Calcium cyanamide

Carcinogenicity

Description of key information

The carcinogenicity database available for calcium cyanamide (CaCN₂) consist of two studies conducted in mice and rats.

In the carcinogenicity study with F344 (Fischer) rats no tumour at any site in the rats could be associated with the administration of the substance. In the carcinogenicity study with B6C3F1 mice calcium cyanamide was not carcinogenic under the conditions of this study.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

- 20 animals were used in the control group instead of at least 50. - Two doses of the test item were used instead of three. - In the first 13 weeks of the test period animals were weight only once per months, instead of once per week. - The analytical determination of the diet was not performed. - The food consumption was not determined. - No blood smears were evaluated 12 and 18 months and prior to sacrifice.

Qualifier:

no guideline followed

Principles of method if other than guideline:

For details on principles of method see materials an methods section in IUCLID5 dossier.

GLP compliance:

no

Remarks:

study is a publication of the U.S. Department of Health, Education and Welfare.

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

water

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The analytical determination of the homogeneity and the correctness of the dietary concentrations was not carried out. However, data from a 2-generation study in rats confirm the homogeneity of Cyanamide in the diet (Koeter, 1986, Doc-No. 553-001).

Duration of treatment / exposure:

107 weeks

Frequency of treatment:

Daily

Post exposure period:

No exposure period

Remarks:

Doses / Concentrations:
The dietary dose levels were 0, 100 and 200 ppm for males and 0, 100 and 400 ppm for females, respectively.
Basis:
nominal in diet

No. of animals per sex per dose:

50 rats per sex per dose. In the control group 20 males and 20 females.

Control animals:

yes, concurrent no treatment

Positive control:

No positive control

Observations and examinations performed and frequency:

Feed consumption was not determined. The body weights were determined at least once per months, except for weeks 50 to 80 where no weights were recorded. The animals state of health was checked each day. Clinical examination and palpation for masses were performed each month. Animals were assessed gross-pathologically followed by histopathological examinations.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

No other examinations

Statistics:

The one-tailed Fisher exact test and Cochran-Armitage test for linear trend in proportions, with continuity correction was used.

Clinical signs:

no effects observed

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

no effects observed

Details on results:

- There were no test substance related mortalities in any of the treatment groups. Mean body weights of the high dose male and female rats were slightly lower than those of the corresponding controls, but are considered not to be compound related.

- A variety of non-neoplastic lesions and disorders were encountered with regularity in both control and dosed groups of rats. Such lesions are considered as common in aged F344 rats and therefore not to be compound related. The sites of neoplasms observed most frequently were the adrenal, pituitary, thyroid and testes. The pituitary neoplasms as well as adenomas (and hyperplasias) of the thyroid and interstitial-cell tumours of the testes occurred with comparable frequency in control and dosed rats. Therefore, they are not considered test substance-related.

- The number of cortical tumours in the dosed animals seemed to be balanced by similarly affected control rats. No statistical significant incidence was observed for this tumour. However, the incidence of phaeochromocytomas in the dosed females appears to be greater than the normal incidence and may be related to the administration of the test compound. Although the incidence of adrenal medullary tumours is high, statistical evaluation indicate neither a significant dose-related trend nor a significance by direct comparison of the dosed groups with the control. Therefore, the increased incidence is not to be considered related to the treatment of Calcium Cyanamide. The authors concluded that “In summary, no tumour at any site in the rats can clearly be associated with the administration of Calcium Cyanamide in this bioassay and that under the conditions of this bioassay, the test formulation of Calcium Cyanamide was not carcinogenic for F344 rats of either sex.”

Relevance of carcinogenic effects / potential:

No relevance of carcinogenic effects / potential

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Carcinogenicity study in rats: adrenal neoplasms:

	Cortical Adenoma	Phaeochromo-cytoma, benign	Phaeochromo-cytoma, malignant
Males Control 100 ppm 200 ppm	0/20 (0 %) 4/20 (20 %) 0/20 (0 %)	3/49 (6 %) 10/49 (20 %) 0/49 (0 %)	3/50 (6 %) 15/50 (30 %) 1/50 (2 %)
Females Control 100 ppm 400 ppm	3/19 (16 %) 0/19 (0 %) 0/19 (0 %)	1/50 (2 %) 4/50 (8 %) 0/50 (0 %)	7/50 (14 %) 6/50 (12 %) 1/50 (2 %)

* The one-tailed Fisher exact test and Cochran-Armitage test for linear trend in proportions, with continuity

correction was used.

