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Administrative data

Type of information:
experimental study
Adequacy of study:
supporting study
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Inappropriate dosing regime (non-standard inhalation expsoure procedure) and irrelevant endpoint (olfaction/sense of smell).

Data source

Reference Type:
Nickel sulfate induces location-dependent atrophy of mouse olfactory epithelium: protective and proliferative role of purinergic receptor activation
Jia C, Roman C, Hegg CC
Bibliographic source:
Toxciological Sciences. 115(2). 547-556.

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Male Swiss Webster mice were intranasally instilled with NiSO4 or saline followed by ATP, purinergic receptor antagonists, or saline. The olfactory epithelium was assessed for NiSO4-induced changes using histology and immunohistochemistry 1-7 days postinstillation and compared results to olfactory bulb ablation-induced toxicity.
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Reference substance name:
Nickel sulfate
Nickel sulfate
Details on test material:
- Name of test material (as cited in study report): nickel sulfate
- Molecular formula (if other than submission substance): NiSO4
- Other: purchased from Sigma-Aldrich

Test animals

Swiss Webster
Details on test animals or test system and environmental conditions:
- Source: Adult male Swiss Webster mice purchased from Charles River (Portage, MI).
- Age at study initiation: 6-8 weeks
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): as libitum
- Other: All procedures were conducted in accordance with the Society of Toxicology's Guiding Principles in the Use of Animals in Toxicology and the National Institutes of Health Guide for the Car and Use of Laboratory Animals as approved by Michigan State University Institutional Animal Care and Use Committee.

- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: intranasal
physiological saline
Details on exposure:
METHOD OF EXPOSURE: Anesthetized mice (4% isoflurane) were bilaterally instilled with saline vehicle or NiSO4 (0.1. 0.5, or 2.5 mg/kg body weight) using a pipettor to place a total volume of 50 µl onto the nares. Mice were held in an upright position at least 1 min following instillation during the recovery from anesthesia, to insure that they inhaled the complete dose. In some studies, purinergic receptor antagonists (suramin, 143 mg/kg, and pyridoxalphosphate-6-azophenyl-20, 40-disulfonic acid [PPADS], 30 mg/kg) or ATP (0.2 mg/kg) were instilled 30 min prior to NiSO4 treatment and daily thereafter.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable.
Duration of treatment / exposure:
A single exposure per animal.
Frequency of treatment:
One treatment.
Doses / concentrationsopen allclose all
Dose / conc.:
0.1 mg/kg bw/day (nominal)
Dose / conc.:
0.5 mg/kg bw/day (nominal)
Dose / conc.:
2.5 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Not specified, other than 3-4 mice within each dose group received intraperitoneal BrdU injections (180 mg/kg) at 18, 20, and 22 hours after the NiSO4 intranasal instillation.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: doses are equivalent to 0.3, 1.5, and 7.6 mM, and approximate the cytotoxic NiSO4 concentration of 1mM used in in vitro studies.


