Calcium cyanamide

Endpoint summary

Description of key information

Skin: According to the results of an in vitro skin corrosion study (human skin model), the test substance calcium cyanamide, technical grade "Kalkstickstoff" is considered to be non-corrosive to skin. In an acute dermal toxicity study with rabbits (2 g/kg bw), erythema and oedema as well as necrotic patches were observed in all animals with signs of irritation being present at the end of the observation period in three of the ten animals tested. Mean irritation scores (mean of 48/72 h) of 2.25 for erythema and 1.7 for oedema were calculated. In conclusion, the study was evaluated to be skin irritating.

Eye: In two in vivo eye irritation studies with calcium cyanamide technical grade ("Kalkstickstoff") and PERLKA, it could be shown that calcium cyanamide is severely irritating to the eye. In addition an in vitro BCOP test confirms the results from the in vivo studies.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989-05-10 to 2014-05-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.3 (Acute Toxicity (Dermal)) (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

Species:

rabbit

Strain:

New Zealand White

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

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Remarks:

moistened with water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 2 g/kg bw

Duration of treatment / exposure:

24 h

Observation period:

15 days

Number of animals:

preliminary study: one male and one female
main study: 5 male and 5 female

Details on study design:

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 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)

The treated areas of the skin were examined daily for signs of dermal irritation and assessed according to the following arbitrary scoring system:

Erythema and eschar formation:
0 no erythema
1 slight erythema
2 well-defined erythema
3 moderate erythema
4 severe erythema (beet redness) to slight eschar formation (injuries in depth)

Oedema formation:
0 no oedema
1 slight oedema
2 well-defined oedema (area well-definite by definite raising)
3 moderate oedema (raised approximately 1 mm)
4 severe oedema (raised more than 1 mm and extending beyond the area of exposure)

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1.6

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #4

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #5

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 11 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #5

Time point:

24/48/72 h

Score:

1.6

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal #6

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal #6

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 11 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal: #7

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 11 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal: #7

Time point:

24/48/72 h

Score:

2.3

Max. score:

4

Reversibility:

fully reversible within: 11 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal: #8

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal: #8

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal: #9

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal: #9

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

erythema score

Basis:

animal: #10

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 10 days

Remarks on result:

positive indication of irritation

Irritation parameter:

edema score

Basis:

animal: #10

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

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).

Other effects:

Mortality
There were no deaths following a single dermal dose of calcium cyanamide, technical grade, at 2 g/kg bw.

Clinical signs
There were no clinical signs of systemic toxicity in response to treatment.

Body weight
A slightly lower bodyweight gain was recorded for one female on Day 15. All other animals achieved anticipated gains throughout the study.

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.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In a acute dermal irritation study with rabbits 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. In conclusion the test item was evaluated to be skin irritating.

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. In conclusion the test item was evaluted to be skin irritating.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-04-27 to 2010-06-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

440/2008/EC

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline No. 431 (In Vitro Skin Corrosion: Human Skin Model Test ), 13 April 2004.

Deviations:

no

GLP compliance:

yes

Species:

other: not applicable (in vitro test)

Strain:

other: not applicable (in vitro test)

Details on test animals or test system and environmental conditions:

MatTek´s EpiDerm System consists of normal, human-derived EpiDermal keratinocytes which have been cultured from a multilayered, highly differentiated model of the human EpiDermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs (R), 10 mm diameter) and shipped as kits, containing 24 tissues on shipping agarose.

Type of coverage:

other: not applicable (in vitro test)

Preparation of test site:

other: not applicable (in vitro test)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

The application spoon was filled with 25 mg finely grounded test substance. The "spoonful" was levelled by gently scratching the excess material away, avoiding compression. 25 μL H2O were added for wetting of the test substance.

