Cyanamide

Carcinogenicity

Description of key information

Originally the study was rated klimisch 2. As read-across to the test substance used in this study is not considered appropriate (please see explanation in the field 'Rationale for reliability incl. deficiencies'), klimisch rating can not be applied.

In a chronic toxicity study with rats (total exposure 91 weeks) also used by RAC to evaluate the carcinogenic potential of the substance no indication for a carcinogenic potential of cyanamide even if tested at clearly systemic toxic doses was noted.

 

In the key carcinogenicity study with cyanamide in drinking water with mice, at the high dose of 600 ppm (equivalent to 39 mg/kg bw/day) which clearly exceeds the MTD, a slight increase in ovarian granulosa-theca cell tumors was noted. Those effects were not considered sufficient for a classification and labelling by the registrant. The interpretation of these tumors with regard to classfication and labelling was controversially discussed at the RAC. Finally the RAC concluded that these findings cannot be ignored and that it justifies a classification and labelling as Canc. Cat 2, In addition It should be mentioned, that the registrant does not support this interpretation, but adopted the classification and labelling accordingly.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.32 (Carcinogenicity Test)

Deviations:

no

GLP compliance:

yes

Species:

mouse

Strain:

CD-1

Sex:

male/female

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

Not indicated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The intended test substance concentrations in the drinking water were achieved throughout the study. The test substance intake is summarised in a table below in "Any other information on material and methods".

Duration of treatment / exposure:

Male mice were exposed to the test substance for 100 weeks and female mice were exposed to the test substance for 104 weeks.

Frequency of treatment:

Daily

Post exposure period:

No post exposure period

Dose / conc.:

70 ppm (nominal)

Remarks:

in water

Dose / conc.:

200 ppm (nominal)

Remarks:

in water

Dose / conc.:

600 ppm (nominal)

Remarks:

in water

No. of animals per sex per dose:

60 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:

All animals were examined twice daily to detect dead or moribund animals. All animals were examined once daily for signs of ill health or overt toxicity and additionally, each animal was given a detailed clinical examination at weekly intervals.
Individual body weights were recorded before treatment on the first day of the study, at weekly intervals up to week 16, at four weekly intervals up to week 100 for males and week 104 for females and at sacrifice.
The food and water consumed by each cage of animals was determined at weekly intervals to week 16 and one week in every four thereafter.
Blood samples for total white cell counts were obtained from all surviving animals in weeks 52, all surviving males in week 100 and all surviving females in week 104.
A full internal and external examination was carried out at the end of administration in all animals.

Sacrifice and pathology:

A full internal and external examination was carried out at the end of administration in all animals.
Organ weights (brain, kidneys, liver and testes) from ten animals per sex per group were obtained.
Samples of all tissues from animals in the control and high dose groups, from animals that died or were killed during the study as well as gross lesions, tissue masses, lungs, livers, kidneys, thyroids and urinary bladders from all animals were fixed, sectioned, stained and evaluated.

Statistics:

Statistical evaluation of survival (Kaplan-Meier technique), body weight gains over the treatment period, food and water intake over selected intervals and all organ weight data (analysis of variance technique for normally distributed error (ANOVA) and t-tests) was carried out. The ovarian granulosa-theca tumours were analysed statistically using the methods described in the IARC annex (Peto, R., 1980 Guidelines for simple sensitive significance tests for carcinogenic effects in long term animal experiments. Annex to IARC monographs on the evaluation of carcinogenic risk of chemicals to humans, supplement 2).
The tumours were all classified as either fatal or non-fatal. The ad-hoc runs method was used to determine suitable time intervals for the analysis of non-fatal tumours. The incidence of fatal tumours was too low to perform statistical analysis, however where appropriate, combined fatal and non-fatal tumour analysis was performed. One directional tests for an increasing dose response among all groups were performed with pairwise one-directional tests for increased incidence between each treated group and its control group.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

no effects observed

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

- About two-thirds of the animals died or were killed because of ill-health during the course of the study. However, the number of animals after a study period of 72 weeks (18 months) was sufficient (survival 73 % and more) and the recommendations of the OECD guideline 451 were fulfilled (survival more than 50 %). In males there was no treatment-related effect on the overall incidence of morbidity and mortality. Non-neoplastic conditions (amyloidosis and urogenital tract lesions) accounted for two-thirds of the death and are considered not to be treatment related.
In females, there was a slight excess of morbidity and mortality in the mid and high dose group. In addition, the pair-wise test of the high dose group against the control (estimated by Kaplan-Meier technique) was statistically significant (p < 0.05). Neoplasia accounted for over half of the morbidity and mortality in the females, but no single neoplastic or non-neoplastic condition accounted for the slightly higher incidences in these dose groups.

