Calcium cyanamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1959-1960

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 1

Reference Type:

study report

Title:

Unnamed

Year:

1960

Report date:

1960

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1960

Report date:

1960

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of rats were fed for 17-50 weeks with dosages of 0.003%, 0.012%, 0.05% and 0.2% calcium cyanamide in the diet. The study was then either terminated or animals were subjected to an additional recovery period of 4 weeks (0.2% dosage) or 4 month (0.05% dosage). Bodyweights were taken twice per week for the first three weeks and once per week thereafter. Clinical observations were recorded once per month for the first 6 month and once every 6 weeks thereafter. At termination, packed cell volume was determined, circulating leucocyte counts and differential counts were made and autopsy was performed including histopathology. Bodyweights and organ weights were examined by rank test method for statistical difference from controls (plain diet).

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Calcium cyanamide

EC Number:

205-861-8

EC Name:

Calcium cyanamide

Cas Number:

156-62-7

Molecular formula:

CN2.Ca

IUPAC Name:

calcium cyanoazanediide

Test material form:

solid: crystalline

Details on test material:

Substance preapred in Laboratory

Specific details on test material used for the study:

Supplier American Cyanamide Company

Test animals

Species:

rat

Strain:

Sherman

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wyckoff breeding colony
- Age at study initiation: 19-23 days
- Weight at study initiation: 30-70 g
- Fasting period before study: no
- Housing: individual in galvanized metal cages with wire mesh floor and front
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): about 24°C
- Humidity (%): 40-60%
- Air changes (per hr): air-conditioned room, no data on ventilation rate
- Photoperiod (hrs dark / hrs light): artificial light for 10 hours each day and 4 hours on Saturday and Sunday

Administration / exposure

Route of administration:

oral: feed

Vehicle:

not specified

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): sufficient amounts of drug diets were mixed weekly. At the end of each week the remaining diet was weight and discarded.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

17-50 weeks

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0.003%, 0.012%, 0.05% and 0.2%
Basis:
nominal in diet

No. of animals per sex per dose:

18/sex/dose

Control animals:

yes, plain diet

Details on study design:

No details on study design are provided in the report.

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes; gross observations were made daily

DETAILED CLINICAL OBSERVATIONS: Yes; Clinical observations on condition of skin, hair, eyes, teeth, feet, tail and behaviour were recorded once a month for the first 6 months and once every 6 weeks thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: twice per week for the first 3 weeks and once per week thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was measured weekly for each group and compound intake (as mg/kg/rat/day) was calculated from food consumption and bodyweight figures.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination of the experiment

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

No data

Statistics:

Bodyweights and organ weights were examined by a rank test method for statistical differences from controls.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
Twelve males and twelve females died in the course of the one-year study period. The mortality rate of the animals at the highest dosage level (0.2% calcium cyanamide) was very high and, therefore, it was to be expected that animals would not survive a one-year drug administration period. Thus, animals were treated for 7 month instead. Of the 18 males and 18 females of the 0.2% group, only 8 and 10 survived until seven month of treatment, respectively. Of the 0.05% group, one male and two females died and of the 0.012% group one female died. All other animals survived until termination one year after start of the study.
Respiratory infections were present in the control animals as well as in the four treatment groups. Weight loss accompanied prolonged infection. Skin lesions were observed in areas around the neck, chin, shoulder girdle and back, which is frequently seen in the rat colony of the performing laboratory. Skin irritation in the mouth/nose area was observed in animals fed the 0.2% diet after the first week of treatment which might be due to the irritating properties of calcium cyanamide. Other signs of intolerance seen at this dosage level were ptosis, excitability, priapism among males during the first month of treatment, poor balance, urinary incontinence, stiffness of the hind quarters, hunched posture and poor coats with bristle-like hair.

BODY WEIGHT AND WEIGHT GAIN
Bodyweights of the animals treated at 0.2% and 0.05% were significantly reduced compared to controls. The weight gain of animals of the 0.003% and 0.012% groups did not differ significantly from controls, although females of the 0.003% group exhibited an average bodyweight greater than controls.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Consumption of food was significantly decreased at the two highest dose levels (0.2% and 0.05%), whereas the lower dosage groups (0.012% and 0.003%) did not show reduction in food consumption except for the males of the 0.012% group. Females of the 0.003% group consumed more food than control animals.
The average drug intake was 1.3-3.8 mg/kg/day for the 0.003% group, 5.5-14.9 mg/kg/day for the 0.012% group, 24-63.9 mg/kg/day for the 0.05% group and 134-229 mg/kg/day for the 0.2% group.
HAEMATOLOGY
Haematology data from treated animals did not differ from those of controls.

