Calcium cyanamide

Endpoint summary

Description of key information

Based on the results of a guinea pig maximisation test (modified according to Maurer and Hess), calcium cyanamide technical grade (Kalkstickstoff) is classified as skin sensitiser.

There are no data from respiratory sensitisation studies on calcium cyanamide in laboratory animals available. However, in a review on human data on calcium cyanamide, the potential sensitising properties of the substance were evaluated. Respiratory sensitisation after exposure towards calcium cyanamide was not described and considered of concern in the MAK 2007 review (ref section 7 .10.4).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-17 to 2010-07-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17 July 1992, (modified according to Maurer & Hess)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Regulation (EC) No 440/2008

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

According to ECHA guidance R.7.a "Guidance on Information Requirements and Chemical Safety Assessment" (December 2016) tests other than LLNA generated or initiated before 11 October 2016 are acceptable, if these tests provide clear results that are adequate for classification. This well documented guideline test has been conducted in 2010 and thus, further in vivo testing is not necessary.

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, 88353 Kisslegg, Germany
- Age at study initiation: Approx. 5 - 7 weeks at the first application
- Weight at study initiation: 318 g - 379 g
- Housing: Group caging in plastic containers (48 cm x 115 cm x 36 cm), partly shaded, 6 (control group) or 11 (test substance group) animals per container. Wood chips (aspen) from Fa. ABEDD Dominik Mayr KEG, 8580 Köflach, Austria. Reduction of microorganisms by autoclaving.
- Diet (e.g. ad libitum): Ssniff Ms-H (Guinea Pig Maintenance Diet V2233), including ascorbic acid (2400 mg/kg), ad libitum, offered in stainless steel containers. Analysis of the feed for ingredients and contaminants are performed randomly by ssniff Spezialdiäten GmbH, Ferdinand-Gabriel-Weg 16, 59494 Soest, Germany.
- Water (e.g. ad libitum): Tap water offered in Makrolon bottles with stainless steel canules ad libitum. Random samples of the water are analysed by the "AGES", 1226 Vienna, Austria to assure that the water fulfills the requirements for drinking water for humans.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Mean of 20.58°C (continuous control and registration).
- Humidity (%): Mean of 56.10 % (continuous control and registration)
- Air changes (per hr): Approx. 12/h
- Photoperiod (hrs dark / hrs light): Only artificial light from 6.00 a.m. to 6.00 p.m

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

Induction exposure on Day 0:
The test substance was applied at a concentration of 50 % (w/w) in white petrolatum. About 0.5 g of test substance formulation or of white petrolatum were applied to each animal. The exposure time was 24 hours.

Induction exposure on Day 7:
The test substance was applied at a concentration of 2.5 % (w/w) in white petrolatum. About 0.5 g of test substance formulation or of white petrolatum were applied to each animal. The exposure time was 48 hours.

Challenge exposure
The test substance was applied at a concentration of 1 % (w/w) in white petrolatum. About 0.5 g of test substance formulation and about 0.6 g of white petrolatum were applied to each animal. The exposure time was 24 hours.

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

The test substance was applied at a concentration of 50 % (w/w) in white petrolatum. About 0.5 g of test substance formulation or of white petrolatum were applied to each animal.

Day(s)/duration:

Day 0 / The exposure time was 24 hours.

Adequacy of challenge:

not specified

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

The test substance was applied at a concentration of 2.5 % (w/w) in white petrolatum. About 0.5 g of test substance formulation or of white petrolatum were applied to each animal.

Day(s)/duration:

Day 7 / The exposure time was 48 hours.

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

The test substance was applied at a concentration of 1 % (w/w) in white petrolatum. About 0.5 g of test substance formulation and about 0.6 g of white petrolatum were applied to each animal

Day(s)/duration:

Challenge exposure / The exposure time was 24 hours.

