Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards.

Data source

Reference
Reference Type:
publication
Title:
In vitro embryotoxicity testing of metals for dental use by differentiation of embryonic stem cell test
Author:
Imai K. and M. Nakamura
Year:
2006
Bibliographic source:
Congenital Anomalies. 46:34–38

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: embryonic stem cell test (EST) protocol (Scholz et al. 1999)
Principles of method if other than guideline:
Test conducted according to: Scholz G, Pohl I, Genschow E, Klemm M, Spielmann H (1999) Embryotoxicity screening using embryonic stem cells in
vitro: Correlation to in vivo teratogenicity. Cells Tissues Organs 165: 203–211.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Metal powder
- Molecular formula (if other than submission substance): Not different than submission substance
- Molecular weight (if other than submission substance): Not different than submission substance
- Smiles notation (if other than submission substance): Not different than submission substance
- InChl (if other than submission substance): Not different than submission substance
- Structural formula attached as image file (if other than submission substance): Not different than submission substance
- Substance type: Pure product
- Physical state: Solid, metal powder
- Other details on test material not reported or not applicable

Test animals

Species:
mouse
Strain:
other: embryonic stem cells (ES) from the D3 mouse cell line
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Embryonic stem cells (ES) from the D3 mouse cell line and clone A31 from Balb/c 3T3 cells (3T3 cells) from mice.
The 3T3 cells were purchased from American Type Culture Collection (ATCC; Cat. No. CCL-163).
- Other information not reported or not applicable

ENVIRONMENTAL CONDITIONS: Not applicable
IN-LIFE DATES: Not applicable

Administration / exposure

Route of administration:
other: Cell culture
Type of inhalation exposure (if applicable):
not specified
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Culture mediums For ES-D3 cells, preheat treated 20% (v/v) fetal calf serum (FCS) (Hyclone, USA) was added to 1% (v/v) nonessential amino acids
(Gibco, USA), 0.1 mM β-mercaptoethanol (Sigma, USA), 2 mM L-glutamine (Gibco), and Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) with
penicillin/streptomycin (Gibco). Except during tests, 1000 U/mL of mouse leukemic inhibitory factor (mLIF) was added to inhibit natural cell
differentiation. For 3T3 cells, a culture medium was prepared by adding 10% (volume ratio) FCS to DMEM with 4 mM L-glutamine and penicillin/
streptomycin.

Differentiation assay: ES-D3 cells were added to each test solutions at a range of solution concentrations. The number of ES-D3 cells were adjusted
to a final concentration of 3.75 × 10^4 cells/mL in each test solution using a hemacytometer, and 20 μL cell suspension was dropped 60–70
times onto the inner side of the lid of a 10-cm diameter Petri dish using a micropipette. Each drop of the cell suspension contained approximately
750 cells. Five ml of sterilized phosphate buffered saline (PBS) was poured into the Petri dish, and the lid was quickly reversed and placed on to the
dish before the cell suspension on the lid could flow down. Suspension culture was carried out for three days in a CO2 incubator (5% CO2 and 95%
air; 37°C) (Sanyo, Japan). Drops of the cell suspension on the inside of the Petri dish lid were then collected into a 6-cm diameter Petri dish. The test
solutions were replaced with fresh ones using a pipette and each test solution was subjected to further reaction for two days in the same condition.
Subsequently, two 24-well multidishes were used for every test solution. Each of the embryoid bodies (EBs) formed were placed into a well with a
micropipette, and the EBs were cultured statically for five days. The presence of beating myocardial cells in each well was examined under an inverted phase difference microscope (IX-70, Olympus, Japan). The number of wells in which beating cells were observed at each test solution concentration was examined, and the ID50 was calculated from the ratio of the number of the wells to the number of wells in which EBs were successfully
disseminated.

Cell viability assay: In order to examine the effects of the test solutions on the two kinds of cells, i.e. 3T3 cells and ES cells, cell suspensions of
1 × 10^4 cells/ mL were prepared in the absence of mLIF, inoculated into a 96-well multidish, and test solutions were added to each well after 2 h.
Finally, the cell viability assay was performed on day 10 of the culture. Cell viability measurements were made using a
(3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl terazolium bromide, Sigma, USA) MTT assay. MTT 100 mg was dissolved in 200 mL PBS, and
20 μL was placed into each well. After 2 h incubation, the MTT solution was removed using a Pasteur-pipette, and exactly 130 ìL
acid isopropanol solution was added to each well in order to measure absorbance at 570 nm by ELISA reader (SpectraMax Plus,
Molecular Device, USA).

DIET PREPARATION: Not applicable
VEHICLE: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of metallic elements in the extracts were measured with a sequential radiofrequency plasma emission spectrometer
(ICPS-1000IV; Shimadzu, Japan), and each test solution was obtained by serial dilution.
Details on mating procedure:
Not applicable
Duration of treatment / exposure:
Not applicable
Frequency of treatment:
Not applicable
Duration of test:
Not applicable
No. of animals per sex per dose:
Not applicable
Control animals:
yes, concurrent no treatment
Details on study design:
Not applicable

Examinations

Maternal examinations:
Not applicable
Ovaries and uterine content:
Not applicable
Fetal examinations:
Not applicable
Statistics:
The results are expressed as the mean and the confidence interval of 95%. Each data point represented four independents experiments
where three independents wells were used for the one experiment. P-values less than 0.05 were regarded as significant.
Indices:
Not applicable
Historical control data:
Not applicable

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no data

Details on maternal toxic effects:
Not applicable

Effect levels (maternal animals)

open allclose all
Dose descriptor:
other: IC50
Effect level:
91 other: uM Ni
Basis for effect level:
other: other:
Dose descriptor:
other: IC50
Effect level:
79.7 other: uM Ni
Basis for effect level:
other: other:
Dose descriptor:
other: ID50
Effect level:
75.8 other: uM Ni
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The authors reported that the test results indicated that nickel has no embryotoxicity and this in conflict with the previous reports.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The authors reported that the test results indicated that nickel has no embryotoxicity.
Executive summary:

STUDY RATED BY AN INDEPENDENT REVIEWER.