Cyanamide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Five reproduction toxicity studies in rats are reported.

In the key study, a two-generation study by Morseth (1990), the following findings were obtained: Reduced body weight and food consumption in parental animals of mid and high dose (F0 and F1 generation), reduced fertility at high dose, retarded body weight gain (F1 pups) and reduced survival (F2 pups) in mid and high dose group. No morphological alterations. Thus, NOAEL of 1.25 mg/kg bw/day pure active ingredient Cyanamide was determied for maternal and developmental parameters and a NOAEL of 3.75 mg/kg bw/day pure active ingredient was determined for reproduction parameters.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

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 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

(1982)

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:

The rats were 7 week old.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

the dosing solutions were prepared weekly by dilution with distilled water and were stored under refrigeration. Approximately 1 hour prior to application the solutions were thawed and stirred during dosing. The application volume was 10 ml/kg bw.

Details on mating procedure:

One female was mated with one male overnight for a period of up to 3 weeks to produce the F1 litter. From the F1 pups 26 males and 26 females/dose group were selected as F1 parental generation to produce the F2 generation.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

14 weeks prior to mating, and dosing continued until termination.

Frequency of treatment:

Daily

Details on study schedule:

F0 males were dosed once daily for approximately 14 weeks prior to mating, and dosing continued until termination. F0 females were dosed once daily for approximately 14 weeks prior to mating and throughout the mating, pregnancy, lactation and post lactation periods. 1 female was mated with 1 male overnight for a period of up to 3 weeks to produce the F1 litter. From the F1 pups 26 males and 26 females/dose group were selected as F1 parental generation to produce the F2 generation following treatment with dose levels of 0, 1.25, 3.75 and 15.0 mg/kg bw/day for at least 14 weeks.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

(corresponding to 2.5 mg/kg bw/day active ingredient) for 12 weeks

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

(corresponding to 7.5 mg/kg bw/day active ingredient) for 12 weeks

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

(corresponding to 30.0 mg/kg bw/day active ingredient) for 12 weeks

Dose / conc.:

2.5 mg/kg bw/day (nominal)

Remarks:

(corresponding to 1.25 mg/kg bw/day pure active ingredient) from week 12 onwards

Dose / conc.:

7.5 mg/kg bw/day (nominal)

Remarks:

(corresponding to 3.75 mg/kg bw/day pure active ingredient) from week 12 onwards

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

(corresponding to 15.0 mg/kg bw/day pure active ingredient) from week 12 onwards

No. of animals per sex per dose:

26 animals per sex per dose

Control animals:

yes, sham-exposed

Details on study design:

From the beginning of week 12 the doses were lowered due to severe impact on health expressed by loss in body weights of the F0 high dose animals.

Positive control:

No positive control

Parental animals: Observations and examinations:

The examination of parental animals included monitoring for clinical symptoms/mortalities, food consumption, body weight development, mating and reproductive performances.

Litter observations:

All pups were examined macroscopically at necropsy (external and organ findings), stillborn pups and pups that died intercurrently were additionally examined for any skeletal findings.Litter size reduction in litters with more than 8 pups was performed on postnatal day 4.

Postmortem examinations (parental animals):

Pathological examination was performed by gross inspection as well as by histopathological examination with special attention to the organs of the reproductive system. Pups were sexed, weighed and monitored with respect to their viability and growth.

Postmortem examinations (offspring):

Culled pups were sacrificed by intraperitoneal injection of sodium pentobarbital and examined for visceral abnormalities.

Statistics:

A significant difference between the control and the treated groups was statistically examined by the Dunnett´s test (p < 0.05, p< 0.01).

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Signs of an effect in the high dose males were apparent after several weeks of treatment and included rough hair coats, thin appearance and substantially lower mean body weight gains compared to the control group. The body weight effect was also evident in the high dose females and mid dose males and females during the F0 growth phase. In these animals a lower mean food consumption tended to parallel the body weight effect in these animals. Thus, dose levels were reduced at week 12 of treatment.
After reduction of the dose levels towards the end of pre-mating, the body weight gain improved and especially the males of all treatment groups gained weight above the respective control during the post-mating period.
Mating ability was unimpaired by treatment in the F0 (and the F1 generation). Most females mated during the first oestrus and the distribution of matings that occurred at second oestrus or later did not indicate a test substance-related effect. A low fertility and gestation index were obtained in the F0 animals at the high dose group.
Gross pathological and histopathological data from F0 (and F1 adults) did not reveal any significant treatment-related changes.

Key result

Dose descriptor:

NOAEL

Remarks:

for maternal and developmental parameters

Effect level:

1.25 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: 1.25 mg/kg bw/day of the pure active ingredient cyanamide.

