK mutation test system.

Justification for classification or non-classification

Categories Display