Duration of treatment / exposure:

Exposure times:
1. 3 minutes
2. 1 hour (incubator, 37°C, 5 % CO2)

Observation period:

not applicable (in vitro test)

Number of animals:

2 Tissues each for 3 min and 1 hour exposure times (3 readings for each tissue).

Details on study design:

Test design
One hour before starting the assay EpiDerm™ tissues were transferred to 6-well plates with assay medium, which was replaced immediately before the test was started. Two tissue replicates were used for each treatment (exposure time), including deionised water as negative and 8N KOH as positive control.

Exposure times:
- 3 minutes
- 1 hour (incubator, 37°C, 5 % CO2)

Time schedule
Day 0: Receipt of EpiDerm™ tissues.
Day 1: Performance of the test. Measurement of the optical density, end of test.

Application of test and reference substances

Reference substances
50 μL of each reference substance were dispensed directly atop the Epi-200 tissue.

Test substance
The application spoon was filled with 25 mg finely grounded test substance. The "spoonful" was levelled by gently scratching the excess material away, avoiding compression. 25 μL H2O were added for wetting of the test substance.

MTT-test
After incubation with the test chemical, the negative (deionised water) and positive control (8N KOH) for 3 minutes or for 1 hour followed by washing with PBS (Phosphate Buffered Saline), the tissues were incubated with MTT medium at 37°C and 5 % CO2. After 3 hours, the MTT medium was aspirated from all wells and the tissues were gently rinsed with PBS (2 times). For extraction, the tissues were incubated with extractant solution (isopropanol) for 2 hours with shaking. After the extraction period, the tissues were pierced with an injection needle and the extract (now a blue formazan solution) was allowed to run into the well from which the tissue was taken. The 24-well plates were placed on a shaker for 15 minutes until the solutions were homogeneous in colour.

Cell viability measurement
Per each tissue 3 × 200μL aliquots of the blue formazan solution were transferred into a 96- well flat bottom microtiter plate (see plate design Figure 1) and the OD was measured using the extractant solution as blank in a plate spectrophotometer at 570 nm, without reference filter.

Assay acceptance criteria
In the protocol INVITTOX n°119 by ECVAM the following assay acceptance criteria are defined:
- The mean OD 570 of the two negative control tissues is >= 0.8.
- The mean tissue viability of the 3 minutes positive control is <= 30 %.
- The inter tissue viability difference is < 30 % between two identically treated tissues.

Interpretation of Results
Corrosivity potential of the test materials is predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues. According to the Regulation (EC) 440/2008: B.40.BIS. and the OECD-Guideline 431 a chemical is classified to be corrosive to skin in any case, if the relative tissue viability after 3 min exposure is less than 50%. In addition, those materials classified to be non corrosive after 3 min (viability ³ 50%) are classified as corrosive if the relative tissue viability after 1 h exposure is less than 15%.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 Minutes

Value:

ca. 84.7

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

ca. 84.7

Other effects / acceptance of results:

Assay acceptance criteria, validity of the test
- The mean OD of the tissues, treated with deionised water (negative control) was 1.983 after 3 minutes, and 2.125 after 1 hour of exposure, that is higher than 0.8, as required by the assay acceptance criteria.
- The mean tissue viability of the 3 minutes positive control was 27,8 %, that is lower than 30 %, as required by the assay acceptance criteria.
- The maximum inter tissue viability differences of the "CALCIUM CYANAMIDE" treated skin discs were 2.1 % for the 3 minutes and 6.0 % for the 1 hour exposure, that is below 30 % as required by the assay acceptance criteria.
The method was acceptable as the concurrent positive and negative control and the maximum inter tissue viability differences fell within the acceptable ranges given by the protocol INVITTOX n°119.

Interpretation of results:

GHS criteria not met

Conclusions:

According to the results of this study, the test substance calcium cyanamide, technical grade "Kalkstickstoff" is considered to be non-corrosive to skin.

Executive summary:

The EpiDerm Skin Corrosivity Test (Model EPI-200) was performed to reveal a possible corrosive potential of calcium cyanamide, technical grade "Kalkstickstoff". The test substance was topically applied for 3 minutes and for 1 hour to the EpiDermal surfaces of three-dimensional human EpiDermis models, followed by immediate determination of the cytotoxic effect.