Key result

Dose descriptor:

NOAEL

Remarks:

for reproduction pararmeters

Effect level:

3.75 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: 3.75 mg/kg bw/day of the pure active ingredient cyanamide.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Clinical observations of the F1 animals did not reveal any evidence of a treatment-related effect. Mean body weight gain values were lower for the high dose F1 males compared to the control animals for the during pre- and post-mating periods. In addition, an effect on body weight gain was also observed for the mid dose males during post-mating and high dose females during premating and gestation but not during lactation. Food consumption of the F1 animals showed lower values in the high dose group compared to the controls during the growth, gestation and lactation phases.
Mating ability was unimpaired by treatment in the F0 and the F1 generation. Most females mated during the first oestrus and the distribution of matings that occurred at second oestrus or later did not indicate a test substance-related effect. Low fertility and gestation indices were obtained in the F1 (and F0).
No substance-induced morphological abnormalities were found in the offspring. Neonatal survival (day 0 - 4) of the F1 and the F2 pups, however, was significantly lower in all treated groups compared to the control group. The investigators do not consider this to be treatment-related because of considerable variability in the historical data base. However, the historical data have not been provided for comparison and the reviewer considers the concurrent control to be more representative for the actual study conditions than historical control ranges.
F1 neonates had reduced birth weights in the mid and high dose group. Although the finding in the intermediate dose could be related to the higher mean live litter size the comparison with the low dose indicates that a test substance effect on foetal growth was present. Weaning weights were also lower in the mid and high dose groups. For the F2 pups at the high dose level a slight effect on birth weight was observed and the weights attained at weaning were marginally lower than in the control.
Birth weights of the F1 and F2 pups were not affected. In the F1 pup growth during lactation was retarded in the mid and high dose groups compared to the control group but not during raise of the F2 pups.
No treatment-related morphological alterations were observed in the F1 and F2 pups during inspection for cervical, thoracic and abdominal visceral abnormalities in the culled, intermittent died or scheduled killed pups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1.25 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

yes

Body weight gain data of F0/F1 parental animals:

Dose level (mg/kg bw/day)	0	2.5	7.5	30
F0 males - pre-mating (week 0-12)	271	249	224**	108**
F0 females
- pre-mating (week 0-12)	129	126	106**	59**
Dose level (mg/kg bw/day)	0	1.25	3.75	15
F0 males - post-mating (week 12-24)	68	92*	106*	99*
F0 females
- pregnancy (GD 0-20)	130	143	139	111
- lactation (PND 0-21)	-1.4	0.6	3.0	25
F 1 males - pre-mating (week 0-12)	461	446	441	391*
F 1 males - postmating (week 12-27)	111	101	87*	36*
F 1 females
- pre-mating (week 0-12)	221	232	222	202**
- pregnancy (GD 0-20)	119	129	126	94**
- lactation	1.4	-7.5	6.6	27**
* p < 0.05; ** P < 0.01 (Dunnett test)

 

Fertility and litter data of F0/F1 generation animals:

Dose level (mg/kg bw/day)	0	1.25	3.75	15
F0 generation
Cohabitated pairs	26	26	26	26
Mated females	26	22	24	23
Pregnant females	20	20	20	15*
Female fertility index (%)	77	91	83	65
Male fertility index (%)	80	91	83	65
Gestation index (%)	100	100	100	80
Mean duration of pregnancy (days)	22.06	22.11	21.89	22.10
Total prenatal litter loss	0	0	0	3
Live litters	20	20	20	12
Pups delivered (mean)	13.15	15.00	14.95	10.83
Live birth index (%)	98	96	96	99
Viability index (alive PND 0-4)	92	83**	88**	84**
(HCD: 94.7%±5.6, Min.: 84% - Max.: 98%, supplied by breeder)
(HCD: % dead pups prior to culling: 2.76% ±2.76, Min.: 0.0% - Max.: 13.1% supplied by MARTA, 1996)
Weaning index (alive PND 4–21)	92	85	87	88
(HCD: 95.2%±10.9, Min.: 73% - Max.: 100%, supplied by breeder)
(HCD: % dead pups post-culling - weaning: 0.71% ±1.28, Min.: 0.0% - Max.: 8.6% supplied by MARTA, 1996)
Male pup weight PND 0 (g)	6.70	6.59	6.49	6.28
Female pup weight PND 0 (g)	6.34	6.27	6.05	5.89
Male pup weight PND 21(g)	50.48	47.13	43.20*	42.62*
Female pup weight PND 21 (g)	49.04	47.03	40.43**	42.26*
F1 generation
Cohabitated pairs	26	26	26	26
Mated females	25	24	26	23
Pregnant females	24	21	23	19
Female fertility index (%)	96	88	88	83
Male fertility index (%)	96	88	88	83
Gestation index (%)	96	95	91	89
Mean duration of pregnancy (days)	21.8	22.0	21.9	22.0
Total prenatal litter loss	0	0	2	2
Live litters	23	20	21	17
Pups delivered (mean)	12.87	13.45	15.05*	12.29
Live birth index (%)	98	98	98	95
Viability index (alive PND 0-4)	93	87*	82**	81**
(HCD: 94.7%±5.6, Min.: 84% - Max.: 98%, supplied by breeder)
(HCD: % dead pups prior to culling: 2.76% ±2.76, Min.: 0.0% - Max.: 13.1% supplied by MARTA, 1996)
Weaning index (alive PND 4–21)	87	81	87	97*
(HCD: 95.2%±10.9, Min.: 73% - Max.: 100%, supplied by breeder)
(HCD: % dead pups postculling - weaning: 0.71% ±1.28, Min.: 0.0% - Max.: 8.6% supplied by MARTA, 1996)
Male pup weight PND 0 (g)	6.42	6.57	6.58	6.20
Female pup weight PND 0 (g)	6.02	6.14	6.24	5.83
Male pup weight PND 21 (g)	44.77	43.11	43.78	42.65
Female pup weight PND 21 (g)	43.22	42.68	41.13	39.99

 

It should be taken in account that due to the change of dose levels just before mating of the F0 animals, the apparent effects on fertility and neonatal growth and survival both in the highest dose group were attributed to treatment but cannot be attributed to a specific dose level. The general health of these animals was influenced and not comparable to that of the control animals at the time of mating and the reproductive effects occurred in the presence of a clear maternal effect.

