Cyanamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cyanamide

EC Number:

206-992-3

EC Name:

Cyanamide

Cas Number:

420-04-2

Molecular formula:

CH2N2

IUPAC Name:

cyanamide

Test material form:

other: aqueous solution

Details on test material:

- Test substance: Aqueous Hydrogen cyanamide
- Purity: 50 % w/w, 53 % w/v active substance
- Appearance: Clear colourless liquid
- Lot/Batch number: 07-07-87

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Hazelton Research Products, Inc., Cumberland, Virginia
- Age at study initiation: 6-8 months old

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Results of analytical chemistry analysis indicated that the test solutions were generally prepared within the acceptable range of target concentrations. Stability evaluation of the low and high-dose solutions were established and showed stability over the entire range.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

for the first two weeks; corresponding to 0 mg/kg bw pure active substance Hydrogen cyanamide

Doses / concentrations 2

Dose / conc.:

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

Remarks:

for the first two weeks; corresponding to 0.1 mg/kg bw pure active substance Hydrogen cyanamide

Doses / concentrations 3

Dose / conc.:

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

Remarks:

for the first two weeks; corresponding to 0.5 mg/kg bw pure active substance Hydrogen cyanamide

Doses / concentrations 4

Dose / conc.:

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

Remarks:

for the first two weeks; corresponding to 2.5 mg/kg bw pure active substance Hydrogen cyanamide

Doses / concentrations 5

Dose / conc.:

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

Remarks:

for the remaining 50 weeks; corresponding to 0 mg/kg bw pure active substance Hydrogen cyanamide

Doses / concentrations 6

Dose / conc.:

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

Remarks:

for the first two weeks; corresponding to 0.2 mg/kg bw pure active substance Hydrogen cyanamide

Doses / concentrations 7

Dose / conc.:

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

Remarks:

for the first two weeks; corresponding to 1 mg/kg bw pure active substance Hydrogen cyanamide

Doses / concentrations 8

Dose / conc.:

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

Remarks:

for the first two weeks; corresponding to 5 mg/kg bw pure active substance Hydrogen cyanamide

No. of animals per sex per dose:

4 animals per sex per dose

Control animals:

other: A concurrent control group administered with water by gavage

Positive control:

No positive control

Examinations

Observations and examinations performed and frequency:

All animals were observed twice daily for mortality and moribundity and once daily for clinical signs. Individual body weights and food consumption were recorded weekly for weeks 0 - 16, and every fourth week thereafter. Ophthalmoscopic examinations were performed pre-treatment to initiation and at termination. Evaluation of clinical pathology parameters (haematology, serum chemistry, thyroid function tests and urinalysis) were evaluated prior to initiation of treatment and at weeks 13, 26 and 52.

Sacrifice and pathology:

After 52 weeks of compound administration all animals were sacrificed and subjected to complete gross examination. Organ weight evaluations (absolute and relative) and histomorphological examinations were performed on selected organs.

Statistics:

The differences between the control groups and the test substance treated groups were statistically examined by the dunnets test criteria p< 0.05.

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):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

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:

no effects observed

Details on results:

General observations:
There were no deaths during the course of this study. As compound related clinical sign, salivations were noted in all high dose animals and in one medium-dosed female as well as tremors were seen in three high-dosed males and two high dose females. At termination of the study, no ophthalmological abnormalities occurred related to the test compound.
Mean body weights were reduced at week 13 and more pronounced in week 52 in both sexes at 5.0 mg/kg bw, when compared to the control groups Compared with the control animals, mean cumulative body weight gain, an indication of growth, was statistical significant decreased in the high-dose groups. Mean food consumption values were generally comparable between each dose group and control.
Compared with the control animals, mean cumulative body weight gain, an indication of growth, was statistical significant decreased in the high-dose groups. Mean food consumption values were generally comparable between each dose group and control.
Haematology and clinical chemistry:
Anaemia was seen in the female dogs of the high dose-group at week 13 with statistical significance based on reduced haemoglobin and haematocrit. The data of the males and of both sexes at week 52 were even suspected for anaemia. All erythrocyte parameters (MCV, MCH, MCHC) were reduced. The statistical significant decreases of MCV and MCH at 1.0 mg/kg bw at week 52 were not contributed to be substance-related, since no anaemia occurred at this dose.
Additionally, lymphocytes were decreased at week 13 and more pronounced at week 52 in the high-dose groups of both sexes. Leucocytes and segmented neutrophiles were reduced in the high dose males. Monocytosis was seen at 5.0 mg/kg bw in both sexes.
Following parameters were decreased in the high dose groups in week 13 and more pronounced in week 52: Serum albumine in both sexes and calcium in the female. Globulin was increased in week 13 and 52. Glucose and urea blood nitrogen was statistical significant decreased in females -and phosphorus in males- when compared to control animals. Statistically significant decreases were noted for aspartate aminotransferase (AST) in the high dose animals and for alanine aminotransferase (ALT) in the high dose males and females only at week 13. A statistical significant increase of cholesterol was seen at week 13 at 1 mg/kg bw in females and at 5 mg/kg bw in both sexes.
Decreased thyroxine values (T4) were observed in the high dose groups with statistically significance in the male at week 52 (-45 % vs –41 % in females). At 13 weeks the decrease was 28 % in the males and 46 % in the females compared to the control dogs. T3 values were lower in the high-dosed groups, but without statistical significance. The data of TSH were not shown, since not all animals were tested in the groups.
Results of urinalysis were generally unremarkable.
Gross pathology, organ weights and histopathology:
A pale area on the spleen was seen in one medium dose male and in two high dose females. Other findings were not related to treatment with the test compound.
The only significant finding in organ weight data was an increased thyroid/parathyroid weight-to-terminal body weight ratio value in the high dose females.
Compound-related histomorphologic alterations were observed in the high-dose groups. Thymic atrophy noted in one high dose female was accompanied by demodicosis. An increased incidence and severity of brown pigment was present in Kupfer cells of the livers in the high dose of both sexes, increased extramedullary hematopoiesis was present in the spleen of two high dose males and microcholeliths were present in the gallbladder of one high dose male and two high dose females. Other findings in high dose dogs were inflammation of the testes and decreased spermatogenic activity. Thymic atrophy was noted in all four high dose males and in one high dose female, that also showed demodicosis.
Other histopathological effects were not attributed to the treatment, since they were incidental and not dose-dependent.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

haematology

other: The NOAEL is 1.0 mg/kg bw/day of the pure active subsatnce cyanamide. Haematological changes which correlated to histomorphological findings, macroscopic findings and histopathological changes at 5 mg/kg bw/day dose group.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

Table 1: Body weight gain at week 13 and 52:

Pure active substance cyanamide (mg/kg bw/day)** (n = 4 each sex)	Males Body weight (kg)		Males Bw gain (kg)		Females Body weight (kg)		Females Bw gain (kg)
Week	week 13	week 52	week 13	week 52	week 13	week 52	week 13	week 52
0	10.9	12.0	1.0	2.1	9.2	9.9	1.3	2.0
0.2	10.9	12.2	0.9	2.2	10.4	11.7	1.9*	3.2
1.0	11.3	12.4	1.1	2.2	8.6	9.1	1.2	1.6
5.0	10.3	10.3	0.6	0.7*	8.5	8.2	0.2*	0.0*

*   Significantly different from control by the Dunnets test criteria, p<0.05

** Dose levels during weeks 0-2 were 0.1, 0.5 and 2.5 mg/kg bw/day pure active ingredient cyanamide

 

Table 2: Haematology in the dog (week 13 and 52):

Pure active substance cyanamide (mg/kg bw/day) (n = 4 each sex)	0		0.2		1.0		5.0
Week	week 13	week 52	week 13	week 52	week 13	week 52	week 13	week 52
Males:
Erythocytes (10^6/µL)	7.07	6.90	6.84	6.93	7.03	7.72	6.68	7.03
Haemoglobin (g/dL)	16.1	16.3	15.5	15.8	15.8	17.0	13.8	14.1
Haematocrit (%)	46.2	46.3	44.1	45.3	45.1	49.0	40.5	41.5
MCV (fl)	65.5	67.0	64.6	65.2	64.2	63.5*	60.7	58.9*
MCH (pg)	22.9	23.6	22.6	22.7*	22.4	22.1*	20.7*	20.1*
MCHC (g/dL)	34.9	35.1	35.1	34.8	35.0	34.8	34.1	34.1*
Leucocytes^+(10^3/µL )	9.8	8.7	nd^#	nd^#	nd^#	nd^#	9.2	7.4*
Segmented neutrophils	6.2	6.3	nd^#	nd^#	nd^#	nd^#	3.3*	3.0*
Lymphocytes	2.8	1.8	nd^#	nd^#	nd^#	nd^#	5.0*	3.6
Monocytes	0.3	0.3	nd^#	nd^#	nd^#	nd^#	0.5	0.6*
Females
Erythocytes (10^6/µL)	6.63	6.16	6.53	6.72	6.92	7.06	6.38	6.36
Haemoglobin (g/dL)	15.6	14.5	15.2	15.6	15.3	15.1	13.5*	13.0
Haematocrit (%)	44.3	41.3	43.5	44.5	43.9	43.9	39.3*	38.4
MCV (fl)	66.8	67.1	66.6	66.3	63.4	62.3*	61.6*	60.4*
MCH (pg)	23.6	23.5	23.3	23.2	22.2	21.5*	21.1*	20.5*
MCHC (g/dL)	35.3	35.0	34.9	35.0	34.9	34.5	34.3*	33.9*
Leucocytes^+(10^3/µL )	9.9	9.0	nd^#	nd^#	nd^#	nd^#	11.3	11.8
Segmented neutrophils	6.5	6.6	nd^#	nd^#	nd^#	nd^#	5.0	7.4
Lymphocytes	2.8	1.6	nd^#	nd^#	nd^#	nd^#	5.2*	3.6*
Monocytes	0.3	0.3	nd^#	nd^#	nd^#	nd^#	0.8	0.9*

* Significantly different from control, by Dunnetts test criteria, p < 0.05

⁺corrected count,^#no data

 

Table 3: Clinical chemistry (week 13 and 52):

Pure active substance cyanamide (mg/kg bw/day) (n = 4 each sex)	0		0.2		1.0		5.0
Week	week 13	week 52	week 13	week 52	week 13	week 52	week 13	week 52
Males:
Total cholesterol (mg/dL)	165	136	176	132	191	153	249*	189
Serum albumine (g/dL)	3.8	3.8	3.7	3.7	3.7	3.6	3.3*	3.2*
Globulin (g/dL)	2.5	2.7	2.7	2.8	2.6	2.8	3.0	3.3
Phosphor (mg/dL)	5.2	3.5	5.4	3.8	5.1	3.8	5.3	4.4*
AST (U/L)	28	30	25	30	25	27	16*	25
ALT (U/L)	54	72	42	41	56	64	26*	55
Females
Total cholesterol (mg/dL)	158	153	195	165	218*	230	222*	215
Serum albumine (g/dL)	3.7	3.6	3.7	3.6	3.6	3.5	3.1*	2.8*
Globulin (g/dL)	2.4	2.8	2.5	2.9	2.8	2.9	3.3*	3.5
Phosphor (mg/dL)	5.2	8.6	5.9	5.6	5.6	5.0	5.6	4.1
Serum glucose (mg/dL)	98	87	102	90	96	88	87	71*
Blood urea nitrogen (mg/dL)	14	15	11	12	13	13	9	8*
Creatinine (mg/dL)	0.9	0.9	0.9	0.8	0.9	0.8	0.8*	0.6*
Calcium (mg/dL)	10.6	9.8	10.6	9.6	10.7	9.8	9.8*	8.9*
AST (U/L)	28	30	27	28	26	37	17*	23
ALT (U/L)	58	45	31	33	33	31	23*	25

