Zinc cyanide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

A study was performed at term and at weaning to verify the toxic effects of the prenatal exposure to potassium cyanide (KCN) and potassium thiocyanate (KSCN) in pregnant Wistar rats. The experiments were carried out in accordance with the ethical principales in animal research adopted by the bioethics committee of the faculty of veterinary medicine and zootechny, University of Sao Paulo. One hundred and forty pregnants rats were randomly assigned to one of seven groups: 6 experimental and 1 control with 20 animals in each. The experimental groups received daily in drinking water the doses: 1, 3 and 30 mg KCN/kg or 0.8, 2.4 and 24 mg KSCN/kg from GD 6 to GD 20 (trial A) or one day after weaning (trial B). The control group received only drinking water. Water consumption was measured daily and fresh KCN or KSCN solutions provided daily. Every two days the amount of KCN and KSCN administered in the drinking water was adjusted to the body weight. During all the experimental period, the general state of the rats and the occurence of abortions were monitored. Maternal body weight was measured on GD 0, 6, 8, 10, 12, 14, 16, 18 and 20 (trial A) or GD 21 (trial B) and individual diet consumption on GD 6-9, 10-12, 13-15 and 16-18 in both trials. On GD 19, two trials (A and B) were performed with 70 rats in each and 10 rats randomly assigned to each group. In the trial A, the food was removed at night and on GD 20, the dams were anesthetized after an 8h fast. Blood samples were taken for biocheminal analyses. After the procedure, all the dams were euthanised with CO2 chamber and the uterine horns were immediately removed and weighted. The number of corpora lutea, implantations, resorptions and live and dead fetuses as recoreded. Fragments of kidney, pancreas, brain, thyroid, spleen, lungs and liver were collected and eximined in microscope. The fetuses were weighed, measured, sexed, and examined for macroscopic external malformations. In trial B, all the dams were allowed to give birth naturally and immediately after birth (PND 1) rats pups from each litter were culled to leave, as close as possible, four males and four females in each litter. DUring the nursing, the weight of the litter was recorded on PND 1, 7, 14 and 21. On PND 21 the rat pups were subjected to euthanasia in a CO2 chamber, sampled for the biochemical analysis and histopathological studies were performed as described for their mothers.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Potassium cyanide

EC Number:

205-792-3

EC Name:

Potassium cyanide

Cas Number:

151-50-8

Molecular formula:

KCN

IUPAC Name:

Potassium cyanide

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

The experiments were carried out in accordance with the ethical principales in animal research adopted by the bioethics committee of the faculty of veterinary medicine and zootechny, University of Sao Paulo. Male and female Winstar rats aged 2 months were supplied by breeding facilities of the School of Veterinary Medicine, University of Sao Paulo. For mating, two females were housed with one male of the same strain overnight. Successful mating was ascertained by the presence of sperm in a vaginal smeat, and the following initial 24h was designated as Day 1 of gestation. The female rats with positive vaginal smears were housed individually in polycarbonate cages with pelleted food and water ad libitum and randomly assigned to either one of the experimental groups or to the control group. They were kept in a room maintained under a 12h light-dark cycle at 23-25°C.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

The experimental groups received KCN or KSCN solution at different concentrations in the drinking water, from GD6 to GD20 in order to obtain the target dose of 1.0, 3.0 and 30.0 mg/kg/day of KCN or 0.8, 2.4, and 24.0 mg/kg/day of KSCN. The control group received only drinking water. Water consumption was measured daily and fresh KCN or KSCN solutions provided daily. Every two days the amount of KCN or KSCN administered in the drinking water was adjusted to the body weight.

Details on mating procedure:

For mating, two females were housed with one male of the same strain overnight. Successful mating was ascertained by the presence of sperm in a vaginal smeat, and the following initial 24h was designated as Day 1 of gestation. The female rats with positive vaginal smears were housed individually in polycarbonate cages with pelleted food and water ad libitum and randomly assigned to either one of the experimental groups or to the control group.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily intake (drinking water)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:

During all the experimental period, the general state of the rats and the occurence of abortions were monitored. Maternal body weight was measured every two days as weel as individual diet consumption. After GD19 (trial A) and GD20 (trial B) blood samples were taken for biocheminal analyses.

Litter observations:

During the nursing, the weight of the litter was recorded on PND 1, 7, 14 and 21.

Postmortem examinations (parental animals):

After the procedure, all the dams were euthanised with CO2 chamber and the uterine horns were immediately removed and weighted. The number of corpora lutea, implantations, resorptions and live and dead fetuses as recoreded. Fragments of kidney, pancreas, brain, thyroid, spleen, lungs and liver were collected and eximined in microscope.

