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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
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 labeling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
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.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - 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.
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - 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.
Qualifier:
according to
Guideline:
other: ICH S2A document April 24, 1996
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
other: ICH S2B document November 21, 1997
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: 86.6%

Method

Target gene:
thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's Medium for Leukemic Cells of Mice
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Toxicity Assay: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 3500 µg/mL
Initial Mutagenesis Assay (cloning): 500, 750, 1000, 1500, 2000 µg/mL (without activation); 150, 250, 500, 600, and 750 µg/mL (with activation)
Extended Treatment Assay (cloning): 250, 500, 600, 750, and 1000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: Based on solubility of the test substance and compatibility with the target cells.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
-S9 treatments
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
+S9 treatments
Details on test system and experimental conditions:
METHOD OF APPLICATION: Treatment was carried out in conical tubes by combining 6 x 10e6 L5178Y/TK+/- cells, F0P
medium or S9 activation mixture, and 1.0 mL dosing solution of test article in solvent or solvent alone in a total volume of 10 mL. The positive controls were treated with 100 μL MMS (at final concentrations in treatment medium of 15 and 20 μg/mL with a 4-hour exposure or 5.0 and 7.5 μg/ml with a 24-hour exposure) or 7,12-DMBA (at final concentrations in treatment medium of 1.0 and 1.25 μg/mL). Treatment tubes were gassed with 5±1% CO2in air, capped tightly, and incubated with mechanical mixing for 4 or 24 hours at 37±1°C. The preparation and addition of the test article dosing solutions were carried out under amber lighting and the cells were incubated in the dark during the exposure period. After the treatment period, the cells were washed twice with F0P or F0P supplemented with 10% horse serum, 2 mM L-glutamine, 100 U penicillin/mL and 100 μg streptomycin/mL (F10P). After the second wash, the cells were resuspended in F10P, gassed with 5±1% CO2 in air and placed on the roller drum apparatus at 37±1°C.


DURATION

- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 48 hours for 4-hour exposure; 72 hours for 24-hour exposure
- Selection time (if incubation with a selection agent): 10-14 days


SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 3x10e6 cells/100 mL; 600 cells/100 mL were plated for a viable count determination.


DETERMINATION OF CYTOTOXICITY
- Method: Exposure in the absence and presence of S9 activation for 4 hours, and without activation for 24 hours. For the 4-hour exposure, cell population density was determined 24 and 48 hours after the exposure to the test article; the cultures were adjusted to 3x10e5 cells/mL after 24 hours only. For the 24-hour exposure, cell population density was determined 24, 48, and 72 hours after the exposure to the test article. The cell population was adjusted to 3 x 10e5 cells/mL immediately after test article removal and 24 hours after test article removal. Cultures with less than 3x10e5 cells/mL were not adjusted. Toxicity was measured as suspension growth of the treated cultures relative to the growth of the
solvent control cultures after 48 hours.



OTHER

EXPRESSION OF THE MUTANT PHENOTYPE: For expression of the mutant phenotype, the cultures were counted using an electronic cell counter and adjusted to 3x10e5 cells/mL at approximately 24 and 48 hours after treatment in 20 and 10 mL total volume, respectively. For the 24-hour exposure, cultures were adjusted to 3x10e5 cells/mL in 20 mL immediately after test article removal, then at 48 and 72 hours after treatment in 20 and 10 mL total volume, respectively. Cultures with less than 3x10e5 cells/mL were not adjusted.

SCORING PROCEDURE: After the incubation period, the VC plates were counted for the total number of colonies per plate and the total relative growth determined. The TFT-resistant colonies were then counted for each culture with ≥ 20% total relative growth (including at least one concentration with ≥ 10% but ≤ 20% total growth). The diameters of the TFT-resistant colonies for the positive and solvent controls and, in the case of a positive response, the test article-treated cultures were determined over a range of approximately 0.2 to 1.1 mm.
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 exhibited mutant frequencies of ≥90 mutants per 10e6 clonable cells over the background level (based on the average mutant frequency of duplicate cultures). If the average solvent control mutant frequency was >90 mutants per 10e6 clonable cells, a doubling of mutant frequency over the background will also be required.

A result was considered negative if the treated cultures exhibited mutant frequencies of less than 90 mutants per 10e6 clonable cells over the background level (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.

