Cyanamide

Developmental toxicity / teratogenicity

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cyanamide

EC Number:

206-992-3

EC Name:

Cyanamide

Cas Number:

420-04-2

Molecular formula:

CH2N2

IUPAC Name:

cyanamide

Test material form:

other: aqueous solution

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day

Remarks:

active substance

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day

Remarks:

active substance

Doses / concentrations 4

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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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).

Maternal developmental toxicity

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.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOEL

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOEL

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

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.

Overall developmental toxicity

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

Any other information on results incl. tables

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)

 

Applicant's summary and conclusion

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.