1,3,5-triazine-2,4,6-triamine phosphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 weeks

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

modified according toTh. Maurer and R. Hess (1989) The maximization test for skin sensitization potential-updating the standard protocol and validation of a modified protocol. Fd Chem. Toxic. Vol. 27, No. 12, p. 807-811.

GLP compliance:

yes (incl. QA statement)

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

Test material information

Constituent 1

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 8620

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: ± 1 week

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
- Microbiological status of animals, when known: Optimal hygienic conditions
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approx. 5 - 7 weeks at the first application
- Weight at study initiation: 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: Ssniff Ms-H (Guinea Pig Maintenance Diet V2233), including ascorbic acid (2400 mg/kg), ad libitum, offered in stainless steel containers
- Water: ap water offered in Makrolon bottles with stainless steel canules ad libitum
- Acclimation period: 13 days (step1), 7days (step2)
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 30 – 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

petrolatum

Induction 2

Concentration / amount:

50 % (w/w) in white petrolatum

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Challenge 2

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), 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".
The treated sites were covered occlusively with foil and kept in place and fixed with Guinea-Pig Jackets. 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, (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.

A skin reaction after the challenge exposure was regarded as positive when the site, where test substance formulation was applied, was more irritated than the area of the site, where the vehicle was applied. The rate of these positively reacting animals in the test substance group minus the rate of positively reacting animals in the negative control group gave the net percentage of sensitised animals. The t-test was used to evaluate differences in the mean body weight between the test substance group and the control group on Days 0 and 24.

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)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 (FCA, white petrolatum)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No positive skin reaction in any animal at any reading time

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

TS, 50 % (w/w) in white petrolatum

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No positive skin reaction in any animal at any reading time

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

FCA & TS, 50 % (w/w) in white petrolatum

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No positive skin reaction in any animal at any reading time

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

TS, 50 % (w/w) in white petrolatum

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No positive skin reaction in any animal at any reading time

Results 5

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

10%

No. with + reactions:

5

Total no. in group:

10

Results 6

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

10%

No. with + reactions:

5

Total no. in group:

10

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met