Tricobalt dicitrate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well documented publication which meets basic scientific principles.

Data source

Reference

Reference Type:

publication

Title:

Effects of cobalt sulfate on prenatal development of mice, rats, and rabbits, and on early postnatal development of rats.

Author:

Szakmary, E. et al.

Year:

2001

Bibliographic source:

Journal of Toxicology and Environmental Health, Part A 62: 367-386

Materials and methods

Principles of method if other than guideline:

The effects of cobalt(II)sulfate heptahydrate administered to pregnant rats were studied on fetal and postnatal offspring.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

10026-24-1

EC Number:

600-050-9

Cas Number:

10026-24-1

IUPAC Name:

10026-24-1

Constituent 2

Reference substance name:

cobalt(II)sulfate heptahydrate

IUPAC Name:

cobalt(II)sulfate heptahydrate

Details on test material:

- Name of test material (as cited in study report): Cobalt(II)sulfate heptahydrate (CoSO4x7H2O, CAS 10026-24-1, REANAL Budapest)
- Analytical purity: no data

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LATI, Gödöllö
- Weight at study initiation: 200-240 g
- Housing: plastic cages, on steam-sterilized, hardwood shavings
- Diet (ad libitum): standard rat pellets
- Water (ad libitum): tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod: 0600-0800 dim light; 0800-1800 daylight; 1800-2000 dimlight; 2000-0600 darkness

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Doses of cobalt(II)sulfate heptahydrate per kilogram body weight were dissolved in 10 mL tap water for rats.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Rats were mated in harem system: Males were placed among the females overnight. Vaginal smears were taken daily and stained with 1% methylene blue solution. The day when sperm were found in the vaginal smear of females in estrus was regarded as the first day of gestation.

Duration of treatment / exposure:

1) GD 1-20
2) single exposure to pregnant rats
3) single exposure on GD 20
4) single exposure on GD 16
5) GD 1-21

Frequency of treatment:

Experiment 1 and 5: once daily

Duration of test:

up to ~ 3 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
1) 0, 25, 50, 100 mg/kg; 2) 100 mg/kg; 3) 0, 100, 200, 300 mg/kg; 4) 0, 0.0625, 0.125, 0.250 mg/kg; 5) 0, 25 mg/kg
Basis:
other: nominal conc., calculated as the anhydrous salt of cobalt(II) sulfate heptahydrate

No. of animals per sex per dose:

1) 3
2) 6
3) 6
4) 3
5) no data

Control animals:

yes

Details on study design:

1) Cobalt concentration was determined by atomic absorption spectrometry in samples of maternal and fetal blood as well as in amniotic fluid taken at the time of the treatment on day 20 and 24 hours after that.
2) Cobalt concentration was determined by atomic absorption spectrometry in blood samples taken at 0, 1, 2, or 3 hours after the treatment.
3) Cobalt concentration was determined by atmoic absorption spectrometry in blood samples taken at 0, 2, 4, 8, 12, or 24 hours after the treatment.
4) Rats were anesthetized on day 16 of gestation; the uterus was exposed and the fetuses were treated intra-amnially with the test substance. The dams were exsanguinated under ether narcosis 24 hours later.
5) Dams were allowed to give birth.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT and Body weight gain: Yes

FOOD CONSUMPTION: Yes
- Time schedule for examinations: daily

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

Autopsy and Histological Examinations:
Findings at autopsy were recorded. Weights of brain, hypophysis, thymus, lungs, heart, liver, spleen, kidneys, adrenals, and ovaries were measured and the relative organ weights were calculated. Histological examination was performed on the excised oragns, the stomach and uterus. Furthermore, all pathologic alterations found in any of the animals were examined histologically (fixation in 4% neutralized formaldehyde, embedding in paraffin, slicing sections of 6 μm, and staining with HE.

Clinical Laboratory tests:
Blood samples were taken from the aorta under ether anesthesia: complete blood cell counts, hemoglobin concentration, mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) values, blood glucose, serum urea nitrogen, creatinine, bilirubin, albumin, serum alkaline phosphate, and aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) activities.