There are some situations where a chemical would be considered negative when there was no culture showing between 10-20% survival: 1) There was no evidence of mutagenicity (e.g. no dose response or increase in mutant frequencies between 45 and 89 mutants per 106 above control) in a series of data points within 100% to 20% survival and there was at least one negative data point between 20% and 25% survival. 2) There was no evidence of mutagenicity (e.g. no dose response or increase in mutant frequencies between 45and 89 mutants per 10e6 above control) in a series of data points between 100% to 25% survival and there was also a negative data point between 10%and 1% survival. In this case it would be acceptable to count the TFT colonies of cultures exhibiting <10% total growth.

Statistics:
The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency (number of mutants per 10e6 surviving cells) for each treatment condition was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies in the three corresponding VC plates and multiplying by the dilution factor (2x10e-4) then multiplying by 10e6. For simplicity, this is described as: (Average # TFT colonies / average # VC colonies) x 200.

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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the cultures treated with 1500, 2000, 2500, 3000, and 3500 μg/mL was adjusted to neutral with 1N hydrochloric acid.
- Effects of osmolality: The osmolality of the solvent control was 245 mmol/kg and the osmolality of the highest soluble concentration, 3500 μg/mL, was 260 mmol/kg. (Tested in preliminary toxicity assay)
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: No visible precipitate was present at any concentration in treatment medium.
- Other confounding effects: none reported


RANGE-FINDING/SCREENING STUDIES: Suspension growth relative to the solvent controls was 0% at 3500 μg/mL without activation with a 4-hour exposure and at ≥ 1500 μg/mL with S9 activation with a 4-hour exposure and without activation with a 24-hour exposure. Based on the results of the toxicity test, the concentrations tested in the mutagenesis assay ranged from 500 to 3500 μg/mL for the non-activated cultures with a 4-hour exposure and 150 and 2000 μg/mL for the S9-activated cultures with a 4-hour exposure and non-activated cultures with a 24-hour exposure.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the nonactivated system, cultures treated with concentrations of 500, 750, 1000, 1500, and 2000 μg/mL were cloned and produced a range in suspension growth from 13% to 92%. In the S9-activated system, cultures treated with concentrations of 150, 250, 500, 600, and 750 μg/mL were cloned aproduced a range in suspension growth from 20% to 102%.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, the test substance was concluded to be negative in the presence and absence of S9 metabolic activation in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay.
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).

Executive summary:

The test substance 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 was used to establish the concentration range for the mutagenesis assay. The mutagenesis assay was used to evaluate the mutagenic potential of the test article. The dosing formulations were adjusted to compensate for the purity of the test article using a correction factor of 1.15.

Sterile distilled water 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 water at approximately 50 mg/mL, the maximum concentration tested.

In the preliminary toxicity assay, the maximum concentration of the test substance in treatment medium was 3500 μg/mL (10 mM). No visible precipitate was present at any concentration in treatment medium. Selection of concentrations for the mutation assay was based on reduction of suspension growth relative to the solvent control. Substantial toxicity, i.e., suspension growth of ≤ 50% of the solvent control, was observed at 3500 μg/mL without activation with a 4-hour exposure, ≥ 1500 μg/mL with S9 activation, and ≥ 1500 μg/mL without activation with a 24-hour exposure.

Based on the results of the preliminary toxicity assay, the concentrations tested in the initial mutagenesis assay ranged from 500 to 3500 μg/mL for the non-activated cultures and 150 to 2000 μg/mL for the S9-activated cultures with a 4-hour exposure. Visible precipitate was not present at any concentration in treatment medium. The concentrations chosen for cloning were 500, 750, 1000, 1500, and 2000 μg/mL without activation and 150, 250, 500, 600, and 750 μg/mL with S9 activation. No cloned cultures exhibited mutant frequencies ≥ 90 mutants per 10e6 clonable cells over that of the solvent control. There was no concentration-related increase in mutant frequency.

Based on the results of the preliminary toxicity assay, the concentrations tested in the extended treatment assay ranged from 150 to 2000 μg/mL for non-activated cultures with a 24-hour exposure. Visible precipitate was not present at any concentration in treatment medium. The concentrations chosen for cloning were 250, 500, 600, 750, and 1000 μg/mL. No cloned cultures exhibited mutant frequencies ≥ 90 mutants per 10e6 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 positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

All criteria for a valid test were met. Under the conditions of this study, the test substance was concluded to be negative in the presence and absence of S9 metabolic activation in the L5178Y/TK+/- Mouse Lymphoma Mutagenesis Assay. The assay was negative.