Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 97%

Method

Target gene:
thymidine kinase locus of L5178Y mouse lymphoma cells
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y/TK+/- mouse lymphoma cells are heterozygous at the normally diploid thymidine kinase (TK) locus. L5178Y/TK+/- cells, clone 3.7.2C, Each batch of frozen cells was tested and found to be free of mycoplasma contamination. L5178Y/TK+/- cells were prepared in 50% conditioned F0P supplemented with 10% horse serum and 2 mM L-glutamine (F10P) and 50% Fischer's Media for Leukemic Cells of Mice with 0.1% Pluronics F-68 (F0P). All media contained antibiotics.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Toxicity Assay - 4.61 to 1180 μg/mL
Mutagenicity Assay - 148, 295, 590, 885 and 1180 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: based on the solubility of the test article and compatibility with the target cells
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (presence and absence of S9), or 24 hours (absence of S9 only)
- Expression time (cells in growth medium): For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium and collected by centrifugation. The cells were resuspended in 20 mL F10P on Day 1 and in 10 mL F10P on Day 2, and incubated at 37 ± 1°C for two days following treatment.
Cell population adjustments to 3x10e5 cells/mL were made as follows:
• 4-hour treatment – 1 and 2 days after treatment.
• 24-hour treatment – immediately after test article removal, and 2 and 3 days after treatment.

SELECTION
- Cells from selected dose levels were cultured in triplicate with 2-4 μg TFT/mL at a density of 1x10e6 cells/100-mm plate in cloning medium containing 0.22 to 0.24% agar. For estimation of cloning efficiency at the time of selection of those same cultures, 200 cells/100-mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 10 or 11 days.
- The total number of colonies per culture was determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate was then determined for those cultures with ≥10% total growth (including at least one concentration between 10 and 20% total growth, if possible). Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures were determined over a range from 0.2 to 1.1 mm.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: The mutant frequency for each treatment condition was calculated by dividing the mean number of colonies on the TFT-plates by the mean number of colonies on the VC-plates and multiplying by the dilution factor (2 x 10-4), and was expressed as TFT-resistant mutants/10e6 surviving cells. The induced mutant frequency (IMF) was defined as the mutant frequency of the treated culture minus the mutant frequency of the vehicle control cultures. The International Workshop on Genotoxicity established a Global Evaluation Factor (GEF) for a positive response at an IMF of ≥90 mutants/10e6 clonable cells at the Aberdeen meeting in 2003

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition relative to the vehicle control
Evaluation criteria:
* A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibit induced mutant frequencies of ≥90 mutants/10e6 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants/10e6 clonable cells, a doubling of mutant frequency over the vehicle would also be required.
* A result was considered negative if the treated cultures exhibit induced mutant frequencies of less than 90 mutants/10e6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.
Statistics:
Cultures treated with a minimum of four concentrations of test article must be evaluated and their mutant frequencies reported. Results may be accepted, with justification, when only three concentrations of test article are evaluated and otherwise meet the other criteria for a valid test. The highest test article concentration must produce 80 to 90% toxicity unless limited by solubility or the maximum required concentration. In the case of a test article with a steep toxicity curve (no concentrations with 10 to 20% survival), the results may be considered acceptable if a concentration spacing of ≤2-fold is used and the highest concentration tested showed <20% survival or total kill

Results and discussion

Test results
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of precipitation/pH: No visible precipitate was observed at the beginning or end of treatment, and the test article had no adverse impact on the pH of the cultures.
- Water solubility: The test article formed a clear solution in sterile water at a maximum concentration of ~50 mg/mL in the solubility test conducted at BioReliance.

RANGE-FINDING/SCREENING STUDIES:
The test item was prepared in sterile distilled water and evaluated in a preliminary toxicity assay at concentrations from 4.61 to 1180 μg/mL [the highest concentration evaluated approximated the 10 mM limit dose for this assay; dose formulations were adjusted for purity (97%) using a correction factor of 1.03]. Relative suspension growth (RSG) was 110, 105 and 101% at a concentration of 1180 μg/mL (4-hour treatments with and without S9, and 24-hour treatment without S9, respectively).




Any other information on results incl. tables

The treated cultures exhibited 86 to 99%, 92 to 101% and 96 to 115% RSG (4-hour treatments with and without S9, and 24-hour treatment without S9, respectively), and were cloned. Relative total growth of the cloned cultures ranged from 75 to 113% (4-hour treatment with S9), 77 to 98% (4-hour treatment without S9) and 105 to 128% (24-hour treatment without S9). No increases in induced mutant frequency ≥90 mutants per 10e6 clonable cells were observed under any treatment condition.

The trifluorothymidine-resistant colonies for the positive and vehicle control cultures were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS and DMBA positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

Applicant's summary and conclusion

Conclusions:
The results indicate the test item was negative in the L5178Y/TK+/- Mouse Lymphoma Assay, in the presence and absence of metabolic activation.
Executive summary:

The test substance was evaluated to determine its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of Aroclor-induced rat liver S9. The test substance was prepared in sterile distilled water and evaluated in a preliminary toxicity assay at concentrations from 4.61 to 1180 μg/mL [the highest concentration evaluated approximated the 10 mM limit dose for this assay; dose formulations were adjusted for purity (97%) using a correction factor of 1.03]. No visible precipitate was observed at the beginning or end of treatment, and the test article had no adverse impact on the pH or osmolality of the cultures. Relative suspension growth (RSG) was 110, 105 and 101% at a concentration of 1180 μg/mL (4-hour treatments with and without S9, and 24-hour treatment without S9, respectively).

Based on the results of the preliminary toxicity assay, cultures were treated at concentrations of 148, 295, 590, 885 and 1180 μg/mL under all three treatment conditions in the mutagenicity assay. No visible precipitate was observed at the beginning or end of treatment, and the test article again had no adverse impact on the pH of the cultures. The treated cultures exhibited 86 to 99%, 92 to 101% and 96 to 115% RSG (4-hour treatments with and without S9, and 24-hour treatment without S9, respectively), and were cloned. Relative total growth of the cloned cultures ranged from 75 to 113% (4-hour treatment with S9), 77 to 98% (4-hour treatment without S9) and 105 to 128% (24-hour treatment without S9). No increases in induced mutant frequency ≥90 mutants per 106 clonable cells were observed under any treatment condition. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met. These results indicate the test substance was negative in the L5178Y/TK+/- Mouse Lymphoma Assay, in the presence and absence of metabolic activation, under the conditions and according to the criteria of the test protocol.