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Administrative data

Description of key information

There are five studies available, in which the acute oral toxicity of calcium cyanamide is examined. One of the studies was performed in rats using Kalkstickstoff concentrations of up to 995 mg/kg bw (calcium cyanamide 20.5%) and was considered the most critical of the studies available and therefore defined as key study. Results of this investigation revealed an acute oral LD50 of 765 mg/kg bw, which is equivalent to 639 mg/kg bw calcium cyanamide technical grade. Another study on Kalkstickstoff (calcium cyanamide 63.5%) was performed in mice and supports the results of the abovementioned rat study with an LD50 of 1800 mg/kg bw. Other supporting studies were performed with PERLKA or pure calcium cyanamide in rats and mice.

The acute inhalation toxicity potential of calcium cyanamide was studied in three studies. In two of the studies, rats were exposed to Kalkstickstoff either in the non-oiled (155 mg/m3 air) or oiled (312 mg/m3 air) form and it was reported that the acute LC50 was above the tested concentrations. A third study was conducted using PERLKA, resulting in an acute inhalation LC50 of 5.1 mg/L (5100 mg/m3), which is equal to 8.22 mg/L (8220 mg/m3) calcium cyanamide technical grade. 

Two studies on the acute dermal toxicity are available using PERLKA at the limit dosage (2000 mg/kg bw). Rabbits showed erythema and oedema as well as necrotic patches and the acute median lethal dermal dosage was established at > 2000 mg/kg bw.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
Deviations:
not specified
GLP compliance:
no
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Young albino rats from the Institute's colony (Wistar derived) males 128 to 240 g and females 128 to 178 g weight were used. They were housed in groups of five in screen-bottomed stainless steel cages, in a well-ventilated room maintained at 25°C. Before dosing the rats were fasted overnight.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
After some preliminary observations the test material was given by gavage as a 10% (w/v) aqueous suspension to groups of five males and five females in single doses of 4.80, 5.76, 8.29 or 9.95 ml per kg body weight.
Doses:
480, 476, 691, 829 and 995 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
After treatment the rats received stock diet and tap water ad libitum. They were observed for signs of intoxication during a 14 days period, after which autopsies were carried out on the survivors. The LD50 was calculated according to the method of Weil (Biometrics 8 (1952) 249-263).
Statistics:
The LD50 was calculated according to the method of Weil (Biometrics 8 (1952) 249-263).
Preliminary study:
not indicated
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
ca. 765 mg/kg bw
95% CL:
> 0.639 - < 0.845
Remarks on result:
other: calculated for calcium cyanamide 20.5%N
Sex:
male/female
Dose descriptor:
LD0
Effect level:
576 mg/kg bw
Remarks on result:
other: calcium cyanamide technical grade (Kalkstickstoff)
Mortality:
Deaths occurred between 5 and 21 hours after treatment.
Clinical signs:
other: Within a few hours after dosing all rats showed decreased activity and humpback behaviour. Tremors in the legs and loss of conscious were frequently observed. The survivors recovered and looked quite healthy again at the end of the observation period.
Gross pathology:
Macroscopic examination of the survivors did not reveal treatment-related gross alterations.
Other findings:
no other findings
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
From the mortality figures the LD50 of technical calcium cyanamide is calculated to be 765 mg/kg bw with 693 and 845 as the 95% confidence limits, which is equal to 639 mg/kg Calcium cyanamide, technical grade.
Executive summary:

In an acute oral toxicity study, groups of fasted, young Wistar rats (5/sex) were given a single oral dose of calcium cyanamide, technical grade in water by gavage at doses of 480, 576, 691, 829 and 995 mg/kg bw and observed for 14 days.

Within a few hours after dosing all rats showed decreased activity and humpback behaviour. Tremors in the legs and loss of consciousness were frequently observed. Deaths occurred between 5 and 21 hours after treatment. The survivors recovered and looked quite healthy again at the end of the observation period. Macroscopic examination of the survivors did not reveal treatment-related gross alterations.