No. of animals per dose:

10

Details on study design:

First induction exposure: Commenced on Day 0.
Four separate intradermal injections of FCA, emulsified with isotonic saline (to enhance a possible sensitisation) were given at an area of approx. 2 x 4 cm in the interscapular region. The injections were followed immediately afterwards by an epicutaneous application of the test substance, incorporated in white petrolatum (test substance group) or plain white petrolatum (negative control group) to the sites of the intradermal injections. Test patches (filter papers), 2 cm x 4 cm, with the test substance preparation (test substance group) or with the vehicle (negative control group), were applied. They were fixed with a strip of "Fixomull stretch" (self-adhesive, non-woven fabric, hypoallergenic, made by Beiersdorf AG, 20245 Hamburg, Germany). The treated sites were covered occlusively with a Teflon ® - foil and kept in place and fixed with Guinea-Pig Jackets (Hugo Sachs Elektronik- Harvard Apparatus GmbH, 79232 March-Hugstetten, Germany). 24 h afterwards (Day 1) the jackets and the patches were removed. Effects of the first induction exposure were checked by a skin examination 24 h after the end of the exposure period (Day 2).

Second induction exposure: Commenced on Day 7.
At the site of the preceding injections of the first induction exposure, an epicutaneous application of the test substance, incorporated in white petrolatum (test substance group) or plain white petrolatum (negative control group) to the sites of the intradermal injections analogously to the first induction exposure. 48 h afterwards (Day 9) the jackets and the patches were removed. Effects of the second induction exposure were checked by a skin examination 24 h after the end of the exposure period (Day 10).

Challenge exposure: Commenced on Day 21.
The challenge exposure consisted of two separate epicutaneous applications, identically given to test substance and negative control group animals: One with a test substance preparation to the left flanks and one with the vehicle (white petrolatum) to the right flanks of all animals. Both exposed sites were apart from the exposure sites of the two induction exposures. Test patches (now only 2 cm x 2 cm) and coverings were the same as in both induction exposures. 24 h afterwards (Day 22) the jackets and the patches were removed. Effects of the challenge exposure were checked by a skin examination 24 h after the end of the exposure period (Day 23) and a second skin examination further 24 h later (Day 24). Positive skin reactions of the test substance treated sites after the challenge exposure indicate a sensitising effect of the test substance, if the scores are higher than those of the vehicle treated sites and if the rate of those - positively reacting - animals is higher than the corresponding percentage of animals in the negative control group.

Challenge controls:

yes

Positive control substance(s):

yes

Remarks:

The sensitivity and the reliability of the experimental procedure is checked separately, twice a year, using α-hexyl cinnamic aldehyde as sensitizer and the same strain of animals and the same experimental procedure as in the present study.

Positive control results:

50 % of the animals had a positive response in this test, which is markedly more than the minimum of 30 %, the threshold for classification requested by the guideline. Thus the results confirm both the sensitivity and the reliability of the experimental techniques.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 % induction, 0 % challenge

No. with + reactions:

1

Total no. in group:

5

Clinical observations:

severe erythema and/or oedema in one animal

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 % induction, 0 % challenge

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 % induction, 1 % challenge

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

all animals showed mild to severe skin reactions

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 % induction, 1 % challenge. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: all animals showed mild to severe skin reactions.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 % induction, 1 % challenge

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

9/10 animals showed mild to severe skin reactions

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 % induction, 1 % challenge. No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: 9/10 animals showed mild to severe skin reactions.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

100% induction, 50% 1st challenge, 10% 2nd challenge

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

very slight to severe erythema and/or oedema

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

100% induction, 50% 1st challenge, 10% 2nd challenge

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

very slight to severe erythema and/or oedema

Remarks on result:

positive indication of skin sensitisation

Mortality

All animals survived till the end of the study.

Body weights

No effects of the test substance on body weights can be derived from the data.

General observations

Immediately after the beginning of all epicutaneous exposures (inductions, challenge) the motor activities of all animals were decreased. This is due to the dressings which restrict the freedom of movement. Soon afterwards the behaviour was regular again. Except of the observations described above no abnormal behaviour or clinical signs were detected during the experiment in the animals.

Skin reactions after the intradermal FCA administration

Cranial and caudal injection sites: Local irritations were observed in all animals beginning on the day after the injections. The irritations started with local erythema, which became more severe and led to ulcerations. Lesions did not heal until the end of the study. These local alterations are known effects of Freund's adjuvant.

