Nickel difluoride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Data generated with the related substance NiSO4 is used for endpoint coverage.

No effects on fertility have been found in studies following oral administration; no data are available for inhalation and dermal contact. The most reliable NOAEL is from the two-generation study (SLI 2000a,b) where the NOAEL is the highest dose investigated, i. e. 2.2 mg Ni/kg bw/day (10 mg nickel sulphate hexahydrate/kg bw/day). A repeated dose toxicity study provides a NOAEC for effects on sperm and oestrus cyclicity of 0.45 mg Ni/m³ (2.0 mg nickel sulphate hexahydrate/m³) for inhalation exposure (Dunnick et al., 1989).

Value used for CSA (route: oral):NOAEL: 10.0 mg nickel sulphate hexahydrate/kg bw/day

Value used for CSA (route: inhalation):NOAEC: 2.0 mg nickel sulphate hexahydrate/m³ air

Link to relevant study records

Reference

Reference 1

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

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

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

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.

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

2.5 mg/kg bw/day (nominal)

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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.

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.

Key result

Critical effects observed:

no

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

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.

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.  

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.  

Reference 2

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1998 to December 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Principles of method if other than guideline:

Similar to OECD 415

GLP compliance:

yes

Limit test:

no

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: 11 wks
- Weight at study initiation: 269-360 g (males) and 187-247 g (females)
- Fasting period before study: not reported
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 25 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 65-79 deg F
- 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: August 4, 1998 To: October 30, 1998

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 16 test animals of each parental group (8 males/8  
females) received the test substance daily by gavage at the following dosage concentrations:  0, 1.0, 2.0, 3.0, 5.0, and 7.5 mg/ml (equivalent to  
dose levels of 0, 10, 20, 30, 50, and 75 mg/kg/day).  Dose volume was 10 ml/kg, adjusted for body weight.  F0 parental animals received the test substance (or vehicle for control group) daily for 14 days prior to mating and F1 generation on postpartum day 22 and dosing continued until one day 
prior to sacrifice.

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.
- 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: 14 days prior to mating through gestation until postnatal day 21
F1: On lactation day 0 through day 21

Frequency of treatment:

daily

Details on study schedule:

not applicable

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

No. of animals per sex per dose:

8 males/8 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: selected to produce a graded response
- Rationale for animal assignment (if not random): random

Positive control:

none reported

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: more detailed clinical observations were made 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: Yes
- Time schedule for examinations: weekly

Oestrous cyclicity (parental animals):

Estrous cycle determinations (length and normality) were made daily prior to mating and during cohabitation.   

Sperm parameters (parental animals):

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

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 6 and 13 postpartum
- If yes, maximum of [8] pups/litter ([8]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: [mortality, general health twice daily]

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS: not reported

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS: not reported

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:

gestation length, litter size, fertility indices

Offspring viability indices:

litter size, sex rations, number of live/dead pups

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:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

DETAILS ON RESULTS:
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.  
Post-implantation loss was significantly higher at 30, 50 and 75 mg/kg/day). No treatment related changes found at gross necropsy. 

Key result

Dose descriptor:

LOAEL

Effect level:

75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The incidence of dead pups on lactation day 0 was significantly increased and mean live litter size was significantly decreased at the 75 mg/kg/day level.

Key result

Dose descriptor:

LOAEL

Effect level:

>= 30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Mean post-implantation loss was significantly increased at dosage levels >= 30 mg/kg/day.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING):
Litter size was significantly decreased at 10, 20, 30, and 75 mg/kg-day. No significant differences in clinical signs, pup weights, weight gain, or
gross necropsy findings.

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Reproductive effects observed:

not specified

Effect level was not reported.

Conclusions:

In conclusion, based on the results of this one-generation reproduction range-finding study, dosage levels of 1.0, 2.5, 5.0 and 10.0 mg/kg-day were selected for a definitive two-generation reproductions study in rats.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

K1 and K2 studies included in the current version of the nickel sulphate dossier were reviewed and included. K3 and K4 studies from the NiSO4 dossier were not included in the NiF2 dossier if new studies were included in the last update of the NiSO4 dossier.

 

ENDPOINT SUMMARY INFORMATION FROM THE 2008/2009 NICKEL SULPHATE RISK ASSESSMENT; In addition, a background document summarizing available human data on developmental and reproductive toxicity of soluble Ni compounds is attached in Sections 7.8 and 7.10.2 of this IUCLID.

 

Two oral multi-generation reproduction studies and a range-finding one-generation study of nickel sulphate are available (Ambrose et al. 1976, SLI 2000a, SLI 2000b). No effects on fertility have been found in these studies following oral administration; no data are available for inhalation and dermal contact. The study by Ambrose et al. (1976) and the one-generation range-finding study (SLI 2000a) indicate NOAELs of 52-80 mg Ni/kg bw/day and 16.8 mg Ni/kg bw/day, respectively. However, the Ambrose et al. study has a limited reporting of data and the range-finding study uses only a limited number of animals (8 per group).While some effects have been reported in studies in mice with soluble nickel (e.g. Pandey and Srivastava, 2000), the European Union Risk Assessment Report (EU RAR) in 2008 did not consider the data sufficient to classify soluble nickel compounds for fertility effects. Therefore, the most reliable NOAEL is from the recent OECD TG 416 two-generation study (SLI 2000b) where the NOAEL is the highest dose investigated, i. e. 2.2 mg Ni/kg bw/day. This value is taken forward to the risk characterization; however, it should be considered that the NOAEL is probably higher.

In addition, a background document summarizing available human data on developmental and reproductive toxicity of soluble Ni compounds is attached in Sections 7.8 and 7.10.2 of IUCLID.

 

Based on the findings of peri-/postnatal death in the multi-generation studies there is not considered to be urgent need for further testing for developmental toxicity if nickel fluoride is classified in Category 2 for developmental toxicity. The potential for effects of nickel fluoride on fertility have not been sufficiently investigated, since the highest dose level for Nickel Sulfate in the recent OECD TG 416 two-generation study did not induce any signs of toxicity in the adult animals. Therefore, to be able to draw clear conclusions regarding the potential for effects of nickel fluoride on fertility further studies using higher dose levels would be relevant. However, there is no reason to expect that such testing would lead to lower NOAELs than the ones already determined for fertility and developmental effects.

 

Ni fluoride is classified as Cat 2 and Repr. 1B; H360D in the 1st ATP to the CLP Regulation.

Effects on developmental toxicity

Description of key information

No standard prenatal developmental toxicity studies with Ni-sulphate via either the oral or inhalation routes were located.The existing studies on soluble nickel compounds provide consistent evidence of developmental toxicity (stillbirth, post-implantation/perinatal death) in rats at dose levels not causing maternal toxicity.No standard PNDT studies with Ni acetate via either the oral or inhalation routes were located. Regarding teratogenicity effects,nickel chloride was shown to cause malformations in a PNDT study in mice at higher doses (Saini et al., 2013).Human data from reproductive epidemiological studies of female refinery workers with high exposures to water soluble nickel compounds (and insoluble ones) have not demonstrated adverse developmental effects such as: 1) male newborns with genital malformations, 2) spontaneous abortions, 3) small-for-gestational-age newborns, or 4) musculosketal effects in newborns of female refinery workers exposed to nickel. The data in these manuscripts showed no correlation between nickel exposures (urinary levels as high as 30-fold over background) and observed reproductive impairment.Based on the increased post-implantation/perinatal lethality in F1 generation in an OECD TG 416 two-generation study (SLI 2000a,b) at 2.2 mg Ni /kg bw/day, the NOAEL used for developmental toxicity for regulatory purposes is set at 1.1 mg Ni/kg bw/day.

 

Value used for CSA (route: oral):NOAEL: 5.0 mg nickel sulphate hexahydrate/kg bw/day

 

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

5 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

K1 and K2 studies included in the current version of the nickel sulphate dossier were reviewed and included. K3 and K4 studies from the NiSO4 dossier were not included in the NiF2 dossier if new studies were included in the last update of the NiSO4 dossier.

 

ENDPOINT SUMMARY INFORMATION FROM THE 2008/2009 NICKEL SULPHATE RISK ASSESSMENT; In addition, a background document summarizing available human data on developmental and reproductive toxicity of soluble Ni compounds is attached in Section 7.8 of IUCLID

 

Two oral multi-generation reproduction studies and a range-finding one-generation study of nickel sulphate are available (Ambrose et al. 1976, SLI 2000a, SLI 2000b).No PNDT studies of nickel sulphate were identified. A comprehensive read-across program based on water solubility and bioaccessibility data in synthetic fluids validated by in vivo toxicokinetics and acute oral toxicity data has been conducted on a series of Ni compounds including Ni sulphate. The results of this program suggest that the developmental toxicity effects of Ni sulphate should be read-across from Ni chloride for oral and/or inhalation systemic exposure. A background document summarizing available human data on developmental and reproductive toxicity of soluble Ni compounds is attached in Sections 7.8.1 and & 7.8.2 of IUCLID.

 

Data requirements for nickel sulphate are fulfilled by the results of a two-generation study with nickel sulphate,a rat prenatal developmental toxicity (PNDT) study with nickel chloride (RTI, 1988b), and a mouse (PNDT) study with nickel chloride (Saini et al., 2013). In the rat study, no evidence of teratogenic effects was reported in a multi-generational study where the F2b generation was subjected to a classical teratology assessment (RTI, 1988a,b). The F2b generation was delivered by Caesarean section on gestation day 20 and were examined for external, internal, and skeletal malformations mimicking the protocol of a stand-alone PNDT study, but with extended exposure of the parental animals. This F2b cohort showed no exposure-related adverse effects. The lack of adverse effects in the F2b generation, examined on gestation day 20, indicated that the developmental effects in the other generational cohorts (i.e., perinatal mortality) were expressed during the perinatal or postnatal periods and not during gestation (RTI, 1988a,b).

 

Saini et al. (2013) studied the effects of oral (gavage) exposure during gestation (GD6-13) of Swiss albino mice to Ni chloride hexahydrate at doses of 0, 46, 92 and 185 mg Ni/kg b.w. per day. Maternal toxicity (decrease feed consumption, water intake and b.w.) was observed at doses ≥ 92 mg Ni/kg b.w. per day and fetotoxicity (decreases in b.w.), embryotoxicity (decrease in the number of live fetuses/dam, increases in post-implantations losses and resorptions at high dose), and teratogenicity (malformations such as open eyelids, club foot, umbilical hernia, ophthalmic anomalies, hydrocephaly, reduced ossification, dose-dependent increase in skeletal anomalies) were observed at doses ≥ 92 mg/kg b.w. per day (microphthalmia already at 46 mg/kg b.w. per day). The NOAEL for maternal toxicity was 46 mg Ni/kg b.w. per day and the LOAEL for developmental toxicity was 46 mg Ni/kg b.w. per day.

 

The multi-generation studies and the one-generation range-finding study provide consistent evidence of developmental toxicity (stillbirth, post-implantation/perinatal death) in rats at dose levels not causing maternal toxicity. Based on the increased post-implantation/perinatal lethality in F1 generation in an OECD TG 416 two-generation study (SLI 2000a,b) at 2.2 mg Ni /kg bw/day, the NOAEL used for developmental toxicity for regulatory purposes is set at 1.1 mg Ni/kg bw/day. This value is taken forward to the risk characterisation.

 

Ni fluoride is classified as Cat 2 and Repr. 1B; H360D in the 1st ATP to the CLP Regulation.

Toxicity to reproduction: other studies

Description of key information

The full document summarizing these data in humans is attached in sections 7.8 and 7.10.2 of IUCLID and inAppendixB4to the CSR.In summary, there was a lack of a correlation between nickel exposure and observed (sex organ and skeletal) malformations in the human reproductive studies of nickel-exposed workers reviewed above (Vaktskjold et al., 2006, 2007; 2008a,b). Specific endpoints, their assessment methods, and the number of subjects included: genital malformations in newborns of female nickel-refinery workers were examined with a register-based, nested case-control study (n= 103 cases; 23,038 controls); small-for-gestational-age newborns of female refinery workers exposed to nickel were examined with a register-based, nested case-control study (n= 2,096 cases; 20,740 controls); spontaneous abortions among nickel-exposed female refinery workers were examined with a case-control study (n=184 cases, 1,691 controls); and maternal nickel exposure and congenital musculoskeletal defects were examined with register-based, nested case-control study (n=341 cases, 22,624 controls). In all studies, nickel exposure was not associated with adverse pregnancy outcome for any of the endpoints examined. Such large studies provide adequate statistical power to support a conclusion to exclude the risk of reproductive effects (e.g. malformations) from exposure to the chemical (EMA/CHMP Guideline, 2006). 

Additional information

K1 and K2 studies included in the current version of the nickel sulphate dossier were reviewed and included. K3 and K4 studies from the NiSO4 dossier were not included in the NiF2 dossier if new studies were included in the last update of the NiSO4 dossier.

 

A reproductive study of female refinery workers has not demonstrated an association between relatively high soluble nickel exposures (worst case scenario with higher blood and urinary levels) and the following reproductive outcomes: genital malformations (hypospadias and cryptorchidism), spontaneous abortions, small-for-gestational-age newborns, and skeletal malformations (Vaktskjoldet al., 2006, 2007, 2008a&b). Researchers created a birth registry for all births occurring in the region of a Russian nickel refinery at Monchegorsk during the period of the study, which included information on 22,836 newborns and 2,793 pregnancy outcomes surveyed for spontaneous abortions (Vaktskjoldet al.2007; 2008a). They also reconstructed the exposures (using air and urinary nickel measurements) for the female workers at the refineries so as to be able to link specific pregnancy outcomes with occupational exposures via inhalation and systemically bioavailable doses of nickel (i.e.urinary nickel levels). The study culminated in a series of manuscripts by Vaktskjoldet al.(2006, 2007, 2008a,b) describing the results of the investigation. The study demonstrated nickel compound and metal exposure was not associated with adverse pregnancy outcome for 1) male newborns with genital malformations, 2) spontaneous abortions, 3) small-for-gestational-age newborns, or 4) musculoskeletal effects in newborns of female refinery workers exposed to nickel. The data in these manuscripts showed no correlation between nickel exposures (urinary levels as high as 30-fold over background^[1]) and observed reproductive impairment. It is important to note thatgenital malformations are considered as one of the most sensitive endpoints for human developmental toxicity while spontaneous abortion in humans would most closely approximate the observation of perinatal lethality associated with nickel exposure in rodents. Further evidence that nickel compound and metal exposure was not adversely affecting the reproduction of these women was provided by the lack of a “small-for-gestational-age” finding and also the lack of an association of male genital malformations with nickel exposure. Both of these findings are considered “sentinel” effects (i.e., sensitive endpoints) for reproductive toxicity in humans.

 

The work by Vaktskjoldet al.(2006, 2007, 2008a,b) is important in demonstrating that no hazard for reproductive impairment from nickel compound and metal exposure exists under the conditions of the study, which were extremely high exposure levels which are no longer present in current refinery operations.^[2]Thus, the reproductive effects observed in rats 1) may not be relevant to humans or 2) may not have been observed in the exposed human population because even the highest achievable female workers’ exposure (179 µg Ni/l in urine) is lower than those achieved in rats at the LOAEL for reproductive effects (2300 µg Ni/L in urine). In either case, for classification and risk assessment purposes, the relevance of the positive results in rats with soluble nickel compounds (at what seem to be unachievable human exposures) needs to be considered together with the negative results in human studies at Monchegorsk (for the highest exposed human population) in a weight of evidence approach. At the very least, the data indicates that humans do not appear to be greatly more sensitive to reproductive effects of nickel ion than rats. While the exact mode of action for the perinatal mortality effects of Ni ion are not known, the existing data demonstrates that these are clearly threshold-mediated effects. Because the study correlated the systemically available nickel levels to reproductive outcomes, the lack of reproductive toxicity effects is relevant to nickel compounds and nickel metal. Importantly, the results from human studies showing no reproductive toxicity effects due to nickel compound and metal exposure reported by Vaktskjoldet al.were not available at the time when nickel compounds were classified as Cat. 1B reproductive toxicants.

 

The mode of action for the reproductive toxicity of soluble nickel ion observed in rodents is not currently known; thus its relevancy for humans is also unknown. What is known is that epidemiological studies of female workers exposed to the highest attainable levels of water soluble nickel compounds (via inhalation) and who had urinary nickel levels up to 30-fold above background failed to show an association between exposures to nickel and observed adverse reproductive effects. To place the animal and human results in context, we can compare the urine nickel levels in the workers’ cohort with the urine levels in rat reproductive studies. Background urinary nickel levels in the female Monchegorsk population had a geometric mean of 5.9 µg/l, the low exposure refinery workers had urinary levels up to 70 µg/l (~12-fold increase in urinary levels) and the high exposure workers had urinary levels between 70 and 179 µg/l (up to 30-fold increase in urinary levels). Urinary levels of 70 µg/l in low exposure workers corresponded to approximately 160 µg soluble nickel inhalable exposure/m³ (>1000-fold over ambient air levels).

 

Comparison of the exposures in female workers to the exposure of rats can be made. In a rat oral study with nickel sulfate (100% bioaccessible nickel), blood and urinary nickel levels were measured after two years of exposure to 2.2 to 10 mg Ni/kg (Heim et al., 2007; Rush, 2005). A linear dose-response between oral intake of nickel and urinary nickel levels was found. An exposure of 2.2 mg Ni/kg corresponded to a mean urine value of 2300 µg Ni/L (males + females) with blood peak levels of ~70 µg Ni/l. Thus, the rat urinary nickel level at the LOAEL for reproductive developmental effects (2.2 mg Ni/kg) is 33-fold and 13-fold higher than those measured in Low and High exposure nickel refinery workers, respectively. Likewise, the rat urinary nickel level at the NOAEL for developmental effects (1.1 mg Ni/kg) are expected to be 16.5-fold and 6.5-fold higher than those measured in Low and High exposure nickel refinery workers, respectively. 

 

Thus, the reproductive effects observed in rats 1) may not be relevant to humans or 2) may not have been observed in the exposed human population because even the highest achievable human exposures are lower than those at which adverse reproductive effects have been observed in rats. In either case, the relevance of the positive results in rats (at unachievable human exposures) needs to be considered together with the negative results in human studies at Monchegorsk (for the highest exposed human population).  

These data show that while a reproductive “hazard” from nickel ion systemic exposure can be demonstrated in animals, this hazard has not been demonstrated in humans.

 

The full document summarizing these data in humans is attached in Sections 7.8 and 7.10.2 of IUCLID.

 

[1]Background urinary nickel levels in female Monchegorsk population had a geometric mean of 5.9 µg/l, the low exposure refinery workers had urinary levels up to 70 µg/l (~12-fold increase in urinary levels) and the high exposure workers had urinary levels between 70 and 179 µg /l (up to 30-fold increase in urinary levels). Urinary nickel levels are better indicators of fetal exposure, as they account for systemically absorbed nickel ion from occupational and non-occupational sources (e.g. diet) by all routes of exposure.

[2]The geometric means of the workers’ exposures in this study ranged from 0.03-0.084 mg Ni/m³ in the low exposure group to 0.15-0.33 mg Ni/m³ in the high exposure group.

 

 

FOR AN EXTENSIVE DISCUSSION, REFER TO THE NICKEL SULFATE DOSSIER WHICH IS BASED ON THE CONCLUSIONS EXPLAINED IN THE 2008/2009 EUROPEAN UNION EXISITING SUBSTANCE RISK ASSESSMENT OF NICKEL (EU RAR) (EEC 793/93)

Justification for classification or non-classification

Based on the read across results from Ni sulfate, Ni difluoride is classified as Repr. 1B; H360D in the 1st ATP to the CLP Regulation. Background information is provided in the discussion sections above and in the background document attached to Sections 7.8.1 and 7.8.2 of IUCLID.