Investigations performed were in conformance with the Regulation (EC) 440/2008: B.40.BIS. "In vitro skin corrosion: human skin model", 30 May 2008 and the OECD-Guideline 431, "In Vitro Skin Corrosion: Human Skin Model Test ", 13 April 2004.

- The mean percentage viability of the treated skin discs after 3 minutes of exposure was 84,5 % which is above the threshold of 50 % for classification.

- The mean percentage viability of the treated skin discs after 1 hour of exposure was 84,7 % which is above the threshold of 15 % for classification.

According to the results of this study, the test substance calcium cyanamide, technical grade "Kalkstickstoff" is considered to be non-corrosive to skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-17 to 2010-07-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method (BCOP)), adopted 2009-09-07

Deviations:

no

GLP compliance:

yes

Species:

other: Bovine corneas

Strain:

not specified

Details on test animals or tissues and environmental conditions:

isolated corneas from the eyes of cows and bulls aged between 18-20 month and free of macroscopic visible defects.
Supplier: Slaughterhouse Klaus Grandits, Ungerbachstr. 10, A-2860 Kirchschlag, Austria.

Vehicle:

water

Controls:

yes, concurrent vehicle

Amount / concentration applied:

750 µL test substance were introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed during exposure (close-chamber method).

Duration of treatment / exposure:

The corneas were exposed in a horizontal position for 4 hours while ensuring that the test substance adequately covered the epithelial surface. Incubation temperature, monitored with a min/max thermometer, was 30.9°C - 33.5°C.

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

Total of 9 corneas: 3 for the test substance, 3 for the negative control and 3 for the positive control

Details on study design:

Bovine corneas were isolated and mounted in cornea holders and equilibrated for one hour to achieve normal metabolic activity. After exclusion of corneas which did not achieve quality criteria, the corneas were distributed into groups (3 per group) and exposed to the test substance, the negative control and the positive control for 4 hours. Then the substances were removed and the corneas were accurately washed and the opacity and permeability of each cornea were recorded. Opacity was measured quantitatively with the aid of an opacimeter. Permeability was determined by the amount of sodium fluorescein dye that penetrated all cornea layers. For this purpose fluoresceine solution was filled into the anterior chambers of the cornea holders followed by an incubation period of 90 minutes. The amount of sodium fluoresceine that crossed into the posterior chambers was quantitatively measured with a spectrophotometer at OD490. Using opacity and permeability data an IVIS was calculated. The positive and negative control groups were simultaneously used for the concurrently performed studies.

Irritation parameter:

in vitro irritation score

Run / experiment:

BCOP

Value:

443.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The negative and positive control substance values are valid.
The opacity value of "Calcium cyanamide" was 356.6, 15 x Permeability value was 86.835 which resulted in an IVIS of 443.4.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The opacity value of calcium cyanamide was 356.6, 15 x Permeability value was 86.835 which resulted in an IVIS of 443.4. Thus calcium cyanamide is regarded to be an ocular corrosive or severe irritant according to the OECD Guideline 437 for the testing of chemical "Bovine Corneal Opacity and Permeability test Method for identifying Ocular Corrosives and Severe Irritants".

Executive summary:

The Bovine Corneal Opacity and Permeability Study (BCOP Test Method) was performed to reveal possible ocular corrosivity and severe irritation of calcium cyanamide.

Fresh isolated and quality checked corneas were mounted in cornea holders and the initial opacity was determined. After equilibration 750 µL of the test substance preparation were topically administered to 3 isolated bovine corneas to the epithelial surfaces for 4 h and the final opacity was measured. Then 1 mL of a fluorescein solution was added on the epithelial site and permeability was measured after 90 minutes.

Two groups of 3 corneas each served as positive and negative controls. Both control substances were administered under identical conditions as the test substance. The following solutions served as control substances (negative control: sterile aqua dest., positive control: 20% imidazole). Finally the IVIS (in Vitro Irritancy Score) was calculated as follows: IVIS = mean opacity value + (15 x permeability).

The opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity value results from subtraction of final opacity from initial opacity. A substance that induces an IVIS >= 55.1 is defined as ocular corrosive or severe irritant.

The IVIS for calcium cyanamide was 443.4. IVIS of the negative control was 4.9 and for the positive control 123.7, thus demonstration validity of the experiment. According to the results of this study and the OECD Guideline 437, the test substance calcium cyanamide is considered to be an ocular corrosive or severe irritant.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

(Annex V of directive 79/831/EEC, Part B, B.5 eye irritation)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Froxfield Farms (U.K.) Ltd., Petersfield, Hampshire, England
- Age at study initiation: approximately 11 weeks
- Weight at study initiation: 2.6 kg
- Housing: individually in metal cage with a perforated floor
- Diet: SDS standard rabbit diet, ad libitum
- Water: ad libitum
- Acclimation period: no information given on period of time for acclimation, but the rabbit selected for the study was acclimated to the laboratory environment


ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 19°C
- Humidity (%): 30-70%
- Air changes (per hr): 19 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light (artificial light: 0700-1900 hours)

Vehicle:

unchanged (no vehicle)

Controls:

other: the untreated eye of the rabbit served as control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 90 mg

Duration of treatment / exposure:

The test substance was placed into the lower everted lid of one eye of the animal and eyelids were gently held together for 1 second before releasing. The test substance was not removed from the eye.

Observation period (in vivo):

1 day

Number of animals or in vitro replicates:

1

Details on study design:

REMOVAL OF TEST SUBSTANCE
The test substance was not removed from the treated eye of the animal.


SCORING SYSTEM:
Cornea:
Opacity: degree of density (area most dense taken for reading)
0 No ulceration or opacity
1 Scattered or diffuse areas of opacity (other than slight dulling of normal lustre)
2 Easily discernible translucent areas, details of iris clearly visible
3 Nacreous areas, no details of iris visible, size of pupil barely discernible
4 Opaque cornea, iris not discernible through the opacity

Iris:
0 Normal
1 Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia or injection, any of these or combination of any thereof, iris still reacting to light (sluggish reaction is positive)
2 No reaction to light, haemorrhage, gross destruction (any or all of these)

Conjunctivae:
- Redness (refers to palpebral and bulbar conjunctivae cornea and iris):
0 Blood vessels normal
1 Some blood vessels definitely hyperaemic (injected)
2 Diffuse, crimson color, individual vessels not easily discernible
3 Diffuse, beefy red
- Chemosis (lids and/or nictitating membranes):
0 No swelling
1 Any swelling above normal
2 Obvious swelling with partial eversion of lids
3 Swelling with lids about half-closed
4 Swelling with lids more than half-closed

TOOL USED TO ASSESS SCORE: hand-held torch

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: 1 - 24 hours

Score:

ca. 4

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: the study was terminated 24 hours after instillation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 1 - 24 hours

Score:

ca. 0

Max. score:

0

Reversibility:

not specified

Remarks on result:

other: iris could not be properly observed due to severity of opacity

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Time point:

other: 1 - 24 hours

Score:

ca. 2

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: the study was terminated 24 hours after instillation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: 1-24 hours

Score:

ca. 2.5

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: the study was terminated 24 hours after instillation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Remarks on result:

not measured/tested

Irritant / corrosive response data:

Severe opacification involving approximately half the corneal surface was present one and 24 hours after instillation. The iris could not be properly observed due to the density of the opacity. A diffuse crimson-red coloration of the conjunctivae accompanied by considerable swelling with the lids about half-closed and copious discharge, were observed. Blanching of the nictitating membrane observed one hour after instillation had developed into necrosis by the following day.
Due to the severity of the response, only one animal was treated with calcium cyanamide, technical grade, and the study was terminated 24 hours after instillation.

Other effects:

No other effects observed.

no remarks

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the results of the study and according to OECD guideline 405, the test substance calcium cyanamide is severely damaging to the eyes.

Executive summary:

The study was designed to assess the eye irritation potential of calcium cyanamide, technical grade.

