Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

chronic toxicity: oral
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study

Data source

Reference Type:

Materials and methods

Test guidelineopen allclose all
according to
other: OECD 451 (Carcinogenicity Studies)
according to
other: EPA OPPTS 870.4200 (Carcinogenicity)
GLP compliance:
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): Nickel sulphate (NiSO4) hexahydrate, CAS No. 10101-97-0
- 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
- Purity: 99.9%
- Other details not reported or not applicable

Test animals

Fischer 344
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina.
- Age at study initiation: 6 weeks
- Weight at study initiation: 118 to 147 g for males and 93 to 112 g for females
- Fasting period before study: not reported
- Housing: housed individually in suspended stainless steel cages that were rotated in a regular fashion
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: acclimated to the laboratory conditions prior to in-life initiation
- Other: The results of the pretest health screen (gross necropsy and serological analyses) conducted prior to in-life initiation indicated that the population of animals was suitable for study use. Serological analyses of blood samples fromfive male and five female sentinel animals conducted by
BioReliance Corporation, Rockville, Maryland, during weeks 25, 51, 77 and 103 did not reveal the presence of any viral infections that would
negatively impact the results of this study.

- Temperature (°C): 70 to 76 °F
- Humidity (%): 29 to 73%
- Air changes (per hr): 10 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-h light/12-h dark cycle

IN-LIFE DATES: not reported

Administration / exposure

Route of administration:
oral: gavage
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: a specified amount of the test article and vehicle was mixed weekly. The mixtures were stirred continuously throughout each exposure period. The appearance of each test article preparation was determined and documented as a clear colorless solution for groups 2 and 3 (10 and 30mg/kg/day) and a clear pale blue solution for group 4 (50 mg/kg/day).

- not applicable

- water
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analyses were conducted by KAR Laboratories, Inc. (Kalamazoo, Michigan) prior to study initiation, during week 51, and following study completion
to confirm the stability and purity of the test substance. Reverse osmosis deionized tap water was used for administration to control animals and in
the preparation of the test article mixtures. Analytical concentration verification analyses conducted throughout the study demonstrated that the
exposure solutions were stable and properly prepared. All analyses were within ±10% of the nominal concentration.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
60 males/60 females per dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Exposures for the 90-day range finding study were selected based on previous gavage studies of nickel sulfate hexahydrate in rats. The 90-day range-finding study of nickel sulfate hexahydrate administered by gavage was conducted using exposures of 0, 50, 75, 100, 125, and 150 mg/kg. Findings from this study are reported in the Results section. Based on the data from the 90-day range-finding study, exposure levels of 10, 30 and 50 mg/kg/day were selected for the 2 year oral gavage carcinogenicity study.

- Rationale for animal assignment (if not random): Sixty female and sixty male animals were assigned to each exposure group using a computer randomization program.
Positive control:
None reported


Observations and examinations performed and frequency:
- Time schedule: General health/mortality/moribundity checks were performed twice daily.

- Time schedule: Detailed clinical observations were performed weekly and on the day of scheduled euthanasia (weeks 104–105). Beginning on week 25, detailed clinical observations included a palpable mass examination (including the occurrence, size, location and description of any palpable masses) followed by persistence or disappearance of these masses being documented at the next weekly clinical observation.

- Time schedule for examinations: Individual body weights were recorded prior to randomization (day −3), on day 0 (i.e., the start of exposure), weekly during the first 13 weeks, once every 4 weeks thereafter and during week 103.

- Individual food consumption (grams/animal/day) was recorded on day 0, weekly during the first 13 weeks and once every 4 weeks thereafter, with the final food consumption measurement during week 103.