ORGAN WEIGHTS
Animals of the 0.2% group showed reduced weights of salivary glands, spleen, heart, lungs, liver, kidneys and brain when compared to controls. No statistical significant differences in organs weights were found in animals of the 0.05%, 0.012% and 0.003% groups.

GROSS PATHOLOGY
Rats of the highest dosage group (0.2%) showed granular appearance of liver surface, enlarged, dark red thyroid and changes related to the reproductive system. Animals treated at 0.05% showed enlarged, red thyroid.

HISTOPATHOLOGY:
Overall, of the effects found in this investigation on calcium cyanamide, the liver cirrhosis, dental anomalies, thyroid hyperplasia, decrease of acidophils and the presence of thyroidectomy cells in the anterior lobe of the pituitary are the only morphological changes, which according to their distribution and/or nature can be attributed to drug treatment. All other pathological findings are spontaneous diseases known to occur in rats and neither their distribution within the groups nor their morphological nature allows their classification as harmful drug effects. Therefore, only those effects related to treatment with calcium cyanamide are described in the following for the different dosage groups.
Extensive morphological activation of the thyroid was seen in animals treated at 0.2% calcium cyanamide. As well, proliferation of bile ducts and mesenchymal elements with liver cell necrosis was found at this dose level. In addition, thyroidectomy cells were found in the anterior lobe of the pituitary of these animals. When calcium cyanamide was discontinued and animals were examined four weeks later, repair processes were observed both in thyroid and liver. Dental anomalies were observed consisting predominantly of worn or broken incisors leading to overgrowth of the respective partner and subsequent ulceration of gums and lips. The high incidence of this finding suggests that this might be a drug effect, although these changes occur spontaneously on rare occasions in the animal colony, but were not observed in the 5-month study on calcium cyanamide. Hypoplasia of the reproductive system was especially pronounced in the males whereas the females were somewhat more resistant. The interpretation of these hypoplastic changes is difficult since the experimental design does not allow for the differentiation between starvation effects and hypothyroid state, both of which are possible triggers of this finding. Therefore, these effects cannot be attributed to treatment with calcium cyanamide alone.
Rats of the 0.05% group showed moderate to extensive morphological activation of the thyroid, thyroidectomy cells and a decreased number of acidophils in the pituitary and mesenchymal infiltration of liver. Rats that were treated at 0.05% calcium cyanamide for one year and were then subjected to a four month recovery period showed transformation of the thyroid into a diffuse colloid-goiter and no signs of malignancy were observed.
At 0.012% morphological activation of the thyroid and thyroidectomy cells in the pituitary were observed.
At 0.003% the only pathologic finding was moderate chronic tracheitis and in rare cases moderate morphological activation of the thyroid.

The high degree of morphological activation of the thyroid which were noted at all dose levels required a more elaborate evaluation, especially since some of the histological findings were indicative of malignant transformation. Destructive infiltration and vascular invasion as well as formation of papillary pattern, which was regarded as secondary, were observed. Animals of the 0.012% dose level did not show any of these signs. In the recovery studies it was shown that at least three animals of the 0.2% group show histological signs of regression as documented by the disappearance of colloid, flattening of the follicular epithelium and absence of capillary hyperamia. In addition, acidophil elements were visible in the pituitaries of these animals and the number of thyroidectomy cells decreased. Additional evidence for this regression was found in the 0.5 % group which were treated with 0.1% sodium iodide in the diet for 4 months for recovery. The three histological signs of malignancy disappeared completely and the thyroids of all animals showed histological pattern indicative of the transformation into a diffuse colloid goiter except for one animal where a large undifferentiated structure was observed. The recovery studies indicate that the pituitary and the thyroid show tendencies towards regression and repair in most of the cases. Therefore, it is extremely difficult, if not impossible, to justify a diagnosis of malignancy on the basis of histological examination alone. Thus, it is necessary that additional biological proof of malignancy of these structures should be given. On the basis of the regression studies and in spite of the histological criteria used for the determination of malignancy the described thyroid changes at the end of the drug feeding period cannot be regarded as malignant.