 

Conclusions:

The following findings were obtained in study: Clinical findings, reduced body weight and food consumption in parental animals of mid and high dose (F0 and F1 generation).
Reduced fertility at high dose. Retarded body weight gain (F1 pups) and reduced survival (F2 pups) in mid and high dose group. No morphological alterations were seen.
Thus, NOAEL of 1.25 mg/kg bw/day pure active ingredient cyanamide was determined for maternal and developmental parameters and NOAEL of 3.75 mg/kg bw/day pure active ingredient was determined for reproduction parameters

Executive summary:

The continuous application of aqueous hydrogen cyanamide to Sprague-Dawley rats over two generations at dose levels of 0, 2.5, 7.5 and 30 mg/kg bw/day pure active ingredient during the first 12 weeks of treatment followed by 0, 1.25, 3.75 and 15 mg/kg bw/day of pure active ingredient orally by gavage affected reproductive performance and fertility of the F0 or F1 parental animals only in form of reduced fertility indices in F0 and F1 parental animals at the top dose group of 30/15 mg/kg bw/day active ingredient. All other observable differences between both of these groups and the concurrent controls were regarded to be incidental in nature and not of toxicological or biological concern.

 

Signs of general, systemic toxicity in both parental generations (F0 and F1) were confined to the rats of the mid and high dose levels. Toxicity was characterized by effects on food consumption and body weights and body weight gain during premating within the first 12 weeks. After reduction of the dose levels prior to mating of the F0 parental animals, the health status improved continuously. However, an impairment of body weight gain was also noted for the F1 parental males at the mid and high dose level (i.e. e. 3.75 and 15 mg/kg bw/day active ingredient) especially in the post-mating period and for the high dose F1 males during pre- and post-mating as well as for the high dose F1 females during premating and gestation. Concerning pathology, none of the recorded gross lesions or microscopic findings in the F0 and F1 generation parental animals was interpreted to represent an adverse treatment related effect.

 

Substance induced signs of developmental toxicity were observed in the progeny of the F0 parental generation in form of retarded body weight gain and in the F2 pups in form of reduced survival during early postnatal live resulting in decreased viability indices in the mid and high dose groups. In none of the F1 or F2 pups substance-induced morphological alterations were noted by external and visceral inspection.

 

Thus, under the conditions of this study the NOAEL (no observed adverse effect level) for reproductive performance and fertility is 3.75 mg/kg bw/day pure active ingredient due to the reduced fertility indices in both generations. The NOAEL for parental and developmental toxicity is considered to be 1.25 mg/kg bw/day pure active ingredient.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

3.75 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline compliant key study and 4 supporting studies.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Five reproduction toxicity studies in rats are reported.

In the most appropriate and valid as key selected study (Morseth, 1990), aqueous hydrogen cyanamide was orally applied by gavage to Sprague-Dawley rats over two generations. The dose levels for the first 12 weeks consisted of 0, 2.5, 7.5 and 30 mg/kg bw/day pure active ingredient but were lowered to 0, 1.25, 3.75 and 15 mg/kg bw/day of pure active ingredient subsequently due to severe effects, e.g. on general health, food consumption and body weight gain. Reproductive performance and fertility of the F0 or F1 parental animals in form of reduced fertility indices was affected in F0 and F1 parental animals at the top dose group, while signs of general systemic toxicity in both parental generations (F0 and F1) were confined to the rats of the mid and high dose levels in form of clinical findings and effects on food consumption and body weights and body weight gain during premating within the first 12 weeks. After reduction of the dose levels the health status improved continuously. Gross pathology and histopathology revealed no adverse treatment related effect. Substance induced signs of developmental toxicity were observed in the progeny of the F0 parental generation in form of retarded body weight gain and in the F2 pups in form of reduced survival during early postnatal live resulting in decreased viability indices in the mid and high dose groups. In none of the F1 or F2 pups substance-induced morphological alterations were noted by external and visceral inspection. The NOAEL for reproductive performance and fertility was 3.75 mg/kg bw/day pure active ingredient, while the NOAEL for parental and developmental toxicity was demonstrated at 1.25 mg/kg bw/day pure active ingredient