Conclusions:

It is concluded that under the conditions of this bioassay, the test formulation of Calcium Cyanamide was not carcinogenic for F344 (Fisher) rats of either sex.

Executive summary:

Calcium Cyanamide was administered in feed to 50 male and 50 female F344 (Fischer) rats per dose level in this oncogenicity study. In the concurrent control group only 20 males and 20 females were used. All surviving animals per sex and dose were sacrificed at 107 weeks. The dietary dose levels were 0, 100 and 200 ppm for males and 0, 100 and 400 ppm for females, respectively.

The body weights were determined at least once per months, except for weeks 50 to 80 where no weights were recorded. The animals state of health was checked each day. Clinical examination and palpation for masses were performed each month. Animals were assessed gross-pathologically followed by histopathological examinations.

Results showed reduced body weight weights in the high dose of male and female rats. High incidences of adrenal medullary tumours revealed neither a dose relation nor a significant difference to the controls and are therefore considered as not treatment related.

It is concluded that under the conditions of this study, the test formulation of Calcium Cyanamide was not carcinogenic for rats of either sex.

Reference 2

Endpoint:

carcinogenicity: oral

Remarks:

oral (gavage/feed) and subcutaneous

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1963 - 1968

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

- study duration: 18 month instead of 24 month due to the hybrid strain of mice chosen - only one dosage (maximum tolerated dosage) was applied - hybrid mice were used in the study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The carcinogenic potential of 130 commercial pesticides, including calcium cyanamide, were evaluated for carcinogenicity in an 18-month study. Groups of two different strains of hybrid mice were treated at the different test substances via the oral route and subcutaneously both at the maximum tolerated dose (calcium cyanamide: 100 mg/kg) and negative, vehicle and positive controls were included in the investigation. Animals either received a single subcutaneous injection of the test substance in the neck or the test substance was administered via intubation for the first two weeks and afterwards via food.

GLP compliance:

not specified

Species:

mouse

Strain:

other: B6AKF1 and B6C3F1 hybrid mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Cumberland View Farms, Clinton, Tennessee
- Age at study initiation: 7 days for oral administration, 28 days for subcutaneous injection
- Weight: 10 - 19 g at 28 days of age
- Housing: a maximum of 6 mice per cage
- Diet: ad libitum
- Water: ad libitum

Two different strains of mice were used in the carcinogenicity study. Both were F1 hybrids from crosses of inbred strains. Hybrids designated B6C3 are the product of mating C57BL/6 females with C3H/Anf males and those designated B6AK derived from C57BL/6 females mated with AKR males.

No information on environmental conditions provided in the report.

Route of administration:

other: oral (gavage/feed) and subcutaneous

Vehicle:

other: 0.5% gelatin

Details on exposure:

ORAL APPLICATION:
The compound was suspended in 0.5% gelatin
Daily administration via stomach tube during Day 7 - 28 of age (100 mg/kg)
Test substance was mixed with the diet for the remaining study period (Day 29 of age onwards). A supply of feed was prepared containing 240 ppm (100 mg/kg) calcium cyanamide and portions of one week's supply for all animals were packed separately in waxed paper bags, which were in turn sealed in polyethylene bags and refrigerated until use.
The calculated dose was not adjusted to the changing bodyweight during the three weeks of stomach tubing but a single adjustment was made at the time of conversion from stomach tube to mixture in the feed.

SUBCUTANEOUS INJECTION:
The compound was suspended in 0.5% gelatin and 100 mg/kg were injected as a single dose subcutaneously (0.05 mL) in the nape of the neck to weanling mice on approximately the 28th day of age.

For both pathways of application, the maximum tolerated dose was given.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatography analyses were used to show that the test substance had been homogeneously dispersed throughout the feed.

Duration of treatment / exposure:

single exposure (subcutaneous injection) and 18 month (oral exposure)

Frequency of treatment:

single treatment (subcutaneous injection) and daily (oral exposure)

Post exposure period:

No post exposure period for animals treated via the oral route and 18 month for animals receiving a single subcutaneous injection.

