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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:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in compliance with OECD GLP regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
No deviations occurred that negatively impacted the integrity of the study.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
MTDID 948
IUPAC Name:
MTDID 948
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): MTDID 948
- Substance type: Mono-constituent
- Physical state: Liquid
- Analytical purity: 94.4%
- Purity test date: 16 April, 2014
- Lot/batch No.: 20014, Unit 027
- Expiration date of the lot/batch: 11 August, 2016
- Storage condition of test material: Ambient temperature

Method

Target gene:
Thymidine kinase (TK) locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes/no
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
2.5, 5, 10, 20, and 30% v/v
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: None, cells were exposed to test article vapor
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Clean air
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) in the absence and presence of S9 mix, respectively
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 5-7 days
- Exposure duration: Without S9: 24 (3 million cells) or 4 (5 million cells) hours. With S9: 4 hours
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent): 10-14 days in selection agent
- Fixation time (start of exposure up to fixation or harvest of cells): 10.5-15 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF CELLS EVALUATED: Cell suspensions were diluted to a density of 10,000 cells/mL in culture medium before selection with TFT.

DETERMINATION OF CYTOTOXICITY
- Method: Relative initial cell yield, relative suspension growth, and the relative total growth.

The cloning efficiency of the cells was calculated from the total number of negative wells on the microtiter plates and the number of cells seeded per well. To assess the cytotoxic effects of the test material or the positive controls on the cells, the initial cell yeild after the treatment period, the RSG and the RTG to that of the negative controls were calculated. The CE of the cells was used, together with the CE and the TFT-containing plates, to calculate the MF. The MF was expressed as the number of TFT-resistant mutants per 1 million clonable cells.
Evaluation criteria:
The following criteria were used to validate the data obtained in the gene mutation assay.
-The average CE of the negative controls should not be less than 60% or more than 40%.
-The average SG of the negative controls should be between 8 and 32.
-The average MF of the negative controls should fall within the range of 40-300 TFT-resistant mutants per 1 million clonable cells.
-The MF of the positive controls should be higher than 400 TFT-resistant mutants per 1 million clonable cells, and should be at least twice that of the corresponding negative control.
-Unless the test substance shows no cytotoxicity at the highest possible concentration (determined by its solubility, pH, and osmolar effects), the highest concentration should result in a clear cytotoxic response. The RTG value of one of the data points should be between 10 and 20%, or one data point between 1 and 10% and another between 20 and 30%.
A response was considered positive if the induced MF (MF of the test material minus that of the vehicle negative control) was more than 126 mutants per 1 million clonable cells. A response was considered to be equivocal if the induced mutant frequency was more than 88 mutants (but not more than 126 mutants) per 1 million clonable cells. Any apparent increase in MF at concentrations of the test material causing more than 90% cytotoxicity and with no evidence of mutagenicity at RTG>10%, was considered to be an artifact and not indicative of genotoxicity.
The test material was considered to be mutagenic in the gene mutation test at the TD-locus if a concentration-related increase in MF was observed, or if a reproducible positive response for at least one of the test material concentrations was observed.
The test material was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the MF nor a reproducible positive response at any of the test material concentrations.

Results and discussion

Test results
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the presence of S9-mix, the initial cell yield and/or relative suspension growth and/or relative total growth were not reduced when compared to the negative control. In the absence of S9-mix after 4 hour treatment, test substance was slightly toxic to the cells resulting in a mean RTG at the highest concentration evaluated (30%) of 84% (first experiment) or 88% (repeat experiment). In the absence of S9-mix after 24 hour treatment however, the inital cell yield and/or relative suspension growth and/or relative total growth were reduced by more than 10% at and above 10% test article (v/v). The three highest dose levels of the test substance evaluated for mutagenicity were 10, 20, and 30% (v/v); the RTG at these doses were 27%, 3%, and 0.2%, respectively.
Remarks on result:
other: strain/cell type: L5178Y
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of the study, the test article is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the presence or absence of metabolic activation (S9 -mix).
Executive summary:

The test article was evaluated for mutagenic potential in the L5178Y mouse Lymphoma Mutagenesis assay in the presence and absence of a metabolic activation system (S9 -mix). The study was performed in compliance with OECD GLP regulations. The test method was based on OECD 476 (1997). The cells were exposed to the test article as a vapor at 2.5, 5, 10, 20, or 30 % (v/v). Two independent tests were conducted. In the first test, five duplicate cultures were treated for 4 hours in the presence and absence of S9 -mix, and 24 hours in the absence of S9 -mix. in the second test (repeat of the 4 hour treatment group), five duplicate cultures were treated in the absence of S9 -mix. Methyl methanesulphonate (MMS) and 3 -methylcholanthrene (MCA) were used as positive control substances in the absence and presence of S9 -mix, respectively and clean air served as the negative control in all tests. In the first test in the 4 hour treatment group in the absence of S9 -mix, the criteria for the positive control were not met. Therefore, this part of the test was repeated. All criteria were met in the second test. In the presence of S9-mix, the initial cell yield and/or relative suspension growth and/or relative total growth were not reduced when compared to the negative control. In the absence of S9-mix after 4 hour treatment, test substance was slightly toxic to the cells resulting in a mean RTG at the highest concentration evaluated (30%) of 84% (first experiment) or 88% (repeat experiment). In the absence of S9-mix after 24 hour treatment however, the initial cell yield and/or relative suspension growth and/or relative total growth were reduced by more than 10% at and above 10% test article (v/v). The three highest dose levels of the test substance evaluated for mutagenicity were 10, 20, and 30% (v/v); the RTG at these doses were 27%, 3%, and 0.2%, respectively. In the first and second experiment, in the absence and presence of S9 -mix after 4 hour treatment, no increase in the mutant frequency by more than 88 or 126 mutants per 1 million clonable cells (no equivocal or positive response) compared to the negative control was observed at any dose level. In the absence of S9 -mix, after 24 hours treatment, at the highest concentration of 30% (v/v), an increase in the mean mutant frequency by more than 88 but less than 126 mutants per 1 million clonable cells was observed. The increase in mutant frequency was, however only observed at a concentration causing more than 99% cytotocicity (RTG value <1%). Therefore, the increase is considered to be not biologically relevant. At concentrations up to 99% cytotoxicity no increase in mutant frequency was observed. Based on the results of the study, the test article is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the presence or absence of metabolic activation (S9 -mix).