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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:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not Applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Nickel dihydroxide
EC Number:
235-008-5
EC Name:
Nickel dihydroxide
Cas Number:
12054-48-7
Molecular formula:
Ni(OH)2
IUPAC Name:
nickel(2+) dihydroxide
Details on test material:
- Name of test material (as cited in study report): Nickel dihydroxide (N106)
- Physical state: solid powder/green
- Active components: >99% nickel dihydroxide
- Stability: stable
- Lot/batch No.: not provided by the study sponsor
- Storage: at room temperature, closed container

Method

Target gene:
TK
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 complete medium (see specific descriptions below for culture, treatment and selective medium preparations)
Complete Culture Medium = RPMI 1640 medium supplemented with 15% horse serum, 100U/100 ug/ml Penicillin/Streptomycin, 1mM sodium pyruvate, 2mM L-glutamine, 25 mM HEPES, 250 ug/ml amphotericin B
Treatment Medium = RPMI 1640 medium supplemented with 3% horse serum, 100U/100 ug/ml Penicillin/Streptomycin, 1mM sodium pyruvate, 2mM L-glutamine, 25 mM HEPES, 250 ug/ml amphotericin B
Selective Medium = RPMI 1640 medium supplemented with TFT (5ug/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes; stock cultures of the cleansed L5178Y cell line were utilized.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver microsome preparation (S9) from phenobarbital- and beta-naphtoflavone-induced rats
Test concentrations with justification for top dose:
The selection of concentrations was based on data from a pre-experiment, which consisted of 6 concentrations (0.5 to 10 mM with and without metabolic activation). Exposure concentrations for the main study were:
With metabolic activation: 2, 3.5, 5, 6, 6.4, 7.6, 8, 8.5 mM
Without metabolic activation: 1, 2, 2.5, 3, 3.5, 4.5, 5, 5.5 mM
Vehicle / solvent:
Nickel dihydroxide was suspended in cell culture medium (RPMI + 3% HS) and diluted prior to treatment. The solvent was compatible with the survival of the cells and the S9 activity.
Controls
Untreated negative controls:
other: solvent used as negative control
Negative solvent / vehicle controls:
yes
Remarks:
used as negative control
True negative controls:
no
Positive controls:
yes
Remarks:
three compounds used as positive controls
Positive control substance:
other: ethylmethanesulfonate (EMS) and methylmethanesulfonate (MMS) used as positive controls without metabolic activation; benzo[a]pyrene used as positive control with metabolic activation
Remarks:
Not Applicable
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1 x 10^7 cells/mL (25cm2) flasks) were suspended in 11mL RPMI medium with 3% horse serum and exposed to designated concentrations of the test compound in the presence or absence of metabolic activation


DURATION/NUMBER OF CELLS/REPLICATIONS EVALUATED:
- Exposure duration: 4 hr
- Expression time (cells in growth medium): cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period of 72 hr at 37°C and 5% CO2/95% humidified air. The cell density was determined each day and adjusted to 3 x 10^5 cells/ml if necessary.
- Selection time (if incubation with a selection agent): Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200uL selective medium with TFT. Plates were scored after an incubation period of 11 to 14 days at 37°C in 5% CO2/95% humidified air.

SELECTION AGENT (mutation assays): TFT (5 ug/mL)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; following the 72 hr expression time, the relative cloning efficiency (RCE) was determined. Cells were seeded in two 96-well plates, 1.6 cells/well (statistical number), and incubated for 6 days at 37°C in a humidified atmosphere with 5% CO2. 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.

DETERMINATION OF MUTATION FREQUENCY
Mutation frequencies were calculated from the data obtained from cultures (4 plates/dose group) used for the cloning efficiency (cultures with non selective medium) and those used for selection (cultures with selective medium): mutation frequency = (-ln[NC/TC (selective medium)])/(-ln[NC/TC(non selective medium)]) x 800.
NC: number of negative cultures
TC: total number of cultures seeded

DETERMINATION OF POTENTIAL CLASTOGENIC EFFECTS
Colony sizing was performed for the highest concentrations of the test substance and for the negative and positive controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone) indicated by a low large/small colony ratio (ratio of 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.
Evaluation criteria:
Several criteria were used in determining a positive result:
(1) clear and dose-related increase in the mutant frequncy
(2) biologically relevant response (at least 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
(3) combined with a positive effect in the mutant frequncy, 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.

A test item is considered to be negative if there is not biologically relevant increase in the induction of mutant cells above concurrent control levels, at any dose level.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
mutagenic, clastogenic
Cytotoxicity / choice of top concentrations:
other: growth inhibition observed
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Pre-tests for toxicity were conducted. Two negative control (vehicle) groups were tested along with 6 concentrations of the test compund (0.5 to 10 mM, with and without metabolic activation). The relative suspension growth (RSG) was 100% in the control group. For the test compound RSG ranged from 5.19% to 93.68% with metabolic activation (dose-response relationship observed), and from 3.38 to 101.4% without metabolic activation (dose-response relationship observed).

