Reaction mass of aluminium fluoride and chromium trifluoride and nickel difluoride

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Not specified

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD TG 451 / EPA OPPTS 870.4200 and GLP compliant study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 451 (Carcinogenicity studies)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.4200 (Carcinogenicity)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nickel sulfate hexahydrate

IUPAC Name:

Nickel sulfate hexahydrate

Constituent 2

Reference substance name:

10101-97-0

EC Number:

600-152-3

Cas Number:

10101-97-0

IUPAC Name:

10101-97-0

Details on test material:

- Name of test material (as cited in study report): Nickel sulfate hexahydrate
- Molecular formula (if other than submission substance): NiSO4.6H2O
- Analytical purity: 99.99%
- Provider: Sigma-Aldrich, USA
- Other: Analyses were conducted by KAR Laboratories, Inc. (Kalamazoo, Michigan) prior to study initiation, during week 51 and following study completion to confirm stability and purity of the test substance.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: 118-147 g (males) / 93-112 g (females)
- Fasting period before study: Not specified
- Housing: individually in suspended stainless steel cages rotated in a regular fashion
- Diet: ad libitum
- Water: ad libitum, by an automatic watering system or bottles when clinical signs warranted
- Acclimation period: Yes (duration not specified)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 29-73
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12-12

IN-LIFE DATES: Not specified

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Reverse osmosis deionized tap water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For each dosing group, a specified amount of test article and vehicle was mixed weekly.
The mixtures were stirred continuously throughout each exposure period.
The appearance of each test article solution was determined and documented as a clear colorless solution for 10 and 30 mg/kg groups and a clear pale blue solution for 50 mg/kg group.

VEHICLE
- Concentration in vehicle: Not specified
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): Not applicable (tap water)
- Purity: Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical concentration verification analyses conducted throughout the study demonstrated that exposure solutions were stable and prepared properly. All analyses were within +/-10% of the nominal concentration.

Duration of treatment / exposure:

104 weeks minimum

Frequency of treatment:

Once daily
NB. Starting during week 24 and continuing through the remainder of the study, oral dosing was delayed from early morning until after 11:00 a.m. in order to allow time for gastric emptying and avoid potential pulmonary toxicity secondary to aspiration of nickel sulfate solution due to a full stomach.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 30 or 50 mg/kg bw/day (corresponding to 0, 2.2, 6.7 and 11 mg nickel/kg bw/day)
Basis:
other: nominal doses

No. of animals per sex per dose:

60

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 90-day range-finding study at 0, 50, 75, 100, 125 and 150 mg/kg bw/day
- Rationale for animal assignment: Computer randomization program
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not applicable

Positive control:

Not included

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly and on the day of scheduled euthanasia in week 104-105

BODY WEIGHT: Yes
- Time schedule for examinations: on days -3 and 0, weekly during first 13 weeks, once every 4 weeks thereafter, and in week 103

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in weeks 54 and 104/105
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals/sex/group
- Parameters examined: according to OECD 451 protocol

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in weeks 54 and 104/105
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 animals/sex/group
- Parameters examined: according to OECD 451 protocol

URINALYSIS: Yes
- Time schedule for collection of urine: in weeks 103, approximately 24 hrs post dosing
- Parameters examined: creatinine and albumin concentrations

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Nickel determination in urine, feces and blood: immediately following dosing on one day during week 103, 5 animals/sex/group were placed in urine collection cages equipped with fecal collection systems and an ice bath for cooling collected urine samples. Urine and fecal samples were collected from each cage approximately 24 hrs post-exposure. Blood was collected from the orbital plexus of each animal approximately 30 min and 24 hrs post-dosing.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (at the time of death or euthanasia)
HISTOPATHOLOGY: Yes (by a board-certified veterinary pathologist followed by a peer-review and a review by a US NTP-style Pathology Working Group in order to confirm potential treatment-related effects and resolve any diagnostic discrepancies arising during initial peer-review)

Other examinations:

No additional examinations

Statistics:

- In-life data (body weight, food consumption, hematology): Levene's test for equality of variance followed by Shapiro-Wilks test for normality. Single-factor ANOVA followed by Dunett's test for comparison between treated groups and respective control, or Kruskal-Wallis non-parametric ANOVA followed by Dunn's multiple comparison test if no equality or normality of variances.
- Survival data: Kaplan-Meier estimates of group survival rates followed by log-rank dose response trend test and log-rank test for survival.
- Tumor analysis: Peto's mortality-prevalence method without continuity correction (incidence of tumors) / Peto's mortality-independent method (mortality-independent tumors) + One-sided trend test, pairwise comparisons with control group, exact permutation test (low tumor incidence) and trend tests for each tumor type.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

mortality only (see below)

Mortality:

mortality observed, treatment-related

Description (incidence):

mortality only (see below)

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY (see Table 1 below)
- Before week 24: higher rate of mortality at 10, 30 or 50 mg/kg in both males and females, considered nickel sulfate-related, likely secondary to nickel-induced pulmonary toxicity due to aspiration of test solution because of relatively full stomach at the time of dosing (rats are nocturnal feeders) as suggested by dark red areas on lungs at necropsy.
- Weeks 24-48: mortality notably reduced by shifting dosing time (after 11:00 a.m.), with only one death in females given 30 mg/kg.
- Over the entire 105-week dosing period: no apparent treatment-related effects on mortality in treated males, increasing exposure-response trend in mortality in females relative to the controls (p<0.008).

BODY WEIGHT AND WEIGHT GAIN (see Table 2 below)
Dose-related decrease in body weight, reaching biological significance (-10%) at 30 mg/kg (males) or 50 mg/kg (females), and statistical significance (p<0.01) at 30 mg/kg (males and females).

