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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-03-24 to 2014-10-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium cyanamide
EC Number:
205-861-8
EC Name:
Calcium cyanamide
Cas Number:
156-62-7
Molecular formula:
CN2.Ca
IUPAC Name:
calcium cyanoazanediide
impurity 1
Chemical structure
Reference substance name:
Calcium oxide
EC Number:
215-138-9
EC Name:
Calcium oxide
Cas Number:
1305-78-8
Molecular formula:
CaO
IUPAC Name:
oxocalcium
impurity 2
Chemical structure
Reference substance name:
Carbon
EC Number:
231-153-3
EC Name:
Carbon
Cas Number:
7440-44-0
Molecular formula:
C
IUPAC Name:
carbon
impurity 3
Reference substance name:
Hematite (Fe2O3)
EC Number:
215-275-4
EC Name:
Hematite (Fe2O3)
Cas Number:
1317-60-8
Molecular formula:
Fe2O3
IUPAC Name:
diiron oxide
impurity 4
Chemical structure
Reference substance name:
Urea
EC Number:
200-315-5
EC Name:
Urea
Cas Number:
57-13-6
Molecular formula:
CH4N2O
IUPAC Name:
urea
impurity 5
Chemical structure
Reference substance name:
Silicon dioxide
EC Number:
231-545-4
EC Name:
Silicon dioxide
Cas Number:
7631-86-9
Molecular formula:
O2Si
IUPAC Name:
dioxosilane
impurity 6
Chemical structure
Reference substance name:
Trisilicon tetranitride
EC Number:
234-796-8
EC Name:
Trisilicon tetranitride
Cas Number:
12033-89-5
Molecular formula:
N4Si3
IUPAC Name:
trisilicon tetranitride
impurity 7
Chemical structure
Reference substance name:
Calcium dihydroxide
EC Number:
215-137-3
EC Name:
Calcium dihydroxide
Cas Number:
1305-62-0
Molecular formula:
CaH2O2
IUPAC Name:
calcium dihydroxide
impurity 8
Chemical structure
Reference substance name:
Aluminium oxide
EC Number:
215-691-6
EC Name:
Aluminium oxide
Cas Number:
1344-28-1
Molecular formula:
Al2O3
IUPAC Name:
aluminium oxide
impurity 9
Chemical structure
Reference substance name:
Cyanoguanidine
EC Number:
207-312-8
EC Name:
Cyanoguanidine
Cas Number:
461-58-5
Molecular formula:
C2H4N4
IUPAC Name:
2-cyanoguanidine
impurity 10
Chemical structure
Reference substance name:
Calcium acetylide
EC Number:
200-848-3
EC Name:
Calcium acetylide
Cas Number:
75-20-7
Molecular formula:
C2Ca
IUPAC Name:
calcium ethynediide
impurity 11
Chemical structure
Reference substance name:
Calcium sulfate
EC Number:
231-900-3
EC Name:
Calcium sulfate
Cas Number:
7778-18-9
Molecular formula:
CaH2O4S
IUPAC Name:
calcium sulfate
impurity 12
Reference substance name:
unknown
IUPAC Name:
unknown
Test material form:
solid: particulate/powder
Details on test material:
Batch 335211