Remarks:

Doses / Concentrations:
100 mg/kg
Basis:
other: actual ingested (stomach tube), nominal in diet (oral/feed), nominal concentration (subcutaneous injection)

No. of animals per sex per dose:

18/sex/strain/route of administration; only one dosage applied

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

Two different strains of hybrid mice (B6C3 and B6AK) were chosen in expectation of a hugh susceptibility to carcinogenic stimuli coupled with the hardiness and longevity characteristic of F1 hybrids. There were no previous data on the incidence of neoplastic or other diseases in these two hybrids. Since one could not be aware of the lifespan of the hybrid strains by the time the study was initiated, a study period of 18 months instead of 24 months was chosen. The study period chosen provided optimum conditions for carcinogenic studies, i.e. maximum exposure and/or observation time with a minimum morbidity and/or mortality due to old age.
For oral administration of the test substance, calcium cyanamide was mixed with the diet from 29 days of age onwards. The potential hazard for man associated with the vast majority of the different chemicals investigated in this study is by the oral route and in many cases admixed with food. The single subcutaneous administration was chosen to assist in disclosing "strong" carcinogens. It has long been known that single exposures to these compounds early in life will result in significant tumour incidence over controls.
The basic concept of the dosage choice was the use of a maximum tolerated dose in both the single subcutaneous and the continuous oral administration studies.

Positive control:

ethyl carbamate, amitrol, ethylene imine, aramite, dihydrosafrole, isosafrole, safrole

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: animals were observed once per week for liver or kidney tumidity

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

no other examinations

Statistics:

The Chi Square Method was employed for the significance of the study data over controls.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

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):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

One female of the B6AKF1 strain and two males of theB6C3F1 strain died in the course of the study after subcutaneous administration of calcium cyanamide. Of the animals in the oral treatment group, one female of the B6AKF1 strain and three males of the B6C3F1 strain died.
No adverse effects on bodyweight gain were noted.
Of the 36 mice examined per strain, four mice of the B6C3F1 strain developed tumours after subcutaneous administration of calcium cyanamide; two reticulum cell sarcoma (type A), one pulmonary adenoma and one adeno/mammary carcinoma were observed. Mice of the other strain receiving calcium cyanamide subcutaneously did not show any tumours at autopsy.
Of the mice treated repeatedly with oral dosages of calcium cyanamide, 23 mice were found with tumors from the B6C3F1 strain and 6 mice with tumors in the B6AKF1 strain; 10 reticulum cell sarcoma (type A), one pulmonary adenoma, 5 hepatoma, 3 angioma and 9 gastric papilloma were noted.
Based on this low tumor incidence, the probability for calcium cyanamide to develop reticulum cell carcinoma was 0.01.

In the control groups, a low incidence of neoplastic diseases were observed. All of the orally applied positive control substances were carcinogenic.

Relevance of carcinogenic effects / potential:

No relevance for carcinogenic effects / potential

Key result

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

no remarks

Conclusions:

It is concluded that under the conditions of the study, calcium cyanamide administered at the maximum tolerable dosage is not carcinogenic for B6C3F1 and B6AKF1 hybrid mice.

Executive summary:

The carcinogenic potential of 130 commercial pesticides, including calcium cyanamide, were evaluated for their carcinogenic potential in an 18-month study. Therefore, groups of 18 male and 18 female hybrid mice of two different strains were treated with each of the test substances using both oral and subcutaneous application. An equal number of mice served as untreated controls and another group of 36 served as control for each solvent used. Two types of studies were conducted: (1) a single subcutaneous injection in 0.05 mL of suspension in the neck at 28 days of age; and (2) daily oral stomach tube administration from Day 7 to 28 of age and thereafter administration via diet. Both for subcutaneous and oral application, the maximum tolerated dose which was determined in a preliminary acute toxicity study, was given (100 mg calcium cyanamide /kg).

A low incidence of spontaneous neoplastic diseases was observed in both control groups and all of the positive controls applied via the oral route were carcinogenic.

The probability of calcium cyanamide to develop reticulum cell carcinoma after oral application was very low. Therefore it is concluded that calcium cyanamide is not carcinogenic in B6C3F1 and B6AKF1 mice when applied at the maximum tolerated dose level.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

Quality of whole database:

The supporting study conforms results from the key study.

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the two studies and in accordance with Regulation (EC) No 1272/2008 (GHS/CLP), the test substance, calcium cyanamide technical grade, is not classified for carcinogenic effects.

