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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
repeated dose toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study (OECD 451)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2008

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 451 (Carcinogenicity Studies)
Principles of method if other than guideline:
This study was conducted in general compliance with the OECD and EPA guidelines for carcinogenicity studies (OECD 451 adopted in
1981) and conformed with the Good Laboratory Practice (GLP) Standards promulgated by the EPA and OECD.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Nickel 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, powder
- Analytical purity: 99.9% (NiPERA)
- Storage: stored at room temperature and stable under this condition
- Other details not reported or not applicable

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles RiverLaboratories, Inc., Raleigh, NC, USA.
- Age at study initiation: 6-weeks
- Weight at study initiation: 129-192 g (males) and 107-155 g (females)
- Fasting period before study: none, no food or water during 6-hour exposured
- Housing: During exposures, the animals were housed individually in stainless steel wire-mesh cage batteries
- Diet (e.g. ad libitum): ad libitum (PMI Nutrition International Certified Rodent Chow® #5002)
- Water (e.g. ad libitum): ad libitum (municipal water)
- Acclimation period: animals were acclimated to the laboratory conditions prior to initiation of exposure, they were observed twice daily and weighed twice.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 19 °C and 25 °C,
- Humidity (%): 30% and 70%,
- Air changes (per hr): There were at least 12–15 chamber air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark photoperiod

IN-LIFE DATES: not reported

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: MMAD = 1.8 micrometers
GSD = 2.4 micrometers
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animal exposures were conducted in four 2.0-m3 stainless steel and glass whole-body exposure chambers.
- Method of holding animals in test chamber: none reported, house individually
- Source and rate of air: not reported
- Method of conditioning air: not reported
- System of generating particulates/aerosols: Nickel metal atmospheres were generated as dust aerosols of elemental nickel using Fluidized Bed Aerosol Generators (Model no. 3400A, TSI, Inc., St. Paul, MN).
- Temperature, humidity, pressure in air chamber: monitored continuously, temperature and humidity daily averages were between 20 °C and 26 °C, and 30% and 70%,
- Air flow rate: not reported
- Air change rate: 12–15 chamber air changes/hour.
- Method of particle size determination: Aerosol particle size determinations for the nickel metal aerosols were conducted weekly during study weeks 0–18 and monthly thereafter using cascade impactors.
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Real-time aerosol monitoring was performed using light scattering aerosol monitors as a guide to adjust concentration during animal exposure. Actual individual exposure concentrations of nickel metal were determined by standard gravimetric methods. For each chamber, at least one set of filters per week were analyzed for nickel by atomic absorption spectroscopy in the Analytical Chemistry Department, WIL Research Laboratories, LLC.
- Samples taken from breathing zone: yes

VEHICLE (if applicable): none reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Actual individual exposure concentrations of nickel metal were determined by standard gravimetric methods. For each chamber, at least one set of filters per week were analyzed for nickel by atomic absorption spectroscopy in the Analytical Chemistry Department, WIL Research Laboratories, LLC.
Duration of treatment / exposure:
2-year exposure period (24 months)
Frequency of treatment:
6 hr/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.1, 0.4, and 1.0 mg Ni/m3
Basis:
nominal conc.
No. of animals per sex per dose:
Main: 50 males/females per dose
Satellite A: groups consisted of 22 animals/sex/group.
Satellite B: 6 animals/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The exposure levels for the carcinogenicity study were selected based on the results from the 13-week range-finding study. The 13- week inhalation range-finding study with nickel metal powder was conducted using exposure levels of 0, 1, 4, 8 mg/m3 (Kirkpatrick 2004)

[Kirkpatrick, D.T., 2004. A 13-week inhalation toxicity study (with recovery) of nickel metal in albino rats (Study No. WIL-437002). WIL Research Laboratories, Inc., Ashland, Ohio, 2004.]