Fetal examinations:

The perinatal [(number of live pups on day 5 / number of live newborns)x100] and the survival index [(number of live pups on day 21 / number of live pups on day 5)x100] were determined. The perinatal and postnatal development of the pups was followed up to postnatal day 21. The total litter size (live and dead pups) was determined and the body weight of the pups was measured on postnatal day 1, 7, 14, and 21. Further, the days of descending of testes, forming of ears, breakthrough of the teeth, and opening of the eyes were recorded.
For monitoring the development of the nervous system, the development of forward movement and swimming was examined according to (Vorhees et al., 1979), and the righting reflex was examined according to (Brunner et al., 1978). The development of muscle strength was also monitored, with remaining on a tilted plane as the parameter according to (Paksy et al., 1982) and cliff avaoidance according to (Brunner et al., 1978). All examinations were carried out on postnatal day 1-21, at times indicated, on 2 male and 2 female pups from each litter.

Statistics:

The litter was regarded as the statistical unit. Parametric data (maternal organ weights, clinical and biochemical parameters, food and fluid consumption, fetal and placental weights) of groups were compared using analysis of variance and the Dunnett test, while nonparametric variables (frequencies of pre- and post-implantational losses, frequencies of body weight, skeletal and internal organ retardations, minor and major anomalies) of groups were compared using a Kruskall-Wallis test. The level of significance was chosen to be 5%.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
Daily food consumption was 1010-1200 J/kg bw in the control group and 790-990 J/kg bw in the group treated with the highest cobalt dose. Cobalt sulfate did not significantly affect maternal weight gain. At the highest dosage, the relative weights of liver, adrenals, and spleen were increased, but not significantly. Significantly increased urea nitrogen and creatinine concentrations and significantly decreased albumin and glucose concentration in the serum or blood were determined. Pathologic histological alterations were not observed in any of the dose groups.
The number of mothers that died during delivery rose in proportion to the increase of doses of cobalt sulfate (0/15, 1/15, 5/15, 12/20 corresponding to 0, 25, 50 and 100 mg/kg bw).

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The frequency of fetuses with retarded body weight and skeletal and visceral retardation significantly increased with the dose of cobalt sulfate. The two higher doses increased the frequency of malformations of skeleton (cranium, vertebrae) and the urogenital system (dilated renal pelvis and ureter, dystopic ovaries and testes) was predominatly affected.
Cobalt salt administered into the amnion proved to be embryotoxic. The frequency of dead fetuses increased and the body weight of the live fetuses decreased. No histopathological changes were found in the live fetuses in the uterus, while the fetuses that died in the uterus showed extensive necrotic, necrobiotic changes of the skin and the parenchymal organs.
The perinatal index decreased significantly from the value of 92.0 ± 7.0 in the control group to 73.3 ± 8.8 following cobalt sulfate treatment. The survival index was not significantly affected.
The body weight of the newborn animals was significantly smaller in the treated group compared to the control on postnatal day 1 and 7, while on postnatal day 14 and 21 there was no difference between the groups.
Eye opening was completed in the usual time period in both groups, while time of appearance of the teeth, descending of the testes, shaping of ears, and development of hearing was delayed in the treated group. The development of muscle strength and of the locomotor system was delayed. All the functions studied (forward movement, swimming, righting reflex) normalized by postnatal day 21, with the exception of muscle strength.
The frequency of fetuses with retarded body weight and skeletal and visceral retardation significantly increased with the dose of cobalt sulfate. The two higher doses increased the frequency of malformations of the skeleton (cranium, vertebrae) and the urogenital system (dilated renal pelvis and ureter, dystopic ovaries and testes) was predominantly affected.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

2

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

3

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Basis for effect level:

other: teratogenicity

4

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Cobalt concentration in the maternal blood was increased in proportion to the administered doses. Cobalt crossed the placenta and appeared in the fetal blood and amniotic fluid. Regardless of the administered dose of cobalt sulfate, cobalt concentration in the blood peaked 2 hours after administration.

Applicant's summary and conclusion

Conclusions:

It is concluded that cobalt sulfate exposure decreases the perinatal viability of the fetuses, but the functions of the surviving fetuses with perinatal retardation become compensated by postnatal week 2-3. The development of fetuses is undisturbed thereafter.