In a second, less valid and appropriate study (Rives and Obdach, 1985), the investigation of potential toxicity to reproduction was carried out in a 2-generation study. An aqueous solution of cyanamide (Colme) was administered orally per gavage in dose levels of 0, 2, 7 and 25 mg/kg bw to P0 male and female rats, before mating, during copulation (hereafter termination for males) and until weaning of resulting pubs (F1). Half of the P0 females was sacrificed on GD13 for a embryotoxicity study, the other half after weaning of F1 pubs. One F1 male and female were selected from each litter for mating and treated until weaning of resulting pubs (F2). Different examinations were carried out: body weight, sperm parameters, daily vaginal smear inspection during mating, organ weight and ratios, histopathological examination of reproductive organs of F0 and F1 males and females, embryotoxicity in females of F0 (number and distribution of embryos in horns, corpora lutea, presence of empty implantation sites, resorptions), behavioural and developmental parameters (litter). The results indicate a decreased fertility rate in male and female rats of generation F0 treated with 25 mg/kg bw of cyanamide. This finding is in accordance with some weight diminutions in the reproductive organs of the F0 generation males treated with the highest dose. Additionally, testicular atrophy was observed, uni- and bilateral in this dose group. Further, alterations in the embryotoxicity study were observed with 25 mg/kg bw. A diminished number of implantations and, consequently, a decreased number of embryos occurred. As no group-specific differences regarding resorptions and the number of dead embryos was recorded, the possible effects of high dose of cyanamide seem to take place previously to the implantations in the uterus. The reproduction study in F0 lead to results that are in line with the ones from the embryotoxicity study: a lower number of implantations and new-born obtained from rats treated with the highest dose of the test item, compared to the control group. The results of the behavioural developmental study in F1-pubs did not show any treatment-relation. Further, no treatment-related effects were observed in the reproduction study of F1. The results indicate no changes in fertility rate and a lack of toxicological effect on development. Similarly, F2 was showing quantitatively irrelevant delays and anticipations. No external and visceral anomalies were induced in the offspring by the treatment with cyanamide.

In conclusion, cyanamide administered at doses of 2 and 7 mg/kg bw in rats did not affect fertility and reproductive or embryotoxic parameters in a two-genereration study in rats.

Cyanamide aqueous solution was also administered via the diet in a supporting 2-generation reproduction toxicity study at concentrations of 0, 20, 60 and 180 ppm to Wistar rats (Koeter, 1986). The only effect on reproductive performance and fertility consisted of a reduction in litter size of the 1st F1 pregnancy at the high dose level and an increased incidence of F1 males with interstitial cell proliferation in the testes at the high dose level. Parental toxic effects occurred in form of reduced food consumption, lower body weights, retarded body weight gains and effects on the thyroid gland due to increased weights and histopathological findings at the high dose level only. Indications for developmental toxicity were only observed in form of initially affected postnatal survival (F2 pups 2nd pregnancy) and reduced birth weights and retarded body weight gain until weaning in both litters at the high dose. In none of the pups substance-induced morphological alterations were noted. The respective NOAELs for reproductive performance and fertility, parental toxicity as well as for developmental toxicity were 60 ppm.

In an additional supporting study cyanamide was administered via oral gavage to Sprague- Dawley CD rats for 2 consecutive generations at dose levels of 0, 2, 7 and 25 mg/kg bw/day (Valles, 1987). The fertility of high dose males and, to a lower extent, high dose females was impaired. The NOAELs for parental animals, reproduction and offspring were 7 mg/kg bw/day, based on the histopathological changes in the testes, the fertility rate and embryo-fetal mortality.

Further, oral (gavage) administration of hydrogen cyanamide to Sprague-Dawley rats from days 6 to 12 of gestation at a dose of 45 mg/kg/day was associated with body weight loss and a reduction in mean food consumption leading to 12 % lower mean terminal body weight compared with control (Pique. 2016). A treatment-related increase in arterial blood pressure before dosing on G 11 was noted. There was also a lower mean zinc concentration in the 45 mg/kg/day group compared with the control. Thyroid follicular-cell hypertrophy, associated with a higher mean TSH concentration, was observed in all animals given 45 mg/kg/day. A treatment-related lower group mean thymus weight, correlated with lymphoid depletion, was noted. There was also a treatment-related haemorrhage in the sternal bone marrow, probably accounting for decreased reticulocyte count, and a reduction in haematopoiesis in the spleen.

Finally, considering the weight of evidence of the recorded effects and the validity and reliability of the different studies the overall NOAELs were derived from the first study as a conservative approach. Namely, 3.75 mg/kg bw/day pure active ingredient for reproductive performance and fertility and 1.25 mg/kg bw/day pure active ingredient for parental and developmental toxicity.

Moreover, it is noteworthy to mention that in all reproduction toxicity studies, irrespectively of application procedure and dose levels, no substance-induced morphological alterations were noted by external and visceral inspection in any of the F1 or F2 pups that died or were killed scheduled for culling or at weaning. There was especially no increased incidence in pups with diaphragma hernia or eye anomalies.

Justification for selection of effect on fertility via oral route: GLP and guideline compliant two-generation study.

