1,3,5-Triazine-2,4,6-triamine, N2,N2'-1,6-hexanediylbis[N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethyl-4-piperidinyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 August 2015 - 01 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

The substance has an irritation potential and therefore the Bühler assay was considered better suitable.

Test material

Test material information

Constituent 1

Reference substance name:

N2-[6-({4,6-bis[butyl(2,2,6,6-tetramethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}(2,2,6,6-tetramethylpiperidin-4-yl)amino)hexyl]-N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine

EC Number:

629-754-4

Cas Number:

210988-99-1

Molecular formula:

C82 H156 N18

IUPAC Name:

N2-[6-({4,6-bis[butyl(2,2,6,6-tetramethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}(2,2,6,6-tetramethylpiperidin-4-yl)amino)hexyl]-N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine

Details on test material:

- Physical state: Solid / white to beige
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: HARLAN, The Netherlands
- Age at study initiation: 3 or 4 weeks
- Weight at study initiation: average weight of 269g
- Housing: in groups of 2 in polycarbonate containers
- Diet: SAFE 106, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C
- Humidity: 30 % to 70 %,
- Air changes: at least 10 cycles per hour
- Photoperiod: 12 hrs day / 12 hrs darkness

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

50%

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

50%

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

25%

No. of animals per dose:

10 in control and 20 in test group

Details on study design:

RANGE FINDING TESTS:
A pre-test was performed to determine the maximal item concentration without risk of an irritant effect during the challenge phase. Three guinea pigs were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 6 hours at 4 different concentrations: diluted at 50%, 25%, 10% and 5% in liquid paraffin. Washing of the skin after removal of the dressing was done with liquid paraffin for the site treated with the test item at 50%. Washing of the skin after removal of the dressing was not necessary for the other sites. The animals treated with the concentrations 50%, 25%, 10% and 5% received 0.5 mL of the corresponding preparation. A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressings. 24 and 48 hours after removal of the patches, no cutaneous reaction was noted at all tested concentrations. In view of these results, the concentration selected was 50% for the 3 inductions of the main study and the concentration selected was 50% for the challenge phase.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 3 local applications on D0, D7 and D14 during 6 hours under occlusive dressing
- Test groups: The animals of the test group received 0.5 mL of 50% in liquid paraffin.
- Control group: The animals of the control group received 0.5 mL of liquid paraffin
- Site: scapular zone
- Concentrations: 50 %
Washing of the skin after removal of the dressing was done with liquid paraffin.

B. CHALLENGE AND RE-CHALLENGE EXPOSURE
- No. of exposures: two challenges
- Day(s) of challenge: D28 (challenge) and D35 (re-challenge)
- Exposure period: 6 hours
- Test groups: 0.5 mL of the test item diluted at 50% (25%) and 0.5 mL of liquid paraffin
- Control group: 0.5 mL of the test item diluted at 50% (25%) 0.5 mL of liquid paraffin
- Site: left flank (test item) and right flank (liquid paraffin)
- Concentrations: 50% (D28), 25% (D35)
- Evaluation (hr after challenge): 24, 48 and 72 hours after removal of the dressing
Washing of the skin after removal of the dressing was done with liquid paraffin.

Positive control substance(s):

yes

Remarks:

the sensitivity is periodically tested with α-Hexylcinnamaldehyde, recent test reports are presented

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

20

Total no. in group:

20

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0%

No. with + reactions:

0

Total no. in group:

20

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50%

No. with + reactions:

6

Total no. in group:

10

Results 4

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Any other information on results incl. tables

Induction phase

No cutaneous reaction was recorded after the first and second induction in the treated group. Discrete erythema (grade 1) was noted in eighteen animals (18/20) and a moderate erythema (grade 2) was noted in two animals (2/20) after the third induction. No cutaneous reaction was recorded during the induction phase in the control group.

Challenge phase

In the treated group (treatment dose of 50%), it was recorded a slight to intense erythema (grade 1-3) in 100% (20/20) of the animals, 24, 48 and 72 hours after the challenge phase, on the treated area. In the control group (associated with the treatment dose of 50%), it was recorded a slight to moderate erythema (grade 1-2) in 60% (6/10), 40% (4/10) and 30% (3/10) of the animals, 24, 48 and 72 hours after the challenge phase, on the treated area. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (control item).

As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. So, a sensitization reaction was noted in 60%, 50% and 20% of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the area challenged with the test item at 50%.

Re-challenge phase

In the treated group (treatment dose of 25%), it was recorded a slight to intense erythema (grade 1-3) in 90% (18/20), 80% (16/20) and 15% (3/20) of the animals, 24, 48 and 72 hours after the re-challenge phase, on the treated area. Additionally, 9 animals in total showed dryness of the skin. In the control group (associated with the treatment dose of 25%), it was recorded a slight to moderate

erythema (grade 1-2) in 70% (7/10), 50% (5/10) and 20% (2/10) of the animals, 24, 48 and 72 hours after the re-challenge phase, on the treated area. Dryness of the skin was observed in 4 animals in total. No cutaneous reaction was recorded in animals from the treated and control groups after the re-challenge phase, on the treated area with liquid paraffin (control item).

