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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP.
Cross-reference
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
other information
Justification for type of information:
The skin sensitization property of the registration substance can be reliably derived based on the read-across to melamine (CAS 108-78-1). The justification is attached below.
Reason / purpose for cross-reference:
assessment report
Interpretation of results:
GHS criteria not met
Executive summary:

The skin sensitization property of the target chemical can be derived based on the read-across consideration.

The source chemical melamine is investigated for its skin sensitization property in guinea pigs according to the OECD Guideline 406 and was found to be a non-sensitizer.

Likewise, the registration substance is a non-sensitizer.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Principles of method if other than guideline:
The "maximisation test" of B. Magnusson and A. M. Kligman, modified according to Maurer & Hess was performed to reveal a possible sensitising potential of melamine. 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, Fd Chem. Toxic. 1989, Vol. 27, No. 12, p. 807-811) instead of the original GPMT.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A LLNA (Local Lymph Node Assay) could not be performed, as the test substance is poorly soluble in the solvents, commonly used for the LLNA.

Test material

Constituent 1
Chemical structure
Reference substance name:
Melamine
EC Number:
203-615-4
EC Name:
Melamine
Cas Number:
108-78-1
Molecular formula:
C3H6N6
IUPAC Name:
1,3,5-triazine-2,4,6-triamine
Details on test material:
- Name of test material (as cited in study report): Melamine
- Analytical purity: 99.96 % by method MSC-TIS 002 of DSM Melamine.
- Impurities (identity and concentrations): Water <0.01 %. Ash <0.01 %.
- Purity test date: 25 January 2010.
- Lot/batch No.: 8620
- Stability under test conditions: >1 year.
- Storage condition of test material: room temperature.

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: Approx. 5 - 7 weeks at the first application
- Weight at the first application: Step 1: 337 g - 370 g. Step 2: 300 g - 344 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.
- 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", A-1226 Vienna, to assure that the water fulfils the requirements for drinking water for humans.
- Acclimation period: 13 days (step1), 7days (step2).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C.
- Humidity (%): 30 – 70% for relative humidity
- 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.

IN-LIFE DATES: From: 22 March 2010 To: 25 May 2010

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
50 % in white petrolatum.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
50 % in white petrolatum.
No. of animals per dose:
The study was performed in two consecutive steps: In the first step 5 control and 10 test substance animals were used and, as after the challenge exposure negative results were obtained, additional 5 control animals and 10 test substance animals were exposed. This procedure is in accordance with the guidelines.
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 groups) or plain white petrolatum (negative control groups) 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).
On day 6, 24 h prior to the second induction exposure, the exposed skin sites were covered in all animals with a preparation of Na-dodecylsulfate, (E. Merck, 64271 Darmstadt, Germany, item No. 13760; approx. 0.5 g, 10 %, w/w, in white petrolatum), to produce a local hyperaemia.

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 groups) or plain white petrolatum (negative control groups) 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:
The challenge exposure consisted of two separate epicutaneous applications, identically given to test substance and negative control group animals.
Positive control substance(s):
yes

Results and discussion

Positive control results:
The net rate of animals with a positive skin reaction, regarded as sensitised by hexyl cinnamic aldehyde was 50 %.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10 %
No. with + reactions:
5
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10 %
No. with + reactions:
5
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

All animals survived till the end of the study.

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

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 (score "3"), which were attributed to the effects of the adjuvant. Skin reactions after the challenge exposure: No positive skin reaction in any animal at any reading time.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Melamine is not a skin sensitizer according to the criteria in 67/548/EEC and 1272/2008.
Executive summary:

The "maximisation test" of B. Magnusson and A. M. Kligman modified according to Maurer & Hess was performed to reveal a possible sensitising potential of melamine.

Method:

Investigations performed were in conformance with the Regulation (EC) 440/2008, the EC-directive 96/54, B.6., "Skin Sensitisation", and the OECD-guideline 406, "Skin Sensitisation".

The study was performed in two consecutive steps. In each step a test substance group with 10 female guinea pigs and a negative control group with 5 female guinea pigs was used.

The administered test substance concentrations were derived from the results of a preliminary test.

Two induction exposures were performed with a one week interval.

First induction exposure: Intracutaneous injection of Freund´s complete adjuvant emulsified with isotonic saline (1:1) and epicutaneous administration of the test substance (50 % in white petrolatum).

Second induction exposure: Preceded by a topical application of 10 % Na-dodecylsulfate in petrolatum for erythema induction 24 h before the epicutaneous administration of the test substance (50 % in white petrolatum).

Control animals were given plain white petrolatum at both inductions.

Challenge exposure: Two weeks after the second induction exposure. Epicutaneous administration of the test substance (50 % in white petrolatum) and the vehicle (white petrolatum), identical for the test substance group and the negative control group.

For the epicutaneous exposures occlusive dressings were used.

Results

Challenge exposure: The control sites of all animals of both groups were normal at each reading time. After the challenge exposures, no animal of the test substance group had positive skin reactions at the test substance treated sites 24 hours and/or 48 hours after the end of the exposure. No adverse skin reactions were observed in the control animals. Therefore no animal of the test substance group was regarded as sensitised.