Effects on developmental toxicity

Description of key information

Three teratogenicity studies examined the effects of cyanamide in rats and in rabbits. In the first rat study (Pique, 2014), a NOEL of 3 mg/kg bw/day for both maternal and develomental toxicity was revealed. The NOAEL for maternal toxicity in the second rat study (Morseth, 1989) was very close to 5 mg/kg bw/day of pure active ingredient based on the effect on maternal body weight only. The NOAEL for prenatal developmental toxicity in rats was 15 mg/kg bw/day of pure active ingredient based on reduced fetal weights and affected fetal morphology (Morseth, 1989). In rabbits the NOAEL for both maternal toxicity and prenatal developmental toxicity in rabbits was considered as 6 mg/kg bw pure active ingredient (Koeter, 1989).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-05-21 to 2013-06-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Rationale for repetition of study: Cyanamide is registered in the USA as a plant growth regulator and is currently reviewed by EPA. In order to support an acceptable reference dose for short-term dermal exposure AlzChem decided to prepare a study according to OECD 414 and OPPTS 870.3700. A previous prenatal development study in the Sprague-Dawley rat with Hydrogen Cyanamide (HLA2319-124) did not establish a clear no effect level for maternal toxicity, so this study was performed in order to further characterize previous findings whilst using a wider range of doses. In addition, the relevant guidelines have been updated since the previous study was conducted. Therefore, this study provides all data as required in the current guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France
- Age at study initiation: age at mating 10 to 13 weeks
- Weight at study initiation: 198 to 245 g at mating
- Housing: One air-conditioned room in a barrier protected unit
- Diet: ad libitum, pelleted commercial complete rodent diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants.
- Water: Filtered (0.2 µm) mains drinking water was available ad libitum (via bottles).
- Acclimation period: 6 days between animal arrival and the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3
- Humidity (%): Between 35 and 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared as a solution in the vehicle at concentrations of 0.3, 1.5 and 4.5 mg Hydrogen Cyanamide/mL based on a content of 526.4 mg/mL which took into account the actual density (1.057 g/cm3 at +20 °C) and concentration of Hydrogen Cyanamide (49.8 % w/w). Dosing solution was prepared weekly.

VEHICLE
- Amount of vehicle: Dose volume: 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplicate 3 mL samples were taken from each newly prepared formulation, including the vehicle, to ensure that the nominal concentrations were in line with the analytically determined concentrations. One set of samples was analysed immediately at the Test Facility using the validated method.
The transfer and validation of the analytical method as well as the stability analysis was the subject of a separate study plan (WIL Research study number AB18598).

Details on mating procedure:

The females were mated at the supplier with a documented day of mating. They were received at the Test Facility on day 0 of gestation.

Duration of treatment / exposure:

From day 6 (G 6) to day 19 (G 19) of gestation inclusive.

Frequency of treatment:

once daily

Duration of test:

Animals were killed on the day after the last treatment, which was day 20 of gestation. Treatment had started on day 6.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

3 mg/kg bw/day

Remarks:

active substance

Dose / conc.:

15 mg/kg bw/day

Remarks:

active substance

Dose / conc.:

45 mg/kg bw/day

Remarks:

active substance

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The mid and high dose levels were selected solely to replicate a previous prenatal development study in the Sprague-Dawley rat (HLA 2319-124) which yielded equivocal results. In that study a marked maternal toxicity (including body weight loss) was elicited in the high dose group. The low dose was selected to try and establish a no effect level for maternal toxicity.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 3, 6, 9, 12, 15, 18 and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The ovaries and uterus of each female were removed and examined.
The placentae were also examined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: -individual foetal weights, foetal sex

Fetal examinations:

- External examinations: Yes [all per litter]
- Soft tissue examinations: Yes [all per litter]
- Skeletal examinations: Yes [appr. 50 % per litter]
- Head examinations: Yes [appr. 50 % per litter]

Statistics:

Statistical analysis was performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon:
-the normality of the data distribution tested by the Shapiro-Wilk's test
-the homogeneity of the variances across groups tested by the Bartlett's test.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
The number of resorptions, number of dead foetuses and all litter-based percentages were analysed using non-parametric methods, i.e. Kruskal-Wallis test followed by non parametric Dunnett’s test if the Kruskal-Wallis was significant.
Selected incidence data were analysed using a chi2 test for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.

Historical control data:

Results were compared to Historical control data; Rat - CD® IGS (Crl:CD[SD]), 1996-2009; Charles River Laboratories Preclinical Services Montreal; All routes of administration; Embryo-fetal development studies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-related and statistically significant reduction in overall mean body weight gain and food consumption during the dosing period in the 15 and 45 mg/kg bw/d groups leading to lower mean terminal body weight (-6 % and -27 % in groups 3 and 4, respectively) compared with the control. The effect in the high dose group was associated with body weight loss during the first 6 days of the dosing period (GD 6 to GD 12). The effect was confirmed by lower mean net body weight change (i.e. maternal body weight change from GD 6 to GD 20 minus gravid uterus weight) in the 15 and 45 mg/kg bw/d groups (27 g and -34 g, respectively) compared with the control (48 g).

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment-related clinical signs were restricted to the 45 mg/kg bw/day group and included piloerection for all females and decreased activity, intermittent whole body tremors and pale faeces for approximately half of the animals. The health status of three females in the high dose group was closely monitored due to severe body weight loss during the first 6 days of the dosing period, in order to decide if an ethical sacrifice was necessary. There was a dose-related and statistically significant reduction in overall mean body weight gain and food consumption during the dosing period (G 6 to G 20) in the 15 and 45 mg/kg bw/day groups leading to lower mean terminal body weight (-6 % and -27 % in groups 3 and 4, respectively) compared with the control. The effect in the high dose group was associated with body weight loss during the first 6 days of the dosing period (G 6 to G 12). The effect was confirmed by lower mean net body weight change (i.e. maternal body weight change from G 6 to G 20 minus gravid uterus weight) in the 15 and 45 mg/kg bw/day groups (27 g and -34 g, respectively) compared with the control (48 g).
There were no treatment-related macroscopic findings in any group.