Additional information

The carcinogenicity database available for calcium cyanamide technical grade consists of two studies in mice and rats (study details and outcomes provided in section below):

- NCI (1979) with B6C3F1 mice for calcium cyanamide technical grade

- NCI (1979) with F344 rats for calcium cyanamide technical grade

This data was used by the Committee for Risk Assessment (RAC, 2015) for the assessment of carcinogenicity of cyanamide (RAC Opinion proposing harmonised classification and labelling at EU level of cyanamide, CLH-O-0000001412-86-67/F, Adopted 5 June 2015). In this process, both studies on calcium cyanamide were used together with the two following studies on cyanamide:

- Carcinogenicity study of Goodyer (1988) with CD-1 mice for cyanamide

- Long-term study of Osheroff (1991) with Sprague-Dawley rats for cyanamide

Even though calcium cyanamide can rapidly hydrolyse to cyanamide (e.g. in aqueous solutions), due to the differences in physicochemical properties (e.g. chemical composition) and dissolution kinetics (e.g. in the stomach under low pH conditions) as well as the dissimilar outcomes between cyanamide and calcium cyanamide observed in some of the toxicity studies (e.g. acute toxicity and developmental toxicity), the use of data from cyanamide as read-across or weight-of-evidence approach for evaluating the carcinogenic potential of calcium cyanamide technical grade is not appropriate (for more information, see report "Scientific Rationale for not using Cyanamide as Read-Across Substance for Calcium Cyanamide on Toxicological Endpoints” in Section 13.2). Furthermore, there is an existing carcinogenicity study of calcium cyanamide (NCI, 1979), which reported that calcium cyanamide technical grade was not carcinogenic for both rats and mice of either sex. Nevertheless, summary of both studies on cyanamide (Goodyer, 1988; Osheroff, 1991) is given in this section below to demonstrate the full consideration of the available toxicological data on cyanamide.

Summary of results including those with cyanamide:

The NCI bioassay (TR 163, 1979) with calcium cyanamide

B6C3F1 mice (50/sex/dose) were exposed via feed over 100 weeks to a commercial formulation with 63% CaCN₂ at 500 and 2000 ppm, corresponding to 157 and 630 ppm each of H₂CN₂ and Ca(2+). In addition, the commercial formulation contained 22% of calcium oxide that might (100% bioavailability assumed) add another 79 and 314 ppm Ca(2+), in total possibly 237 and 944 ppm Ca(2+). As feed consumption was not recorded exposure in mg/kg bw/d cannot be calculated. The control group only comprised 20 mice/sex. Dose selection was based on a 7-week feeding study with 5 animals /sex/dose at dose levels of 1500-30000 ppm (commercial formulation). No mortality occurred up to 16000 ppm and mean body weights after week 7 were in the range of 92% for males and 85-87% for females in comparison to controls leading to 2000 ppm as high dose for the carcinogenicity study. Histopathology showed trace amounts of bile duct hyperplasia in both sexes at 16000 ppm with focal hepatic necrosis in some females.

In the carcinogenicity study mean body weight was not affected in low dose females and was slightly reduced (approximately <10%) in the other dose groups. Mortality showed a significant dose related trend (p=0.005) in male mice (survival 100, 90 and 76% for control, low and high dose), while in females no dose related mortality was observed (survival 90, 86 and 92%). These data indicate that the MTD was reached or even slightly exceeded for high dose male mice but not for female mice. In the NCI report it is stated that mice may have been able to tolerate higher doses without differentiation between sexes. But the conclusion about the MTD is associated with some uncertainty due to the low size of the control group. The non-neoplastic histopathological observations did not indicate any specific organ possibly being affected by treatment.

Hemangiosarcomas were present in male mice (5, 4 and 20% for control, low and high dose) and malignant lymphomas or leukaemias in females (5, 24 and 36%). For hemangiosarcomas the trend test was statistically significant (p=0.006) but the Fisher exact test for dose wise comparison was not. The mean historical control incidence was 4% with a highest incidence of 10% (related to a control group of about 19 animals). Thus, the incidence in high dose males exceeded the historical control range. The trend test was also statistically significant for the incidence of lymphomas and leukaemias (p=0.009) as was the Fisher exact test for the high dose. The mean historical control incidence of the laboratory was 21% for lymphomas and leukaemias, but a range was not provided. As the incidence in the concurrent control group was clearly lower than the mean historical incidence this may indicate that the comparison to the concurrent control group may have been the cause for the statistical significances. Nevertheless, it cannot be excluded that the incidence in high dose females exceeded or was in the upper range of the historical incidence span. Based on these findings the NCI report concluded that CaCN₂ may be carcinogenic to male and female mice. But the interpretation of this bioassay is surrounded by some uncertainties, namely the low number of control animals and the limited historical control data provided. It should also be mentioned that the study on CaCN₂ was conducted in the same room as feeding studies with other chemicals that might impact tumour incidences in case of cross contamination, like 2 -chloroethyl)trimethylammonium chloride or 2,4 -diaminotoluene and others.