Oral LD50 = 765 mg/kg bw (693-845 mg/kg bw 95% C.I.), equal to 639 mg/kg Calcium cyanamide, technical grade.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
639 mg/kg bw
Quality of whole database:
Several studies for this endpoint are available showing with comparable results.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
not specified
GLP compliance:
no
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
other: Wistar derived, albino CPB-rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
A group of five male and five female SPF-rated, Wistar derived, albino CPB-rats, 9 weeks old, was used. The average weight of the males was 223 g, that of the females 153 g. After exposure the rats were returned to their living cages and provided with stock diet and tap water ad libitum during an observation period of 14 days.
Route of administration:
inhalation: dust
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
The powdered substance was continuously dispersed in air by means of a "Wright" Dust Feed Mechanism at a maximum rate of 17 g/m3. The dustladen air was passed into a 1.5 m3 stainless steel exposure chamber in which the experimental animals were housed individually in wire-screen cages. The animals could be observed continuously through the hard glass entrance door. The temperature and relative humidity inside the exposure chamber were 23°C and 45-50% respectively.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
The concentration of the dispersed Kalkstickstoff was determined gravimetrically by passing a measured amount of the test atmosphere through a glassfibre filter. Particle size determinations and counts were carried out in samples taken from the atmosphere in the chamber with a cascade impactor.
The group of the five male and female was exposed to a dispersion of Kalkstickstoff in air at a maximum attainable concentration of 155 mg/m3. Particle size determination and particle counts in air revealed a 99 % particle size range of 0.8 - 3.3 µm.
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
no further details given
Statistics:
not reported
Preliminary study:
no preliminary study
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 155 mg/m³ air (analytical)
Exp. duration:
4 h
Mortality:
no mortality
Clinical signs:
other: During the first 45 minutes of the exposure period the rats were quiet and their behaviour was normal. During the next two hours the rats were slightly restless, but these signs of irritation gradually disappeared. The last hour of the exposure period all
Body weight:
not reported
Gross pathology:
not reported
Other findings:
none
Interpretation of results:
GHS criteria not met
Conclusions:
It was concluded that the 4-hour LC50 of Kalkstickstoff was higher than 155 mg/m3 of air.
Executive summary:

The group of the five male and female was exposed to a dispersion of Kalkstickstoff in air at a maximum attainable concentration of 155 mg/m3. Particle size determination and particle counts in air revealed a 99 % particle size range of 0.8 - 3.3 µm.

During the first 45 minutes of the exposure period the rats were quiet and their behaviour was normal. During the next two hours the rats were slightly restless, but these signs of irritation gradually disappeared. The last hour of the exposure period all rats were completely quiet and seemed to be asleep. No mortality and no signs of intoxication were observed.

The results of the present acute inhalation toxicity study lead to the conclusion that the 4 -hour LC50 of Kalkstickstoff is higher than 155 mg/m3 of air.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-05-10 to 1989-05-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Version / remarks:
(Annex V of the 6th amendment of the EEC directive of 18th September, 1979, Part B, 4.1.3 Acute toxicity dermal)
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: A. Smith, Warlingham, Surrey, England
- Age at study initiation: approximately 9 - 12 weeks
- Weight at study initiation: weight range of 2.7 - 3.1 kg
- Fasting period before study: no
- Housing: individually in metal cages with perforated floors
- Diet: standard laboratory diet ad libitum
- Water: ad libitum
- Acclimation period: 14 days prior to start of the main study


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21°C
- Humidity (%): 54%
- Air changes (per hr): 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light in each 24 hour period (i.e. 12 hours dark / 12 hours light)


Type of coverage:
occlusive
Vehicle:
water
Details on dermal exposure:
TEST SITE
- Area of exposure: dorso-lumbar region
- % coverage: 10% of total body surface
- Type of wrap if used: the treated area was covered with gauze, held in place with an impermeable dressing encircled firmly around the trunk


REMOVAL OF TEST SUBSTANCE
- Washing: washing with warm (30-40°C) water and blotting dry with absorbent paper
- Time after start of exposure: 24 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2 g/kg bw, both for preliminary and main study
- Concentration (if solution): test substance moistened with distilled water at 1 mL/kg
- Constant volume or concentration used: yes
- For solids, paste formed: yes, test substance moistened with distilled water at 1 mL/kg