Key result

Dose descriptor:

NOEL

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOEL

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Consistent with the marked maternal toxicity, there was clear evidence of general developmental toxicity (delay in development) in the 45 mg/kg bw/day group with markedly reduced mean foetal weight (-38 %) (resulting in lower mean gravid uterus weight), widespread incomplete/absent ossification of axial and appendicular skeletal bones and thickened and/or wavy ribs (7 % and 31 %, respectively) compared with the control. Also consistent with the low foetal weight and the delayed development, there were also 7 foetuses from the same number of litters with a malpositioned (not completely descended) testis. It is worthy of note that these malformations were observed in only one female out of the three which had the most pronounced maternal toxicity.
Other malformations noted in the high dose group including 10 (7) foetuses (litters) with a small diaphragmatic hernia (no impact on the lung size) and 4 (4) foetuses (litters) with defects of the great blood vessels can not be clearly attributed to the observed maternal toxicity. However, due to the severity of the maternal toxicity in the high dose group (27 % lower terminal mean body weight compared with the control), foetal well-being was compromised, so the regulatory value of the data in this group are likely to be of limited value for determining developmental effects.
In the 15 mg/kg bw/day dose group, there was also a treatment-related lower mean foetal weight in comparison with the concurrent control (-8 %) (resulting in a lower mean gravid uterus weight of 13 %). The presence of a single foetus (1/312 foetuses – 0.3 % incidence) in the mid dose group with great blood vessel defects is probably incidental since great vessels defects are also seen at a low frequency (0.00 - 0.69 %) in the historical control data for the Sprague-Dawley rat.

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

other: malpositioned testes, diaphragmatic hernia, defects of the great bood vessels

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

visceral/soft tissue: cardiovascular

visceral/soft tissue: male reproductive system

Description (incidence and severity):

There was clear evidence of general developmental toxicity (delay in development) in the 45 mg/kg bw/d group with markedly reduced mean foetal weight (-38 %) (resulting in lower mean gravid uterus weight), widespread incomplete/absent ossification of axial and appendicular skeletal bones and thickened and/or wavy ribs (7 % and 31 %, respectively) compared with the control. There were also 7 foetuses from the same number of litters with a malpositioned (not completely descended) testis. Other malformations noted in the high dose group included 10 (7) foetuses (litters) with a small diaphragmatic hernia (no impact on the lung size) and 4 (4) foetuses (litters) with defects of the great blood vessels.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

45 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Table 1: Summary of maternal and litter data

Dose level (mg/kg bw/d)	0	5	15	45
No of females in study	25	25	25	25
Pregnant dams	25	24	25	23
Corpora lutea (mean)	15.2	14.7	14.6	14.6
Implantation sites (mean)	14.1 ± 2.0	14.0 ± 2.5	14.0 ± 2.0	14.1 ± 1.7
Early resorptions (%)	6.96 ± 8.18	8.63 ± 9.98	10.23 ± 7.25	8.06 ± 8.07
Late resorptions (mean % ± SD)	0.87 ± 2.42	3.40 ± 12.30	0.77 ± 2.87	3.14 ± 8.39
Pre-implantation loss (mean % ± SD)	6.51 ± 9.30	5.36 ± 8.75	3.72 ± 7.48	3.2 ± 4.78
Post implantation loss (mean %)	7.83 ± 9.27	12.03 ± 14.13	11.00 ± 7.88	11.20 ± 13.27
Body weight gain day 6-16 (g)	50.2	40.9*	31.9*	6.2*
Body weight gain day 6-20 (g)	105.8	94.2*	81.4*	51.0*
Food consumption day 6-16 (% of control)	100	96	89*	76*
Food consumption day 16-20 (% of control)	100	96	92*	77*
Carcass weight (adjusted) (g)	315.7	315.2	300.7*	236.6*
Net weight change (adjusted) from day GD6-20 (g)	47.75 ± 10.67	43.76 ± 10.59	27.15 ± 11.24**	-34.34 ± 20.30***
Gravid uterus weight (g)	80.18	75.48	69.80*	51.39***
Live litter size (mean)	13 ± 2.4	12.5 ± 2.9	12.5 ± 2.2	12.6 ± 2.7
Mean foetal weight (G)	4.01 ± 0.21	3.97 ± 0.30	3.67 ± 0.31**	2.47 ± 0.35***
Dead foetuses (%)	0	0	0	0
% male foetuses	47.49	50.97	50.14	44.70

*/** statistically significant (p-values not indicated)

 

Table 2: Summary of relevant foetal observations

Dose level (mg/kg bw/d)	0	3	15	45
Diaphragma hernia	0	0	0	10 (7)**
Testis malpositioned	0	0	0	7 (7)**
Great blood vessel defects - aortic arch narrowed - multiple abnormalities	0 0 0	0 0 0	1 (1) 0 1 (1)	4 (4) 1 (1) 3 (3)
Fused sternebrae	0	0	0	2 (1)
Hemisterebrae	0	0	0	3 (3)
Wavy/bent ribs	0	0	0	47 (19)**
Incomplete ossification - skull - limbs - sternebrae - ribs - vertebrae - pervic girdle	- - - - - - -	- - - - - - -	- - - - - - -	↑↑↑** ↑↑↑** ↑↑↑** ↑↑↑** ↑↑↑** ↑↑↑** ↑↑↑**
Thickened ribs	0	0	0	10 (16)*
Short rib	0	0	0	4 (4)
Thoracic Vertebra: bipartite ossification of centrum	1 (1)	1 (1)	2 (2)	14 (10)**
Hemicentrum	0	0	0	2 (1)**