Skin reactions after the first and the second induction exposure

All animals of both groups had severe erythema and oedema in the interscapular region, which were attributed to the effects of the adjuvant.

Skin reactions after the challenge exposure

These are the reactions of interest for the grading of an allergenic potency of the test substance.

 

Negative control group
Vehicle site	No positive skin reaction in any animal at any reading time
Test substance site	1/5 animals (20 %) had a severe erythema and/or oedema 24 hours after the end of the challenge exposure.
Test substance group
Vehicle site	No positive skin reaction in any animal at any reading time.
Test substance site	10/10 animals (100 %) had very slight to severe erythema and/or oedema 24 and/or 48 hours after the end of the challenge exposure.

The net rate of animals with a positive skin reaction, regarded as sensitised, was therefore 80 % (100 % - 20 %).

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the results of this guinea pig maximation test (modified according to Maurer and Hess), calcium cyanamide, technical grade (Kalkstickstoff) has to be classified as skin sensitizer.

Executive summary:

A "maximisation test" of B. Magnusson and A. M. Kligman modified according to Maurer & Hess was performed to reveal a possible sensitising potential of calcium cyanamide, technical grade ("Kalkstickstoff"). The use of this test is justified by the fact, that an LLNA (Local Lymph Node Assay) cannot be performed, as the test substance is poorly soluble in the solvents, commonly used for the LLNA. The same cause is the reason for choosing the modified test (Maurer and Hess) instead of the original GPMT.

Investigations performed were in conformance with the Regulation (EC) 440/2008, Part B: Methods for the determination of toxicity and other health effects: Skin Sensitization; Official Journal of the European Union, No. L 142 and the OECD-guideline 406, "Skin Sensitisation", EC-directive 2001/59 for classification and OECD principles of GLP. 10 female guinea pigs (Dunkin Hartley, HsdPoc:DH) were used as a test substance group and another 5 females were used as a negative control group. No repetition with another 10 + 5 animals was performed due to positive results. Immediately after the injection of Freund´s complete adjuvant the test substance was administered epicutaneously on the same area. One week later a second epicutaneous induction exposure followed and two weeks afterwards the epicutaneous challenge exposure.

Test substance concentrations selected for the main study were derived from the results of preliminary tests. Test substance concentrations were:

50 % (w/w) in white petrolatum for the first epicutaneous induction exposure (maximum possible concentration),

2.5 % (w/w) white petrolatum for the epicutaneous second induction exposure and

1 % (w/w) in white petrolatum for the epicutaneous challenge exposure.

For the epicutaneous exposures occlusive dressings were used.

All animals survived till the end of the study. Intradermal injections of Freund's adjuvant caused severe local reactions in all animals, a known effect of the adjuvant skin. Sensitisation excluded, no other adverse effects were noted. The results of these skin examinations were decisive for the evaluation of skin senitising potential. The control sites of all animals of both groups were normal at each reading time. 24 and/or 48 hours after the end of the challenge exposure, 10/10 animals (100 %) of the test substance group had mild to severe erythema and/or oedema at the test substance treated sites. A severe erythema and/or oedema was observed in 1/5 of the control group animals (20 %) 24 hours after the end of the challenge exposure. Therefore the net rate of animals with positive skin reactions (80%), regarded as sensitistised by calcium cyanamide, was >>30%.

In conclusion, calcium cyanamide has to be classified as skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A "maximisation test" of B. Magnusson and A. M. Kligman modified according to Maurer & Hess was performed to reveal a possible sensitising potential of calcium cyanamide, technical grade ("Kalkstickstoff"). The use of this test is justified by the fact, that an LLNA (Local Lymph Node Assay) cannot be performed, as the test substance is poorly soluble in the solvents, commonly used for the LLNA. The same cause is the reason for choosing the modified test (Maurer and Hess) instead of the original GPMT.

Investigations performed were in conformance with the Regulation (EC) 440/2008, Part B: Methods for the determination of toxicity and other health effects: Skin Sensitization; Official Journal of the European Union, No. L 142 and the OECD-guideline 406, "Skin Sensitisation", EC-directive 2001/59 for classification and OECD principles of GLP. 10 female guinea pigs (Dunkin Hartley, HsdPoc:DH) were used as a test substance group and another 5 females were used as a negative control group. No repetition with another 10 + 5 animals was performed due to positive results. Immediately after the injection of Freund´s complete adjuvant the test substance was administered epicutaneously on the same area. One week later a second epicutaneous induction exposure followed and two weeks afterwards the epicutaneous challenge exposure.