VEHICLE
- Amount(s) applied (volume or weight with unit): test substance moistened with distilled water at 1 mL/kg
Duration of exposure:
24 hours
Doses:
2 g/kg bw
No. of animals per sex per dose:
preliminary study: one male and one female
main study: 5 male and 5 female
Control animals:
no
Details on study design:
- Duration of observation period following administration: Animals of the preliminary and main studies were observed for 5 and 14 days, respectively, after dosing.
- Frequency of observations and weighing: Animals were observed soon after dosing, then at frequent intervals for the remainder of Day 1 and at subsequent days at least twice per day. Individual bodyweights of rabbits in the main study were recorded on Days 1, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs were recorded at each observation (nature, severity, approximate time of onset and duration of toxic sign), body weight, macroscopic post mortem examination (opening the abdominal and thoracic cavities)
Statistics:
No data
Preliminary study:
The results of the preliminary study performed on two rabbits (one male and one female) indicated that the acute median lethal dermal dose to male and female rabbits of calcium cyanamide, technical grade, was greater than 2 g/kg bw.
Key result
Sex:
male/female
Dose descriptor:
LD0
Effect level:
> 2 000 mg/kg bw
Remarks on result:
other: no mortality occurred
Mortality:
There were no deaths following a single dermal dose of calcium cyanamide, technical grade, at 2 g/kg bw.
Clinical signs:
other: There were no clinical signs of systemic toxicity in response to treatment.
Gross pathology:
At terminal autopsy, red streaks were observed in the left ventricle of the heart of one male rabbit. Small cysts were also observed on the outer surface of the kidney of the same male. A large fluid-filled cyst (10 x 10 cm) was also present in the kidney. Small cysts were also observed on the surface of the kidneys of one other male.
Other findings:
Dermal responses:
Well-defined to severe erythema accompanied by well-defined to moderate erythema were observed in all treated animals after removal of the dressings. Irritation had resolved in two male and two female rabbits by Day 10 or 11 and in further two animals (one male and one female) by Day 14. Irritation was still present in the remaining two males and two females at the end of the study (Day 15).
Small areas of necrosis were present in one male and two females from Day 2 until the end of the study. Necrotic patches were observed in a further three males and two females from Day 3 and until Day 9 (with the exception of one male where the necrotic patch was still present at the end of the study).

no remarks

Interpretation of results:
GHS criteria not met
Conclusions:
The acute lethal dose to rabbits of calcium cyanamide, technical grade, was found to be > 2 g/kg bw.
Executive summary:

The study was designed to assess the acute dermal toxicity of calcium cyanamide, technical grade, following a single dose onto rabbit skin. The test substance was applied as supplied, moistened sufficiently with distilled water to ensure good contact with the skin.

A preliminary study (trial test) was performed in one male and one female rabbit using 2 g/kg bw of test substance. Animals were observed for 5 days after dosing and results indicated that the acute median lethal dose of the test substance was > 2g /kg bw.

For the main study, a group of ten rabbits (5 male and 5 female) were treated at 2 g/kg bw of calcium cyanamide.

One day prior to treatment, hair was removed from the dorso-lumbar region with electric clippers exposing an area equivalent to 10% of the total body surface. No shaving or chemical depilation was used. The test substance was spread evenly over the prepared skin and was then covered with gauze which was held in place with impermeable dressing encircled firmly around the trunk.

At the end of the 24 hour exposure period, the dressings were carefully removed and the treated area decontaminated by washing with warm water and blotting with absorbent paper.

Animals were observed for skin irritation and clinical signs for 14 days after dosing.

All animals of the main study were killed on Day 15 by an intravenous overdose of phenobarbitone sodium and were subjected to macroscopic post mortem examination.

There were no death and no clinical signs of systemic toxicity observed throughout the study period. Erythema and edema as well as necrotic patches were observed in all of the animals with signs of irritation being present at the end of the observation period in three of the ten animals tested.

A slightly lower bodyweight gain was recorded for one female on Day 15 and two males showed cysts on the surface of the kidney at terminal autopsy. All other animals were without findings regarding bodyweight and post mortem examination.

The acute lethal dose to rabbits of calcium cyanamide, technical grade, was found to be > 2 g/kg bw for both males and females.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

There are several studies on the acute toxicity (5 studies on oral, 3 on inhalation and 2 on dermal exposures) of calcium cyanamide available. For each of the different exposure routes, the most critical study was chosen to determine an acute reference value, i.e. the acute LD50 value. Please refer to the statement in section 13 for a detailed description of the composition of the different test materials.

