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Toxicological information

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
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
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
modified according toTh. Maurer and R. Hess (1989) The maximization test for skin sensitisation potential-updating the standard protocol and validation of a modified protocol. Fd Chem. T oxic. Vol. 27, No. 12, p. 807-811.
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study already existed when the data gap analysis for this substance was performed.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: "MELAMINE"
Chemical name: 2,4,6-Triamino-1,3,5-Triamine
Trade name: Melamine
Supplier: Sponsor
Molecular formula: C3H6N6
Batch No. : 8620
CAS Nr. 108-78-1
Appearance: Powder (crystalline)
pH: 8.2 -9.4 (10 % con.)
Purity: See Certificate of Analysis, Appendix 1
Solubility in water: 0.3 g/100 mL (20 °C)
Conditions of storage: Room temperature
Stability at conditions of storage: > 1 year
Stability of an aqueous solution/suspension: ± 1 week

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
Supplier: Charles River Laboratories, 88353 Kisslegg, Germany
Sex, specification: Female healthy young adult and non pregnant animals
Age of the animals Approx. 5 - 7 weeks at the first application
Weight range of the animals at the first application:
Step 1: 337 g - 370 g.
Step 2: 300 g - 344 g
Number of the animals in the main study: 10 + 10 animals for the test substance group, 5 + 5 animals for the control group
Spare animals: One additional animal per group was kept and administered under the same conditions as the other animals of the respective group. Findings on the spare animals were only to be incorporated into this report if other animals of the test substance group or of the control group would have died spontaneously. Otherwise, the skin reactions of these animals were not used for the interpretation of the results.
Hygiene: Optimal hygienic conditions
Room number: EH1-23 (both steps)
Room temperature / relative humidity: The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 20 – 24°C for temperature and of 30 – 70% for relative humidity. There were no deviations from these ranges, which influenced the results of the study.
Air exchange: Approx. 12/h
Light: Only artificial light from 6.00 a.m. to 6.00 p.m
Cages: 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
Feed: 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.
Bedding material: Wood chips (aspen) from Fa. ABEDD Dominik Mayr KEG, A-8580 Köflach.
Reduction of microorganisms by autoclaving.
Water: 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.
Identification of the animals: Numbers tattooed in the pinna of the right ear
Acclimatisation: 13 days (step1), 7days (step2)

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
petrolatum
Concentration / amount:
50% (w/w) in white petrolatum
Challenge
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
50% (w/w) in white petrolatum
No. of animals per dose:
Number of the animals in the main study:
10 + 10 animals for the test substance group
5 + 5 animals for the control group
Spare animals: One additional animal per group was kept and administered under the same conditions as the other animals of the respective group. Findings on the spare animals were only to be incorporated into this report if other animals of the test substance group or of the control group would have died spontaneously. Otherwise, the skin reactions of these animals were not used for the interpretation of the results.
Details on study design:
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.
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 sensitivity and the reliability of the experimental procedure is checked separately, twice a year, using a-hexyl cinnamic aldehyde as sensitizer and the same strain of animals and the same experimental procedure as in the present study.
Positive control substance(s):
yes
Remarks:
a-hexyl cinnamic aldehyde

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0% induction, 50% challenge
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No positive skin reaction in any animal at any reading time
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0% induction, 50% challenge
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No positive skin reaction in any animal at any reading time
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% induction and challenge
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No positive skin reaction in any animal at any reading time
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% induction and challenge
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No positive skin reaction in any animal at any reading time
Group:
positive control
Remarks on result:
positive indication of skin sensitisation
Remarks:
checked separately, twice a year

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria are not met, no classification is required according to Regulations (EC) No 1272/2008.