Therefore, 90 mg of the test substance was placed into the lower lid of one eye of a rabbit and the other eye served as control. Eyes were examined for corneal opacity, redness and chemosis of conjunctivae and changes related to iris, aided by the use of a hand-held torch.

Severe ocular lesions were observed at one and 24 hours after instillation of the test substance. Severe opacification involving approximately half of the corneal surface and a diffuse crimson-red coloration of the conjunctivae accompanied by considerable swelling of the lids were observed at one and 24 hours. The iris could not be properly observed due to the density of the opacity. Blanching of the nictitating membrane observed one hour after instillation, had developed into necrosis the following day.

Due to the severity of the response only one rabbit was treated with calcium cyanamide, technical grade, and the study was terminated 24 hours after instillation.

It was concluded that calcium cyanamide, technical grade, is severely damaging to the rabbit eye under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin:

In vitro skin corrosion study with calcium cyanamide, technical grade "Kalkstickstoff":

The EpiDerm Skin Corrosivity Test (Model EPI-200) was performed to reveal a possible corrosive potential of calcium cyanamide, technical grade "Kalkstickstoff". The test substance was topically applied for 3 minutes and for 1 hour to the EpiDermal surfaces of three-dimensional human EpiDermis models, followed by immediate determination of the cytotoxic effect.

Investigations performed were in conformance with the Regulation (EC) 440/2008: B.40.BIS. "In vitro skin corrosion: human skin model", 30 May 2008 and the OECD-Guideline 431, "In Vitro Skin Corrosion: Human Skin Model Test ", 13 April 2004.

- The mean percentage viability of the treated skin discs after 3 minutes of exposure was 84.5 % which is above the threshold of 50% for classification.

- The mean percentage viability of the treated skin discs after 1 hour of exposure was 84.7% which is above the threshold of 15% for classification.

According to the results of this study, the test substance calcium cyanamide, technical grade "Kalkstickstoff" is considered to be non-corrosive to skin.

In an acute dermal toxicity study in rabbits, 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 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. 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 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 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 body weight and post-mortem examination. Mean irritation scores (mean of 48/72 h) of 2.25 for erythema and 1.7 for oedema were calculated. In conclusion, the study was evaluated to be skin irritating.

In an in vivo skin irritation study (disregarded due to major methodological deficiencies; Klimisch score of 3), 0.5 g of calcium cyanamide technical grade (Kalkstickstoff) was moistened with water and applied to the dorso-lumbar region of three rabbits. The test area was covered with a 2.5 cm² gauze pad and elastic adhesive dressing. After 4 hours exposure, the semi-occlusive dressing was removed and the treatment site washed with water to remove any residual test substance. The treated skin was examined at Day 1 (approximately 30 minutes after removal of the test substance) and on Days 2, 3 and 4 and dermal reactions were graded and scored. No dermal reactions were seen throughout the observation period. Therefore, it was concluded that under the conditions of the study, calcium cyanamide technical grade (Kalkstickstoff) is not irritating to skin. This study is not taken into account for classification and labelling as results from acute dermal toxicity and experience in handling and use of the substance indicate a skin irritating potential of the substance.