 

Oral:

To determine the acute oral LD50, the study performed by de Groot (1976) was chosen as key study. "Kalkstickstoff 20.5%N" was used as the test substance and the study was performed in rats. Groups of rats were administered dosages of 480, 576, 691, 829 and 995 mg/kg bw by oral gavage and observed for 14 days. Within a few hours after dosing, all rats showed decreased activity and humpback behaviour. Tremors in the legs and loss of conscious were frequently observed. Deaths occurred between 5 and 21 hours after treatment. The survivors recovered and looked quite healthy again at the end of the observation period. Macroscopic examination of the survivors did not reveal treatment-related gross alterations. Based on these findings, the acute oral LD50 for Kalkstickstoff 20.5% was established at 765 mg/kg bw in this study, which is equivalent to 639 mg/kg calcium cyanamide technical grade.

Another study (Allan, 1992) was performed using Kalkstickstoff, of which the results support the acute toxicity data of the abovementioned study of de Groot (1976). Groups of fasted mice were treated at a single dose of Kalkstickstoff by gavage, using dose levels of 0.5, 0.8, 1.26, 1.6 and 2.0 g/kg bw. Animals were observed for 15 days and those surviving treatment were killed and examined macroscopically at the end of the observation period. There were deaths among male and female mice dosed at 1.6 g/kg body weight and above. Deaths occurred at intervals from Days 2 to 5 and slight body weight losses were recorded for all animals that died. Macroscopic examination of these animals revealed abnormalities of the stomach. Clinical signs of reaction to treatment included pilo-erection, abnormal body carriage (hunched posture), abnormal gait (waddling), lethargy, decreased respiratory rate, ptosis and pallor of the extremities. Recovery of surviving mice, as judged by external appearance and behaviour, was complete by Day 2 (three males and three females at 1.6 g/kg), Day 3 (0.5, 0.8 and 1.26 g/kg), Day 5 or 6 (one male and one female at 1.6 g/kg), Day 7 (one female at 1.26 g/kg) or at intervals from Day 10 to 13 (2.0 g/kg). Among survivors, no change in bodyweight was recorded for one female dosed at 2.0 g/kg and body weight losses were recorded for one male and one female dosed at 2.0 g/kg on Day 8; these mice achieved satisfactory gains on Day 15. All other mice achieved satisfactory body weight gains throughout the study. Macroscopic examination on Day 15 revealed blister-like appearances on the limiting ridge in the stomach surrounded by white raised areas for one male and one female dosed at 0.8 g/kg; thickening of the stomach wall with the stomach adhered to the liver for one male and two females dosed at 2.0 g/kg. Based on these observations, the acute oral LD50 was determined at 1.8 g/kg bw (1800 mg/kg bw), which is above the one stated in the De Groot (1976) rat study.

Another acute oral toxicity study was performed in rats using PERLKA at dosages of 700, 1000, 900 or 1200 mg/kg bw (Osherhoff, 1990). The test substance was administered by oral gavage. Substance-related signs of toxicity, besides mortality, included depression, tremors, respiratory difficulty, red stains on nose and/or eyes, salivation, compound-coloured faeces, soft faeces, prostration and/or ataxia, urine stains, low body temperature, cyanotic appearance, and/or lacrimation. In general, these signs were of greater severity and/or more frequently observed with increasing dose. In addition, the females tended to display these signs with greater severity than the males administered the same dose level. Based on the results of this study, the estimated LD50 for PERLKA is 711 mg/kg bw in males (equals to 441.2 mg/kg calcium cyanamide technical grade), 492 mg/kg bw in females (305.3 mg/kg calcium cyanamide technical grade) and 594 mg/kg bw in both sexes combined (368.6 mg/kg calcium cyanamide technical grade). This study was not used for classification and labelling and risk assessment because the study was conducted with PERLKA (a mixture of calcium cyanamide), whereas an acute oral toxicity study with Kalkstickstoff (calcium cyanamide technical grade) is available.

In a food repellent study performed by Reng (1976), Kalkstickstoff 20.5%N was mixed with a grated biscuit and water and fed to fasted mice at dosages of 4500, 5000, 5500, 6000, 7000, 10000 and 12000 mg/kg bw. Results revealed that mice reject food with a content of more than 10000 mg/kg bw (equal to 8353 mg/kg calcium cyanamide technical grade). An LD50 could not be determined in this study. No mortality occurred in groups fed 4500, 5000, 5500 and 6000 mg/kg bw. A mortality rate of 10% (male) and 25 % (female) was observed at 10000 mg/kg bw. Food with 12000 mg/kg bw was refused by the animals, and the mice died within 13 days.

A study was performed on pure calcium cyanamide which was administered to groups of fasted Wistar rats at doses of 500, 600, 650, 700, 800 and 900 mg/kg bw in water with 2% starch suspension (Berger, 1966). Animals were observed for 7 days and the most prominent signs of toxicity following oral administration of calcium cyanamide were respiratory depression, tremors, ataxia, and prostration prior to death. All deaths in rats receiving 900 mg/kg bw of calcium cyanamide occurred within 24 h after dosing. At 650-800 mg/kg bw most deaths occurred within 24 hours. Some delayed deaths (2-4 days after dosing) were also noted. No deaths were observed with 600 mg/kg bw, but one delayed death was recorded with 500 mg/kg bw on the third day after dosing. Based on the results obtained, the acute LD50 was determined at 690 mg/kg bw (equals to 1015 mg/kg calcium cyanamide technical grade). This study was not used for classification and labeling because a study with Kalkstickstoff (calcium cyanamide technical grade) is available and the relevant impurities of the substance must also be considered. Furthermore, calcium cyanamide undergoes rapid hydrolysis to form cyanamide in aqueous solutions; therefore, there is uncertainty whether the treated animals are rather exposed to cyanamide than calcium cyanamide, which makes the study not suitable as a key study.

In summary, regarding the results obtained in the different acute oral toxicity studies, the overall LD50 for calcium cyanamide technical grade is derived at 639 mg/kg bw based on the investigation in rats using Kalkstickstoff 20.5%N since this study from De Groot (1976) was considered the most critical and relevant key study. Results on the other investigations support this LD50 because either the other LD50 values are higher than the one established in the De Groot (1976) rat study or at the highest dosage applied, no mortalities occurred.

Disregarded studies on cyanamide: Two studies, in which the acute oral toxicity of cyanamide, is examined are available. The key study equivalent to the OECD Guideline 401 administered cyanamide (supplied as 50 % aqueous solution) via oral gavage to male and female Albino Wistar rats (Engel, 1973). Mortality was observed at all dose levels (100, 125, 150, and 175 mg/kg – body weight measured on day 1 only) and most incidences occurred during the first day after dosing. No gross pathology was found during necropsy. A supporting study (Daamen, 1994) dosed 5 male and 5 female rats per dose group with up to 400 mg/kg bw (50 % aqueous solution) cyanamide via oral gavage, resulting in dose-dependent mortality which occurred mainly on day 2. Haemorrhages and thickened areas in the digestive tract were revealed during necropsy in addition to damage in other organs. An oral LD50 of 223 mg/kg bw (pure active cyanamide) for both sexes was determined. As there are existing reliable studies available on calcium cyanamide that can be used for evaluation as well as the differences in physicochemical properties and toxicokinetics between calcium cyanamide and cyanamide (see report "Scientific Rationale for Not Using Cyanamide as Read-Across Substance for Calcium Cyanamide on Toxicological Endpoints" in Section 13.2 for more details), a read-across approach using cyanamide is not necessary and also not suitable for calcium cyanamide.

Inhalation:

Regarding acute inhalation toxicity, one study on (non-oiled) Kalkstickstoff, one on oiled Kalkstickstoff and one using PERLKA are available. The study performed by Appelman (Appelman, 1978) using calcium cyanamide technical grade was defined as key study. Groups of rats were exposed to a dispersion of (non-oiled) calcium cyanamide technical grade in air at a maximum attainable concentration of 155 mg/m3. Particle size determination and particle counts in air revealed a 99 % particle size range of 0.8 - 3.3 µm. During the first 45 minutes of exposure, rats were quiet and their behaviour was normal. During the next two hours the rats were slightly restless, but these signs of irritation gradually disappeared. The last hour of the exposure period all rats were completely quiet and seemed to be asleep. No mortality and no signs of intoxication were observed. The results of this acute inhalation toxicity study lead to the conclusion that the 4-hour LC50 of calcium cyanamide technical grade is greater than 155 mg/m3 of air.

In the second study, rats were exposed to a dispersion of 0.5% oiled calcium cyanamide technical grade in air at a maximum attainable concentration of 312 mg/m3 (Appelman, 1979). Particle size determinations and particle counts in the atmosphere samples revealed 95% of the particles to have a diameter of less than 5.9 µm. No mortality occurred during the 4-hour exposure and the subsequent 14-day observation period. During the 4-hour period the rats were quiet and had closed their eyes; their behaviour was normal. The results of the present acute inhalation toxicity study lead to the conclusion that the 4-hour LC50 of 0.5% oiled calcium cyanamide technical grade is higher than 312 mg/m3 of air.

For the third acute inhalation toxicity study, rats were exposed to PERLKA dust at a concentration of 5.10 mg/L for a single four-hour exposure. Treatment-related effects included one death, increased secretory responses, respiratory distress, tremors, prostration, behavioural effects, compound on fur, general signs of poor health, and loss of body weight. Possible treatment-related findings exhibited by the animal found dead 30 min after exposure included mottled liver and lungs, failure of lungs to collapse, dark nasal turbinates containing dark material, pale spleen, material in the lumen of stomach and trachea, and dark area on the tongue. Gross postmortem evaluations at terminal sacrifice revealed respiratory findings, including dark lungs or dark areas of the lungs, dark material in the nasal turbinates, and ocular lesions including pale, dark and/or depressed area(s) of the external eye, dry eye(s), or globe ruptured ante mortem. This study was initiated as a limit test. Because only one mortality was observed, it was concluded that the LC50 for the rat with PERLKA dust in a four-hour exposure is higher than the dose tested of 5.10 mg/L, which is equal to 8.22 mg/L calcium cyanamide technical grade.

The overall LD50 of calcium cyanamide, technical grade was established as > 155 mg/m3 as this is the maximum attainable concentration of Kalkstickstoff and no animals died at this concentration. The data with PERLKA support a LC50 >= 8.22 mg/L and consequently, no classification and labelling is needed for acute inhalation toxicity.

Disregarded studies on cyanamide: One study is available that examines cyanamide in an acute inhalation toxicity experiment (Kruysse, 1973). The toxicity of cyanamide (a 50 % aqueous solution) by the inhalation route (whole-body) was investigated in five male and five female Wistar rats. The rats were exposed to the mist of technical active cyanamide in a concentration of 2.0 mg/L of air (corresponding to the highest technical attainable concentration) for four hours. No mortalities were recorded during the study. During the exposure, a lumback behaviour and a rapid shallow respiration with frequent coughing and swallowing were observed. Within a few hours after exposure all animals were completely recovered and showed normal behaviour. No visible lesions were observed at gross necropsy. The inhalation LC50 of cyanamide was > 1 mg/L in male and female rats after 4 hour inhalation (corresponding to technical active Cyanamid L500 > 2.0 mg/L). As there are existing acute inhalation studies available on calcium cyanamide that can be used for evaluation, a read-across approach using cyanamide for acute inhalation toxicity is not necessary.

 

Dermal:

There are two acute dermal toxicity studies available using calcium cyanamide technical grade or PERLKA.

In the first study, calcium cyanamide technical grade was moistened sufficiently with distilled water to ensure good contact with the rabbit skin (Ligget and Allan, 1989). A preliminary study (trial test) was performed in one male and one female rabbit using 2 g/kg bw of test substance. Animals were observed for 5 days after dosing and results indicated that the acute LD50 of the test substance was > 2g /kg bw. For the main study, a group of ten rabbits (5 male and 5 female) were treated at 2 g/kg bw of calcium cyanamide technical grade. One day prior to treatment, hair was removed from the dorso-lumbar region with electric clippers exposing an area equivalent to 10% of the total body surface. The test substance was spread evenly over the prepared skin and was then covered with gauze which was held in place with impermeable dressing encircled firmly around the trunk. At the end of the 24-hour exposure period, the dressings were carefully removed and the treated area decontaminated by washing with warm water and blotting with absorbent paper. Animals were observed for skin irritation and clinical signs for 14 days after dosing. All animals of the main study were killed on day 15 and were subjected to macroscopic post-mortem examination. There were no death and no clinical signs of systemic toxicity observed throughout the study period. Erythema and oedema as well as necrotic patches were observed in all of the animals with signs of irritation being present at the end of the observation period in three of the ten animals tested. A slightly lower body weight gain was recorded for one female on day 15 and two males showed cysts on the surface of the kidney at terminal autopsy. All other animals were without findings regarding body weight and post-mortem examination. Based on the results obtained, the acute dermal LD50 was established at > 2 g/kg bw.

In the second study, rabbits were treated with PERLKA at a limit dose level of 2000 mg/kg bw, which remained on the skin for approximately 24 hours (Osheroff, 1990). After attempting to remove the test material with tap water and gauze, most of the compound remained on the skin. Animals were observed for mortality and/or moribidity twice daily and for obvious indications of a toxic effect once daily. Dermal irritation was graded and scored according to the Draize technique and body weights were recorded. After 14 days of observation, all rabbits were humanely sacrificed and subjected to a complete gross necropsy. All animals survived to the scheduled termination of the study. With the exception of one female, all animals gained weight during the study period. Clinical observation revealed soft faeces and/or anorexia frequently in one female between days 7 and 14 and in another female soft faeces on day 14 only. All rabbits displayed erythema on day 1 (grade 2 - 4), and with the exception of one male that demonstrated decreasing severity on subsequent days. By day 14, two rabbits failed to display any evidence of erythema and the remaining animals were noted with grades 1 and 2 only. Although oedema was noted in 9 of the 10 rabbits on day 1 post-dose, the condition appeared to subside at subsequent time periods. Other skin effects recorded included substance-like material on the application site, thickening, blanching, green colouration on application site, fissuring with bleeding and/or necrosis. At termination, gross necropsy revealed the intestines of one female distended with air and containing yellow mucoid-like material. All other animals were without findings. It may therefore be concluded that the acute dermal LD50 of PERLKA is > 2000 mg/kg bw, which is equivalent to 1241.2 mg/kg calcium cyanamide technical grade.

The overall acute dermal LD50 is determined at > 2000 mg/kg bw for PERLKA and for calcium cyanamide technical grade as no mortalities were noted in neither of the two studies.

Disregarded studies on cyanamide: There is one study available that examines the acute dermal toxicity of cyanamide (Osheroff, 1988). The test substance cyanamide (supplied as a 50 % aqueous solution) was applied dermally to the shaved intact skin of five male and five female New Zealand White rabbits per dose group. In particular, doses of 50 % w/w hydrogen cyanamide technical solution at 1.0, 2.0 and 4.0 mL/kg bw (corresponding to 530, 1060 and 2120 mg/kg bw pure active substance cyanamide, respectively) were applied. The liquid test material was held in contact with the skin for a 24-hour period with impervious rubber damming. The animals were evaluated for treatment related effects on the day of dosing and for a subsequent 14-day observation period. Clinical signs included tremors, ataxia, anorexia, depression were observed at the low and high dose group. No signs of dermal irritations were noted in the low dose group, whereas all surviving animals of the high dose group showed a well defined erythema and a very slightly oedema on day 1. All other animals died before primary dermal irritation scoring could be performed. In rabbits that survived no pathologic changes at necropsy were observed. In rabbits found dead during the observation period changes mainly concerning the lung were observed (dark red lungs or dark red lung areas). In one animal of the high dose group a pale liver was noted. The acute dermal LD50 was 901 mg/kg bw in males, 742 mg/kg bw in females and 848 mg/kg bw combined for the sexes pure active substance cyanamide in rabbits (corresponding to about 1.7 mL/kg bw for males, 1.4 mL/kg bw for females and 1.6 mL/kg bw for the sexes combined of the 50 % aqueous hydrogen cyanamide solution). As there are existing acute dermal studies available on calcium cyanamide that can be used for evaluation, a read-across approach using cyanamide for acute dermal toxicity is not necessary.

Justification for classification or non-classification

Based on the results of the acute oral toxicity studies (LD50 of 765 mg/kg bw of Kalkstickstoff 20.5%N; equivalent to 639 mg/kg bw for calcium cyanamide technical grade) the test substance is classified as category 4 for acute oral toxic effects and labeled as H302 according to Regulation (EC) No 1272/2008 (CLP/GHS).

Based on the results of the acute inhalation toxicity study (LC50 greater than the maximum attainable concentration of 155 mg/m3 Kalkstickstoff) calcium cyanamide is not classified with respect to acute inhalation toxicity. PERLKA data revealing a LC50 >= 8.22 mg/L for calcium cyanamide technical grade supports this conclusion.

In addition, the test substance calcium cyanamide is not classified for acute dermal toxicity according to the CLP/GHS and 67/548/EEC based on the obtained LD50 values (LD50 > 2000 mg/kg calcium cyanamide technical grade).