Cyanamide

Endpoint summary

Description of key information

Results in subacute studies:

1. Oral (gavage), 28 days, rat: Depression in body weights, body weight gains and food consumption at 20 and 40 mg/kg bw/day. Histopathological findings in thyroid, liver and spleen at 10, 20 and 40 mg/kg bw/day. No effects in the lowest dose. NOAEL: 5 mg/kg bw/day


2. Dermal 21 days, rabbit: Substance related effects in organ weights (heart, liver, kidney and testes) and microscopic and macroscopic changes in some organs in both sexes. No relevant treatment related effects at lower doses. NOAEL: 25 mg/kg bw/day


3. Inhalation 14 days, rat: Decrease in weights and variations in food consumption were observed in all dose groups treated with the substance. Macroscopic and microscopic effects in brain, lungs, heart and liver were observed at the high dose group in m/f rats. No relevant treatment related effects at the low and mid-dose group. NOAEL: 0.2629 g/m³

 

Results in subchronic studies:

1. Oral (feeding), 90 days, rat: Marked effects in the thyroid in males and in females at 4.5 mg/kg bw/day; additionally increases in erythrocyte counts. NOAEL: 1.5 mg/kg bw/day


2. Oral (gavage), 90 days, dogs: Slight anemia correlated with a slight hypothyroidism and a slight monocytosis found at 2 and 6 mg/kg bw in both sexes. Microscopic examination revealed marked changes at 6 mg/kg bw in the testes together with a reduced testes weight. No substance related effects at 0.6 mg/kg bw/day in male and female dogs. NOAEL: 0.6 mg/kg bw/day


3. Oral (gavage), 90 days, dogs: Decrease of body weight and testes weight; microscopic and macroscopic changes were observed at the higher dose group. No substance related effects at 0.6 mg/kg bw/day in male and female dogs. NOAEL: 0.6 mg/kg bw/day

 

Results in chronic studies:

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


2. The NOAEL of pure active substance cyanamide is 1.0 mg/kg bw/day in Sprague-Dawley rats.

Peer Review

Review of the studies support that the NOAEL of 1 mg/kg bw/day observed in the one year dog study is the relevant starting point for a DNEL derivation.

Classification as STOT RE2 (thyroid) is triggered based on a detailed evaluation by RAC considering siginificant effects on thyroid particular in the rat but also in the dog. The conclusion takes into account the particular sensitivity of rats as well as evidence of probable higher sensitivity of dogs compared to humans. The apparent lack of adverse effects on the thyroid from the cyanamide treated human population is also considered as supportive information. In addition, a relation between the effects on thyroid observed in vivo and the effects observed in current TPO inhibition studies on rat/dog/humans in vitro cannot be excluded.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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

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

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

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

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

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

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

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

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

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.

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.

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.

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

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

 

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1975

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Stability of the test substance, correctness of the dietary preparations as well as homogeneity of the test substance in the dietary preparation not proven; no individual data; no ophthalmological examination; determination of food consumption during the first 4 weeks and in week 10 to 12, instead of a weekly determination throughout the study; no determination of water consumption; no determination of haematocrit, platelet count and blood clotting time/potential; no determination of sodium, potassium, glucose, total cholesterol, urea, blood urea nitrogen, creatinine; no determination. of volume, osmolality, specific gravity and blood/blood cells during urinalysis; no absolute organ weights; no histopathological investigation of brain, pituitary parathyroid, thymus, spleen, heart, female mammary gland, skin and bone marrow. Although according to the study report the following organs were investigated (but no findings were observed): brain, thymus, spleen and heart.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

see above in "rationale for relaibility".

GLP compliance:

no

Remarks:

study was conducted prior to the implementation of GLP.

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Weanling albino rats from the CIVO colony (Wistar derived)

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Not indicated

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analytical verification was made

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily intake

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

20 ppm (equivalent to 10 ppm pure active substance Cyanamide) corresponding to mean daily intake of 1 mg/kg bw/day (equivalent to 0.5 mg/kg bw/day pure active substance Cyanamide).

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

60 ppm (equivalent to 30 ppm pure active substance Cyanamide) corresponding to mean daily intake of 3 mg/kg bw/day (equivalent to 1.5 mg/kg bw/day pure active substance Cyanamide)

Dose / conc.:

9 mg/kg bw/day (nominal)

Remarks:

180 ppm (equivalent to 90 ppm pure active substance Cyanamide) corresponding to mean daily intake of 9 mg/kg bw/day (equivalent to 4.5 mg/kg bw/day pure active substance Cyanamide).

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

Not indicated

Positive control:

No positive control

Observations and examinations performed and frequency:

The animals were observed for clinical symptoms and mortalities. Body weights were recorded weekly and food intake of each group was measured during the first four weeks and in week 10 to 12. Haematological investigations were performed during week 12 in all rats (erythrocyte count, haemoglobin, packed cell volume, leucocytes, lymphocytes, differential blood count), urinanalysis (appearance, pH, glucose, protein, occult blood, ketones and microscopy of the sediment) was done in week 13. At autopsy in week 14 clinical chemical investigations were performed in all rats: glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase, alkaline phosphatase, total serum protein and serum albumin. All animals were subjected to gross macroscopic investigation. The weights of the heart, kidneys, liver, spleen, brain, gonads, thymus, thyroid and adrenals were determined. Histopathology was confined to all rats of the highest dosage group and of the control groups.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Other examinations:

No other examinations

Statistics:

The statistical test was not indicated

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):

no effects observed

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

General observations:
A test substance-related mortality did not occur. No increase in the incidence of clinical signs was observed in any of the groups fed with cyanamide in the diet.
The body weights of all test groups were generally slightly higher compared to the control groups in correspondence with the food consumption but without a tendency of a dose-related response.

The results of the haematological investigations showed a significant increase in erythrocyte counts in males of the highest dose group. A slight decreased haematocrit value in males was only obvious in the mid dose group. No statistical significant changes in the leucocyte and differential blood count were seen.

Clinical chemical investigations demonstrated no significant changes in serum enzyme activities or in serum protein values. There were no effects on urinalysis.

Gross pathology, organ weights and histopathology:
No test substance-related gross lesions were observed at necropsy. Organ weight determinations revealed a significant increase in the relative liver weight in males and a significant decrease in the relative weight of the thymus in female rats receiving 4.5 mg/kg bw/day.

Histopathological investigations revealed treatment-related changes in the thyroids.
Thyroid effects were clear seen in the high dose groups. The effects in one male at 1.5 mg/kg bw were not seen as an adverse effect, since the incidence was 1/20 rats. The histopathological changes observed in other organs were equally distributed between control and test groups or occurred only in a single animal.

Key result

Dose descriptor:

NOAEL

Effect level:

1.5 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: The NOAEL (1.5 mg/kg bw/day pure active substance cyanamide, equivalent to 30 ppm in the diet) is based on the histopathological findings in the thyroid at 4.5 mg/kg bw/day cyanamide (equivalent to 90 ppm in the diet) in males and females.

Key result

Dose descriptor:

LOAEL

Effect level:

4.5 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4.5 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Body weights

Pure active substance cyanamide (mg/kg bw/day) (n = 10 each sex)	Males Body weight (g) day 0	Males Body weight (g) day 91	Females Body weight (g) day 0	Females Body weight (g) day 91
0	54.8	311	51.7	187
0.5	54.9	326	51.6	193
1.5	54.8	327	51.5	196
4.5	54.8	320	51.3	193

 

Food consumption (in g)

 

Pure active substance cyanamide (mg/kg bw/day) (n = 10 each sex)	Males week 0-2	Males week 3-4	Males week 11-12	Females week 0-2	Females week 3-4	Females week 11-12
0	20.2	31.2	31.5	15.7	21.7	21.7
0.5	20.9	33.2	33.5	17.0	22.6	23.1
1.5	21.5	31.3	32.0	17.5	23.1	23.4
4.5	21.7	33.2	33.5	17.1	23.7	24.9

Erythrocytes indices

 

 

Organ weights

 

Pure active substance cyanamide (mg/kg bw/day) (n = 10 each sex)	0	0.5	1.5	4.5
Males:
Relative^aliver weight ( % )	3.26	3.35	3.28	3.44*
Relative^athymus weight ( % )	0.93	0.92	0.103	0.86
Females:
Relative^aliver weight ( % )	3.05	3.00	3.06	3.18
Relative^athymus weight ( % )	0.148	0.152	0.142	0.123*

 

Incidences of histopathological findings in the thyroid

 

Pure active substance cyanamide (mg/kg bw/day) (n = 10 each sex)	0	0.5	1.5	4.5
Males:
Thyroid
Predominantly small follicular lumens without colloid, separated by proliferating epithelial cells and interfollicular cells	0	0	1	3
Females:
Thyroid
Predominantly small follicular Lumens without colloid, separated by proliferating epithelial cells and interfollicular cells	0	0	0	2

 

Conclusions:

The NOAEL in the 90-day feeding study in rats was 1.5 mg/kg bw/day pure active substance cyanamide (equivalent to 30 ppm in the diet) based on the histopathological findings in the thyroid at 4.5 mg/kg bw/day cyanamide (equivalent to 90 ppm in the diet) in males and females.

Executive summary:

In a 90-day feeding study, Cyanamid L500 was administered to weanling albino rats from the CIVO colony (Wistar derived). 10 males and 10 females were used per concentration at dietary dose levels of 0, 20, 60 and 180 ppm (equivalent to 0, 10, 30 and 90 ppm pure active substance cyanamide) corresponding to mean daily intake of 0, 1, 3 and 9 mg/kg bw/day (equivalent to 0, 0.5, 1.5 and 4.5 mg/kg bw/day pure active substance cyanamide). A test substance-related mortality did not occur. Marked effects were observed in the thyroid in males and in females (non-neoplastic) at 4.5 mg/kg bw/day. Significant increase in erythrocyte counts was also observed in the highest dose group. The NOAEL in the presented study was therefore detemined to be 1.5 mg/kg bw/day pure active substance cyanamide (equivalent to 30 ppm in the diet).

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Not indicated

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test substance was prepared weekly and administered daily by gavage at a dose volume of 10 ml/kg.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the dosing solution over a 8 h period was demonstrated. The correctness of the concentrations in the vehicle was analytically verified.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily by gavage

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

corresponding to 5 mg/kg bw/day active substance Hydrogen cyanamide.

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

corresponding to 10 mg/kg bw/day active substance Hydrogen cyanamide.

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

corresponding to 20 mg/kg bw/day active substance Hydrogen cyanamide.

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

corresponding to 40 mg/kg bw/day active substance Hydrogen cyanamide.

No. of animals per sex per dose:

5 rats per sex per concentration

Control animals:

yes, concurrent vehicle

Details on study design:

Not indicated

Positive control:

An additional group of 5 males and 5 females received the vehicle (distilled water) and served as the concurrent control group.

Observations and examinations performed and frequency:

Animals were observed twice daily for mortality and/or moribundity and once daily for overt signs of toxicity. Body weight and food consumption measurements were performed once weekly.

Sacrifice and pathology:

Following 28 days of treatment, all animals were sacrificed and subjected to a complete gross necropsy. Organ weight evaluations (absolute and relative) and histopathological examination was performed on all animals.

Other examinations:

Clinical pathology parameters (haematology and clinical chemistry and thyroid function test) were evaluated.

Statistics:

The difference in different examined parameters (body weights, food consumption , haematology, clinical chemistry, thyroid function, organ weights) between the dose-treated groups and the control groups was tested statistically by teh Dunnets test, criteria p< 0.05.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

General observations:
The death of one female animal at 40 mg/kg bw/day on day 29 is considered to be due to the general decrease in its health status. Rough hair coat was observed in three males and one female in the high dose group at the week 4 physical examination. All other clinical observations noted appeared to be incidental in nature and were not contributed to the administration of cyanamide.
The mean body weights at termination were statistical significant decreased at 20 mg/kg bw/day in males (-15 %) and at 40 mg/kg bw/day in both sexes (-25.4 % for males and -19.6 % for females), when compared to the control groups. Mean body weight gain values for weeks 0 - 4 were significantly decreased for males and females at 20 (- 30.3 % in males, -35.9 % in females) and at 40 mg/kg bw/day (-53.0 %/-57.3 %) when compared to the corresponding control.
Total food consumption for weeks 1 - 4 was decreased at 20 and 40 mg/kg bw/day in females and males when compared to the corresponding control.

Haematology and clinical chemistry:
Evaluation of the haematology data revealed a decrease in red cell parameters (RBC, Hct and Hb) in the high-dose animals at week 4, that was statistically significant in the males and in the females (except RBC in females). This reduction was associated with statistical significant decreases in MCH and MCHC in males of the highest dose. Monocytes were statistical significant increased in the high dose group of males and females.
Several statistical significant changes were noted in clinical chemistry data: the male mean globulin levels were decreased at 20 and 40 mg/kg bw/day, while the mean total bilirubin and blood urea nitrogen values were elevated at 40 mg/kg bw/day. Those effects were seen in females without statistical significance. Other incidental changes included a decrease in thrombocytes and an increase in WBC and lymphocytes almost most pronounced in the highest dose groups of both sexes. Decreased total protein in males and decreased calcium in females were statistically significant decreased only at 20 mg/kg bw/day.
At 40 mg/kg bw/day the mean TSH values were increased by 100 %, whereas T4 concentrations were decreased by 28 % in both sexes compared to control animals.

Gross pathology, organ weights and histopathology:
No gross pathology findings were attributed to the administration of cyanamide
Mean relative kidney weights (females and males) and mean relative liver weights (males) were statistical significantly increased already at 20 mg/kg bw/day, whereas the absolute testes weight was statistical significantly decreased. Most of those effects were pronounced in the high dose group. Additionally the mean relative weight of the brain with stem in male rats and the mean relative liver weight in females were statistically significantly increased in the highest dose.
Compound related histopathological lesions were observed in sections of thyroid, spleen and liver. In the thyroid, the incidence of follicular cell hyperplasia was increased at 10 mg/kg bw/day and higher in male rats. A decreased colloid content and small and closely packed follicles were seen already, even though to a marginal extent, at 5 mg/kg bw/day in the males. The females were less sensitive: thyroid effects were seen dose dependent at 20 and 40 mg/kg bw/day. In the spleen, the incidence of pigmented macrophages was increased in the high dose group in male rats and in the female rats at 10, 20 and 40 mg/kg bw/day. In the liver, the incidence of biliary hyperplasia was increased dose dependent at 10 mg/kg bw/day and higher in male rats. No other compound related histomorphological lesions were observed.

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: findings in thyroid and liver

Key result

Dose descriptor:

LOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

histopathology: neoplastic

histopathology: non-neoplastic

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

Conclusions:

NOAEL (subacute oral rat)= 5 mg/kg bw/day

Executive summary:

In a 28-day study, Cyanamid L500 was administered by gavage (10 mL/kg) to male and female Sprague Dawley rats (5 rats/sex/concentration) at dose levels of 10, 20, 40, and 80 mg/kg bw/day, corresponding to 5, 10, 20 and 40 mg/kg bw/day active substance hydrogen cyanamide. An additional group of 5 males and 5 females received the vehicle (distilled water) and served as the concurrent control group. Animals were observed twice daily for mortality and once daily for overt signs of toxicity. Body weight and food consumption measurements were performed once weekly. At termination clinical pathology parameters (haematology and clinical chemistry and thyroid function test) were evaluated. Following 28 days of treatment, all animals were sacrificed and subjected to a complete gross necropsy. Organ weight evaluations (absolute and relative) and histopathological examination were performed on all animals. The NOAEL was determined to be 5 mg/kg bw/day of pure active substance cyanamide based on the findings in the thyroid seen in all males at 10 mg/kg bw/day and higher. In contrast, marginal histopathological effects seen at 5 mg/kg bw/day were not accompanied by follicular cell hyperplasia and were therefore not assessed as adverse. In females, thyroid changes were obvious in the mid and high dose group with a dose-dependent increase of incidences. At 10 mg/kg bw/day and above, additional histopathological findings were found in the liver of males (bile duct hyperplasia) and in splenic pigmentation in females and males but only at the highest dose of cyanamide. Anaemia occurred in both sexes at 40 mg/kg bw/day with statistical significance. Thyroid function tests demonstrated increased thyroid stimulating hormone (TSH) as well as decreased thyroxine (T4) at 40 mg/kg. The LOAEL for cyanamide was considered to be 10 mg/kg bw/d, based on the more significant thyroid effects in males (follicular cell hyperplasia, decreased colloid content and small, densly packed follicles).

In a 28-day study, Cyanamid L500 was administered by gavage (10 mL/kg) to male and female Sprague Dawley rats (5 rats/sex/concentration) at dose levels of 10, 20, 40, and 80 mg/kg bw/day, corresponding to 5, 10, 20 and 40 mg/kg bw/day active substance hydrogen cyanamide. An additional group of 5 males and 5 females received the vehicle (distilled water) and served as the concurrent control group. Animals were observed twice daily for mortality and once daily for overt signs of toxicity. Body weight and food consumption measurements were performed once weekly. At termination clinical pathology parameters (haematology and clinical chemistry and thyroid function test) were evaluated. Following 28 days of treatment, all animals were sacrificed and subjected to a complete gross necropsy. Organ weight evaluations (absolute and relative) and histopathological examination were performed on all animals. The NOAEL was determined to be 5 mg/kg bw/day of pure active substance cyanamide based on the findings in the thyroid seen in all males at 10 mg/kg bw/day and higher. In contrast, marginal histopathological effects seen at 5 mg/kg bw/day were not accompanied by follicular cell hyperplasia and were therefore not assessed as adverse. In females, thyroid changes were obvious in the mid and high dose group with a dose-dependent increase of incidences. At 10 mg/kg bw/day and above, additional histopathological findings were found in the liver of males (bile duct hyperplasia) and in splenic pigmentation in females and males but only at the highest dose of cyanamide. Anaemia occurred in both sexes at 40 mg/kg bw/day with statistical significance. Thyroid function tests demonstrated increased thyroid stimulating hormone (TSH) as well as decreased thyroxine (T4) at 40 mg/kg. The LOAEL for cyanamide was considered to be 10 mg/kg bw/d, based on the more significant thyroid effects in males (follicular cell hyperplasia, decreased colloid content and small, densly packed follicles).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 mg/kg bw/day

Study duration:

chronic

Species:

dog

Quality of whole database:

6 studies (acute to chronic) with aqueous solution of cyanamide in two species (rat and dog)

Organ:

thyroid gland

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only 14 days of exposure instead of 28 days; Only 3 animals of each sex of the intermediate dose groups; actual concentration spaces are to narrow between the low and mid dose groups.

Qualifier:

according to guideline

Guideline:

other: Gaitonde committee guidelines

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

only 3 animals of each sex of the intermediate dose groups; actual concentration spaces are to narrow between the low and mid dose groups, satellite group.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

head only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Particles generated size: < 5 µm in diameter

Details on inhalation exposure:

The aerosol of the test substance was generated by infusing it through the nebulizer which generates particles less than 5 µm in diameter. During the exposure, samplings of the atmosphere from the inhalation chamber were done for the HPLC determination of actual concentrations of Hydrogen cyanamide.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During the exposure, samplings of the atmosphere from the inhalation chamber were done for the HPLC determination of actual concentrations of Hydrogen cyanamide.

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

a daily treatment: 6 hours/ per day

Dose / conc.:

0.148 mg/L air (analytical)

Remarks:

range 0.0848-0.1916; nominal: 2.5 g/m³ of Hydrogen cyanamide

Dose / conc.:

0.263 mg/L air (analytical)

Remarks:

range 0.1489-0.3270; nominal: 5 g/m³ of Hydrogen cyanamide

Dose / conc.:

0.799 mg/L air (analytical)

Remarks:

range 0.4080-1.1184:; nominal: 9.375 g/m³ of Hydrogen cyanamide

No. of animals per sex per dose:

10 rats (5 males and 5 females) were exposed to the low and high doses whereas 16 rats (8 males and 8 females) were exposed to the intermediate dose. An additional group of 8 males and 8 females served as the concurrent control group.

Control animals:

yes, concurrent no treatment

Details on study design:

On day 15 after start of the exposure 5 males and 5 females of each group were necropsied. The remaining animals (3 males and 3 females) of the control and intermediate dose group were maintained without any exposure for a further period of 2 weeks (satellite group, to investigate recovery).

Positive control:

No positive control

Observations and examinations performed and frequency:

Animals were observed daily for mortality and/or moribundity and on hourly basis during exposure for toxic symptoms. Body weight and food consumption measurements were performed daily. Clinical pathology parameters (haematology and clinical chemistry) were determined in rats after 15 days of exposure in the main groups and after 29 days in the animals of the satellite group (control and intermediate dose).

Sacrifice and pathology:

On day 15 after start of the exposure 5 males and 5 females of each group were necropsied. The remaining animals (3 males and 3 females) of the control and intermediate dose group were maintained without any exposure for a further period of 2 weeks (satellite group, to investigate recovery). After the scheduled exposure regimen all animals of the low and high dose group and 5 males and 5 females each from the control and intermediate group and on day 29 the remaining animals were necropsied and the lesions were noted grossly. Organ weight evaluations (absolute and relative) and histopathological examination were performed on all animals. The same procedure was performed in the remaining animals at day 29.

Statistics:

The difference in body weight and organ weight between the animals of the control groups and the test substance treated groups was examined by the Student´s test criteria, p< 0.01 or p<0.05.

Clinical signs:

no effects observed

Mortality:

no mortality observed

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:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

No mortality was observed in rats belonging to any of the groups throughout the study period. No clinical signs of toxicity were found in rats exposed to Hydrogen cyanamide.

Statistically significantly decreased body weight could be observed for all treatment groups. However, the reduction in the males showed no dose-response-relationship. The extent of body weight decrease was below 10 % for the low and high dose males as well as for the low dose females. Subsequently to a withdrawal period of another 14 days, the body weights of the satellite group (mid dose animals) were in the control range (5 % below the body weight of controls) indicating a clear recovery. Consequently, the findings in low and mid dose are considered to be not adverse.

Total food consumption was not mentioned. Clear substance-related effects were not seen in food consumption. No compound-related effects were found in haematological and blood chemistry parameters.

Several absolute and relative organ weights in all treated groups in males and females were statistical significant different from the control group.In the satellite group the absolute and relative liver and kidney was still increased in male animals. In female animals an increase in absolute and relative lung weight and an increase in relative kidney weight was obtained.

Gross pathological lesions were observed in animals of all dose groups including the controls.

Histopathological changes revealed consistent recurring lesions in the brain, liver, heart and lungs in the high dose group in both sexes.

Key result

Dose descriptor:

NOAEL

Effect level:

0.26 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Key result

Critical effects observed:

no

No remarks

Conclusions:

NOAEL (rat, inhalation): 0.26 mg/L / 0.2629 g/m³ (analytical concentration; active ingredient)

Executive summary:

In a subacute inhalation study, Dormex (Hydrogen cyanamide, 50 % w/w formulation) was administered by inhalation (head only exposure) to male and female Wistar rats for 14 days. The nominal concentrations of the active ingredient, Hydrogen cyanamide were 2.5, 5 and 9.375 g/m³ to which the low, the intermediate and high dose group were exposed. 10 rats (5 males and 5 females) were exposed to the low and high doses whereas 16 rats (8 males and 8 females) were exposed to the intermediate dose. An additional group of 8 males and 8 females served as the concurrent control group. The aerosol of the test substance was generated by infusing it through the nebulizer which generates particles less than 5µm in diameter. During the exposure, samplings of the atmosphere from the inhalation chamber were done for the HPLC determination of actual concentrations of Hydrogen cyanamide. Animals were exposed for 6 hours per day, 5 days a week for 2 weeks. On day 15 after start of the exposure 5 males and 5 females of each group were necropsied. The remaining animals (3 males and 3 females) of the control and intermediate dose group were maintained without any exposure for a further period of 2 weeks (satellite group, to investigate recovery). After the scheduled exposure regimen all animals of the low and high dose group and 5 males and 5 females each from the control and intermediate group and on day 29 the remaining animals were necropsied and the lesions were noted grossly. Organ weight evaluations (absolute and relative) and histopathological examination were performed on all animals. The same procedure was performed in the remaining animals at day 29.

No mortality was observed in rats belonging to any of the groups throughout the study period. No clinical signs of toxicity were found in rats exposed to Hydrogen cyanamide.

Statistically significantly decreased body weight could be observed for all treatment groups. However, the reduction in the males showed no dose-response-relationship. The extent of body weight decrease was below 10 % for the low and high dose males as well as for the low dose females. Subsequently to a withdrawal period of another 14 days, the body weights of the satellite group (mid dose animals) were in the control range (5 % below the body weight of controls) indicating a clear recovery. Consequently, the findings in low and mid dose are considered to be not adverse.

Total food consumption was not mentioned. Clear substance-related effects were not seen in food consumption. No compound-related effects were found in haematological and blood chemistry parameters.

Several absolute and relative organ weights in all treated groups in males and females were statistical significant different from the control group.In the satellite group the absolute and relative liver and kidney was still increased in male animals. In female animals an increase in absolute and relative lung weight and an increase in relative kidney weight was obtained.

Gross pathological lesions were observed in animals of all dose groups including the controls.

Histopathological changes revealed consistent recurring lesions in the brain, liver, heart and lungs in the high dose group in both sexes.

Considering the lack of dose-response-relationship in the males and the recovery in the mid dose group, the NOAEL should be the mid dose, i.e. 0.26 mg/L.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.263 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable scientifically acceptable study

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Stability, homogeneity and correctness of the concentrations not verified analytically; 3 males and 3 females used per concentration instead of 5 males and 5 females; no sacrifice immediately after end of application, but after 14-day; body weight 1200 g ± 200 g instead of 2000 – 3000 g at the beginning of treatment; housing temperature 24 ± 2 °C instead of 20 ± 3 °C; no summary or individual data of clinical findings; less haematological and blood chemistry parameter compared to OECD 410; no individual organ weight data were submitted.

Qualifier:

according to guideline

Guideline:

other: Gaitonde Committee guidelines

Deviations:

no

GLP compliance:

no

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Body weight: 1200 ± 200 g
- Housing temperature: 24 ± 2 °C

Type of coverage:

not specified

Vehicle:

unchanged (no vehicle)

Details on exposure:

Not indicated

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analytical verification of doses or concentrations was performed

Duration of treatment / exposure:

six hours per day on 5 days per week for three consecutive weeks followed by a 14 day post-application observation period.

Frequency of treatment:

Daily treatment

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

equivalent to 12.5 mg/kg bw/day active substance Cyanamide
nominal per unit body weight

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

equivalent to 25 mg/kg bw/day active substance Cyanamide
nominal per unit body weight

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

equivalent to 37.5 mg/kg bw/day active substance Cyanamide
nominal per unit body weight

No. of animals per sex per dose:

3 animals per sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- The concentrations were selected according to the results of a pilot study

Positive control:

No positive control

Observations and examinations performed and frequency:

The animals were examined once daily for mortalities, clinical symptoms and local skin irritations. Data of body weight and food consumption of individual rabbits were recorded weekly. Clinical-chemical and haematological investigation and urinalysis were performed on day 0, 22 and 35.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

No other examinations

Statistics:

The difference between the control groups and the test-dose groups were examined by the Student´s test criteria, p< 0.05.

Clinical signs:

effects observed, treatment-related

Dermal irritation:

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):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

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:

no effects observed

Details on results:

There were no mortalities in any groups of the test material or post-application observation period. At the highest concentration severe erythema of the skin and reduction in the fur density around the areas of application were observed. Slight erythema during the application period were observed at the mid dose of 25 mg/kg bw/day pure active substance cyanamide which recovered one day after application. There were no significant differences in the body weight and feed consumption of animals treated with Dormex when compared to the control animals. None of the haematological, clinical chemical and urine parameters of the treated groups showed any statistical significant variation as compared to the controls at each time of measurement.
After the recovery period some organ weights revealed a statistical significance in the low, mid or high dose males. However, due to the fact that there are no consistent dose-dependent changes the findings are considered to be incidental.
The gross pathological and histopathological examinations showed no consistent findings in the low and mid dose animals. Some high dose animals showed findings even after the 14 d recovery period.

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

37.5 mg/kg bw/day (nominal)

System:

other: histopathological examinations revealed lesions in several tissues/organs, no clear target system

Table 1: Subacute (21-Day) dermal toxicity study in Rabbits: Organ Weights

Pure active ingredient cyanamide	0 mg/kg bw/day	12.5 mg/kg bw/day	25.0 mg/kg bw/day	37.5 mg/kg bw/day
Males:
Absolute heart weight (g)	3.925	3.058*	3.848	3.041*
Relative^aheart weight ( %)	0.257	0.162	0.226	0.171
Absolute liver weight (g)	31.667	45.333*	58.000*	45.667*
Relative^aliver weight ( %)	2.074	2.410	3.433*	2.566
Absolute kidney weight – left (g)	5.315	3.897**	5.207	4.165**
Relative^akidney weight – left ( %)	0.347	0.208**	0.307	0.234
Absolute kidney weight – right (g)	5.515	4.230*	5.137*	4.206*
Relative^akidney weight – right ( %)	0.360	0.226**	0.303	0.236**
Absolute gonad weight – left (g)	0.917	1.620	1.064	2.099
Relative^agonad weight – left ( %)	0.059	0.086	0.063	0.118*
Absolute gonad weight – right (g)	0.929	1.587*	1.254	2.095
Relative^agonad weight – right ( %)	0.060	0.084	0.068	0.118*
Females:
Absolute heart weight (g)	4.383	2.941	4.329	3.310
Relative^aheart weight ( %)	0.261	0.153	0.217	0.180
Absolute liver weight (g)	41.000	48.333	54.000	39.667
Relative^aliver weight ( %)	2.480	2.521	2.704	2.152
Absolute kidney weight – left (g)	4.950	3.819	4.777	3.847
Relative^akidney weight – left ( %)	0.290	0.200	0.241	0.209
Absolute kidney weight – right (g)	4.775	3.739	4.856	4.215
Relative^akidney weight – right ( %)	0.280	0.196	0.245	0.230
Absolute gonad weight – left (g)	0.178	0.119	0.172	0.185
Relative^agonad weight – left ( %)	0.010	0.006	0.009	0.010
Absolute gonad weight – right (g)	0.187	0.137	0.187	0.173
Relative^agonad weight – right ( %)	0.011	0.007	0.010	0.009

 

 

Table 2: Subacute (21-Day) dermal toxicity study in Rabbits: Gross pathological lesions

 

Pure active ingredient cyanamide (mg/kg bw/day)	0	12.5	25	37.5
Number of rabbits/group	3	3	3	3
Males:
Skin
Alopecic	0	0	1	0
Sparse fur growth	0	0	0	1
Lung
Collapsed areas	0	0	0	0
Sanguineous exudate	0	0	0	1
Petechiaeted	0	0	0	2
Small intestine
Catarrhal enteritis	0	0	0	0
Liver
Mottled	0	0	1	0
Congested	0	0	1	0
Kidneys
Medullary congestion	0	0	0	1
Cortico-medullary congestion	0	0	1	1
Cotical grey specks	0	0	0	1
Heart
Apex with petechiae	0	0	0	1
Spleen
Congested	0	0	0	0
Testes
Bulged edges on section	0	0	0	1
Females:
Skin
Alopecic	0	0	0	1
Sparse fur growth	0	0	1	2
Lung
Collapsed areas	0	0	0	1
Sanguineous exudate	0	0	0	1
Petechiaeted	0	0	0	0
Small intestine
Catarrhal enteritis	0	0	0	1
Liver
Mottled	0	0	0	0
Congested	0	0	0	1
Kidneys
Medullary congestion	0	0	0	1
Cortico-medullary congestion	0	0	0	0
Cotical grey specks	0	0	0	0
Heart
Apex with petechiae	0	0	0	0
Spleen
Congested	0	0	0	1
Ovaries
Dark spots	0	0	0	1

 

 

Table 3: Subacute (21-Day) dermal toxicity study in Rabbits: Incidences of microscopic effects

Pure active ingredient cyanamide (mg/kg bw/day)	0	12.5	25	37.5
Number of rabbits/group	3	3	3	3
Males:
Brain-Cerebrum
Microcavitations (Oedema)	0	0	1	2
Brain-Cerebellum
Microcavitations	0	0	0	1
Heart
Focal Hyalinisation of Myocardial cells	0	1	0	1
Foci of calcification	0	0	0	1
Focal haemorrhage	0	0	0	1
Small intestine
Catarrh (Jejunum)	0	0	0	0
Hyperanemia	0	0	0	1
Kidneys
Cysts	0	0	0	0
Eosinophilic material in some Convoluted tubules	0	1	0	3
Few Foci of mononuclear cells	0	0	0	1
Liver
Hyperaemia	0	0	0	0
Lungs
Focal Oedema	0	0	0	2
Atelectasis (focal)	0	0	0	0
Perivasular cuffing	0	1	0	2
Testes
Focal interstitial oedema	0	0	0	1
Females:
Brain-Cerebrum
Microcavitations (Oedema)	0	0	0	2
Brain-Cerebellum
Microcavitations	0	0	0	1
Heart
Focal Hyalinisation of Myocardial cells	0	0	0	0
Foci of calcification	0	0	0	0
Focal haemorrhage	0	0	0	0
Small intestine
Catarrh (Jejunum)	0	0	0	1
Hyperanemia	0	0	0	1
Kidneys
Cysts	0	0	0	1
Eosinophilic material in some Convoluted tubules	0	0	1	1
Few Foci of mononuclear cells	0	0	0	0
Liver
Hyperaemia	0	1	1	1
Lungs
Focal Oedema	1	0	0	1
Atelectasis (focal)	0	1	0	1
Perivasular cuffing	0	0	0	0

 

Conclusions:

The NOAEL for dermal effects in rabbits: 25 mg/kg bw
The NOAEL for systemic effects in rabbits: 25 mg/kg bw

Executive summary:

Dormex (Hydrogen cyanamide, aqueous solution) was applied undiluted to the skin of three male and three female New Zealand White rabbits per dose level for a period of six hours per day on 5 days per week for three consecutive weeks followed by a 14 day post-application observation period. The concentrations were 0, 25, 50 and 75 mg/kg bw/day equivalent to 12.5, 25 and 37.5 mg/kg bw/day active substance Cyanamide, which were selected according to the results of a pilot study. The control group did not receive any treatment of the test substance.

There were no mortalities in any groups of the test material or post-application observation period. At the highest concentration severe erythema of the skin and reduction in the fur density around the areas of application were observed. Slight erythema during the application period were observed at the mid dose of 25 mg/kg bw/day pure active substance cyanamide which recovered one day after application. There were no significant differences in the body weight and feed consumption of animals treated with Dormex when compared to the control animals. None of the haematological, clinical chemical and urine parameters of the treated groups showed any statistical significant variation as compared to the controls at each time of measurement.

After the recovery period some organ weights revealed a statistical significance in the low, mid or high dose males. However, due to the fact that there are no consistent dose-dependent changes the findings are considered to be incidental.

The gross pathological and histopathological examinations showed no consistent findings in the low and mid dose animals. Some high dose animals showed findings even after the 14 d recovery period.

Due to the irreversibility of the local skin effects at 37.5 mg/kg bw and the reversibility of the skin findings at 25 mg/kg bw the NOAEL for dermal effects is 25 mg/kg bw of ai cyanamide in rabbits. Due to the fact that there were no systemic findings in the low and mid dose animals attributable to treatment with cyanamide the NOAEL for systemic effects was deduced at the mid dose of 25 mg/kg bw based on the histopathological findings at the high dose.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Only one reliable study available.

Additional information

Oral administration of cyanamide

The 28-day short term oral toxicity of cyanamide in the rat is characterised by a depression in body weight, body weight gain and food consumption at the upper dose levels. A mild decrease in the red cell mass of the high dose group was obtained in males and females which correlates to splenic pigmentation found at higher doses in both sexes. Histopathological findings were obtained in the thyroid, liver and spleen at 10, 20 and 40 mg/kg bw/day. The NOAEL in this study is 5 mg/kg/day active ingredient hydrogen cyanamide.

 

A 90-day oral toxicity study was performed in rats with 0.5, 1.5 and 4.5 mg/kg bw/day of cyanamide (corresponding to 10, 30, 90 ppm; feeding study). At 4.5 mg/kg bw/day thyroid effects were seen in males as well in females. The changes in the thyroid were comparable to the effects found in the 4 week study. Histopathological changes in the thyroids in only one male (1/20) were not contributed due to an adverse effect of cyanamide at 1.5 mg/kg bw/day. Additionally, male rats showed in contrast to the 28 day study a slight increase in erythrocyte counts. The NOAEL was 1.5 mg/kg bw/day pure active substance cyanamide (equivalent to 30 ppm in the diet) in males and females.

 

A 90-day oral toxicity study was performed in dogs with 0.6, 2 and 6 mg/kg bw/day of cyanamide. The NOAEL in the 90-day gavage study in dogs was 0.6 mg/kg bw/day pure active ingredient cyanamide for males and females. This NOAEL was based on a slight anemia found in the mid and high dose group which may be correlated with the slight hypothyroidism (indicated by a increased plasma cholesterol level) found in the same dose groups. Also a slight monocytosis in the mid and top dose group was found in both sexes. Microscopic examination revealed changes in the testes which were accompanied by reduced testes weights in the high dose group. These changes were found in the low, the mid and more severe in the high dose group. The severity of the changes observed in the high dose group exceeds that of the changes observed in the low and intermediate dose groups and also the severity of the changes observed in control dogs in other experiments (historical data). Therefore, it is justified to relate only the more severe changes in the high dose group to the administration of the test substance, whereas the slight changes found in the lower dose groups are regarded as findings unrelated to treatment since giant cells were also observed in controls and no dose-related effect was obtained and additionally the incidence of spermatogenesis was not clearly dose-related.

 

In the 1-year toxicity study in dogs cyanamide was administered in doses of 0, 0.2, 1, 5 mg/kg bw/day via oral gavage for 52 weeks. Compound-related clinical pathology changes were found in males and females in the intermediate and high dose group. A gross macroscopic finding was obtained in the spleen in one medium dose male and two high dose females. Organ weight data revealed a significant increase in thyroid/parathyroid organ-to-terminal body weight ratio in the high dose females. Histopathological alterations were observed in the high dose group in males and females in the liver, in the spleen of two high dose males, gallbladder in one high dose male and two high dose females, in testes in high dose dogs and in the thymus in all four high dose males and one high dose female. Compound-related effects are allocated to dogs administered 5.0 mg/kg bw/day of pure aqueous ingredient cyanamide. The macroscopic finding in the spleen in the intermediate dose is considered not to be toxicological relevant, since this effect was not obtained at the high dose group. The increase in total cholesterol content and the decrease in the mean cell volume and mean cell haemoglobin concentration at the medium dose were also not considered to be compound-related since no other changes in parameters characteristic for thyroidal effects and anaemic effects, respectively were obtained at this dose level. Therefore, 1.0 mg/kg bw/day active ingredient cyanamide is considered to be the NOAEL. The target organs were the thyroid, the liver, the spleen, the gallbladder, the testes and the thymus.

 

In a further chronic toxicity study the test material, aqueous hydrogen cyanamide (50% w/w, 53% w/v active ingredient hydrogen cyanamide) was administered, via oral gavage, to three groups of male and female Sprague-Dawley rats. Twenty rats/sex received aqueous hydrogen cyanamide at 2.5, 7.5 and 30 mg/kg bw/d for the first 16 weeks. Due to poor health status during the first weeks of treatment, the dose was reduced to 1, 2.5 and 7.5 mg/kg bw/d from week 17 to 91. The pattern of mortalities did not appear related to treatment. Clinical signs of toxicity (tremors, rough hair, hunched posture) diminished after the doses were reduced from week 16 onwards. Body weight gain was significantly adversely affected in rats in the mid (7.5 mg/kg) and high dose (30 mg/kg) groups up to week 16, and in the (reduced) high dose group (7.5 mg/kg) only, from week 17 until termination. T3 was significantly reduced in females from 2.5 mg/kg bw/d and above, and in males at 7.5 mg/kg at termination and T4 was reduced in high dose males only at termination. Decreases in mean terminal brain, kidney, liver and testis and epididymides weight were considered related to the considerably reduced mean body weights at terminal sacrifice. The only treatment related histopathological findings consisted of decreased colloid in the thyroids of mid and high dose males and high dose females, consistent with the findings in the sub chronic studies. A NOAEL of 1 mg/kg bw/day active ingredient hydrogen cyanamide after oral administration of aqueous hydrogen cyanamide to male and female Sprague-Dawley rats for at least 91 weeks was deduced.

 

Dermal administration of cyanamide

The 21 day dermal study in rabbits indicated a clear skin irritation potential of cyanamide at the highest concentration. Significant changes in absolute and relative organ weights were found in heart, liver, kidneys and testes in male animals in all dose groups. Gross macroscopic examinations showed lesions in the high dose group in various organs in both sexes. Recurrent histopathological changes were found in brain, heart, small intestine, kidneys, liver, lungs and testes in the highest dose group. Since no histopathological changes were found in the low and mid-dose group the NOAEL is 25 mg/kg bw/day pure active ingredient cyanamide.

 

Administration of cyanamide via inhalation

In the 14-day inhalation study a decrease in body weight was obtained in all dose groups in male and female rats during the exposure period. Also some variations were found in feed consumption in the groups treated with the test substance. In the high dose macroscopic and microscopic lesions were found in some organs (brain, lungs, heart and liver) indicating treatment related effects. The inhalation NOAEL of hydrogen cyanamide for Wistar rats is 0.2629 g/m³ active ingredient cyanamide, referring to approximately 70 mg/kg bw/day pure active ingredient cyanamide.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item requires classification for STOT RE according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.

 

As proposed by RAC, (33 RAC-Meeting, 1-5 June 2015, Helsiniki) the substance was classified as STOT RE 2 under Regulation (EC) No 1272/2008 for the following reasons:

For STOT RE classification the dose level leading to an adverse effect plays a dominant role in contrast to the pure hazard classifications, i.e. for carcinogenicity or mutagenicity. Therefore it has to be considered whether and to what extent the animal model may reflect the sensitivity of humans. If nothing is known in this respect, as in the majority of cases, the dose limits for RE1 and RE2 would apply. But for the thyroidal effects of the test substance there is strong evidence that humans are clearly less sensitive than the rat or the dog, although the extent is unknown. The broad database in humans (human data rely on about 700.000 patients under treatment for alcoholism) and the high sensitivity of rats and dogs in comparison to humans should be taken into account. The epidemiological NOAEL in humans should be compared with the NOAEL in the chronic rat study using the metabolic scaling factor of 4 to extrapolate from humans to rats. Thereby it can be shown that humans are at least by a factor of 4 less sensitive than rats for effects on the thyroid gland. This difference in sensitivity must be taken into due account for extrapolating the experimental LOAELs to humans. It is shown that these extrapolated LOAELs are above the classification limit for STOT RE1 regardless of whether the effects are considered as “severe” or “significant”. Therefore STOT RE1 is not justified and STOT RE2 would be appropriate.

 

The substance is therefore classified and labeled as STOT RE2, H373: May cause damage to organs through prolonged or repeated exposure (thyroid). The route of exposure was not specified by RAC.