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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to international guideline(s), GLP-compliant, performed in recognized contract research organization, no restrictions, fully adequate for assessment.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
of 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: U.S. Food and Drug Administration (FDA), Redbook 2000: Toxicological Principles for the Safety of Food Ingredients, IV.C.1.c Mouse Lymphoma Thymidine Kinase Gene Mutation Assay, 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Target gene:
TK
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell line used: L5178Y tk +/- clone 3.7.2C
The chromosome number of these cells is 40 (stable aneuploid karyotype, 2n = 40). The cells were obtained from MRC Cell Mutation Unit, University of Sussex, Falmer, Brighton, East Sussex, BN1 9RR, UK.

- Type and identity of media: RPMI 1640 medium (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum (10% v/v for growing in flasks, and 20% for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: a karyotype check was carried out in 1995
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (liver fraction of Aroclor 1254-induced male Wistar rats) supplemented with a NADPH-generating system)
Test concentrations with justification for top dose:
ASSESSMENT OF CYTOGENICITY AND MUTAGENICITY
The highest concentrations tested were 108 and 500 µg/mL in the absence and presence of S9-mix, respectively.
The highest concentrations evaluated for mutagenicity were 8.4 and 75 µg/mL in the absence and presence of S9-mix, respectively.

- Without metabolic activation:
First assay: 108, 65, 39, 23, 14, 8.4*, 5.0*, 3.0*, 1.8*, 1.1*, 0.65*, 0.39*, 0.24*, and 0.14⁺ µg/mL, single cultures
Second assay: 15, 12, 9.6, 8.2⁺, 6.9*, 5.9*, 5.0*, 4.0*, 3.2, 2.4*, 1.8, 1.4*, 0.68*, 0.34, 0.17*, and 0.08* µg/mL, single cultures
- With metabolic activation:
First assay: 500, 300, 180, 108, 65⁺, 39*, 23*, 14*, 8.4*, 5.0*, 3.0*, 1.8*, 1.1*, and 0.65 µg/mL, single cultures
Second assay: 30⁺, 24*, 19*, 16*, 14, 12*, 10, 8.0*, 6.4, 4.8*, 3.6, 2.7⁺, 1.4⁺, 0.68, and 0.34 µg/mL, single cultures
Third assay: 75*, 60*, 48*, 38*, 31, 25*, 20, and 16 µg/mL, duplicate cultures

* Assessed for mutagenicity (both replicates of the third assay)
⁺ Scored for small and large colonies

Dose related slight turbidity was observed at the start of treatment at concentrations above 23 µg/mL; at the end of treatment, slight turbidity was observed at concentrations above 8.4 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The test material could be suspended in THF up to 50 mg/mL, resulting in a homogeneous white turbid suspension
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Remarks:
MMS and MCA were used as positive control substances in the absence and presence of the S9-mix, respectively. Final concnetartions: 0.1 mM (11 µg/mL, MMS) and 10 µg/mL (MCA).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (with metabolic activation) , 24 hours (without metabolic activation)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT), final concentration 4 µg/mL

NUMBER OF REPLICATIONS:
- Exposure phase:
first and second assay and positive controls: single cultures;
third assay and vehicle controls: duplicates cultures.
- Assessment of cloning efficiency and mutant potential:
two 96-well plates from each culture (four wells if a reduced cloning efficiency (<50%) was expected)

DETERMINATION OF CYTOTOXICITY
- Method: initial cell yield, relative total growth (RSG) and relative total growth (RTG)

OTHER EXAMINATIONS:
- Scoring of small and large colonies (negative and positive controls and of some test material dose levels)

GROWTH CONDITIONS
- at ca. 37 °C and ca. 5% CO2 in a humidified incubator
Evaluation criteria:
A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 100 mutants per 1,000,000 clonable cells*. A response was considered to be equivocal if the induced mutant frequency was more than 50 mutants per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity was considered to be an artefact and not indicative of genotoxicity.
* Aaron et al, 1994, Mutation Res. 312:235-239; Clive et al., 1995, Environ. Molec. Mutagen. 25:165-168

The test substance was considered to be mutagenic in the gene mutation test at the TK -locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.

The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test points.

Both numerical significance and biological relevance were considered together in the evaluation.
Statistics:
No statistical analysis was performed.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see 'Additional information on results'
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material was cytotoxic to the L5178Y cells in both the absence and the presence of S9-mix. The initial cell yield, relative suspension growth (RSG), and relative total growth (RTG) were decreased above concentrations of 3 µg/mL and 30 µg/mL in the absence and presence of S9-mix, respectively. The RTG values (according to OECD and FDA guidelines) at the highest concentrations evaluated for mutagenicity were below 20%.

Any other information on results incl. tables

In the first and second assay, a slight increase of 53 -75 mutants per 10E6 clonable cells compared to the negative control was observed at three single concentrations in both the absence and presence of metabolic activation. In the third assay no increase of the mutant frequency was observed at any dose level, either at toxic or non-toxic dose levels. Since the observed increases of the mutant frequencies were not reproducible they were considered as accidental occurrence and not indicative for mutagenicity.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative