Nickel(II) carbonate hydroxide tetrahydrate was studied for oral toxicity in female rats in a single dose toxicity test at doses of 300 and 2000 mg/kg. The chemical was classified into category 5 of the GHS concerning acute toxicity.

With regard to repeated dose toxicity, one dam in the 10 mg/kg group died during delivery on day 23 of gestation and showed soiled fur, involution of spleen and thymus, congestion and hydropic degenetration of lung. On detailed clinical examination, males of the 10 mg/kg group demonstrated increase in the number that were unwilling to be taken out of their home cages, or be handled. The NOAEL for the repeated dose toxicity is considered to be 2 mg/kg/day for both sexes.

With regard to reproductive/developmental toxicity, there were no treatment related toxic reproductive effects on males and pregnant and lactating females or their offspring. The NOAELs for reproductive/developmental performance and offspring development are considered to be 10 mg/kg/day.

Reverse mutation tests using microorganisms (Salmonella typhimurium, Escherichia coli) were conducted to assess the potential of nickel(II) carbonate hydroxide tetrahydrate to induce gene mutations.

Nickel(II) carbonate hydroxide tetrahydrate did not induce gene mutations in bacteria under the conditions of this study.

In vitro chromosomal aberration tests using cultured Chinese hamster cells (CHL/IU) were conducted to assess the potential of nickel(II) carbonate hydroxide tetrahydrate to induce chromosomal aberrations.

Nickel(II) carbonate hydroxide tetrahydrate did not induce chromosomal aberrations in cultured cells under the conditions of this study.

1. Single Dose Oral Toxicity 1)

Purity	:	94.7 %
Test species/strain		Rat/Crj: CD(SD)
Test method	:	OECD Test Guideline 423
Route	:	Oral(gavage)
Dosage	:	300 and 2000 mg/kg
Number of animals/group	:	Females, 6
Vehicle	:	0.5 % Sodium carboxymethylcellulose solution
GLP	:	Yes

　Test results:

One of 6 animals died with 2000 mg/kg, but no deaths were evident with 300 mg/kg during the observation period. No abnormalities of general condition were observed in any animals during the observation period.

The LD50 was found to be higher than 2000 mg/kg, so that the chemical was classified into category 5 of the GHS concerning acute toxicity.

2. Repeated Dose and Reproductive/Developmental Toxicity 2)

Purity	:	94.7 %
Test species/strain	:	Rats/Crj:CD(SD)IGS
Test method	:	OECD Test Guideline 422
Route	:	Oral(gavage)
Dosage	:	0, 0.4, 2, 10 mg/kg
Number of animals/group	:	Males, 12(5 for recovery); females, 12; satellite females, 5 (0 and 10 mg/kg) Males, 12; females, 12(0.4 and 2.0 mg/kg)
Vehicle	:	0.5 % Sodium carboxymethylcellulose solution
Administration period	:	Males, 42 days Females, from 14 days before mating to day 4 of lactation Females(satellite), 42 days
Terminal killing	:	Males, day 43 of treatment and day 15 of recovery Females, day 5 of lactation Females(satellite), day 15 of recovery Offspring, 4 days after birth
GLP	:	Yes

　Test results:

<Repeated dose toxicity>

On the morning of the 23rd day of pregnancy, one female rat of 10 mg/kg dose group was found dead after 40 administrations. This female showed soiled fur, involution of spleen and thymus and congestion and hydropic degeneration of lung. These histopathological findings were not considered as the direct cause of death, but possibly influenced by dosing with nickel(II) carbonate hydroxide tetrahydrate.

On detailed clinical examination, males of the 10 mg/kg group demonstrated increase in the number that were unwilling to be taken out of their home cages, or be handled. This finding could not be concluded to as an effect of nickel(II) carbonate hydroxide tetrahydrate. No effects ascribable to the compound were found with reference to the results of function tests, body weight change, food consumption, and hematological or blood biochemical analysis. No effects ascribable to the compound were found in the males and females of the 2 mg/kg or 0.4 mg/kg dose groups.

<Reproductive/developmental toxicity>

No animals showed abnormal estrous cycling during the dosage period before copulation, and all examined animals copulated and conceived. No effects were detected with regard to reproductive organ weights, or autopsy or histopathological findings in either sex, and there were no effects of nickel(II) carbonate hydroxide tetrahydrate on the pups born, birth index, number of dead pups, delivery index, sex ratio, viability index, external anomalies, body weight or necropsy findings.

<Evaluation>

From the above results, the no observed adverse effect level (NOAEL) for repeat dose toxicity of nickel(II) carbonate hydroxide tetrahydrate is considered to be 2 mg/kg/day for both sexes. And the NOAELs for reproductive/developmental performance and offspring development are considered to be 10 mg/kg/day.

3. Genetic Toxicity

3-1. Bacterial test 1)

Purity	:	94.7 %
Test species/strains	:	Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan) and OECD Test Guideline 471
Procedures	:	Pre-incubation method
Vehicle	:	Water for injection
Positive controls	:	-S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, TA98, WP2 uvrA), sodium azide (TA1535) and 9-aminoacridine hydrochloride (TA1537) +S9 mix; 2-Aminoanthracene (all strains)
Dosage	:	[Dose-finding study] -S9 mix; 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains) +S9 mix; 0, 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains) [Main study] -S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 μg/plate (all strains) +S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 μg/plate (all strains) [Confirmative study] +S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 μg/plate (TA1535)
S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	3
Number of replicates	:	3
GLP	:	Yes

　Test results:

No increase in revertant colonies was observed in the test with either the non-activation method (-S9 mix) or the activation method (+S9 mix).

Genetic effects:

Salmonella typhimurium TA100, TA1535, TA98, TA1537

	+	?	-
Without metabolic activation	[ ]	[ ]	[*]
With metabolic activation	[ ]	[ ]	[*]

Escherichia coli WP2 uvrA

	+	?	-
Without metabolic activation	[ ]	[ ]	[*]
With metabolic activation	[ ]	[ ]	[*]

3-2. Non-bacterial in vitro test (chromosomal aberration test) 1)

Purity	:	94.7 %
Type of cell used	:	Chinese hamster lung (CHL/IU) cells
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan) and OECD Test Guideline 473
Vehicle	:	Isotonic sodium chloride solution
Positive controls	:	-S9 mix; Mitomycin C +S9 mix; Cyclophosphamide
Dosage	:	-S9 mix (short-term treatment); 0, 47.1, 67.2, 96.0 μg/mL +S9 mix (short-term treatment); 0, 120, 172, 245, 350 μg/mL -S9 mix (continuous treatment); 0, 5.94, 11.9, 23.8, 47.5 μg/mL
S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	2
GLP	:	Yes

　Test results:

No increase in chromosomal aberrations was observed with either the short-term treatment (-S9 mix and +S9 mix) or the continuous treatment.

Genetic effects :

	clastogenicity			polyploidy
	+	?	-	+	?	-
Without metabolic activation:	[ ]	[ ]	[*]	[ ]	[ ]	[*]
With metabolic activation:	[ ]	[ ]	[*]	[ ]	[ ]	[*]

1)	The tests were performed by the Biosafety Research Center, Foods, Drugs and Pesticides (An-pyo Center), 582-2 Shioshinden, Iwata-shi, Shizuoka, 437-1213, Japan. Tel +81-538-58-1266, Fax +81-538-58-1393
2)	The test was performed by the Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano- shi, Kanagawa, 257-8523, Japan. Tel +81-463-82-4751, Fax +81-463-82-9627.