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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From December 07, 2005 to February 17, 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study is according to OECD Guideline 476 and EU Method B.17 in compliance with Good Laboratory Practices of UK
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

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:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC
Deviations:
no
Qualifier:
according to
Guideline:
other: United Kingdom Environmental Mutagen Society
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
according to UK GLP
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Physical state: Clear colourless slightly viscous liquid
- Lot/batch No.: 5010121200
- Storage condition of test material: Stored at room temperature in the dark

Method

Target gene:
Thymidine Kinase (TK)

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr J Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically "cleansed" against high spontaneous background: Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Pβ/PNF S9 (2% )
Test concentrations with justification for top dose:
Preliminary toxicity test:
- 4 h without S9: 0, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, 20 and 40 µg/mL
- 4 h with S9: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL
- 24 h without S9: 0, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL
Mutagenicity test:
Experiment 1:
- Without S9: 0, 0.13, 0.25, 0.5, 1, 2, 3, 4 and 5 µg/mL;
- With S9: 0, 1.25, 2.5, 5, 7.5, 10, 20, 30, 40, 50 and 60 µg/mL
Experiment 2:
- Without S9: 0, 0.13, 0.25, 0.5, 1, 2, 3, 4 and 5 µg/mL;
- With S9: 0, 10, 20, 30, 35, 40, 45, 50 and 55 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
absence of metabolic activation

Migrated to IUCLID6: 400 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
presence of metabolic activation

Migrated to IUCLID6: 1.25 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: In suspension

DURATION
-Exposure duration: 24 h
Preliminary study:
- Expression duration- Preliminary test: 4 h (with and without S9); 24 h (without S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24- 48 h
Experiment 1: 4 h incubation
Experiment 2: 48 h incubation
- Expression time (cells in growth medium): 48 h

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
STAIN (for cytogenetic assays): MTT solution

NUMBER OF REPLICATIONS: Two




Evaluation criteria:
- Vehicle control values greater than 250 x 10(-6) mutant frequency per survivor are not normally acceptable and will be repeated.
- Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
- Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the Global Evaluation Factor (GEF) of 126 x 10(-6) will be considered positive.
Statistics:
No data

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No


RANGE-FINDING/SCREENING STUDIES: The dose range in the preliminary toxicity test was 0.16 to 40 µg/mL in the 4 h without S9 cultures, 19.53 to 5000 µg/mL in the 4 h with S9 cultures and 0.08 to 20 µg/mL in the 24 h without S9 cultures.
Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 µg/mL or 10 mM.
ii) The presence of excessive precipitate where no test material-induced toxicity was observed.
iii) Test material-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).

As the test material produced cytotoxicity, therefore, criteria (iii) was used for dose selection in the main study.

RESULT DETAILS: IRR 560 induced weak but reproducible toxicologically significant dose-related increases in the mutant frequency both with and without metabolic activation, in the first and second experiment. The mutagenic response was only observed in dose levels approaching the limit of acceptable toxicity. The increase in mutant frequency was predominantly due to small colony formation, indicating clastogenic activity resulting in structural chromosome damage.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation

"confidential substance name" was considered to be mutagenic to L5178Y cells with and without metabolic activation.
Executive summary:

A study was conducted to assess the mutagenic potential of "confidential substance name" on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The study was conducted according to OECD Guideline 476 and EU Method B.17 in compliance with principles of GLP UK.

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at up to ten dose levels, in duplicate, together with vehicle (solvent) and positive controls. The entire experiment was repeated to confirm the result of the first experiment. 4 h exposures were used both with and without activation in Experiment 1 and Experiment 2.

The dose range of the test substance, plated for expression of mutant colonies, was selected following the results of a preliminary toxicity test and was 0.13 to 4 µg/mL without activation and 7.5 to 50 µg/mL with activation for the first experiment. For the second experiment the dose range was 0.13 to 5 µg/mL without activation and 10 to 55 µg/mL with activation.

The maximum dose level used was limited by the test substance induced toxicity. No precipitate of the test substance was observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test substance induced weak but reproducible toxicologically significant dose-related increases in the mutant frequency both with and without metabolic activation, in the first and second experiment. The mutagenic response was only observed in dose levels approaching the limit of acceptable toxicity. The increase in mutant frequency was predominantly due to small colony formation, indicating clastogenic activity resulting in structural chromosome damage.

In conclusion, the test substance was considered to be mutagenic to L5178Y cells with and without metabolic activation.