The postnecrotic liver cirrhosis which was found at the 0.2% and 0.05% diet levels in this investigation and found at the 0.2% group in the 5-month study, puts calcium cyanamide in the group of chemical substances which cause liver cirrhosis in rats. Most of these compounds are carcinogens. It is, however, very unlikely that calcium cyanamide, when given at the 0.2% dose level will ultimately cause hepatic neoplasms, since these animals will probably die before developing liver tumours. Since the maximum dose level for clinical uses in humans is 2 mg/kg/day, no development of hepatic tumours is to be expected as the highest dose administered to rats corresponds to 180 mg/kg/day which will never be applied to human beings. Although the incidence of liver cirrhosis in the 0.05% group (corresponding to 45 mg/kg/day) is very low, it cannot be stated that this dosage is safe regarding hepatotoxicity.

Effect levels

1

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 0.012 other: %

Sex:

male/female

Basis for effect level:

other: Corresponding to 11 mg/kg bw/day. Based on the histopathological findings in thyroid and liver observed at the next higher dosage level.

2

Dose descriptor:

NOAEL

Effect level:

0.003 other: %

Sex:

male/female

Basis for effect level:

other: Corresponding to 1.3 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Conclusions:

The 0.012 % dose level causes a few cases of thyroid hyperplasia and was, thus, considered the borderline adverse effect level (LOAEL). This dosage corresponds to a daily intake of 11 mg/kg bw Calcium cyanamide, purum, considering the bodyweights of rats and daily food consumption, which is equal to 16 mg/kg Calcium cyanamide, technical grade.

Executive summary:

A previous one-year feeding experiment of calcium cyanamide had to be terminated after 5 months because of an accident in the animal quarters. This study was therefore performed to investigate the potential toxicological properties of calcium cyanamide when fed to rats for a period of one year.

Dosages of 0.2, 0.05, 0.012 and 0.003 % calcium cyanamide, purum were applied to rats with the diet for a period of up to one year. In addition, a recovery period of 4 weeks (in case of the highest dosage) or 4 months (for animals treated at 0.05 %) was added for some of the animals to investigate for reversibility of effects induced by treatment.

In the course of the one-year study period, twelve animals died, most of them being treated at the highest dosage (0.2 %). After seven months of treatment it was decided to examine the effects of treatment in the animals of the 0.2% group, because of their poor condition. The other dosage groups were kept on treatment for the remaining time.

Both, bodyweights and food consumption were significantly decreased in the 0.2 and 0.05 % groups. The lower dosage groups did not show reduction in food consumption or bodyweight gain.

Results revealed that oral administration of 0.2 % calcium cyanamide (180 mg/kg/day) up to 7 months caused extreme degree of thyroid hyperplasia, thyroidectomy cells and decrease of acidophilic cells in the anterior pituitary, postnecrotic liver cirrhosis and dental disorders. Similar findings but less pronounced and with lower frequency were observed in animals receiving 0.05 % (45 mg/kg/day) for one year.

The 0.012 % dose level (11 mg/kg/day) caused a few cases of thyroid hyperplasia with the occurrence of thyroidectomy cells and the decrease of acidoplhilic cells in the anterior pituitary.

Thyroid hyperplasia was in rare instances noticeable in animals receiving the 0.003 % diet, but was not considered as treatment related effect, but reflecting biological variation.

The significance of hyperplasia of the reproductive system and the decrease of bodyweight gain both found in animals of the 0.2% and the 0.05% group cannot be clearly attributed to treatment since two mechanisms, the decrease of food consumption and hypothyroidism, can be responsible for these phenomena.

The liver changes observed at the two highest dose levels have been defined as postnecrotic cirrhosis.

The occurrence of thyroidectomy cells and the decrease of acidophils in the anterior pituitary indicate a complete inhibition of the thyroid. The thyroids of the 0.2 % group were extremely hyperplastic and showed histological signs of malignancy after 7 months of drug administration. Similar thyroid hyperplasia was found in the 0.05 % group. The recovery studies showed that all histological signs of malignancy disappeared after the discontinuation of drug treatment, a phenomenon which is not compatible with the biological definition of a malignant neoplasm.

Under the conditions of the study, and regarding the histopathological changes in thyroid and liver observed at the two highest dose levels, a dosage of 0.012 % Calcium cyanamide, purum in the diet can be regarded as borderline effect level (LOAEL), which is equal to 0.017 % Calcium cyanamide, technical grade.