Nickel difluoride

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 1999 to December 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Principles of method if other than guideline:

Similar to OECD 416

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Nickel sulphate

EC Number:

232-104-9

EC Name:

Nickel sulphate

Cas Number:

7786-81-4

Molecular formula:

NiSO4

IUPAC Name:

nickel(2+) sulfate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, NY, USA
- Age at study initiation: 7 weeks
- Weight at study initiation: 205-257 g (males) and 147-213 g (females)
- Fasting period before study: not reported
- Housing: individually
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26  deg. C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 per hour
- Photoperiod (hrs dark / hrs light): 12-h light/dark photoperiod.

IN-LIFE DATES: From: February 1, 1999 To: October 15, 1999

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: not applicable

Vehicle:

other: reverse osmosis deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved in reverse osmosis deionized water.  Control animals received appropriate volumes of water only.  The  
homogeneity and stability of the test substance in dosing solutions were determined.  The concentration of the test substance in dosing solutions  
was analytically confirmed at different time intervals during the study period. Groups of 56 test animals of each parental group (28 males/28  
females) received the test substance daily by gavage at the following dosage concentrations:  0, 0.1, 0.25, 0.50, and 1.00 mg/ml (equivalent to  
dose levels of 0, 1.0, 2.5, 5.0, and 10.0 mg/kg/day).  Dose volume was 10 ml/kg, adjusted for body weight.  F0 parents and F1 pups selected as the  
parental group for the F2 generation received the test substance daily by gavage.  F0 parental animals received the test substance (or vehicle for  
control group) daily for 10 weeks, starting 70 days prior to mating.  F1 offspring selected to produce the F2 generation received the test  
substance starting on postpartum day 22 and dosing continued until one day prior to sacrifice.

DIET PREPARATION
- not applicable

VEHICLE
- deionized water

Details on mating procedure:

- M/F ratio per cage: Animals were caged as mating pairs (1:1) until copulation was confirmed, then transferred to individual cages.
- Proof of pregnancy:The presence of a vaginal plug or sperm was designated as day 0 of gestation.  
- Estrous cycle determinations (length and normality) were made daily prior to mating and during cohabitation. 
- Dams and pups were caged together during lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyzed by AAS

Duration of treatment / exposure:

Exposure period: F0: before and during mating, pregnancy, and through weaning of F1 offspring. F1: after weaning,
during growth, mating, production and weaning of F2 offspring
Premating exposure period (males): 70 days
Premating exposure period (females): 70 days
Duration of test: 2 generations

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until 70 days after selected from the F1 litters.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

28 males/28 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:   Based on the results of 1 generation study, the doses of 0, 1, 2.5, 5.0, and 10 mg/kg-day were selected
- Rationale for animal assignment (if not random): random

Positive control:

none reported

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- During gestation and lactation, F0 and F1 females were examined daily for clinical signs of toxicity.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

Oestrous cyclicity (parental animals):

Daily vaginal smears were collected for a minimum of 3 weeks prior to mating

Sperm parameters (parental animals):

Parameters examined in F0 and F1 parental males [sperm count, concentration, motility, and morphology)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes]
- If yes, maximum of [8] pups/litter ([4]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were recorded for each pup during lactation: viability, external examinations, sex determinations, and body weights.  F1 and F2 litters were randomly adjusted to 4 males and 4 males on lactation day 4. F1 pups used as the parental animals for the F2 generation were selected between postpartum days 4 and 21. A total of 28 male and 28 female F1 pups were selected.  The remaining F1 pups and F2 pups were sacrificed and gross necropsy examinations were performed.  Surviving F0 and F1 parental animals were sacrificed and an assessment was made of reproductive performance.  

Postmortem examinations (parental animals):

HISTOPATHOLOGY / ORGAN WEIGHTS:
The following organs from surviving F0 and F1 parental animals were preserved for histopathological examination:  adrenal glands, brain, gross 
lesions, kidneys, liver, ovaries, pituitary, prostate, right epididymis, testes, seminal vesicles, spleen, uterus, and  
vagina.  The following organs from surviving F0 and F1 parental animals were weighed and recorded:  adrenal glands, brain, epididymides, kidneys,  
testes, ovaries, pituitary, prostate, seminal vesicles, spleen, and uterus. 

Postmortem examinations (offspring):

HISTOPATHOLOGY / ORGAN WEIGHTS:
The following organs from surviving F1 parental animals were preserved for histopathological examination:  adrenal glands, brain, gross 
lesions, kidneys, liver, ovaries, pituitary, prostate, right epididymis, testes, seminal vesicles, spleen, uterus, and  
vagina.  The following organs from surviving F0 and F1 parental animals were weighed and recorded:  adrenal glands, brain, epididymides, kidneys,  
testes, ovaries, pituitary, prostate, seminal vesicles, spleen, and uterus.

Statistics:

One-way analysis of variance (ANOVA) was used to analyze parental and pup body weight, body weight gain, food consumption, organ weights, 
length of gestation and estrous cycle, and litter size.  If significance was detected, Dunnett's test was performed to compare control and treatment  
groups.  Copulation and fertility indices, pup sex ratios, numbers of live and dead pups, and pup survival were evaluated by Chi-Square test.   
Post-implantation loss was evaluated using the Mann-Whitney U test.  The level of significance was 5% (p<0.05).

Reproductive indices:

Copulation and fertility indices, pup sex ratios, numbers of live and dead pups, and pup survival

Offspring viability indices:

Copulation and fertility indices, pup sex ratios, numbers of live and dead pups, and pup survival

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
F0 generation: No test substance related mortality of clinical signs of toxicity.  

ALL PARAMETERS:
There were no toxicologically meaningful differences in body weight gain or food consumption, copulation and fertility indices, gestation length, implantation and post-implantation loss, or sperm parameters.  The numbers of live pups of treated female on lactation day 0 were not significantly different from that control group.  Post-implantation loss  was higher at 10 mg/kg/day (2.1), but was not statistically different from the control group (0.9). No treatment related changes found at gross necropsy.  Statistically significant differences in organ weights included decreased absolute and relative livers weights in males at 10 mg/kg/day, decreased absolute brain weight in females at 2.5 mg/kg/day, and increased relative liver weight in females at 1.0, 2.5, and 10.0 mg/kg/day.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The reproductive NOAEL (2.2 mg Ni/kg BW/day) was based on the absence of any effects on reproduction at the highest expsoure used in the study.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

F1 generation: There were no toxicologically meaningful differences in pup viability data or pup body weights during lactation. Mean litter size on 
lactation day 0 ranged from 13.6 pups/litter (1.0 mg/kg/day group) to 11.4 pups per litter (10 mg/kg/day group).  Post-implantation loss was 
slightly higher at 10 mg/kg/day (1.2), but was not statistically different from the control group (0.9). Clinical signs were noted during lactation, 
but were not considered to be test-substance related.  Clinical signs were of low incidence and sporadically distributed among treatment groups. 
Vaginal opening and completion of preputial separation differences were not considered to be treatment related. Vaginal opening of control and 10 mg/kg/day pups occurred by postpartum day 35.  Preputial separation of control and male pups in the 10 mg/kg/day group was completed by 
postpartum day 46.  Gross necropsy observations included atelectasis and absence of milk in the stomach.  Other necropsy findings were of low 
incidence and sporadically  distributed among treatment groups.

Of F1 animals selected to produce the F2 generation, no test substance related mortality of clinical signs of toxicity were noted.  Clinical  
signs were of low incidence and sporadically distributed among treatment  groups and were, therefore, not considered test substance related.  
There were no toxicologically meaningful differences in body weight or body weight gains during the growth phase, however, mean body weight 
gain was significantly lower in the 1.0 and 5.0 mg/kg/day treatment groups during lactation days 14-21.  There were no toxicologically meaningful  
differences in food consumption.  No statistically significant differences were found in copulation or fertility indices, estrous cycle  
determinations, precoital intervals, or gestation lengths.  There were no  statistically significant differences in mean implantation scar counts,  
mean number of live pups on lactation day 0, or mean post-implantation loss. Statistically significant differences in organ weights included  
decreased absolute pituitary weight in 1.0 mg/kg/day males, increased relative adrenal weight and decreased relative liver weight in 5.0 and  
10.0 mg/kg/day males, and decreased relative liver weight in 2.5 and 10.0 mg/kg/day females.  There were no toxicologically meaningful 
differences in sperm parameters of 10 mg/kg/day males.  No test substance related microscopic histopathological changes were noted.

F2 generation: No test substance related clinical signs of toxicity were noted.  There were no statistically significant differences in body weights 
during  lactation. Gross necropsy observations included atelectasis and absence of milk in the stomach.  Other necropsy findings were of low 
incidence  and sporadically distributed among treatment groups.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

>= 10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no toxicologically meaningful differences in pup viability data or pup body weights during lactation

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

The study was conducted to evaluate the potential effects of nickel sulfate hexahydrate administered to Sprague-Dawley rats in a  

two-generation study.  Oral (gavage) administration of nickel sulfate hexahydrate at dose levels up to 10 mg/kg/day (2.2 mg Ni/kg) had no  

effect on F0 or F1 survival, growth, mating behavior, fertility, gestation, parturition, or lactation.  There was no test substance  

related mortality or clinical signs of toxicity in F0 and F1 rats or their offspring.  Pup viability and growth was not affected.  There were  

no toxicologically meaningful differences in estrous cycling, sperm parameters, copulation, and fertility indices, precoital intervals,  

gestation lengths, gross necropsy findings, or the onset of sexual maturation in F1 rats.  Histopathological examinations did not reveal any  

test article-related changes in the liver, reproductive organs, or other tissues examined.  Statistically significant reductions in absolute  

and/or relative liver weights in F0 males at 10 mg/kg/day and in F1 males at 5.0 and 10.0 mg/kg/day were not regarded as toxicologically  significant.  

Relative liver weight values were less than 10% different  from the respective control values.  

Applicant's summary and conclusion

Conclusions:

Based on these results, 10.0 mg/kg/day is considered a No-Observed-Adverse-Effect Level (NOAEL) for oral administration of nickel sulfate hexahydrate over two generations in rats.

Executive summary:

ROBUST SUMMARY DEVELOPED BY AN INDEPENDENT REVIEWER.

Robust Summary for Siglin (2000)

Male and female Sprague-Dawley rats were obtained from Charles River Laboratories, NY, USA, and acclimated to laboratory conditions. Food and  

water were provided ad libitum during the study period.  Environmental conditions during the study were maintained at a temperature of 18-26  

deg. C, a relative humidity of 30-70%, and a 12-h light/dark photoperiod.

Animals were caged as mating pairs (1:1) until copulation was confirmed, then transferred to individual cages.  Estrous cycle determinations  

(length and normality) were made daily prior to mating and during cohabitation.  The presence of a vaginal plug or sperm was designated as  day 0 of gestation.  

Dams and pups were caged together during lactation.

The test substance was dissolved in reverse osmosis deionized water.  Control animals received appropriate volumes of water only.  The  

homogeneity and stability of the test substance in dosing solutions were determined.  The concentration of the test substance in dosing solutions  

was analytically confirmed at different time intervals during the study period. Groups of 56 test animals of each parental group (28 males/28  females) 

received the test substance daily by gavage at the following dosage concentrations:  0, 0.1, 0.25, 0.50, and 1.00 mg/ml (equivalent to  

dose levels of 0, 1.0, 2.5, 5.0, and 10.0 mg/kg/day).  Dose volume was 10 ml/kg, adjusted for body weight.  F0 parents and F1 pups selected as the  

parental group for the F2 generation received the test substance daily by gavage.  F0 parental animals received the test substance (or vehicle for  

control group) daily for 10 weeks, starting 70 days prior to mating.  F1 offspring selected to produce the F2 generation received the test  

substance starting on postpartum day 22 and dosing continued until one  day prior to sacrifice.

General health checks of F0 and F1 parental animals were made twice daily, and more detailed clinical observations were made weekly. During  

gestation and lactation, F0 and F1 females were examined daily for clinical signs of toxicity.

Individual body weights were measured weekly in F0 and F1 parental animals. Mated females and females that delivered were weighed on days 0,  

7, 14, and 20 during gestation, and on days 1, 4, 7, 14, and 21 during lactation.  Food consumption was recorded weekly, except during  

cohabitation and lactation.   The following parameters were recorded for each pup during lactation: viability, external examinations, sex  

determinations, and body weights.  F1 and F2 litters were randomly adjusted to 4 males and 4 males on lactation day 4.

F1 pups used as the parental animals for the F2 generation were selected between postpartum days 4 and 21. A total of 28 male and 28 female F1  

pups were selected.  The remaining F1 pups and F2 pups were sacrificed and gross necropsy examinations were performed.  Surviving F0 and F1  

parental animals were sacrificed and an assessment was made of reproductive performance.  The following organs from surviving F0 and F1  

parental animals were preserved for histopathological examination:  adrenal glands, brain, gross lesions, kidneys, liver, ovaries, pituitary,  

prostate, right epididymis, testes, seminal vesicles, spleen, uterus, and  vagina.  The following organs from surviving F0 and F1 parental animals  

were weighed and recorded:  adrenal glands, brain, epididymides, kidneys,  testes, ovaries, pituitary, prostate, seminal vesicles, spleen, and  

uterus. Sperm was collected from F0 and F1 parental males and examined for sperm count, concentration, motility, and morphology.

One-way analysis of variance (ANOVA) was used to analyze parental and pup body weight, body weight gain, food consumption, organ weights, length of  

gestation and estrous cycle, and litter size.  If significance was detected, Dunnett's test was performed to compare control and treatment  

groups.  Copulation and fertility indices, pup sex ratios, numbers of live and dead pups, and pup survival were evaluated by Chi-Square test.   

Post-implantation loss was evaluated using the Mann-Whitney U test.  The level of significance was 5% (p0.05).

Homogeneity and stability of the test substance in gavage dosing solutions was found to be stable for 24 hours at room temperature and up  

to 21 days under refrigeration.

F0 generation:
No test substance related mortality of clinical signs of toxicity.  There were no toxicologically meaningful differences in body weight gain or  

food consumption, copulation and fertility indices, gestation length, implantation and post-implantation loss, or sperm parameters.  The  

numbers of live pups of treated female on lactation day 0 were not significantly different from that control group.  Post-implantation loss  

was higher at 10 mg/kg/day (2.1), but was not statistically different from the control group (0.9).

No treatment related changes found at gross necropsy.  Statistically significant differences in organ weights included decreased absolute and  

relative livers weights in males at 10 mg/kg/day, decreased absolute brain weight in females at 2.5 mg/kg/day, and increased relative liver  

weight in females at 1.0, 2.5, and 10.0 mg/kg/day.

F1 generation:
There were no toxicologically meaningful differences in pup viability data or pup body weights during lactation. Mean litter size on lactation  

day 0 ranged from 13.6 pups/litter (1.0 mg/kg/day group) to 11.4 pups per litter (10 mg/kg/day group).  Post-implantation loss was slightly higher  

at 10 mg/kg/day (1.2), but was not statistically different from the control group (0.9).

Clinical signs were noted during lactation, but were not considered to be test-substance related.  Clinical signs were of low incidence and  

sporadically distributed among treatment groups. Vaginal opening and completion of preputial separation differences were not considered to be  

treatment related. Vaginal opening of control and 10 mg/kg/day pups occurred by postpartum day 35.  Preputial separation of control and male  

pups in the 10 mg/kg/day group was completed by postpartum day 46.  Gross necropsy observations included atelectasis and absence of milk in the stomach.  

Other necropsy findings were of low incidence and sporadically  distributed among treatment groups.

Of F1 animals selected to produce the F2 generation, no test substance related mortality of clinical signs of toxicity were noted.  Clinical  

signs were of low incidence and sporadically distributed among treatment groups and were, therefore, not considered test substance related.  There  

were no toxicologically meaningful differences in body weight or body weight gains during the growth phase, however, mean body weight gain was  

significantly lower in the 1.0 and 5.0 mg/kg/day treatment groups during lactation days 14-21.  There were no toxicologically meaningful  

differences in food consumption.  No statistically significant differences were found in copulation or fertility indices, estrous cycle  

determinations, precoital intervals, or gestation lengths.  There were no statistically significant differences in mean implantation scar counts,  

mean number of live pups on lactation day 0, or mean post-implantation loss. Statistically significant differences in organ weights included  

decreased absolute pituitary weight in 1.0 mg/kg/day males, increased relative adrenal weight and decreased relative liver weight in 5.0 and  

10.0 mg/kg/day males, and decreased relative liver weight in 2.5 and 10.0 mg/kg/day females.  There were no toxicologically meaningful differences  

in sperm parameters of 10 mg/kg/day males.  No test substance related microscopic histopathological changes were noted.

F2 generation:
No test substance related clinical signs of toxicity were noted.  There were no statistically significant differences in body weights during  

lactation. Gross necropsy observations included atelectasis and absence of milk in the stomach.  Other necropsy findings were of low incidence  

and sporadically distributed among treatment groups.

The study was conducted to evaluate the potential effects of nickel sulfate hexahydrate administered to Sprague-Dawley rats in a  

two-generation study.  Oral (gavage) administration of nickel sulfate hexahydrate at dose levels up to 10 mg/kg/day (2.2 mg Ni/kg) had no  

effect on F0 or F1 survival, growth, mating behavior, fertility, gestation, parturition, or lactation.  There was no test substance  

related mortality or clinical signs of toxicity in F0 and F1 rats or their offspring.  Pup viability and growth was not affected.  There were  

no toxicologically meaningful differences in estrous cycling, sperm parameters, copulation, and fertility indices, precoital intervals,  

gestation lengths, gross necropsy findings, or the onset of sexual maturation in F1 rats.  Histopathological examinations did not reveal any  

test article-related changes in the liver, reproductive organs, or other tissues examined.  Statistically significant reductions in absolute  

and/or relative liver weights in F0 males at 10 mg/kg/day and in F1 males at 5.0 and 10.0 mg/kg/day were not regarded as toxicologically  significant.  

Relative liver weight values were less than 10% different from the respective control values.