- Selected hematological parameters were measured in blood samples collected from 10 animals/sex/group during week 54 (via tail vein) and prior to scheduled euthanasia during week 104/105 (via orbital plexus). Hematology and clinical chemistry parameters were measured according to the OECD 451 protocol.
Sacrifice and pathology:
All animals were subjected to a complete gross necropsy examination at the time of death or euthanasia. Tissues collected at necropsy from all
animals were processed for histopathological evaluation. Slides were prepared by Histo Techniques (Powell, Ohio) and Charles River Laboratories-
Pathology Associates (Frederick, Maryland) and were examined microscopically by a Charles River Laboratories board-certified veterinary
Other examinations:
Near the end of the study (week 103), additional biological sampling was performed to provide data on nickel in urine, feces and blood. Immediately
following exposure on 1 day during week 103, five females and five males from each exposure group were placed in urine collection cages equipped with fecal collection screens, and an ice bath for cooling collected urine samples. Blood was collected from the orbital plexus of each animal
approximately 30 min and 24 h post-exposure and sent to WIL Research Laboratories, Inc. (Ashland, Ohio) for analysis of blood nickel
concentration. Urine and fecal samples were collected from each cage approximately 24 h post-exposure and sent to KAR Laboratories, Inc. for
urine and fecal analysis of nickel concentrations. Urine was analyzed also for creatinine and albumin concentrations. Other standard hematology
and clinical chemistry parameters for blood as well as other standard urinalysis parameters for urine were measured by Charles River Laboratories.
In-life data: The data were initially tested for normality using Levene's test for equality of variance followed by the Shapiro–Wilks test for normality.
A p≤0.001 level of significance was required for either test to reject the assumptions. If both assumptions were fulfilled, a singlefactor
ANOVA was applied, with animal grouping as the factor, utilizing a p≤0.05 level of significance. If the parametric ANOVA was significant at p≤0.05,
Dunnett's test was used to identify statistically significant differences between the control group and each nickel sulfate-treated group at the 0.05,
0.01 and 0.001 levels of significance. If either of the parametric assumptions was not satisfied, then the Kruskal–Wallis nonparametric ANOVA
procedure was used to evaluate intergroup differences (p≤0.05). The Dunn's multiple comparison test was applied if this ANOVA was significant,
again utilizing significance levels of p≤0.05, 0.01 and 0.001.

Survival Data: Kaplan–Meier estimates of group survival rates were calculated, by sex, and shown graphically. A log-rank dose response trend test
of survival rates was performed utilizing dose coefficients. In addition, a log-rank test for survival was used to make pairwise comparisons of each
treated group with the control group. Both the trend test and pairwise comparisons were conducted at the 0.05 significance level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Not reported.
mortality observed, treatment-related
Description (incidence):
Group (Dose) Males (deaths/N, % mortality) Females(deaths/N, % mortality)
1 (0 mg/kg/day) 36/60 (60) 14/60 (23)
2 (10 mg/kg/day) 29/60 (48) 20/60 (33)
3 (30 mg/kg/day) 30/60 (50) 26/60 (43)
4 (50 mg/kg/day) 34/60 (57) 27/60 (45)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights decreased in a exposure-dependent manner, with significantly decreased body weights observed in the two highest exposure groups for males and females. Reductions in weight gain relative to controls at study week 103 reached the level of biological significance (i.e., >10% decrease) in the group 3 and 4 males and the group 4 females. This significant weight reduction indicates that the Maximum Tolerated Dose was reached in this study for both males and females.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
A few statistically significant differences in the hematology data were observed in the nickel sulfate-treated males and females. However, none of these differences was suggestive of a hyperplastic (i.e., leukemia) response and none of these changes was considered toxicologically meaningful since they were small and did not follow a consistent exposure-related pattern.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No notable differences were observed between controls and treated animals for the biochemistry parameters measured during the toxicokinetic satellite study.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No notable differences were observed between controls and treated animals for the urinalysis parameters measured during the toxicokinetic satellite study.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):

The pathology report, pathology peer-review and the pathology working group concurred that nickel sulfate hexahydrate did not
cause any carcinogenic effects in this study. Analysis of the tumor data revealed only one statistically significant (p<0.001) increase in tumors
corresponding to keratoacanthoma (tail) in the group 2 males. However, this finding is of questionable toxicologic significance since there was no
exposure–response relationship, the incidence rate in the group 2 males (15%) was only slightly higher than the upper end of Haseman's historical
control incidence for this tumor type (0–14%) and the incidence rate in the remaining control and treated groups (0–7%) was within the range of the
CRL-Ohio historical incidence (0–2%) and the Haseman historical incidence (0–14%). No other tumor finding in this study was statistically significant.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Gross necropsy and histopathology observations: Numerous gross necropsy findings were observed for animals in the control and nickel
sulfate-treated groups but the type and incidence of these findings observed for the treated animals were comparable to those observed in the
control group, and were consistent with findings commonly seen in aging rats in a longterm study. None of the neoplastic or non-neoplastic
microscopic findings was considered to be related to the experimental exposures. The non-neoplastic findings were either considered to
be secondary to toxicity or incidental background occurrences rather than a direct effect of nickel sulfate.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Dose descriptor:
Effect level:
2.2 other: mg Ni/kg bw/day
Basis for effect level:
other: significant decrease in body weight
Dose descriptor:
Effect level:
6.7 other: mg Ni/kg bw/day
Basis for effect level:
other: significant decrease in body weight

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion