Registration Dossier

Diss Factsheets

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
Study period:
2009-06-05 through 2009-10-02
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:
2009
Report date:
2009

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
Reference substance name:
Matte, nickel
EC Number:
273-749-6
EC Name:
Matte, nickel
Cas Number:
69012-50-6
Molecular formula:
Not applicable
IUPAC Name:
Nickel matte
Details on test material:
- Name of test material (as cited in study report): Nickel matte (N123-PTL)
- Physical state: granules/gray, black
- Composition of test material, percentage of components: Nickel Subsulfides <60%, Nickel Iron Sulfides <40%, Copper >10%, Cobalt <1%, Arsenic <0.1%, and Magnesium Oxide 4%.
- Stability: stable
- Lot/batch No.: not applicable
- Storage: at room temperature

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: 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 the concentrations was based on data from the pre-experiments. In experiment 1 1000ug/mL (with metabolic activation) and
800 ug/mL (without metabolic activation) were selected as the highest concentrations. In experiment II 1000 uglmL (with metabolic activation) and
80 uglmL (without metabolic activation) were selected as the highest concentrations. Experiment II without metabolic activation was performed as a
24 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I
with metabolic activation:
300, 400, 500, 600, 700, 800, 900 and 1000 uglmL
and without metabolic activation:
100, 300, 400, 540, 620, 650, 680 and 800 uglmL

Experiment II
with metabolic activation:
100, 200, 400, 500, 600, 800, 925 and 1000 uglmL
and without metabolic activation:
0.5, 1, 2, 5, 10, 20, 40 and 80 uglmL

According to OECD Guidelines at least 8 concentrations of the test item were set up in the experiments with and without metabolic activation.

Vehicle / solvent:
Nickel matte was suspended in cell culture medium (RPMI + 3% HS)
Controls
Untreated negative controls:
other: solvent or vehicle alone used as negative control
Negative solvent / vehicle controls:
yes
Remarks:
used as negative control
True negative controls:
no
Positive controls:
yes
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
Details on test system and experimental conditions:
METHOD OF APPLICATION: For short-term exposure, 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. For long-term exposure, 1 x 10^7 cells/mL (175cm2) flasks) were suspended in 50mL RPMI medium with 7.5% 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 durations: 4 hr and 24 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 (for short-term exposure) or 48 hr (for long term exposure) 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): RPMI 1640 complete culture medium supplemented with TFT (5 uglmL)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; following the 72 hr or 48 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:
negative
Remarks:
non-mutagenic, non-clastogenic
Cytotoxicity / choice of top concentrations:
other: Growth inhibition observed in all experiments. Precipitation of the test item was noted in all concentrations in experiment I with and without metabolic activation and in experiment II with metabolic activation. In experiment II without metabolic activat
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
For both short- and long-term exposure, mutant values of the negative controls were within historical control data of the test facility. Mutant values from the all groups with and without metabolic activation were also within the range of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
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 0.08% for the highest concentration (1000 ug/mL) evaluated. The highest concentration evaluated without metabolic activation was 800 ug/mL with a RTG of 32.06 %. In experiment II with metabolic activation the relative total growth (RTG) was 0.39% for the highest concentration (1000 ug/mL) evaluated. The highest concentration evaluated without metabolic activation was 80 ug/mL with a RTG of 18.44%.

CLASTOGENICITY:
In experiment I (short-term exposure) and II (long-term exposure), with or without metabolic activation, colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).
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 - Experiment I (short-term exposure)

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

Test Group Concentration (ug/mL)   RCE (%) Mean* Number of Cultures/96 wells   Mutants/10^6 cells (Mutation Frequency)  Mutation factor
NC1  0 100.00 33.25 126.33 not applicable
NC2  0 100.00 33.75 117.28 not applicable
1 (P)  300 93.63 29.00 135.59 1.11
2 (P)  400 95.84 33.75 149.82 1.23
3 (P)  500 91.41 34.00 178.30 1.46
4 (P)  600 92.52 34.25 173.16 1.42
5 (P)  700 94.74 33.50 155.15 1.27
6 (P)  800 63.16 19.00 195.87 1.61
7 (P)  900 15.51 3.25 174.79 1.44
8 (P)  1000 0.55 0.00 0.00 0.00
S[aIP  3.5 95.29 66.00 411.41 3.38

*Based on 4 plates

NC: Negative Control

(P): Precipitation

B[a]P: Benz(a)pyrene

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

 Test Group  Concentration (ug/mL)  Quotient Large/Small
NC1  0 7.31
NC2  0 5.43
5 (P)  700 5.09
6 (P)  800 3.75
7 (P)  900 3.33
8 (P)  1000

n.c. 

B[aIP  3.5 1.64

NC: Negative Control

(P): Precipitation

B[a]P: Benz(a)pyrene

RESULTS WITHOUT METABOLIC ACTIVATION - Experiment I (short-term exposure)

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

Test Group Concentration (ug/mL)   RCE (%) Mean* Number of Cultures/96 wells   Mutants/10^6 cells (Mutation Frequency)  Mutation factor
NC1  0 100.00 22.75 124.99 not applicable
NC2  0 100.00

 23.50

94.89 not applicable
3 (P)  100 98.19 27.00 139.77 1.27
5 (P)  300 104.82 28.50 119.04 1.08
6 (P)  400 99.40 23.25 113.10 1.03
8 (P)  540 112.05 30.75 89.13 0.81
9 (P)  620 93.37 31.00 189.46 1.72
10 (P)  650 102.41 24.75 110.10 1.00
11 (P)  680 97.59 18.00 89.49 0.81
13 (P)  800 79.52 21.25 172.08 1.57
EMS  500 80.12 81.75 1293.33 11.76
MMS  10 91.57 64.00 560.30 5.10

*Based on 4 plates

NC: Negative Control

(P): Precipitation

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

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

 Test Group  Concentration (ug/mL)  Quotient Large/Small
NC1  0 14.17
NC2  0 17.8
10 (P)  650 13.14
11 (P)  680 6.2
13 (P)  800 3.05
MMS  10 1.13

NC: Negative Control

(P): Precipitation

MMS: Methylmethanesulfonate

RESULTS WITH METABOLIC ACTIVATION - Experiment II (long-term exposure)

Mutagenicity Data with metabolic activation (see Table 10 in original study report)

Test Group Concentration (ug/mL)   RCE (%) Mean* Number of Cultures/96 wells   Mutants/10^6 cells (Mutation Frequency)  Mutation factor
NC1  0 96.70 28.50 154.52 not applicable
NC2  0 103.30 33.75 153.22 not applicable
1 (P)  100 99.10 30.25 154.35 1.00
2 (P)  200 96.10 29.75 165.61 1.08
4 (P)  400 105.11 32.25 135.09 0.88
5 (P)  500 94.29 31.75 188.73 1.23
6 (P)  600 92.49 27.50 166.68 1.08
10 (P)  800 73.27 12.50 110.61 0.72
13 (P)  925 46.25 6.75 113.79 0.74
14 (P)  1000 1.80 0.25 132.46 0.86
B[aIP  3.5 94.29 70.25 618.47 4.02

*Based on 4 plates

NC: Negative Control

(P): Precipitation

B[a]P: Benz(a)pyrene

Colony Sizing, with metabolic activation (see Table 11 in original study report)

 Test Group  Concentration (ug/mL)  Quotient Large/Small
NC1  0 4.43
NC2  0 3.5
6 (P)  600 3.23
10 (P)  800 1.94
13 (P)  925 1.25
14 (P)  1000

n.c 

8[a)P  3.5 0.98

NC: Negative Control

(P): Precipitation

B[a]P: Benz(a)pyrene

RESULTS WITHOUT METABOLIC ACTIVATION - Experiment II (long-term exposure)

Mutagenicity Data without metabolic activation (see Table 14 in original study report)

Test Group Concentration (ug/mL)   RCE (%) Mean* Number of Cultures/96 wells   Mutants/10^6 cells (Mutation Frequency)  Mutation factor
NC1  0 100.00 24.50 131.56 not applicable
NC2  0 100.00 28.25 189.25 not applicable

1

0.5 107.79 28.25 139.45 0.87

2

1

101.30 30.25 180.88 1.13
3 (P)  2 90.91 27.25 204.48 1.27
4 (P)  5 92.21 28.50 209.43 1.31
5 (P)  10 104.55 23.50 123.17 0.77
6 (P)  20 106.49 25.75 129.76 0.81
7 (P)  40 89.61 31.25 248.36 1.55
8 (P)  80 85.71 28.50 242.25 1.51
EMS  200 55.84 87.75 3304.92 20.60
MMS  10 48.70 75.00 2454.69 15.30

*Based on 4 plates

NC: Negative Control

(P): Precipitation

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

Colony Sizing, without metabolic activation (see Table 15 in original study report)

 Test Group  Concentration (ug/mL)  Quotient Large/Small
NC1  0 3.08
NC2  0 3.52
6 (P)  20 3.9
7 (P)  40 3.63
8 (P)  80 1.53
MMS  10 0.73

NC: Negative Control

(P): Precipitation

MMS: Methylmethanesulfonate

Applicant's summary and conclusion

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

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Nickel matte (N123) is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
Executive summary:

STUDY RATED BY AN INDEPENDENT REVIEWER