COMPARISON WITH HISTORICAL CONTROL DATA:
Mutant values of the negative controls were within historical control data of the test facility. Mutant values from the two low dose groups with metabolic activation were also within the range of historical control data, though dose groups of 5 mM and higher were outside of the data. Without metabolic activation, mutant values of the dose groups evaluated up to 3mM were within historical data, but above for dose groups from 3.5 mM and higher.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Growth inhibition was observed with and without metabolic activation. The relative total growth (RTG) with metabolic activation ranged from 18.06% to 84.66%; without metabolic activation, the RTG ranged from 14.05% to 86.3%. Dose-response relationships were observed in datasets associated with and without metabolic activation.

POTENIAL FOR CLASTOGENICITY:
A clastogenic effect was observed for the test substance both with and without metabolic activation (see tables below). The positive control compounds induced a significant increase of the mutant frequency and biologically significant increase of small colonies, thus proving the ability of the test system to indicate potential clastogenic effects.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS WITH METABOLIC ACTIVATION

Mutagenicity Data with metabolic activation (see Table 4 in study report)

 Test Group Concentration (mM)   RCE (%) Mean* Number of Cultures/96 wells   Mutants/10^6 cells (Mutation Frequency)  Mutation factor
 NC1  0 100   21.5  101.45 not applicable 
 NC2  0  100  22.25  119.78  not applicable
 1  2 94.15  26  158.53  1.43
 3  3.5  101.54  29.75  151.26  1.37
 6  5  108.31  40.25  174.97  1.58
 8  6  110.77  44.5  179.69  1.62
 9  6.4  84.92  42.5  368.73  3.33
 12  7.6  92.31  46.75  351.32  3.18
 13  8  96.62  45.75  304.25  2.75
 14  8.5  102.77  51.00  297.33  2.69
 B[a}P  2.5 ug/ml  108.92  54.75  265.06  2.40

*Based on 4 plates

Colony Sizing, with metabolic activation (see Table 5 in study report)

 Test Group  Concentration (mM)  Quotient Large/Small
 NC1  0  1.15
 NC2  0  1.70
 12  7.6  0.53
 13  8  0.54
 14  8.5  0.31
 B[a]P  2.5 ug/ml  0.74

RESULTS WITHOUT METABOLIC ACTIVATION

Mutagenicity Data without metabolic activation (see Table 7 in study report)

 Test Group Concentration (mM)   RCE (%) Mean* Number of Cultures/96 wells   Mutants/10^6 cells (Mutation Frequency)  Mutation factor
 NC1  0  100  19  71  not applicable
 NC2  0  100  18.75  73.44  not applicable
 5  1  100.57  18.25  67.88  0.94
 7  2  92  20.75  106.84  1.48
9  2.5  101.14  20.25  74.34  1.03
 10  3  101.14  24.75  93.56  1.30
 11  3.5  90.29  34.75  207.67  2.88
 13  4.5  89.14  34  208.95  2.89
14  5  95.43  39.75  209.77  2.9
 15 5.5   96.57  41.5  213.44  2.96
 EMS  500 ug/ml  88.57  79.5  855.59  11.85
 MMS  10 ug/ml  88.57  70.75  648.87  8.98

*Based on 4 plates

Colony Sizing, without metabolic activation (see Table 8 in study report)

 Test Group  Concentration (mM)  Quotient Large/Small
 NC1  0  5.33
 NC2  0  2.41
 13  4.5  0.77
14  5  0.53
 15  5.5  0.61
MMS  10 ug/ml  0.55

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive mutagenic in the mouse lymphoma thymidine kinase locus

The study report concluded that based on the mutagenicity test under the experimental conditions reported, the test item Nickel dihydroxide (N106) is considered to be mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L51787.
Executive summary:

This GLP in vitro mammalian cell gene mutation study was conducted in accordance with OECD Guideline No. 476, EEC Directive Annex 4E, B 17, and EPA OPPTS 870.5300. The mouse lymphoma cell line L5178Y was used to evaluate the potential for Nickel dihydroxide (N106) to induce mutations at the thymidine kinase locus, both in the presence and absence of metabolic activation. Cells were exposed to the test substance (in suspension) for 4 hours; 8 concentrations were used (2, 3.5, 5, 6, 6.4, 7.6, 8, 8.5 mM with metabolic activation and 1, 2, 2.5, 3, 3.5, 4.5, 5, 5.5 mM without metabolic activation; dose selection based on findings from a pre-experiment). EMS and MMS were used as positive controls for studies without metabolic activation, and B[a]P was used in studies with activation. Vehicle exposure served as the negative controls. Following designated expression and selection times, cytotoxicity and mutagenicity were evaluated based on relative total growth (RTG) and mutation frequency, respectively. Growth inhibition was observed with and without metabolic activation in a dose-dependent manner. The relative total growth (RTG) with metabolic activation ranged from 18.06% to 84.66%; without metabolic activation, the RTG ranged from 14.05% to 86.3%. Similarly, an increased mutation frequency was observed; mutation factors ranged from 1.37 to 3.33 with metabolic activation, and 0.94 to 2.96 without metabolic activation. The potential for inducing clastogenic effects was also assessed by comparing the ratios of large to small colonies in the high dose groups. Results indicated the potential for clastogenic effects both with and without metabolic activation. The study report concluded that based on the mutagenicity test under the experimental conditions reported, the test item Nickel dihydroxide (N106) is considered to be mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L51787. STUDY RATED BY AN INDEPENDENT REVIEWER