Under consideration of the limited reliability of the comparison of tumour incidences in dosed vs. control animals (due to the small group size of the latter), the RAC (2015) listed historical control data from 20 NTP studies conducted in the 7 years up to 1995. For hemangiosarcomas in male B6C3F1 mice the mean historical incidence in the NTP database was 5.6% with a total range between 2 and 14%. Thus, the incidence in high dose males was also beyond this upper historical incidence. For malignant lymphomas in females the mean historical incidence was 17.3% with a span between 6 and 30%. Again, the incidence in high dose females was outside this historical control range. But these comparisons with historical data must be considered with caution. To avoid potential confounding by changes of the background rate over time or due to other influences (e.g. of the diet, different laboratories etc.), historical data should embrace the study under consideration by a relatively narrow time span, e.g. about +/-5 years. In contrast, the historical data collected by the RAC (2015) were derived from the years 1988-1995 while the NCI study was conducted prior to 1979.

F344 rats (50/sex/dose) were exposed via feed over 107 weeks to a commercial formulation with 63% CaCN₂ at 100 and 200 ppm (males) and 100 and 400 ppm (females). These exposures corresponded to 32 and 63 ppm each of H₂CN₂ and Ca(2+) for males and 32 and 126 ppm for females. In addition, the commercial formulation contained 22% of calcium oxide that might (100% bioavailability assumed) add another 16, 31 and 63 ppm Ca(2+), in total possibly 48, 94 and 189 ppm Ca(2+) for the different dose levels. As feed consumption was not recorded exposure in mg/kg bw/d cannot be calculated. The control group only comprised 20 mice/sex. Dose selection was based on two 7-week feeding studies with 5 animals /sex/dose at dose levels between 400 and 30000 ppm (commercial formulation). No mortality occurred up to 4000 ppm and mean body weights after week 7 at 400 ppm were 89% for males and 97% for females in comparison to controls leading to 200 ppm as high dose for males and 400 ppm for females the carcinogenicity study. By histopathology bile duct hyperplasia and slight to moderate extramedullary hematopoiesis were noted in males and females at ≥15000 ppm. Clearly enlarged thyroid glands occurred at 4000 ppm in both sexes. Alterations of the thyroid gland already started at 400 ppm and increased in severity with dose and these findings were the predominant reason for dose selection for the carcinogenicity study.

In the carcinogenicity study mean body weight was only slightly and inconsistently affected in low dose females and even in high dosed animals generally did not reach 10%. Survival did not indicate to a dose related effect (70, 70 and 78% for control, low and high dose males and 90, 82 and 82% for females). These data indicate that the MTD possibly was not reached. This is also indicated in the NCI report by the statement that the animals may have been able to tolerate higher doses. The non-neoplastic histopathological observations did not indicate to any specific organ possibly being affected by treatment.

The following tumour incidences (in controls, low and high dosed animals in %) were noted in the report

- Cortical adenomas: 0, 6, 6 % for males; 16, 2, 14 % for females

- Pheochromocytomas: 20, 20, 30 % for males; 0, 8, 12 % for females.

The incidence of pheochromocytomas was not statistically significant by the trend test or by pairwise comparison, but it was stated without further specification that the incidence appeared to be greater than normal and may have been related to treatment. The overall conclusion was that CaCN₂ was not clearly carcinogenic in rats. But the interpretation of this bioassay is surrounded by some uncertainties, namely the low number of control animals and the lack of historical control data. It should also be mentioned that the study on CaCN₂ was conducted in the same room as feeding studies with 4-chloro-o-toluidine and N-nitrosodiphenylamine.

Under consideration of the limited reliability of the comparison of tumour incidences in dosed vs. control animals (due to the small group size of the latter), the RAC (2015) listed historical control data from 20 NTP studies conducted in the 7 years up to 1995. For male rats the following control incidences were reported:

- Cortical adenomas 0.9% and for pheochromocytomas mean 24.4 % (range 10-46%)

and for females:

- Cortical adenomas 1.3% and for pheochromocytomas mean 2.7 % (range 0-6%)

 

Carcinogenicity in mice (Goodyer, 1988) with cyanamide (disregarded for evaluation of calcium cyanamide)

Crl:CD-1 mice (60 mice/sex/dose) were exposed to H₂CN₂ in drinking water at 70, 200 and 600 ppm of an aqueous formulation containing 50% H₂CN₂, i.e. 35, 100 and 300 ppm H₂CN₂. If this study would be used for read-across to CaCN₂, the additional exposures to calcium should be calculated taking into consideration that technical CaCN₂ also contains CaO. The amount of CaO is assumed to be in the range of 22%, similar to the material used in the NCI study. This leads to concentrations of about 40.5, 116 and 366 ppm for calcium at the exposure levels of H₂CN₂. The dose levels of H₂CN₂ corresponded to 4.2 -6.5, 12.2 -17.4 and 29.5 -46.1 mg/kg bw/d for males and to 5.5-8.2, 13.4-21.8 and 39-62.1 mg/kg bw/ for females. Initially the study aimed at a duration of 80 weeks but it was extended to 100 weeks for males and 104 weeks for females due to good survival in control and low dose animals. Over the first 6 weeks body weight was significantly depressed in all male dose groups and in mid and high dose females. Subsequently body weight gain was comparable to controls apart from high dose males. At the end of the study mean body weight gain of high dose males was only 62% compared to controls with 90% for high dose females. At termination, body weights were clearly lower in mid and high dose males and high dose females as compared to controls. This corresponded to an increased absolute and relative brain weight and relative testes weight in high dose males. Water consumption was reduced in mid and high dose mice that was on occasions statistically significant from controls. By the end of study survival was not impaired in males while at week 104 24, 20, 14 and 14 of 60 females had survived in control, low, mid and high dose groups. Therefore, the MTD was exceeded for high dose females based on survival and possibly and body weight, too. For high and mid dose males the MTD was reached based on body weight In high dose females there was a statistically significant increase of granulosa-theca cell tumours when one necrotic tumour in the control group was excluded from statistical assessment; after inclusion of this tumour the increased incidence no longer was significant. The incidences were 3/60, 1/59, 6/55 and 8/57 for the control, low, mid and high diose groups. The historical control incidence was 0-3/51 cases in 10 previous control groups. One of the 3 tumours in the concurrent control group showed a necrotic lesion and therefore the diagnosis was equivocal.

 

Chronic toxicity in rats (Osheroff, 1991) with cyanamide (disregarded for evaluation of calcium cyanamide)

20 Sprague-Dawley rats/sex/dose were exposed by gavage to daily doses of 2.5, 7.5 and 30 mg/kg bw/d H₂CN₂ in water. Due to poor general health status, dose levels were reduced at week 17 to 1, 2.5 and 7.5 mg/kg bw/d. Apart from standard parameters for such a chronic study, serum chemistry included T3, T4 and TSH. Survival was not affected until week 52 but at termination at week 92 survival was significantly reduced for mid dose females. Clinical observations at the beginning of exposure demonstrated debilitation of health for high dose males and females (hunched appearance, tremors, rough hair coat). After lowering of dose levels clinical signs subsided. The initial doses led to significant depression of body weight and body weight gain (mid and high dose males and females). At termination body weight were significantly depressed as were body weight gains (week 0-52) for high dose animals (both sexes). Also, food consumption was significantly reduced for high dose males and females. At the high dose until week 16, clinical chemistry (predominantly in high dose animals) showed decreases of the erythron, platelets, parameters potentially related to reduced feed uptake (glucose, albumin, globulin) and thyroxine while white blood cell counts were increased. After dose reduction, at termination levels of T4 (high dose males) and T3 (high and mid dose males, high dose females) were decreased as the most relevant finding. Relative organ weights were increased in high dose animals for brain and testes (probably related to reduced body weight) and for liver and kidney (males) and thyroid (males and females). Histopathological alterations were only observed in the thyroid gland of mid and high dose males and high dose females characterized by microfollicles with reduced colloid. In conclusion, the original high dose level of 30 mg/kg bw/d of H₂CN₂ exceeded the MTD and the NOAEL for at least 91 weeks was 1 mg/kg bw/d.

In relation to the ovary as potential target organ in the mouse carcinogenicity study, sertoliform tubular hyperplasia as possible preneoplastic lesion was analysed. The total incidence in unscheduled and scheduled deaths amounted to 8, 4, 6 and 9 out of 20 for control, low, mid and high dose females with no increase in the grading for severity. Therefore, H₂CN₂ was not associated with a neoplastic response in the ovary of rats.