As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. So, a sensitization reaction was noted in 10%, 5% and 5% of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the area challenged with the test item

at 25%.

Weight evolution

No abnormalities and no differences in the body weight between the control and the treated group were observed.

Mortality

No mortality was registered during the main test.

Clinical signs

No abnormal clinical signs related to the administration of the test item were observed.

ASSESSMENT OF THE SENSITISING POTENTIAL

1st CHALLENGE

Groups	Reading Time	Concentrations	0	1	2	3	% of positive responses ≥ 1	% of animal sensitized
Control group	24 h	50%	4	4	2	0	60%
	48 h	50%	6	3	1	0	40%
	72 h	50%	7	2	1	0	30%
	24 h	0%	10	0	0	0	0%
	48 h	0%	10	0	0	0	0%
	72 h	0%	10	0	0	0	0%
Treated Group	24 h	50%	0	2	6	12	100%	60%
	48 h	50%	0	1	9	10	100%	50%
	72 h	50%	0	1	15	4	100%	20%
	24 h	0%	20	0	0	0	0%	0%
	48 h	0%	20	0	0	0	0%	0%
	72 h	0%	20	0	0	0	0%	0%

RE-CHALLENGE

Groups	Reading Time	Concentrations	0	1	2	3	% of positive responses ≥ 1	% of animal sensitized
Control group	24 h	25%	3	4	3	0	70%
	48 h	25%	5	3	2	0	50%
	72 h	25%	8	2	0	0	20%
	24 h	0%	10	0	0	0	0%
	48 h	0%	10	0	0	0	0%
	72 h	0%	10	0	0	0	0%
Treated Group	24 h	25%	2	5	11	2	90%	10%
	48 h	25%	4	13	2	1	80%	5%
	72 h	25%	17	2	1	0	15%	5%
	24 h	0%	20	0	0	0	0%	0%
	48 h	0%	20	0	0	0	0%	0%
	72 h	0%	20	0	0	0	0%	0%

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item was considered as a skin sensitizer, taking into account the result of the 1st challenge.

Executive summary:

The aim of the study was to evaluate the possible allergenic activity of the test item after topical administration in guinea pigs. After induction of 20 guinea-pigs by 3 topical applications with the test item applied diluted at 50% in liquid paraffin under occlusive dressing and a 13-day rest phase, the challenge phase, under occlusive dressing for 6 hours, consisted of a single topical application of the test item diluted at 50% in liquid paraffin and of a negative control (liquid paraffin). The experimental protocol was established from the O.E.C.D. Test Guideline No.406 dated July 17th, 1992, and the method B.6 of the Council regulation No. 440/2008 and EPA.

The concentrations selected for the induction phase and the challenge phase were based on the results of a pre-test. In the treated group (treatment dose of 50%), it was recorded a slight to intense erythema in 100% (20/20) of the animals, 24, 48 and 72 hours after the challenge phase, on the treated area. In the control group (associated with the treatment dose of 50%), it was recorded a slight to moderate erythema in 60% (6/10), 40% (4/10) and 30% (3/10) of the animals, 24, 48 and 72 hours after the challenge phase, on the treated area. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (control item).

As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. So, a sensitization reaction was noted in 60%, 50% and 20% of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the area challenged with the test item at 50%. The severity of the reactions were 2.5, 2.45 and 2.15 in the treated group versus 0.8, 0.5 and 0.4 in the control group 24, 48 and 72 hours after the challenge phase, respectively.

To confirm the results obtained with the test item, a second challenge phase (re-challenge) was performed with the test item diluted at 25% in liquid paraffin after a 6 day rest phase. In the treated group (treatment dose of 25%), it was recorded a slight to intense erythema in 90% (18/20), 80% (16/20) and 15% (3/20) of the animals, 24, 48 and 72 hours after the re-challenge phase, on

the treated area. Additionally, 9 animals in total showed dryness of the skin. In the control group (associated with the treatment dose of 25%), it was recorded a slight to moderate erythema in 70% (7/10), 50% (5/10) and 20% (2/10) of the animals, 24, 48 and 72 hours after the re-challenge phase, on the treated area. Dryness of the skin was observed in 4 animals in total. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (control item). As irritation was observed in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. So, a sensitization reaction was noted in 10%, 5% and 5% of the animals from the treated group, 24, 48 and 72 hours after the challenge phase, on the area challenged with the test item

at 25%. The severity of the reactions were 1.65, 1.0 and 0.2 in the treated group versus 1.0, 0.7 and 0.2 in the control group 24, 48 and 72 hours after the challenge phase, respectively. In conclusion, the test item was considered to be a skin sensitizer, taking into account the result of the 1st challenge.