Observations and clinical examinations performed and frequency:
Not applicable.
Specific biochemical examinations:
Not applicable.
Neurobehavioural examinations performed and frequency:
Not applicable.
Sacrifice and (histo)pathology:
- Time point of sacrifice: 1, 3, 5, or 7 days post-treatment.
- Parameters measured: thickness of olfactory epithelium in nasal septum and turbinates.
- Procedures for perfusion: Transcardial perfusion with ice-cold 0.1M PBS followed by 4% paraformaldehyde, and decapitated. The lower jaw and skin were removed and tissue was postfixed overnight in 4% paraformaldehyde. For the bulbectomy study, tissue was placed in RDO-Rapid Decalcifier for 4 h (Apex Engineering Products. Aurora. IL), and for the caspase-3 and TUNEL study, tissue was placed in 0.5M EDTA (pH 8.0) for 4-5 days. After decalcification, the tissues were cryoprotected with 20% sucrose and embedded in Tissue Tek OCT (Sakura Finetek, Torrance, CA). Frozen coronal sections of olfactory epithelium (OE) (20 µm) were collected from levels 2-6 of the mouse olfactory epithelium. Tissue was always compared from equivalent levels between treatment groups.
- Tissues evaluated: olfactory epithelium in nasal septum and turbinates.
- Type of staining: Immunohistochemistry, hematoxylin and eosin
- Methodology of preparation of sections: Tissue sections were rehydrated with 0.1M PBS, permeabilized with 0.3% Triton X-100, and blocked with 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA) in 0.1M phosphate-buffered saline for 1 h. Tissue sections were incubated overnight at 4°C either singly or as a mixture with rabbit anti cleaved caspase-3 antibody (ASP175; 1:200, Cell Signaling, Danvers, MA) and goat anti-olfactory marker protein (OMP) antibody (1:1000; Wako Chemical, Plano, TX) followed by incubation with fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG and/or Cy3-conjugated donkey anti-goat IgG (1:200 and 1:50; Jackson ImmunoResearch Laboratory). BrdU immunohistochemistry was performed. Nuclei were visualized by using mounting medium with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Immunoreactivity was visualized on an Olympus FV1000 confocal microscope (Olympus, Center Valley, PA). No immunoreactivity was observed when the primary antibody or secondary antibody was omitted, providing evidence for antibody specificity.
Other examinations:
1. Terminal dUTP nick-end labeling assay: Terminal dUTP nick-end labeling (TUNEL) was performed with In Situ Cell Death Detection Kit, TMR red (Roche, Indianapolis, IN) following manufacturer's instructions.

2. Western blot: Rabbit anti-cleaved caspase-3 (Asp175, 1:1000), rabbit anti-caspase-3 (1:1000; Cell Signaling), goat anti-olfactory marker protein (OMP) (1:2500; Wako Chemicals, Richmond, VA), or mouse anti-actin antibody (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) was used to probe for proteins, and immunoreactive proteins were detected. Films were analyzed by Image J (; Wayne Rasband; NIH, Bethesda, MD). Integrated optimal density per microgram protein was expressed as a percentage of integrated optimal density per microgram protein of vehicle-treated animals. The value of activated caspase-3, caspase-3, and OMP for each animal was then normalized to the value of actin. Each sample was measured on three independent gels.
Positive control:
Not applicable
A one-way ANOVA followed by the Bonferroni post hoc test was performed using Prism 5 (GraphPad Software, San Diego, CA) unless specified otherwise. Significance was considered p < 0.05.

Results and discussion

Results of examinations

Clinical signs:
not specified
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Clinical biochemistry findings:
not specified
Behaviour (functional findings):
not specified
Gross pathological findings:
not specified
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
Reduction in turbinate olfactory epithelium thickness.
Other effects:
not specified
Description (incidence and severity):
Migrated information from 'Further observations for developmental neurotoxicity study'

Details on results (for developmental neurotoxicity):Not applicable. (migrated information)
Details on results:
Reduction in turbinate OE thickness due to sustentacular cell loss and olfactory sensory neuron apoptosis, followed by an increase in cell proliferation.

-Treatment with purinergic receptor antagonists significantly reduced NiSO4-induced cell proliferation and posttreatment with ATP significantly increased cell proliferation.
-Posttreatment with ATP had no effect on sustentacular cell viability but significantly reduced caspase-3-dependent neuronal apoptosis.
-ATP was released following NiSO4-induced apoptosis and has neuroproliferative and neuroprotective functions.

Effect levels

Dose descriptor:
Effect level:
> 0.1 mg/kg bw/day
Basis for effect level:
other: NiSO4 at doses greater than 0.1 mg/kg produced a dose- and time-dependent reduction in the thickness of turbinate olfactory epithelium. The cell loss was mediated by apoptosis, and was followed by an increase in cell proliferation.
Remarks on result:

Any other information on results incl. tables

Not applicable.

Applicant's summary and conclusion

The authors concluded that one-time intranasal instillation of NiSO4 at doses greater than 0.1 mg/kg produced a dose- and time-dependent reduction in the thickness of turbinate olfactory epithelium. The cell loss was mediated by apoptosis, and was followed by an increase in cell proliferation.
Executive summary:

Study rated by an independent reviewer.

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