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: 10 to 13 weeks old
- Weight at study initiation: 187 to 281 g
- Housing: Females were individually housed in plastic cages with autoclaved sawdust and shredded paper as bedding
- Diet: Rat pelleted commercial complete rodent diet ad libitum
- Water: Softened and 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: 22 + 3 °C
- Humidity: Between 35 and 70 %
- Air changes: At least 10 air changes per hour
- Photoperiod: 12 hours light (artificial)/12 hours dark (except when required for technical acts).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Explanation for suitabillity of vehicle "Corn oil" see "any other information on materials and methodes incl. tables"
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Preparation: The test item was prepared as a suspension in the vehicle, at the concentrations of 3.5, 10.5 and 24.5 mg/mL
- Frequency: once daily
- Method: Gavage using a plastic canula. Formulations were brought to room temperature for at least 15 minutes prior to dosing
- Volume of administration: 2 mL/kg/day. Individual dose volumes were calculated using the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability, Homogeneity and formulation analyses were conducted.
- Analysis of formulations: 4 x 3 g samples were taken under magnetic stirring from the top, middle and bottom of each formulation (middle only for control), used on one of the first days of treatment and again during the last week of dosing.
In addition, 5 x 3 g samples were taken from the middle of Groups 2 and 3 formulations for additional analysis.
One set (2 x 3 g) of samples was analysed at the Test Facility using a validated method (WIL study no. AB20445).
- Results: All formulations at 3.5, 10.5 and 24.5 mg/mL of Calcium Cyanamide used for treatment on the first and last days of treatment were in agreement with acceptance criteria since the deviations from the nominal concentrations ranged from -12.3 % to +9.4 %. No test item was detected in control samples.

Details on mating procedure:
The female animals (n= 100) were mated at the supplier and delivered the same day to the test facility (day 0 of gestation (GD 0)).
Duration of treatment / exposure:
From day 6 (G6) to day 19 (G19) of gestation inclusive
Frequency of treatment:
Once daily, in the morning, at approximately the same time of day (+ 30 minutes), except for the first treatment on 31 March 2014 when treatment was performed in the afternoon. This delay was due to late availability of the first analytical results required prior to the start of dosing.
Duration of test:
Experimental starting date (first animal arrival): 25 March 2014
Experimental completion date (last caesarean): 17 April 2014
No. of animals per sex per dose:
25 female animals
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the study sponsor based on the results of the dose range-finding study by the oral route (gavage) in the pregnant rat

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed daily for clinical signs. During the treatment period, the animals were observed before and at least once after dosing to detect any clinical signs or reaction to treatment. A full clinical examination was performed on each weighing day

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed individually on days 0, 6, 9, 12, 15, 18 and 20 of gestation.

FOOD CONSUMPTION: Yes
- Individual food consumption of each animal was measured for the periods (days) 0 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20 during gestation.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Animals were dissected and examined for macroscopic pathological changes. 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

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number and distribution of live foetuses
- Individual foetal weights
- Foetal sex

The uterus of all females was placed in ammonium sulphide solution in order to stain any previously undetected implantation sites.
Fetal examinations:
- External examinations: Yes, each foetus
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
Approximately one half of each litter was examined for fresh visceral anomalies and then processed for skeletal examination. The ossified skeleton was stained with Alizarin red following maceration of the soft tissues in potassium hydroxide solution. The stained specimens were preserved in glycerol

- Head examinations: Yes: half per litter
The remaining half of the foetuses in each litter was fixed in Harrison’s fixative for subsequent examination of the head only by serial sectioning. The remaining foetal carcasses were retained fixed but not examined further.
Statistics:
Statistical analysis was performed, where appropriate, by the data acquisition software, as follows:
- the data were checked for homogeneity of variance using Bartlett’s test
- homogenous data were then analysed by ANOVA and resulting intergroup differences were analysed with a Dunnett's t-test
- non-homogenous data were analysed with a Kruskal-Wallis test followed by Dunn’s test if the Kruskal-Wallis was significant.

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.
Indices:
For each group, the following parameters were calculated:

Pre-implantation loss (in %): Number of corpora lutea - Number of implantations x 100 / Number of corpora lutea

Post-implantation loss (in %): Number of implantations - Number of viable foetuses x 100 / Number of implantations
Historical control data:
The data concerning the control pregnant females and the historical data were used to evaluate the effects of the test item.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: Dose-related reductions in overall mean body weight gain and food consumption in the 21 and 49 mg/kg/day groups

Details on maternal toxic effects:
- Mortality
There was no mortality in any group.

- Clinical signs
There were no treatment related clinical signs in any group.
One control female had a mass in the abdominal region. Scab(s), chromodacryorrhea, localised hairloss and white teeth occasionally observed amongst treated groups were considered incidental.

- Body weight
On average, there was a dose-related and statistically significant lower mean body weight gain during the dosing period (G 6 to G 20) in the 21 and 49 mg/kg bw/day groups (-9 % and -23 %, respectively) compared with the control. This finding was confirmed for net mean body weight gain following adjustment for gravid uterus weight (-23 % and -43 %, respectively compared with the control). Mean terminal body weight and carcass weight were only slightly affected in the 49 mg/kg bw/day group (-6 % compared with the control). Mean body weight gain in the 7 mg/kg bw/day group was comparable with that in the control.

- Food consumption
On average, there was a dose-related and statistically significant reduction in mean food consumption during the dosing period (days 6 to 20 of gestation) in the 21 and 49 mg/kg bw/day groups (-6 and -10 %, respectively) compared with the control. Mean food consumption in the 7 mg/kg bw/day group was comparable with that in the control.

-Necropsy findings of adult females
There was no treatment-related macroscopic finding at necropsy. Consistent with the clinical observations, one control female had a mass in the abdominal region.

-Gravid uterus weight
Consistent with lower mean foetal weight, mean gravid uterus weight in the 49 mg/kg bw/day group was slightly lower (-7 %) than in the control. Despite lower mean foetal weight, there was no similar finding in the 21 mg/kg bw/day group due to a marginally superior litter size (see 10.7.3). Mean gravid uterus weight in the lower dose groups was therefore comparable with that in the control.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 7 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 49 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Pregnancy incidence
There were 24, 25, 23 and 24 pregnant females at the terminal caesarean sections in groups 1 to 4, respectively, all of which had viable foetuses.

- Pre-implantation data
The pre-implantation data (mean numbers of corpora lutea and implantation sites and the percentage pre-implantation loss) were comparable in all groups.

- Post-implantation data
There was no obvious influence of treatment on embryo-foetal survival in any group. Two females in the 49 mg/kg bw/day group had 1 or 3 late resorptions and another had a dead foetus compared with one female with a single late resorption in the control. In the absence of a similar trend amongst the other females in the high dose group, and since the percentage post-implantation loss remained comparable with that in the concurrent control and historical control data, these isolated cases was considered incidental. Mean live litter size was comparable with, or marginally superior to, that in the control group for all treated groups.

- Foetal data
There was a slight but statistically significant lower mean foetal weight in the 21 and 49 mg/kg bw/day groups (3.7 and 3.5 g corresponding to -7 % and -11 %, respectively) in comparison with the concurrent control (3.9 g) and historical control data (4.1 g). There was no effect of treatment on mean foetal weight in the 7 mg/kg bw/day dose group. Foetal sex ratio in treated groups was comparable with that in the control.

- External observations
There was no treatment-related external malformation in any group. One foetus from control female no. 4 had micrognathia.

- Soft tissue findings
There was no foetal visceral malformation noted in any treated group. The incidences of less severe visceral anomalies and variations such as absent innominate artery, pulmonary arteries with a common origin, discolored adrenal gland, transposed umbilical artery and dilated and/or convoluted ureter(s), did not suggest any association with treatment.

- Skeletal observations
There were three foetuses, one in the control group and two from separate litters in the 7 mg/kg bw/day group, with skeletal malformations.
The foetus from the control group had a short mandible confirmed with fused symphysis and one foetus from the 7 mg/kg bw/day group had a short premaxilla and misshapen maxilla. One other foetus from the 7 mg/kg bw/day group had multiple abnormalities of the cervical and thoracic vertebrae leading to scoliosis. These isolated findings in the control and low dose groups were considered to be incidental due to the absence of any similar findings in the higher dose groups.
There was a slightly higher incidence of increased ossification of the 1st centrum of the cervical vertebrae and bipartite ossification of thoracic vertebrae centra in the 49 mg/kg bw/day group compared with the control and historical control data. Although possibly associated with treatment, these minor changes are considered to be of no physiological or toxicological significance and represent normal variation that exists in the rate of development of some skeletal elements.
There were no other differences between the treated groups and the control amongst the remaining minor skeletal changes to suggest any association with treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
49 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effect of treatment on morphological development at any dose

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Summary of Malformations (includes external, visceral and skeletal examinations:

 Dose level (mg/kg bw/day)  Female number  Foetus number Malformations 
 0  4  2 Micrognathia (lower jaw) Cervical vertebrae with multiple abnormalities (absent or misshapen arches)
 7   31  1  Cervical vertebra with an absent archThoracic vertebrae with multiple abnormalitiesof the centra (scoliosis)
 35  1  Upper jaw with multiple abnormalities(misshapen maxilla and short premaxilla)
21  -  -  -
 49  -  -  -

Applicant's summary and conclusion

Conclusions:
Oral (gavage) administration of calcium cyanamide technical grade to Sprague-Dawley rats from days 6 to 19 of gestation at doses of 7, 21 and 49 mg/kg/day (performed in accordance with OECD Test Guideline 414) was associated with dose-related reductions in overall mean body weight gain and food consumption in the 21 and 49 mg/kg/day groups. The low dose of 7 mg/kg/day was considered a No Observed Adverse Effect Level (NOAEL) for maternal toxicity. Evidence of embryo-foetal effects was restricted to dose-related slightly lower mean foetal weight in the 21 and 49 mg/kg/day groups. There was no adverse effect of treatment on morphological development at any dose. Since there were no other findings, including the absence of any adverse effect on morphological development, associated with the slightly lower mean foetal weight, the NOAEL for embryo-foetal toxicity was therefore 49 mg/kg/day.
Executive summary:

A developmental toxicity study was conducted according to OECD TG 414 to evaluate the effects of the test item on embryonic and foetal development of the Sprague-Dawley rat when administered by the oral route (gavage) from implantation to the day before caesarean section.

The test item, Calcium Cyanamide (technical grade), was administered by daily gavage at dose levels of 7, 21 and 49 mg/kg bw/day to groups of 25 mated female Sprague-Dawley rats. A control group received the same volume (2 mL/kg bw) of the vehicle, corn oil. The females were treated from days 6 to 19 of gestation inclusive. Clinical condition, body weight and food consumption were monitored throughout the study. The females were submitted to a caesarean examination on day 20 of gestation and litter parameters were recorded. At necropsy, the females were examined macroscopically, and the gravid uterus and all foetuses were weighed. All live foetuses were examined for external abnormalities. Half of the foetuses in each litter were then examined internally and sexed prior to processing for skeletal examination. The remaining foetuses (approximately one half of the foetuses in each litter) were fixed for examination of the head only by serial sectioning.

There were no unscheduled deaths and no treatment-related clinical signs during the study in any group. There was a dose-related reduction in body weight gain and food consumption during the dosing period (G 6 to G 19) in the 21 and 49 mg/kg bw/day group leading to lower terminal body weight in the 49 mg/kg bw/day group only compared with the control. There was no treatment-related macroscopic observation in any group. There were 24, 25, 23 and 24 pregnant females in Groups 1 to 4, respectively, at the terminal caesarean sections, all of which had viable foetuses. There was no effect of treatment on embryo-foetal survival in any group. There was a slight dose-related reduction in mean foetal weight in the 21 and 49 mg/kg/day groups leading to slightly lower mean gravid uterus weight in the 49 mg/kg bw/day group only. Foetal sex ratio was normal in all groups. There were 1 (1) and 2 (1) malformed foetuses (litters) in Groups 1 and 2, respectively, none of which were considered to be associated with treatment.

The low dose of 7 mg/kg/day was considered a No Observed Adverse Effect Level (NOAEL) for maternal toxicity. Evidence of embryo-foetal effects was restricted to dose-related slightly lower mean foetal weight in the 21 and 49 mg/kg bw/day groups. There was no adverse effect of treatment on morphological development at any dose. Since there were no other findings, including the absence of any adverse effect on morphological development, associated with the slightly lower mean foetal weight, the NOAEL for embryo-foetal toxicity was therefore 49 mg/kg bw/day.