Disregarded studies on cyanamide: Five studies investigated the skin irritation and corrosive effects of varying forms of cyanamide. An in vitro study using a human skin model concluded that cyanamide is corrosive (Reus, 2011). Three out of four in vivo skin irritation and corrosion studies in rabbits reported that cyanamide (a 50% aqueous solution), cyanamide as crystalline substance, and as 25% substance were skin irritants. In the study with the crystalline form (van Beek, 1982), mean skin irritation scores on intact, unabraded skin were 3.0 for erythema and 2.8 for edema, on average 5.8, after 4 hours’ exposure. Fifty-two hours after dosing, erythema was scored at 3.7 and edema was 1.5 for an average of 5.2. The authors concluded that cyanamide is a severe primary skin irritant and corrosive to the skin after four hours’ contact (van Beek, 1982). A similar study testing cyanamide (van Beek, 1982) reported mean skin irritation scores on intact, unabraded skin were 2.3 for erythema and 2.3 for edema after 4 hours for an average of 4.7. During observations at 52 hours post-dosing, erythema was scored at 3.3 and edema had faded to 1.3 for an average skin irritation score of 4.7. cyanamide was classified as a skin irritant and corrosive (van Beek, 1982). The third study (van Beek, 1982) with 1%, 5%, and 25% cyanamide reported a mean score (24-72 hours) for erythema at the 25% cyanamide concentration was 1.2 and the oedema mean score was 0.2. It was concluded, that this formulation was a skin irritant in this study at the concentration of 25 % cyanamide, though the lower concentrations were not (van Beek, 1984). The fourth in vivo study (Ligget, 1989) reported no evidence of dermal irritant or corrosive effects in six rabbits exposed to cyanamide for four hours on clipped skin following OECD guideline 404. 24 hours after application, no oedema were observed. The oedema and erythema reactions which were observed 0.5 hour/ 24 hours (maximum determined score of 1) had resolved by the following day (24/48 hours after end of exposure).

As there are existing skin irritation and corrosion studies available on calcium cyanamide, which indicates its skin irritating potential, a read-across approach using cyanamide for is not necessary and the skin irritation/corrosion studies for cyanamide are disregarded for the skin irritation/corrosion evaluation of calcium cyanamide.

Eye:

In the key study, 90 mg of calcium cyanamide technical grade "Kalkstickstoff" was placed into the lower lid of one eye of a rabbit and the other eye served as control. Eyes were examined for corneal opacity, redness and chemosis of conjunctivae and changes related to iris, aided by the use of a hand-held torch. Severe ocular lesions were observed at 1 and 24 hours after instillation of the test substance. Severe opacification involving approximately half of the corneal surface and a diffuse crimson-red colouration of the conjunctivae accompanied by considerable swelling of the lids were observed at one and 24 hours. The iris could not be properly observed due to the density of the opacity. Blanching of the nictitating membrane, observed one hour after instillation, had developed into necrosis the following day. Due to the severity of the response, only one rabbit was treated and the study was terminated 24 hours after instillation. It was concluded that calcium cyanamide technical grade "Kalkstickstoff" is corrosive to the rabbit eye under the conditions of the study.

In addition to the in vivo data, a new in vitro test (Bovine Corneal Opacity and Permeability Study or BCOP) was conducted, which confirms the in vivo findings. The BCOP Test Method was performed to reveal possible ocular corrosivity and severe irritation of calcium cyanamide. Freshly isolated and quality-checked corneas were mounted in cornea holders and the initial opacity was determined. After equilibration 750 µL of the test substance preparation were topically administered to 3 isolated bovine corneas to the epithelial surfaces for 4 h and the final opacity was measured. Then 1 mL of a fluorescein solution was added on the epithelial site and permeability was measured after 90 minutes. Two groups of 3 corneas each served as positive and negative controls. Both control substances were administered under identical conditions as the test substance. The following solutions served as control substances (negative control: sterile aqua dest., positive control: 20% imidazole). Finally the IVIS (In Vitro Irritancy Score) was calculated as follows: IVIS = mean opacity value + (15 x permeability).

The opacity and mean permeability values were corrected for background opacity and the negative control permeability values. The mean opacity value results from subtraction of final opacity from initial opacity. A substance that induces an IVIS >= 55.1 is defined as ocular corrosive or severe irritant. The IVIS for calcium cyanamide was 443.4. IVIS of the negative control was 4.9 and for the positive control 123.7, hereby demonstrating the validity of the experiment. According to the results of this study and the OECD Guideline 437, the test substance calcium cyanamide is considered to be an ocular corrosive or severe irritant.

There is also one supporting study on the potential eye irritating properties of calcium cyanamide (PERLKA) available. In this study, PERLKA was ground to a powder and 100 mg thereof were administered into the interior conjunctival sac of the right eye of a single rabbit. Ocular examinations were performed in conjunctiva, iris and cornea according to the Draize scale at 1, 24, 48 and 72 hours and at Day 7 after instillation. Severe ocular lesions were observed at all of the examination time points and no reduction in the severity was observed until Day 7. Therefore, the experiment was terminated on Day 7 and no further animal was treated. From the results obtained under the conditions of the study, the test substance PERLKA is corrosive to the eye.

Disregarded studies on cyanamide: There are two in vivo studies investigating the potential eye irritation of cyanamide, the first according to OECD Guideline 405 (Acute Eye Irritation / Corrosion) in 1974, the second according to EPA OPP 81-4 (Acute Eye Irritation) in 1991. The test substance, cyanamide (supplied as aqueous solution) was applied in a single dose of a 0.1 mL aliquot into the lower everted lid of one eye of six adult New Zealand white rabbits. The contralateral eye remained untreated and served as a control (van Beek, 1974). The eyes were not washed following instillation. The eyes were examined 24, 48, 72 hours and 7 days after the end of exposure. The test substance caused eye irritation consisting of light opacity, mild iritis, moderate redness and moderate to severe swelling of the conjunctiva in all test rabbits after 24 hours. One week after the end of exposure some recovery was observed. After seven days, slight conjunctivitis was still noted in all animals. Mean scores (24-72 hours) were as follows: cornea: 1.1, iris: 0.9, conjunctiva-redness: 1.9, conjunctiva-chemosis: 2.3. Under the study´s experimental conditions, cyanamide was irritating to the eyes of New Zealand white rabbits according to the criteria specified in council directive 67/548/EEC.

In the second study (Ligget, 1991), was applied in a single dose of a 0.1 mL aliquot into the lower everted lid of one eye of an adult New Zealand white rabbit (body weight 3 kg, 14 weeks old). The contralateral eye remained untreated and served as a control. Examination of the eye was made 1 hour as well as 1, 2, 3, 4 and 7 day after the end of exposure. The animal was observed daily for clinical signs and behaviour. One hour after the end of exposure the cornea was dull and by the following day an opacity had developed which persisted for the following three days and resolved seven days after instillation. Iritis was observed two and three days after the end of exposure. A diffuse conjunctiva redness was accompanied by swelling with the lids half closed and a copious discharge. There was also evidence of haemorrhage on the nictating membrane. All lesions had resolved seven days after the end of exposure. The mean scores (24- 72 hours) for the examined parameters were as follows: cornea: 2, iris: 0.67, conjunctiva redness: 2, chemosis: 2.3. Due to the severity of the reaction no further animals were exposed to the test substance. Under the test conditions Cyanamid L500 examined in a rabbit can be considered as an eye irritant.

As there are existing eye irritation and damage studies available on calcium cyanamide, which indicates its eye damaging potential, a read-across approach using cyanamide for is not necessary and the eye irritation/damage studies for cyanamide are disregarded for the eye irritation/damage evaluation of calcium cyanamide.

Justification for classification or non-classification

Skin:

Based on the results of an in vitro skin corrosion test and an acute dermal toxicity study, the test substance calcium cyanamide technical grade is classified as skin irritating cat. 2 (H315) according to Regulation (EC) No 1272/2008 (CLP/GHS). This classification was based on the skin irritation observed in an acute dermal toxicity study and on experience in handling and use during application of the substance in the production plant. The dermal toxicity study was conducted with 2 g calcium cyanamide per kg bw, and may therefore overestimates the skin irritating potential of calcium cyanamide. However, as a worst case assumption, classification as Skin irrit.2 (H315) is considered justified.

Eye:

Based on the results of the two in vivo and one in vitro eye irritation studies, the test substance calcium cyanamide technical grade is classified as eye damage category 1 (H318, causes serious eye damage) according to Regulation (EC) No 1272/2008 (CLP/GHS).

 

Respiratory system:

In accordance with Annex VI of Regulation (EC) No 1272/2008 calcium cyanamide is classified STOT Single Exp. 3 (H335: May cause respiratory irritation) according to CLP/GHS.