Test substance concentrations selected for the main study were derived from the results of preliminary tests. Test substance concentrations were:

50 % (w/w) in white petrolatum for the first epicutaneous induction exposure (maximum possible concentration),

2.5 % (w/w) white petrolatum for the epicutaneous second induction exposure and

1 % (w/w) in white petrolatum for the epicutaneous challenge exposure.

For the epicutaneous exposures occlusive dressings were used.

All animals survived till the end of the study. Intradermal injections of Freund's adjuvant caused severe local reactions in all animals, a known effect of the adjuvant skin. Sensitisation excluded, no other adverse effects were noted. The results of these skin examinations were decisive for the evaluation of skin sensitising potential. The control sites of all animals of both groups were normal at each reading time. 24 and/or 48 hours after the end of the challenge exposure, 10/10 animals (100 %) of the test substance group had mild to severe erythema and/or oedema at the test substance treated sites. A severe erythema and/or oedema was observed in 1/5 of the control group animals (20 %) 24 hours after the end of the challenge exposure. Therefore the net rate of animals with positive skin reactions, regarded as sensitised by calcium cyanamide, was >>30%.

In conclusion, calcium cyanamide has to be classified as skin sensitiser.

Disregarded studies: Cyanamide was evaluated in two in vivo studies with guinea pigs. The first, according to the Buehler method and in accordance with OECD Guideline 406 (Skin sensitisation), tested 20 guinea pigs with 0.5 mL of a 40% solution of Cyanamid L500 in distilled water on day 1, 8, and 15, and for the challenge on day 29. In both sexes, there were no signs of clinical intoxication or impairment of body weight development; no oedema or erythema were observed during the induction period. Skin reactions did not occur after the challenge in most of the treated animals; two animals of each sex in the treated group showed slight erythema and one orthoergic type reaction was found during the histopathological examinations. Cyanamide induced some skin reactions after challenge application but the result does not clearly indicate a sensitising potential of cyanamide.

Next, the Guinea Pig Maximisation Test according to the method of Magnusson and Kligman was conducted on 15 male albino guinea pigs. The intradermal injection caused slight erythema and abscesses in all test animals; the dermal applications induced slight erythema in 4 out of 10 test animals. After 24 and 28 hours, most of the test animals showed slight to moderate erythema but no test substance-related clinical signs of toxicity were observed. The challenge treatment of the left flank (1 % dilution) two weeks after dermal induction provoked slight to moderate erythema in all test animals. Challenge treatment of 2.5 % dilution of cyanamide resulted in well-defined to severe erythema immediately after removing the dressing and no test substance-related clinical signs of toxicity were observed. Based on the results of this test, cyanamide is a skin sensitiser.

As there exists an OECD test guideline (406) study for calcium cyanamide, which indicates its skin sensitising potential, read-across from cyanamide to calcium cyanamide for its evaluation is not necessary.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the MAK value documentation of 2007, no signs for respiratory sensitisation in humans were found based on epidemiological data (section 7.10.4). An investigation was performed on 65 workers of a calcium cyanamide production plant who were between 33 and 65 years old and had been working in the plant between 5 and 41 years. Extensive physical and clinico-chemical examination were performed and the analytically determined calcium cyanamide workplace concentration was 0.23 - 8.36 mg/m³. The study did not provide evidence of adverse effects on health or disorders caused by calcium cyanamide in relation to exposure. Thus, calcium cyanamide is no respiratory sensitiser in humans and does not need to be classified and labelled with respect to respiratory sensitisation.

Justification for classification or non-classification

Based on the available data calcium cyanamide has to be classified and labelled as skin sensitising cat.1 according to Regulation (EC) No 1272/2008 (CLP).

Calcium cyanamide is not a respiratory sensitiser in humans and does not need to be classified and labelled with respect to respiratory sensitisation according to the CLP (Regulation (EC) No 1272/2008).