Postmortem examinations (offspring):

Offspring trial A: The fetuses were weighed, measured, sexed, and examined for macroscopic external malformations.
Offspring trial B: On PND 21 the rat pups were subjected to euthanasia in a CO2 chamber, sampled for the biochemical analysis and histopathological studies were performed as described for their mothers.

Statistics:

The litter was the statistic unit. Data on maternal body weight, food consumption, fetal body weight, placenta weight were subjected to one-way analyses of variance (ANOVA), followed by the Dunnett test to compare the mean differences between experimental groups. The numbers of corpora lutea, total implantations, live and dead fetuses, and pre- and post-implantation losses were statistically evaluated using the Kruskal-Wallis test, followed by the Mann-Whitney U-test when appropriate. Incidence data, such as pregnancy rate, external, visceral, and skeletal alterations were compared using Fischer's exact test. A differene was considered statistically significant at p<0.05. All analyses were performed using the sofware GraphPad Instat v.2.01.
The intensity of the lesions, observed by the histopathological study of the groups of rats, was characterized through scores: (-) no lesion, (+) mild lesions, (++) moderate lesions, and (+++) severe lesions.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No significant alterations were found in the skeletal variations between control and experimental groups. Nevertheless, a higher incidence in the number of fetuses with visceral alteration was observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Serum levels of glucose were significantly higher in the dams from the KCN - 30mg/kg/day.
- Serum levels of thiocyanate in trial A were higher in all experimental groups in comparison to the control group while dams from trial B presented no differences.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The microscopique examination of the brain revealed interesting lesions characterized by focal neuronal necrosis and focal nodular gliosis. The cerebellum microscopy showed mild conjestion and mild vacuolization of white matter.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The microscopic changes were dose and time related, thus the lesions were more conspicuous in dams given 30 mg/kg/day of KCN and in dams with 24 mg/kg/day of KSCN.
- The pancreas section revealed moderate vacualization of islet cells in the dams.
- The liver of dams with high doses of KCN and KSCN revealed diffuse microvesicular vacuolization of hepatocytes.
- The kidney showed rare areas of congestion.
- No histological alterations in the lung and spleen.

Histopathological findings: neoplastic:

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

Description (incidence and severity):

KCN and KSCN in drinking water did not cause any change in the number of corpora luea, preimplantation loss, postimplantation loss, fetal and placental weight or fetal lengt. There were no dams with total litter resorptions.

Effect levels (P0)

Dose descriptor:

LOAEC

Effect level:

1 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

clinical biochemistry

neuropathology

histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

1 mg/kg bw/day (actual dose received)

Organ:

brain

kidney

liver

pancreas

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pups have the same lesions in liver and brain as described for their mothers. However, the lesions were milder.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

LOAEC

Generation:

F1

Effect level:

1 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

neuropathology

histopathology: non-neoplastic

Target system / organ toxicity (F1)

Critical effects observed:

yes

Lowest effective dose / conc.:

1 mg/kg bw/day (actual dose received)

Organ:

brain

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

In this study, 1.0, 3.0, and 30.0 mg/kg/day of KCN and 0.8, 2.4, and 24.0 mg/kg/day of KSCN were administered to pregnant Winstar rats via drinking water during GD6 to GD20 (Trial A) or GD6 to GD21 (Trial B).
No changes in feed consumption or body weight gain in experimental dams treated with KCN or KSCN throughout the gestation period were observed in this study. One explanation for this fact may be that physiological status (gestation), sexual dimorphism, age or a combination of these factors, could interfere in CN toxicity when considering weight alone. Regarding biochemical functions, this study demonstrated that there were significant alterations in glucose concentrations in dams from the 30 mg/kg/KCN group. These results suggest that CN could induce pancreatic diabetes during the gestation period in rats. In addition, as no changes were detected in any group of dams treated with KSCN, it could be theorized that it is CN and not its principal metabolite which induces a diabetogenic effect.
Concerning histopathological study, it reaveald increases in the number of vacualors in the folicular colloid of the thyroid gland from all experimental dams, no difference was found in cholesterol levels between control and experimental rats, a parameter indicative of thyroid function. Plus histopathological study demonstated changes in liver and kidney in experimental dams mainly in those treated with the highest doses of both KCN and KSCN.
No alterations were found in the skeletal morphology of th fetuses. Regarding the visceral analysis, the data revealed significance only in the number of fetuses with visceral variations, but not in the number of affected litters, and only in those fetuses of dams exposed to the largest dose of KCN. In contrast, althought no differences in the weight gain were observed during the postnatal period between control and experimental offspring, the histological evaluations revealed that pups from dams treated during gestation with the largest doses of KCN and KSCN showed evident alterations, principally in the liver and brain.
Taken as a whole, the data obtained in this study strongly support that CN and its principal metabolite, SCN, can affect the neonate and that this form of intoxiation may have importante consequences for human and animals.