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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:
07 June 2002 - 21 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4E, dated May 19, 2000.
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Health and Welfare, 1988, Guidelines for Toxicity Studies of Drugs, Chapter 4, "Mutagenicity Study".
Deviations:
no
Qualifier:
according to
Guideline:
other: "U.S. FDA Center for Food Safety & Applied Nutrition Office of Premarket Approval, Redbook 2000, Toxicological Principles for the Safety of Food Ingredients, IV.C.1.c. Mouse Lymphoma Thymidine Kinase Gene Mutation Assay", July 07, 2000.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TCD
- Physical state: Liquid
- Analytical purity: 99.6%
- Lot/batch No.: 011107D
- Expiration date of the lot/batch: 30 September 2002
- Storage condition of test material: Stored in a refrigerator (nominally 4 - 8°C) under nitrogen.

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 Medium
- 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:
S9 Mix
Test concentrations with justification for top dose:
Experiment I
Without S9 Mix: 3.1, 6.3, 12.5, 25.0, 37.5, and 50 µg/mL.

Experiment II
Without S9 Mix: 6.3, 12.5, 25.0, 50.0, 75.0, and 100.0 µg/mL.
With S9 Mix: 3.1, 6.3, 12.5, 25.0, 37.5, and 50.0 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: This solvent was chosen due to its solubility properties, and its relative non-toxicity to the cell cultures.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Used in the absence of S9 mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
Used in the presence of S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours in the first experiment, 24 hours in the second experiment.
- Expression time (cells in growth medium): 72 hours (48 hours in the second experiment without metabolic activation).
- Selection time (if incubation with a selection agent): 10 - 15 days
- Fixation time (start of exposure up to fixation or harvest of cells): 72 hours

SELECTION AGENT (mutation assays): RPMI 1640 medium containing 5 µg/mL Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Two plates were seeded per culture.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.

A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.


A significant response is described as follows:
The test item is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.

The test item is classified as mutagenic if there is a· reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.

However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.

Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 is less than 10 Ok of the solvent control or the cloning efficiency 2 after the expression period is less than 20 %.

Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations and c1astogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was seen at 50 µg/mL with and without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effect relative to the control
- Effects of osmolality: Osmolarity of solvent control = 275; osmolarity of TCD formulation, 1600 µg/mL = 321
- Precipitation: In the pre-experiment, precipitation occurred at 50 µg/mL and above without metabolic activation, and at 100 µg/mL with metabolic activation durinf pulse treatment, and at 400 µg/mL and above at continuous treatment.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

Under the experimental conditions used the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.