Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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: GLP guideline study.
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:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
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): NiO green (N112); Nickel oxide sinter
- Physical state: granules / dark grey
- Composition of test material, percentage of components: nickel oxide: 98%, cobaltous oxide: 1.5%
- Lot/batch No.: not provided by the sponsor
- Stability under test conditions: stable
- Storage condition of test material: at room temperature, closed container

Method

Target gene:
Thymidine kinase (TK)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Mammalian microsomal fraction S9 mix
Test concentrations with justification for top dose:
Experiment I (with and without metabolic activation): 1,3,5,6, 7, 8,9, 10 mM
Experiment II (with metabolic activation): 1.5,3.5,4.5,5.5, 7.5, 8.5, 9.5, 10 mM
Experiment II (without metabolic activation): 1,3,5,6, 7, 8, 9, 10 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [cell culture medium]
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
vehicle
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
vehicle
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
vehicle
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension


DURATION
- Preincubation period: not applicable
- Exposure duration: 4 or 24 hr
- Expression time (cells in growth medium): 48-72 hr
- Selection time (if incubation with a selection agent): 11-14 days


SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)



DETERMINATION OF CYTOTOXICITY
- Method: After the expression period the relative cloning efficiency (RCE; percentage cloning efficiency of the test group in relation to the negative control) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well-plates. The cells were incubated for 6 days at 37°C in a humidified atmosphere with 5% C02. Analysis of the results was based on the number of cultures with cell growth (positive cultures) and/or those without cell growth (negative cultures) compared to the total number of cultures seeded. Relative suspension and total growth
(RSG and RTG; RTG= [RSG x RCE]/100) of the treated cell cultures were
calculated according to the method of Clive and Spector.

Evaluation criteria:
There are several criteria for determining a positive result:
- clear and dose-related increase in the mutant frequency,
- biologically relevant response (at least a 2-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) for at least one of the dose groups,
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/small colonies ratio (ratio of the clastogenic controls MMS and/or B[a]P with a coefficient of 1.5) is an indication for potential clastogenic effects and/or chromosomal aberrations.
Statistics:
Not applicable.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The selection of the concentrations was based on data from the pre-experiment.



ADDITIONAL INFORMATION ON CYTOTOXICITY: No growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I with metabolic activation the relative total growth (RTG) was 100.09% for the highest concentration (10 mM) evaluated. The highest concentration evaluated without metabolic activation was 10 mM with a
RTG of 115.77 %. In experiment II with metabolic activation the relative total growth (RTG) was 115.04% for the highest concentration (10 mM) evaluated. The highest concentration evaluated without metabolic activation was 10 mM with a RTG of79.90%.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Toxicity:

No growth inhibition was observed in experiment I and II with and without metabolic activation.

Mutagenicity:

The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Clastogenicity (small colonies):

All dose groups were considered as not clastogenic.

Applicant's summary and conclusion

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

The study authors concluded that in the described mutagenicity test under the experimental conditions reported, the test item Nickel oxide green (N112) is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
Executive summary:

BSL Bioservice Scientific Laboratories (BSL) GmbH conducted an in vitro mammalian cell gene mutation assay according to OECD Test #476 – In vitro Mammalian Cell Gene Mutation Test guidelines and using GLP standards. The study was completed on 2008-06-10 and the final report issued on 2008-11-27. The test substance, “Nickel oxide green (sample ID N112)”, was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase (TK) locus using the cell line L5178Y. The selection of the concentrations was based on data from the pre-experiment. Four sets of experiments were run where cells were exposed to NiO for 4 or 24 hr with or without metabolic activation. EMS and MMS were used as positive controls for studies without metabolic activation, and B[a]P was used in studies with metabolic activation. Exposure to vehicle or solvents alone served as the negative controls. In Experiment I (4 hours, with and without metabolic activation), the test concentrations were: 1, 3, 5, 6, 7, 8, 9, 10 mM. In Experiment II (24 hours, referred to as longterm exposure) with and without metabolic activation, the concentrations were respectively: 1.5, 3.5, 4.5, 5.5, 7.5, 8.5, 9.5, 10 mM and 1, 3, 5, 6, 7, 8, 9, 10 mM.

Findings indicated that i) no significant growth inhibition was observed at any concentrations in Experiments I and II, with and without metabolic activation, ii) mutation frequencies (in all experiments) did not show biologically relevant increases as compared to the negative controls, and iii) all dose groups were considered as not clastogenic based on colony size. The study authors concluded that Nickel oxide green (sample ID N112) was considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y. STUDY RATED BY AN INDEPENDENT REVIEWER