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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
A gene mutation test conducted on mouse lymphoma L5178Y cells reported no significant reduction in cell count after 3 and 24 hours of exposure to test substance within and without S9-mix metabolic activation. The test substance did not induce the mutant frequency at the TK locus with and without S9-mix.
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:
2 August to 8 November 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)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Mouse lymphoma cells were cultured in F10 complete culture medium. Cell density was preferably kept below 7 x 10^5 cells/mL. F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 10% (v/v) horse serum, L-glutamine (2 mM) and penicillin/streptomycin (50 U/mL and 50 µg/mL respectively).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically "cleansed" against high spontaneous background: Yes, Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in F10 complete culture medium containing 10^-4 M hypoxanthine, 2x10^-7 M aminopterin and 1.6 x10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on medium containing hypoxanthine and thymidine only. After this period cells were returned to normal medium at least for 1 day before starting the experiment.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-fraction
Test concentrations with justification for top dose:
Within and without S9-mix: 0.1, 0.3, 0.56, 1.0, 1.8, 3.3, 5.6, and 10 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulphoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: Dimethylnitrosamine (DMN)
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium, F10 complete culture medium.

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 5 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): F10 complete culture medium, supplemented with 10% (v/v) horse serum and 5µg/mL TFT-Selection
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): 0.5 mg/mL MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma)

NUMBER OF REPLICATIONS: 9.6 x 10^5 cells/concentrations, each well containing 2500 cells in selective medium (TFT-selection)

NUMBER OF CELLS EVALUATED: 8 x 10^6 cells (10^6/mL), if test substance concentration was expected to be toxic, 16 x 10^6 cells (10^6/mL) were used per culture.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency (CE)
Evaluation criteria:
Colonies were divided in small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severe affected mutant cells have grown at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appeared to result from mutants with single gene mutation (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphological dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphological dense colonies with a hazy contour and with a diameter larger than a quarter of a well.
Statistics:
Calculation of the cloning efficiency (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 (equation below).
The mutation frequency (MF) was expressed as the number of mutants per 10^5 surviving cells (equation below).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the exposition medium at a concentration of 10 µg/mL.

RANGE-FINDING/SCREENING STUDIES: In the absence of S9-mix after 3 hours of treatment, the toxicity in the suspension growth was 32% at the test substance concentration of 10 µg/mL compared to the suspension growth of the solvent control.
In the absence of S9-mix after 24 hours treatment, the toxicity in the suspension growth was 34% at the test substance concentration of 10 µg/mL compared to the suspension growth of the solvent control.
In the presence of S9-mix after 3 hours treatment, no toxicity in the suspension growth was observed in all concentrations tested compared to the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: Cloning efficiency of the remaining cells was comparable to the solvent controls even at the highest tested dose.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Both in the absence and presence of S9 -mix, after 3 hours treatment no significant reduction in the cell count was observed even in the highest dose level tested.

The test substance did not induce the mutant frequency at the TK locus either in the absence or in the presence of S9 -mix.

Conclusions:
Interpretation of results (migrated information):
negative

Under the current test conditions the test substance was concluded to be non-mutagenic in mouse lymphoma L5178Y.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Test System Application Effect
Mouse lymphoma L5178Y cells In vitro Mammalian Cell Gene Mutation Test
Mutagenicity - In vitro Mammalian Cell Gene Mutation Test
Test substance exposure up to 24 hours within and without metabolic activation (S9-mix) in presence of Dimethylsulphoxide at 0.1, 0.3, 0.56, 1.0, 1.8, 3.3, 5.6, and 10µg/mL
Negative

Test System Application Effect
S. typhimuriumTA 1535, TA 1537, TA 98, TA 100 and E. coli WP2  Ames - Bacterial Reverse Mutation Assay
S. typhimurium TA 98 - with and without metabolic activation,
S. typhimurium TA 100 - with metabolic activation,
S. typhimurium TA 100 - without metabolica activation, and
S. typhimurium TA 1535, TA 1537 and E. coli WP2uvrA

Postive,
Postive,
Negative,
Negative


Justification for selection of genetic toxicity endpoint
Test substance is the same as registration substance (Pymordiol). Reliable test (Guideline and GLP) test data indicated that pymordiol is not genotoxic.

Justification for classification or non-classification

Taking into account all data, it is not expected that pymordiol exhibits any genotoxic/mutagenic potential.

Based on the results there is no classification required according to Directive 67/548/EEC and Regulation (EC) 1272/2008 (CLP.