- No treatment- related changes in clinical conditions or behaviour were noted. The incidence of all types of palpable mass was low in the treated groups and comparable to that of the control groups.

- Over the first week of treatment 16 males from the high dose group lost body weight, compared to two control males. In week 2 the weight loss continued for three males from group 4 and one of the control males. The remaining animals gaining or maintaining body weight. There was a statistically significant reduction in the mean body weight gain for treated male groups over the first 6 weeks, when compared to the control gain. Subsequently the body weight gain for the low and intermediate groups was comparable to or better than the control. Over the first week of the study 12 females from the high dose group lost body weight, compared to seven control females. In week two, one animal from the high dose group and one animal form the control continued to loose weight, whereas the remaining animals gained weight. In females the body weight gain over the first six weeks of the study was treatment-related reduced for the medium and high dose group, with statistical significant difference from the control. Subsequently the weight gain for the intermediate and high dose groups was comparable to or better than the control.

- The food consumption in the male treated groups was reduced over the majority of the study, but the reduction was only statistically significant from the control over the first six weeks of dosing at the intermediate and high dose groups. In females the reduction of food intake was considered to be slight and the food consumption for all groups was considered to be satisfactory throughout the study.

- There was a treatment-related statistically significant reduction in water consumption in the medium and high dose groups of males and females. Over the periods weeks 1 to 6 and 13 to 16 there was a statistically significant reduction in water consumption when compared to the control consumption. In addition, water consumption was also reduced significantly for the high dose females over week 24 to 28, for medium group males over the period weeks 1 to 6 and for group 3 females over the periods weeks 1 to 6, 13 to 16 and weeks 24 to 28.

- No significant changes were found in the food conversion efficiency for any of the treated groups throughout the study.

- No significant changes were found in the food conversion efficiency for any of the treated groups throughout the study.

- The mean absolute and relative brain weight (statistically significant) and the mean relative testes weight for the high dose males were increased when compared to the control. However, these organ changes were considered to be related to the reduction in body weight seen in the high dose males. All other organ weights for treated groups were comparable to the control.

- No treatment-related histopathological effects were found in the low dose group. In the medium and high dose groups there was a dose-related chronic cystitis in the urinary bladder, accompanied in the high dose group by a marginal increase in the incidence and severity of atrophic basophilic tubules in the kidney.

- The tumour profile in the controls was generally consistent with that expected in ageing mice. The tumour in the low and intermediate groups was comparable to that seen in the control. In the high dose males there was a decrease in the incidence of liver tumours which was probably attributable to the reduced body weight gain of this group.

- In females there was an increased incidence of proliferative lesions in the stromal/luteal tissues of the ovary in the high dose group. The hyperplasias were predominantly of the luteal type, as were the granulosa-theca tumours. The 8 cases of granulosa-theca tumours in group 4 exceeded the range of 0 to 3 cases per group of 51 mice in 10 previous control groups. Statistically, there was a significant dose response trend in granulosa-theca tumours across the 4 groups (p < 0.01) regardless of whether the analysis included or excluded equivocal necrotic control case. Pairwise statistical comparison between groups 1 and 4 was significant (p < 0.05) if the necrotic control case was excluded but not significant (p > 0.05) if this case was included in the analysis.

Relevance of carcinogenic effects / potential:

No relevant carcinogenic effects / potential

Key result

Dose descriptor:

NOAEL

Effect level:

8.4 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: overall effects

Key result

Dose descriptor:

NOAEL

Effect level:

11 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: overall effects

Table 1: Carcinogenicity study in mice: Group survival (%):

Week of study	0 ppm	70 ppm	200 ppm	600 ppm
Males
72	83.3	81.7	83.3	73.3
100	36.7	38.3	43.3	36.7
Females
72	88.3	83.3	81.7	75.0
104	40.0	33.3	23.3	23.3

 

 

Table 2: Carcinogenicity study in mice: Body weight gain:

 

Group/sex	Mean body weight gain over weeks 0 – 6	Mean body weight gain over weeks 6-100/104^+	Mean body weight gain over weeks 0-100/104^+
Males
0	6.6	5.8	12.4
70	5.5**	7.7	13.2
200	5.4**	6.0	11.4
600	4.8***	2.9*	7.7**
Females
0	5.1	8.7	13.7
70	4.7	9.8	14.5
200	4.4*	9.3	13.7
600	3.8***	8.5	12.3

⁺  males sacrificed week 101, females week 105

*   statistically significant by t-test (p< 0.05

**  statistically significant by t-test (p< 0.01)

*** statistically significant by t-test (p< 0.001)

 

Table 3: Carcinogenicity study in mice: Group mean cage food consumption (g/animal/week over period):