FOOD CONSUMPTION
Statistically significant variations from controls observed sporadically in nickel sulfate-treated groups, with no relationship to the decrease in body weight gains.

HAEMATOLOGY
A few statistically significant differences observed in sulfate-treated groups (e.g. increases in red blood cells, heamtocrit and/or hemoglobin at 30 and 50 mg/kg) which may be associated with dehydration or could be related to nickel effects on gene expression of erythropoietin. None of these changes considered toxicologically meaningful.

GROSS PATHOLOGY
Findings of same type and incidence between all groups including controls, consistent with findings commonly seen in aging rats in a long-term study.

HISTOPATHOLOGY: NON-NEOPLASTIC
Non-neoplastic findings considered to be secondary to toxicity or incidental background occurences rather than a direct effect of nickel sulfate.

HISTOPATHOLOGY: NEOPLASTIC (see Table 3 below)
- The pathology report, pathology peer-review and pathology working group concurred that nickel sulfate hexahydrate did not cause any carcinogenic effects.
- Analysis of tumor data revealed only onse statistically significant (p<0.001) increase in tumors corresponding to keratoacanthoma (tail) in males given 10 mg/kg. As there was no dose-response relationship, as the incidence rate in males given 10 mg/kg (15%) was only slightly higher than the upper end of Haseman's historical control incidence for this tumor type (0-14%), and as the incidence rate in the remaining control and treated groups (0-7%) was within the range of the test facility historical incidence (0-2%) and the Haseman historical incidence (0-14%), this finding was considered incidental.

OTHER FINDINGS
Toxicokinetics:
- Blood levels of nickel at the end of the study indicated that steady state levels of nickel in blood increased with exposure, in excess from the control levels by a factor of approximately 100 for 10 mg/kg group and over 350 for 30 and 50 mg/kg groups.
- 24-hour urinary nickel levels also increased with exposure and correlated with blood levels.
- Nickel levels in feces increased in an exposure-dependent manner in treated males and females. The relatively high fecal levels compared to the blood and urinary nickel levels demonstrated that the majority of nickel was not systematically absorbed but excreted in the feces.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

2.2 mg/kg bw/day (nominal)

Based on:

other: nickel content

Basis for effect level:

other: Decreased body weights

2

Dose descriptor:

LOAEL

Effect level:

6.7 mg/kg bw/day (nominal)

Based on:

other: nickel content

Basis for effect level:

other: Decreased body weights

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Male and female mortality during weeks 0 to 105

Dose (mg/kg/day)	Male deaths/total (% mortality)	Female deaths/total (% mortality)*
0	36/60 (60)	14/60 (23)
10	29/60 (48)	20/60 (33)
30	30/60 (50)	26/60 (43)
50	34/60 (57)	27/60 (45)
* Increasing exposure-response trend in mortality relative to controls (p<0.008)

Table 2: Summary of body weights at week 103

Dose (mg/kg/day)	Male [grams +/- SD] (% of controls)	Female [grams +/- SD] (% of controls)
0	397 +/- 49	285 +/- 23
10	376 +/- 49 (95%)	273 +/- 31 (96%)
30	355 +/- 46* (89%)	263 +/- 27* (92%)
50	348 +/- 42* (88%)	257 +/- 21* (90%)
* p<0.01 vs. controls

Table 3: Tumor summary table

Dose (mg/kg/day)	Males*		Females*
	Total number of malignant tumors	Total number of malignant tumors with metastasis	Total number of malignant tumors	Total number of malignant tumors with metastasis
0	28	23	15	11
10	21	11	14	9
30	20	9	11	6
50	17	11	13	7
* 60 animals per group

Applicant's summary and conclusion

Conclusions:

- Nickel sulfate hexahydrate is not carcinogenic in rats up to the MTD (Maximum Tolerated Dose) through the oral route of exposure
- The NOAEL is set at 10 mg/kg bw/day (equivalent to 2.2 mg nickel/kg bw/day) based on significantly decreased body weight at higher dose levels. The dose of 30 mg/kg bw/day (equivalent to 6.7 mg nickel/kg bw/day) can be considered as a LOAEL.

Executive summary:

Nickel sulfate hexahydrate was administered daily to 6 -week-old Fischer 344 rats (60/sex/group) by once daily oral gavage (10 mL/kg) for 2 years (104 or 105 weeks) at exposure levels of 0 (vehicle: reverse osmosis deionized tap water), 10, 30 or 50 mg/kg bw/day in an OECD TG 451, EPA OPPTS 870.4200 and GLP-compliant study.

The administration of nickel sulfate hexahydrate produced a statistically significant reduction in body weight of male and female rats, compared to controls, in a dose -related manner at 30 and 50 mg/kg/day. An exposure-dependent increase in mortality was observed in female rats. However, the overall study survival rate (males and females) was at least 25 animals per group (compliant with OECD guidelines) in the treated animals.

Daily oral administration of nickel sulfate hexahydrate did not produce a dose-related increase in any common tumor type or an increase in any rare tumors. One tumor type was statistically increased in a nickel sulfate-treated group compared to the study controls (keratoacanthoma in males given 10 mg/kg bw/day), but there was no exposure-response relationship for this common tumor type and this was therefore considered to be incidental.

This study achieved sufficient toxicity to reach the Maximum Tolerated Dose (MTD) while maintaining a sufficiently high survival rate to allow evaluation for carcinogenicity. The present study indicated that nickel sulfate hexahydrate does not have the potential to cause carcinogenicity by the oral route of exposure in the Fischer 344 rat. The dose of 10 mg/kg bw/day (equivalent to 2.2 mg nickel/kg bw/day) was a NOAEL, based on body weight decreases, and the dose of 30 mg/kg bw/day (equivalent to 6.7 mg nickel/kg bw/day) was a LOAEL.