 

Table 4: Thyroid hormones:

Pure active substance cyanamide (mg/kg bw/day) (n = 4 each sex)	0			0.2			1.0			5.0
Week	-2	13	52	-2	13	52	-2	13	52	-2	13	52
Males
T3 (ng/dL)	75.6	61.9	54.0	58.9	66.3	56.7	66.0	64.6	66.2	75.5	48.3	62.6
T4 (µg/dL)	3.1	2.5	1.8	2.6	2.5	1.9	2.9	2.2	1.6	2.6	1.8	1.0*
Females
T3 (ng/dL)	72	56.1	56.0	75.3	69.7	61.5	77.4	62.6	65.8	77.8	39.3	41.4
T4 (µg/dL)	3	2.6	2.2	3.3	3.2	2.3	2.8	2.4	2.4	3.8	1.4	1.3

 

Table 5: Organ weights:

Pure active substance cyanamide (mg/kg bw/day) (n = 4 each sex)	0	0.2**	1**	5**
Males:
Absolute liver weight (g)	287	266	278	291
Relative liver weight (%)	2.5	2.2	2.3	2.9
Absolute thyroid/parathyroid weight (g)	1.16	0.88	0.93	1.13
Relative^athyroid/parathyroid weight ( %)	0.010	0.007	0.009	0.011
Females:
Absolute liver weight (g)	214	250	207	221
Relative liver weight (%)	2.3	2.2	2.3	2.9
Absolute thyroid/parathyroid weight (g)	0.87	0.92	0.84	1.08
Relative^athyroid/parathyroid weight ( %)	0.009	0.008	0.009	0.014*

 ^aRelative weight is defined as the organ to body weight ratio.

* Significantly different from control by the Dunnets test criteria, p<0.05.

** Dose levels during weeks 0-2 were 0.1, 0.5 and 2.5 mg/kg bw/day pure active ingredient cyanamide

 

Applicant's summary and conclusion

Conclusions:

The NOAEL of pure active substance cyanamide is 1.0 mg/kg bw/day in beagle dogs.

Executive summary:

Aqueous Hydrogen cyanamide was administered via gavage over a period of one year to 4 male and 4 female dogs of each sex and group, which were 6 - 8 months old at study begin. For the first two weeks the dose levels were 0, 0.2, 1.0 and 5.0 mg/kg bw/day corresponding to 0, 0.1, 0.5 and 2.5 mg/kg bw/day pure active substance Hydrogen cyanamide. The remaining 50 weeks doses of 0, 0.4, 2.0 and 10.0 mg/kg bw/day (corresponding to 0, 0.2, 1.0 and 5.0 mg/kg bw/day pure active substance Hydrogen cyanamide) were administered. An additional group of 4 dogs/sex was administered the vehicle (distilled water) and thus served as the concurrent control group. During the study time all animals were observed for mortality and moribidity, for clinical signs, individual body weights and food consumption, ophthalmoscopic examinations and clinical pathology parameters. After 52 weeks of compound administration all animals were sacrificed and subjected to complete gross examination. Organ weight evaluations (absolute and relative) and histomorphological examinations were performed on selected organs. Clinical pathology changes in the high dose group in males and females. Gross macroscopic findings in the spleen in one medium dose male and two high dose females. Increase in thyroid/parathyroid organ-to terminal body weight ratio in the high dose females. Histopathological effects in the high dose in the liver in males and females, in the spleen in two males, gallbladder in one male and two females, in the testes and in the thymus in 4 males and one female. The NOAEL was therefore determined to be 1 mg/kg bw/day pure active ingredient cyanamide for dogs based on the significantly reduced body weight/gain in both sexes, the occurrence of significant anaemia in female dogs (reduced red blood cell (RBC) parameters also in males) and significantly reduced T4 in dogs at 5 mg/kg bw/d and reduced (not statistically significant) T3. Whether the testicular findings in one dog were treatment-related could not be excluded with certainty.