*/** statistically significant (p-values not indicated)

 

Conclusions:

Oral (gavage) administration of hydrogen cyanamide to Sprague-Dawley rats from days 6 to 19 of gestation at doses of 3, 15 and 45 mg/kg bw/day active ingredient was associated with dose-related lower mean body weight gain and food consumption in the mid and high dose groups leading to 6 % and 27 % lower mean terminal body weight (effects tending to be more pronounced between G 6 and G 12), respectively, compared with the control. In the high dose group, the effect led to a net body weight loss (-34 g compared with +48 g in the control) and it was associated with marked clinical effects (i.e. piloerection, decreased activity, intermittent whole body tremors and pale faeces). Although there was no mortality, a premature sacrifice was considered for three females in the high dose group (45 mg/kg bw/day) due to the severe maternal toxicity.
Foetal development was clearly compromised in the high dose group (45 mg/kg bw/day) with a marked reduction in foetal weight, a delayed testicular descent, a delay in ossification of axial and appendicular skeletal bones and rib anomalies. However, within the context of this study, it is not possible to establish if other developmental effects (small diaphragmatic hernia and great blood vessel changes) can be attributed to the marked maternal toxicity and the compromised foetal well-being. Therefore, the regulatory value of the data in this group is likely to be of limited value for determining developmental effects.
The only developmental effect clearly attributable to treatment in the mid dose group of 15 mg/kg bw/day group was a slight reduction in mean foetal weight.
The low dose of 3 mg/kg bw/day is a no observed effect level (NOEL) for both maternal and embryo-foetal toxicity.

Executive summary:

The test item, hydrogen cyanamide, was administered by gavage at dose levels of 3, 15 and 45 mg/kg bw/day (active ingredient) to groups of 25 mated female Sprague-Dawley rats from days 6 to 19 of gestation inclusive. A control group received the same dose volume (10 mL/kg bw) of the vehicle (water for injection). Clinical condition, body weights and food consumption were monitored throughout the study. The females were submitted to a caesarean examination on day 20 of gestation. At necropsy, the females were examined macroscopically, the gravid uterus was weighed and litter parameters were recorded. All foetuses were weighed, sexed and examined for external andfresh visceral (abdominal and thoracic cavities) abnormalities.The heads of approximately half of the foetuses/litter were removedand subsequently submitted to fixed soft tissue examinationby serial sectioning. The eviscerated carcasses of the remaining half of the foetuses were submitted to skeletal examination. There was no mortality in any group.

Treatment-related clinical signs were restricted to the 45 mg/kg bw/day group and included piloerection for all females and decreased activity, intermittent whole body tremors and pale faeces for approximately half of the animals. The health status of three females in the high dose group was closely monitored due to severe body weight loss during the first 6 days of the dosing period, in order to decide if an ethical sacrifice was necessary.

There was a dose-related and statistically significant reduction in overall mean body weight gain and food consumption during the dosing period (G 6 to G 20) in the 15 and 45 mg/kg bw/day groups leading to lower mean terminal body weight (-6 % and -27 % in groups 3 and 4, respectively) compared with the control. The effect in the high dose group was associated with body weight loss during the first 6 days of the dosing period (G 6 to G 12). The effect was confirmed by lower mean net body weight change (i.e. maternal body weight change from G 6 to G 20 minus gravid uterus weight) in the 15 and 45 mg/kg bw/day groups (27 g and -34 g, respectively) compared with the control (48 g).

There were no treatment-related macroscopic findings in any group.

There were 25, 24, 25 and 23 pregnant females in the control, low, mid and high dose groups, respectively, at the terminal caesarean examinations, all of which had viable foetuses.

There was no influence of treatment on embryo-foetal survival or on sex ratio in any group.

Consistent with the marked maternal toxicity, there was clear evidence of general developmental toxicity (delay in development) in the 45 mg/kg bw/day group with markedly reduced mean foetal weight (-38 %) (resulting in lower mean gravid uterus weight), widespread incomplete/absent ossification of axial and appendicular skeletal bones and thickened and/or wavy ribs (7 % and 31 %, respectively) compared with the control. Also consistent with the low foetal weight and the delayed development, there were also 7 foetuses from the same number of litters with a malpositioned (not completely descended) testis. It is worthy of note that these malformations were observed in only one female out of the three which had the most pronounced maternal toxicity.

Other malformations noted in the high dose group including 10 (7) foetuses (litters) with a small diaphragmatic hernia (no impact on the lung size) and 4 (4) foetuses (litters) with defects of the great blood vessels can not be clearly attributed to the observed maternal toxicity. However, due to the severity of the maternal toxicity in the high dose group (27 % lower terminal mean body weight compared with the control), foetal well-being was compromised, so the regulatory value of the data in this group are likely to be of limited value for determining developmental effects.

In the 15 mg/kg bw/day dose group, there was also a treatment-related lower mean foetal weight in comparison with the concurrent control (-8 %) (resulting in a lower mean gravid uterus weight of 13 %). The presence of a single foetus (1/312 foetuses – 0.3 % incidence) in the mid dose group with great blood vessel defects is probably incidental since great vessels defects are also seen at a low frequency (0.00 - 0.69 %) in the historical control data for the Sprague-Dawley rat.

Based on the observed findings, the low dose of 3 mg/kg bw/day is a no observed effect level (NOEL) for both maternal and embryo-foetal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

3 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

3 GLP and studys available with two different species (rabbit and rat).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Three developmental toxicity studies are available, two conducted on rats and one conducted on rabbits.

In the first rat study (Pique, 2014), oral (gavage) administration of hydrogen cyanamide to Sprague-Dawley rats from days 6 to 19 of gestation at doses of 3, 15 and 45 mg/kg bw/day active ingredient was associated with dose-related lower mean body weight gain and food consumption in the mid and high dose groups leading to 6 % and 27 % lower mean terminal body weight (effects tending to be more pronounced between G 6 and G 12), respectively, compared with the control. In the high dose group, the effect led to a net body weight loss (-34 g compared with +48 g in the control) and it was associated with marked clinical effects (i.e. piloerection, decreased activity, intermittent whole body tremors and pale faeces). Although there was no mortality, a premature sacrifice was considered for three females in the high dose group (45 mg/kg bw/day) due to the severe maternal toxicity.

Foetal development was clearly compromised in the high dose group (45 mg/kg bw/day) with a marked reduction in foetal weight, a delayed testicular descent, a delay in ossification of axial and appendicular skeletal bones and rib anomalies. However, within the context of this study, it is not possible to establish if other developmental effects (small diaphragmatic hernia and great blood vessel changes) can be attributed to the marked maternal toxicity and the compromised foetal well-being. Therefore, the regulatory value of the data in this group is likely to be of limited value for determining developmental effects.

The only developmental effect clearly attributable to treatment in the mid dose group of 15 mg/kg bw/day group was a slight reduction in mean foetal weight.

The low dose of 3 mg/kg bw/day is a no observed effect level (NOEL) for both maternal and embryo-foetal toxicity (Pique, 2014).

 

In the second rat study (Morseth, 1989), dose levels referring to 0, 5, 15 and 45 mg/kg bw/day pure active ingredient led to dose-dependently retarded body weight gain during substance application at all dose levels accompanied a statistically significant reduction in the net body weight change at the mid and high dose level. In addition, initial clinical findings and a reduction in food consumption was noted at these dose levels. An influence on gestational parameter was not noted.

Signs of prenatal developmental toxicity consisted of reduced fetal weights and affected fetal morphology at the high dose level due to an increased incidence of soft tissue malformation in form of diaphragmatic hernia, isolated cases of skeletal malformations and a variety of skeletal variations were observed at 45 mg/kg bw/day, a dose with considerable maternal toxicity.

Thus, although a clear NOAEL for maternal toxicity was not achieved at the low dose level, an appropriate NOAEL can be regarded as very close to 5 mg/kg bw/day of pure active ingredient based on the minor severity of the noted effect on maternal body weight only.

The NOAEL for prenatal developmental toxicity was determined to be 15 mg/kg bw/day of pure active ingredient (Morseth, 1989).

In the rabbit study (Koeter, 1989), the investigated dose levels of 0, 2, 6 and 18 mg/kg bw/day pure active ingredient elucidated maternal toxicity in form of losses in body weight and adjusted body weight during the period of substance application as well as maternal stress indicated by total litter loss in one female at this dose level due to resorption. Gestation was affected at the high dose level due to an increase in early resorption with the consequence of a higher post-implantation loss.

Prenatal developmental toxicity consisted of increases in dead and smaller fetuses, lower number of fetuses and fetal body weight as well as an increase in some soft tissue anomalies (i.e. variations) at the high dose level only. A treatment related effect on the fetal skeleton was not observed and there was no indication for teratogenicity up to and including the high dose level.

Therefore, the NOAEL for maternal toxicity and prenatal developmental toxicity in rabbits was considered as 6 mg/kg bw pure active ingredient (Koeter, 1989).

 

Conclusion

The findings in the high dose groups of both rat studies (Piquet, 214and Morseth, 1989) must be seen in conjunction with the severe maternal toxicity leading to massive reductions of net body weight gain in both studies associated with an extremely compromised health status of some animals in one of these studies. Due to the severe maternal toxicity the findings observed at 45 mg/kg bw/d should not be considered for developmental toxicity classification. The observations at the mid dose level (15 mg/kg bw/d), where still clear maternal toxicity was observed, would per se not warrant a classification for developmental toxicity. However, taking into account the critical effect noted in the high dose groups of both studies (diaphragmatic hernias), a classification into Category 2 (H361d) was concluded by RAC (2015).

Justification for selection of Effect on developmental toxicity: via oral route: Most reliable GLP and guideline compliant study was chosen.

Justification for classification or non-classification

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

The available experimental test data are reliable and sufficient for classification purposes according to Regulation (EC) No 1272/2008 (CLP). Thus, based on the results of the reproductive and developmental toxicity studies discussed above, the substance is classified for developmental toxicity and toxicity to reproduction with cat. 2 (H361fd Suspected of damaging fertility or the unborn child) in accordance Regulation (EC) No 1272/2008, as amended for the eighteenth time in Regulation (EU) 2022/692, also in accordance with the conclusion of the 33 RAC-Meeting (1-5 June, 2015).