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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:
18 June 2009 - 06 July 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepetd scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the relevant results.

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(However, the study protocol (subsection 7.11 Independent Repeat Assay) was written, "Negative results will not be verified unless justification can be provided." )
Deviations:
yes
Remarks:
yes (1. No second experiment was undertaken. 2.The text of the report mentions that the osmolality of the solvent control and the highest soluble concentration in treatment medium were determined.)
GLP compliance:
yes
Remarks:
yes (incl. certificate) (The certificate was signed on the 18th of December 2009)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Test Article Purity: 97.8% (Provided by Sponsor)
Test Article Lot No.: T48220409
Test Article I.D.: 98-06-1163
Test Article Description: Clear colorless liquid
Storage Conditions: Room temperature; protected from light

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y cells, clone 3.7.2C, were obtained from Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC on 14 August 1995. Each lot of cryopreserved cells was tested using the agar culture and Hoechst staining procedures and found to be free of mycoplasma contamination. Prior to use in the assay, L5178Y cells were cleansed of spontaneous TK-/- cells by culturing in a restrictive medium (Clive and Spector, 1975).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary toxicity test: 0.5 to 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on solubility of the test
article and compatibility with the target cells. The test article was soluble in DMSO at approximately 500 mg/mL, the maximum concentration tested.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
(tested concurrently)
Positive control substance:
other: MMS for the non-activated test and DMBA for the S9 activated test system
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 and 48 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): Each lot of cryopreserved cells was tested using the agar culture and Hoechst staining procedures and found to be free of mycoplasma contamination.

NUMBER OF REPLICATIONS:
Only one experiment was undertaken

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method cloning efficiency
The average cloning efficiency of the solvent (or vehicle) controls must be between 65% and 120% and the total suspension growth between 8-32 for the 4-hour exposure

OTHER EXAMINATIONS:
none

OTHER:
Evaluation criteria:
Criteria for a Valid Test The following criteria must be met for the mutagenesis assay to be considered valid:

Negative Controls:
The average spontaneous mutant frequency of the solvent (or vehicle) control cultures must be within 35 to 140 TFT-resistant mutants per 106 surviving cells. Low spontaneous mutant frequencies, i.e., 20 to 34 mutants per 106 surviving cells, are considered acceptable if small colony recovery is demonstrated (Mitchell et al., 1997). The average cloning efficiency of the solvent (or vehicle) controls must be between 65% and 120% and the total suspension growth between 8-32 for the 4-hour exposure (Moore, et al., 2002; 2006; 2007).

Positive Controls
The mutant frequency for at least one dose of the positive controls must meet the criteria for a positive response and induce an increase in small colony mutants according to the following criteria: Induced Mutant Frequency (IMF) positive control = 300 x 10-6 mutants with 40% small colonies or small colony IMF for positive control = 150 x 10-6 (Moore, et al., 2002; 2006).

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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not stated
- Effects of osmolality: The osmolality of the solvent control was 432 mmol/kg and the osmolality of the highest soluble concentration, 50 µg/mL, was 427 mmol/kg.
- Evaporation from medium: not reported
- Water solubility: see section 4.8
- Precipitation: Visible precipitate was present in treatment medium at 200 µg/mL in the main study.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Assay
The results of the preliminary toxicity assay are presented in Table 1 (data not shown). The maximum concentration tested in the preliminary toxicity assay was 5000 µg/mL. Visible precipitate was present at concentrations = 150 µg/mL in treatment medium. The osmolality of the solvent control was 432 mmol/kg and the osmolality of the highest soluble concentration, 50 µg/mL, was 427 mmol/kg. Suspension growth relative to the solvent controls was 88% at µg/mL without activation and 25% at 5000 µg/mL with S9 activation. Based on the results of the toxicity test, the concentrations treated in the mutagenesis assay ranged from 5.0 to 200 µg/mL for both the non-activated and S9-activated cultures.

COMPARISON WITH HISTORICAL CONTROL DATA:
Not stated in report

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results of the main test:

The results of the mutagenesis assay are presented in Tables 2 and 3 (data not shown). Visible precipitate was present in treatment medium at 200 µg/mL. In the non-activated system, cultures treated with concentrations from 50 to 200 µg/mL were cloned and produced a range in suspension growth from 95% to 104%. In the S9-activated system, cultures treated with concentrations from 50 to 200 µg/mL were cloned and produced a range in suspension growth from 60% to 98%.

No cloned cultures exhibited mutant frequencies that were = 90 mutants per 106 clonable cells over that of the solvent control. No concentration-related increase in mutant frequency was observed in the non-activated or S9-activated systems. The total growth ranged from 78% to 98% for the nonactivated cultures at concentrations from 50 to 200 µg/mL, and 58% to 88% for the S9-activated cultures at concentrations from 50 to 200 µg/mL.

The TFT-resistant colonies for the positive and solvent control cultures from both assays 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:
Interpretation of results (migrated information):
negative

All criteria for a valid study were met as described in the protocol. Under the conditions of this study, test article 98-06-1163 was concluded to be negative in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.
Executive summary:

The test article, 98-06-1163, was tested in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay established the concentration range for the mutagenesis assays. The mutagenesis assay was used to evaluate the mutagenic potential of the test article. Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in DMSO at approximately 500 mg/mL, the maximum concentration tested. In the preliminary toxicity assay, the maximum concentration of 98-06-1163 in treatment medium was 5000 µg/mL. Visible precipitate was present at concentrations = 150 µg/mL in treatment medium. Selection of concentrations for the mutation assay was based on the precipitation profile. Substantial toxicity, i.e., suspension growth of = 50% of the solvent control, was observed at = 150 µg/mL only with S9 activation. Based on the results of the preliminary toxicity assay, the concentrations tested in the initial mutagenesis assay ranged from 5.0 to 200 µg/mL for both the non-activated and S9-activated cultures with a 4-hour exposure. Visible precipitate was present at a concentration of 200 µg/mL in treatment medium. The concentrations chosen for cloning were 50, 75, 100, 150, and 200 µg/mL No cloned cultures exhibited mutant frequencies = 90 mutants per 106 clonable cells over that of the solvent control. There was no concentration-related increase in mutant frequency. The trifluorothymidine-resistant colonies for the positive and solvent control cultures from both assays 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. Under the conditions of this study, test article 98-06-1163 was concluded to be negative in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.