- Rationale for animal assignment (if not random): animals were assigned to each exposure group using a computer randomization program.
- Rationale for selecting satellite groups: Satellite A animals had exposures identical to the Core animals but were scheduled for interim
euthanasia to measure nickel lung burdens, blood nickel levels and/ or to isolate bronchiolar-alveolar lung fluid (BALF) for analysis. Satellite B animals for the purpose of investigating lung clearance of nickel metal particles.
- Post-exposure recovery period in satellite groups: none reported
- Section schedule rationale (if not random): random
Positive control:
- Dose selection rationale: The exposure levels for the carcinogenicity study were selected based on the results from the 13-week range-finding study. The 13- week inhalation range-finding study with nickel metal powder was conducted using exposure levels of 0, 1, 4, 8 mg/m3 (Kirkpatrick 2004)

[Kirkpatrick, D.T., 2004. A 13-week inhalation toxicity study (with recovery) of nickel metal in albino rats (Study No. WIL-437002). WIL Research Laboratories, Inc., Ashland, Ohio, 2004.]

- Rationale for animal assignment (if not random): animals were assigned to each exposure group using a computer randomization program.
- Rationale for selecting satellite groups: Satellite A animals had exposures identical to the Core animals but were scheduled for interim
euthanasia to measure nickel lung burdens, blood nickel levels and/ or to isolate bronchiolar-alveolar lung fluid (BALF) for analysis. Satellite B animals for the purpose of investigating lung clearance of nickel metal particles.
- Post-exposure recovery period in satellite groups: none reported
- Section schedule rationale (if not random): random

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly or bi-weekly

FOOD CONSUMPTION: yes
- Time schedule for examinations: weekly or bi-weekly

FOOD EFFICIENCY: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice, and at weeks 52, 78, and 131
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 40-48 per sex per dose

CLINICAL CHEMISTRY: Yes
- After 6 and 12 months of exposure, BALF samples were collected. After six months of exposure, neutrophil numbers, LDH levels and
protein content were increased in all exposed groups compared to control animals.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Blood nickel levels were measured after 3 and 12 months of exposure.
Lung nickel levels were measured after 3, 12, and 24 months of exposure for control, 0.1 and 0.4 (males only) mg/m3. For 0.4 mg/m3 females and 1.0 mg/m3 males and females data are available only for 3 and 12 months of exposure.
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for a minimum significance level of 5% and/or 1%, comparing each nickel metal-exposed group solely to the control group by sex. The Bonferroni inequality was used to control the falsepositive error rate at the overall 0.05 level when several pair-wise comparisonswere made. Statistical analyseswere not conducted if the number of animals was 2 or less.

Body weight, body weight change, food consumption, white blood cell absolute and differential counts, organ weight and BALF data were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistical significance (pb0.05), Dunnett's test (Dunnett, 1964) was used, except in the case of BALF parameters where the Tukey test (Zar, 1999) was used.
The generalized Wilcoxon test (Gehan, 1965) for survival was used to compare the homogeneity of survival rates across the groups. If the rates were significantly different (p<0.05), the generalized Wilcoxon test (Gehan, 1965) was used to make pair-wise comparisons, using Bonferroni adjusted significance levels. A log-rank dose-response trend test of survival rates was also performed.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY:
Survival at end of exposure (103 weeks) by dose and sex:
0 mg Ni/m3: 82% (males), 76% (females)
0.1 mg Ni/m3: 82% (males), 76% (females)
0.4 mg Ni/m3: 72% (males), 48% (females)
Disproportionate mortality in the 1.0 mg/m3 males and females began during study weeks 40 and 53, respectively and was considered related to nickel exposure, resulting in early termination of this group.

Increased respiratory rate and “blue” tinged (cyanosis) extremities, body, and/or facial area were the most common and persistent clinical
findings noted in the nickel-exposed animals in the 0.4 and 1.0 mg/m3 groups. Other exposure-related clinical observations included cool/pale extremities and/or body, dermal atonia, thinness, decreased defecation, and labored respiration (data not shown). With the exception of cyanosis of the extremities and/or facial area, these findings were also noted in the 0.1 mg/m3 group with incidences thatwere similar, or slightly higher, than in the control group.

BODY WEIGHT AND WEIGHT GAIN
Significant nickel metal exposure-related effects on body weights were noted in the 0.1 (males only), 0.4 and 1.0 mg/m3 groups. At the end of the scheduled exposure period (week 103), mean body weights in the 0.4 mg/m3 group males and females were 27% and 18% lower than controls, respectively. In the 0.1 mg/m3 exposure group, significant reduction in body weight (11%) was noted only for the males.