 

Week of study	0 ppm	70 ppm	200 ppm	600 ppm
Males
1 to 6	39.8	38.7	37.8*	38.1*
13 to 16	39.7	38.2	38.5	38.7
24 to 28	39.4	37.1	38.5	37.6
48 to 52	36.5	35.0	36.4	35.9
96 to 100	37.5	37.5	35.4	35.6
Females
1 to 6	41.5	41.1	40.3	39.7
13 to 16	43.8	44.9	42.3	42.0
24 to 28	43.1	42.7	40.7*	40.3*
48 to 52	37.9	38.1	35.8	35.8
100 to 104	37.7	42.1	42.0	36.2

* statistically significant by t-test (p< 0.05)

 

Table 4: Carcinogenicity study in mice: Group mean cage water consumption (g/animal/week over period):  

Week of study	0 ppm	70 ppm	200 ppm	600 ppm
Males
1 to 6	39.6	38.7	34.1**	31.5**
13 to 16	38.1	37.3	33.5	29.6**
24 to 28	39.6	38.5	38.6	32.0
48 to 52	38.0	43.2	39.5	37.9
96 to 100	43.2	41.9	37.5	33.5
Females
1 to 6	37.9	37.7	34.0***	29.7***
13 to 16	39.2	38.5	33.8**	30.2***
24 to 28	47.3	45.6	39.1***	37.7***
48 to 52	47.7	44.8	42.9	40.2
100 to 104	44.7	58.7	41.9	39.8

** statistically significant by t-test (p< 0.01) 

*** statistically significant by t-test (p< 0.001)

 

 

Table 5: Carcinogenicity study in mice: Incidences of microscopic effects:

Hydrogen cyanamide (mg/kg/day):	0	70	200	600
Number of mice examined
Males:
Urinary bladder	56	56	57	57
Chronic cystitis
Minimal	3	4	11	18
Slight	0	2	6	10
Moderate	2	1	0	11
Total	5	7	17	39
Kidney	59	59	59	58
Atrophic/basophilic tubules
Minimal	22	19	25	16
Slight	4	9	4	12
Moderate	2	0	0	5
Total	28	28	29	36
Females:
Urinary bladder	59	54	55	56
Chronic cystitis
Minimal	6	8	18	16
Slight	0	0	5	16
Moderate	0	0	0	4
Total	6	8	23	36
Kidney	60	60	59	58
Atrophic/basophilic tubules
Minimal	16	14	12	15
Slight	4	5	3	6
Moderate	0	0	0	3
Total	20	19	15	25

 

 

Table 6: Carcinogenicity study in mice: Incidences of hyperplastic and neoplastic lesions:

Hydrogen cyanamide (mg/kg/day):	0	70	200	600
Number of mice examined
Males:
Liver	59	57	57	58
Adenoma±Carcinoma	18	11	7	8
Females:
Liver	60	59	55	57
Adenoma±Carcinoma	0	0	1	1
Ovary	60	59	60	58
Stromal/Luteal hyperplasia	16	13	13	22
Granulosa-theca tumour	3*	1	6	8

 

 

 

Conclusions:

Cyanamide was considered not to have affected the incidence and morphology of tumours following oral administration to the mouse. However, as the RAC came to a different conclusion, the proposal to classify the substance as Canc. Cat 2 was followed.

Executive summary:

Hydrogen cyanamide was administered to 240 male and 240 female mice [Crl:CD-1 (ICR) BR] in this carcinogenicity study. 60 males and 60 females received the test substance in water at concentrations of 70, 200 or 600 ppm for 100 weeks (males) and 104 weeks (females), respectively. A further group of mice (60 males and 60 females) received drinking water and acted as the control group. The obtained results were as follows: A slight increase in the morbidity and mortality in the medium and high dose group in females. Reduced food and water consumption in the medium and high dose groups of males and females. Mean absolute and relative brain weight and the mean relative testes weight for the high dose males were increased. A chronic cystis in the urinary bladder in the medium and high dose group was obtained accompanied by an increase in the incidence of atrophic basophilic tubules in the kidney in the male animals. No adverse effect was seen at the low dose (70 ppm) throughout the study and this was considered to be the NOAEL in terms of toxicity of Hydrogen cyanamide referring to 4.2 mg/kg bw/day active substance cyanamide. The magnitude of the changes seen at the high dose level in body weight gain, survival, urinary bladder and the kidney demonstrated that the maximum tolerated dose (MTD) was clearly exceeded at 600 ppm. The only treatment-related changes in the tumour profile were the reduction in the incidence of liver tumours in the high dose males and a slight increase in ovarian granulosa-theca tumours in high dose females. The reduction in liver tumours was probably attributable to the reduction in body weight gain seen. There were no changes in the tumour profile at 200 ppm referring to 12.2 to 17.4 mg/kg bw/day (for males) and 13.4 to 21.3 mg/kg bw/day (for females) pure active substance cyanamide, respectively. Cyanamide was considered not to have affected the incidence and morphology of tumours following oral administration to the mouse if tested at systemic doses up to the MTD.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

8.4 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

Guideline study, reliable without restrictions

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

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

Based on the results of the available data and in accordance with the GHS/CLP regulations, the test substance, cyanamide, was not classified for carcinogenic effects based on the evaluation by the registrant. However, the RAC finally concluded that the observed effects above the MTD (in the key study) justify a classification and labelling as Canc. Cat 2. Therefore, this classification and labelling was adopted accordingly.

Additional information

Key study 

In the key carcinogenicity study with cyanamide in drinking water with mice, the NOEL (toxicity) was 70 ppm referring to 4.2 to 6.5 mg/kg bw/day for males and 5.5 to 8.2 mg/kg bw/day for females, based on increased mortality, reduction in body weight gain, food consumption and histopathological effects (cystis in urinary bladder and atrophic basophilic tubules in kidney) in the mid and high dose. At the high dose of 600 ppm (equivalent to 39 mg/kg bw/day) which clearly exceeds the MTD, a slight increase in ovarian granulosa-theca cell tumors was noted. Those effects are not considered sufficient for a classification and labelling by the registrant. The interpretation of these tumors with regard to classification and labelling was controversially discussed at the RAC.

Finally the RAC concluded that these findings cannot be ignored and that it justifies a classification and labelling as Canc. Cat 2. It should be mentioned, that the registrant does not support this interpretation, but adopted the classification and labelling accordingly.

The carcinogenicity study examined the effect of the test substance cyanamide in a comprehensive drinking water study in mice (Goodyer, 1990, Doc. No. 555-001).

In this carcinogenicity study cyanamide was administered via the drinking water (in concentrations of 70, 200 and 600 ppm) a slight increase in morbidity and mortality in the intermediate and high dose groups was obtained. In the first weeks of the study the body weight gain, the food and water consumption was reduced in the intermediate and high dose groups. Histopathological effects were obtained in the medium and high dose groups evidenced by a dose-related chronic cystitis in the urinary bladder and in the high dose group by atrophic basophilic tubules in the kidney.

The NOEL was 70 ppm referring to 4.2 to 6.5 mg/kg bw/day for male and 5.5 to 8.2 mg/kg bw/day for females, based increased mortality, reduction in body weight gain, food consumption and histopathological effects (cystis in urinary bladder and atrophic basophilic tubules in kidney) in the mid and high dose. In the high dose males there was a decrease in the incidence of liver tumours related to the reduced body weight gain of this group. In the high dose in females a slight increase in granulosa-theca tumours was found. The evaluation of the significance and relevance of the granulosa-theca tumours is complicated by the level of toxicity seen at the high dose as the Maximum Tolerable Dose (MTD) has clearly been exceeded. Since there were no treatment-related changes in the tumor profile at 200 ppm referring to 12.2 to 17.4 mg/kg bw/day for males and 13.4 to 21.3 mg/kg bw/day for females active ingredient cyanamide, respectively, hydrogen cyanamide was considered not to affect the incidence and morphology of tumors following administration of doses up to the MTD in the mouse.

The RAC, however came to a different conclusion.

Justification for selection of carcinogenicity via oral route endpoint: relevant study for classification and labelling

 

 

Supporting study

In a chronic toxicity study (supporting study) with rats (total exposure 91 weeks) also used by RAC to evaluate the carcinogenic potential of the substance no indication at all for a carcinogenic potential of cyanamide even if tested at clearly systemic toxic doses was noted. (Osheroff, 1991, Doc. No. 537-001). 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.

Justification for selection of carcinogenicity via oral route endpoint: Relevant exposure and also considered by RAC to assess the carcinogenic potential.

Based on the results of this study in rats and in accordance with the GHS/CLP regulations, the test substance, cyanamide, has not to be classified for carcinogenic effects.

 

 

Disregarded studies

Additionally two studies with calcium cyanamide are available (NCI-DCCP-CG-1973-1-1, NCI Carcinogenesis Technical Report Series 163 (1979)), which were conducted with mice and rats, respectively.

These studies were disregarded, as calcium cyanamide is not considered a suitable read across substance for cyanamide. This conclusion is drawn based on the composition and on the comparison of toxicological studies from endpoints, where experimental results are available for both compounds, calcium cyanamide and cyanamide.

Therefore, the available data on calcium cyanamide are not considered for evaluation of the carcinogenic potential of cyanamide.