Calcium cyanamide

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

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

Deviations:

no

Test guideline 2

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.

Test material

Test material information

Constituent 1

Reference substance name:

Calcium cyanamide

EC Number:

205-861-8

EC Name:

Calcium cyanamide

Cas Number:

156-62-7

Molecular formula:

CN2.Ca

IUPAC Name:

calcium cyanoazanediide

impurity 1

Reference substance name:

Calcium oxide

EC Number:

215-138-9

EC Name:

Calcium oxide

Cas Number:

1305-78-8

Molecular formula:

CaO

IUPAC Name:

oxocalcium

impurity 2

Reference substance name:

Carbon

EC Number:

231-153-3

EC Name:

Carbon

Cas Number:

7440-44-0

Molecular formula:

C

IUPAC Name:

carbon

impurity 3

Reference substance name:

Hematite (Fe2O3)

EC Number:

215-275-4

EC Name:

Hematite (Fe2O3)

Cas Number:

1317-60-8

Molecular formula:

Fe2O3

IUPAC Name:

diiron oxide

impurity 4

Reference substance name:

Urea

EC Number:

200-315-5

EC Name:

Urea

Cas Number:

57-13-6

Molecular formula:

CH4N2O

IUPAC Name:

urea

impurity 5

Reference substance name:

Silicon dioxide

EC Number:

231-545-4

EC Name:

Silicon dioxide

Cas Number:

7631-86-9

Molecular formula:

O2Si

IUPAC Name:

dioxosilane

impurity 6

Reference substance name:

Trisilicon tetranitride

EC Number:

234-796-8

EC Name:

Trisilicon tetranitride

Cas Number:

12033-89-5

Molecular formula:

N4Si3

IUPAC Name:

trisilicon tetranitride

impurity 7

Reference substance name:

Calcium dihydroxide

EC Number:

215-137-3

EC Name:

Calcium dihydroxide

Cas Number:

1305-62-0

Molecular formula:

CaH2O2

IUPAC Name:

calcium dihydroxide

impurity 8

Reference substance name:

Aluminium oxide

EC Number:

215-691-6

EC Name:

Aluminium oxide

Cas Number:

1344-28-1

Molecular formula:

Al2O3

IUPAC Name:

aluminium oxide

impurity 9

Reference substance name:

Cyanoguanidine

EC Number:

207-312-8

EC Name:

Cyanoguanidine

Cas Number:

461-58-5

Molecular formula:

C2H4N4

IUPAC Name:

2-cyanoguanidine

impurity 10

Reference substance name:

Calcium acetylide

EC Number:

200-848-3

EC Name:

Calcium acetylide

Cas Number:

75-20-7

Molecular formula:

C2Ca

IUPAC Name:

calcium ethynediide

impurity 11

Reference substance name:

unknown

IUPAC Name:

unknown

Test material form:

solid: particulate/powder

In vivo test system

Test animals

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

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Induction 2

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.

Challenge

Challenge 1

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

Challenge 2

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.

Challenge 3

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.

Results and discussion

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.

In vivo (non-LLNA)

Results

Results 1

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

Results 2

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

Results 3

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.

Results 4

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.

Results 5

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

Results 6

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

Any other information on results incl. tables

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 %).

Applicant's summary and conclusion

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.