FOOD CONSUMPTION
Exposure-related effects on food consumption were noted for the 0.4 and 1.0 mg/m3 males and females.

HAEMATOLOGY
At the 78-week evaluation (18 months), higher (p<0.01) mean red blood cell counts, hemoglobin levels and hematocrit values were noted in the 0.1 and 0.4 mg/m3 group males and the 0.4 mg/m3 group females. Slightly higher total leukocyte counts were seen at one or more intervals.

CLINICAL CHEMISTRY
After six months of exposure, neutrophil numbers, LDH levels and protein content were increased in all exposed groups compared to
control animals, but only reached statistical significance in the 0.4 and 1.0 mg/m3 groups.

GROSS PATHOLOGY
Exposure-related alterations including pale and mottled lungs which did not collapse upon excision in both genders; enlarged and/or swollen spleens in females; and enlarged adrenal glands in males. Increases in absolute and relative weight of lungs (males and females) and adrenal glands
(males) among exposed animals were considered to be nickel exposure-related. All other organ weight changes appeared to be a result of decreases in final body weight.

HISTOPATHOLOGY: NON-NEOPLASTIC
Granular brown pigment in the kidneys, extramedullary hematopoiesis in the spleen, and hypercellularity of the sternum and femoral bone marrows were also considered to be related to nickel exposure. In the adrenal glands, increased angiectasis in the adrenal cortex of the 0.4 mg/m3 group females was observed.

Representative histopathological findings included lung lesions of alveolar proteinosis, alveolar histiocytosis and chronic or chronic-active inflammation were clearly exposure-related, as was bronchiolar-alveolar hyperplasia.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
A significant dose-related increase in pheochromocytomas (benign, malignant and combined) was observed in the adrenal medulla of male rats. The incidence of pheochromocytomas in the 0.4 mg/m3 exposure group males was statistically significant for total benign neoplasms (incidence=38%), malignant neoplasms (incidence=10%) and combined benign/malignant neoplasms (incidence= 42%). In the historical control database (30-month
studies) compiled by Bomhard (1992), the incidence of spontaneous pheochromocytomas ranged from 10–54.3% (males) and 0–10% (females) for benign tumors and 0–8% (males) and 0–2% (females) for malignant tumors. Therefore the incidences of pheochromocytomas in the males control group in the present study (0%) can be considered low.

In females, therewas a significant dose-related increase incombined adenoma/carcinoma of the adrenal cortex. In the 0.4 mg/m3 exposure group, there was an statistically significant increase for the combined incidence of adenomas or carcinomas (13%), but not for adenomas or carcinomas when analyzed separately (7% and 6%, respectively). Bomhard (1992) noted a wide variation in the percentage of control animals with spontaneous adrenal cortical adenomas in 30-month studies (0–38% in males and 0–31% in females). The 7.4% incidence of adrenal cortical adenoma in the females in the present study fallswithin Bomhard's range. Poteracki and Walsh (1998) report a 0–7% spontaneous incidence of cortical adenomas and a 0–1.7% incidence of spontaneous cortical carcinomas for females in 24-month studies.

The carcinogenicity assessment included examination of the complete respiratory tract including the nasal tissues, larynx, trachea and lungs (bronchial and alveolar regions) as well as all other tissues required in OECD 451 protocol. No increased incidence
was observed for any neoplasm at any level of the respiratory tract.

OTHER FINDINGS
Nickel levels in blood. Blood nickel levels were measured after 3 and 12 months of exposure. The shape of the dose-response suggests that steady state levels may have been reached by 12 months of exposure.
Data indicate that steady state levels of nickel in the lung were reached by 12 months of exposure for the 0.1 and 0.4 mg/m3 groups.

Effect levels

Dose descriptor:
LOAEC
Effect level:
0.1 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: The LOAEC for respiratory effects associated with inhalation exposure to nickel metal powder (MMAD=1.8 μm, GSD=2.4 ) was 0.1 mg/m³.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion