N4,N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 October 2020 - 05 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N4,N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]

EC Number:

835-183-3

Cas Number:

83420-16-0

Molecular formula:

C56 H102 Cl2 N14

IUPAC Name:

N4,N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch number of test material:SUQIAN UNITECH CORP., LTD; 190701
- Expiration date of the lot/batch: 24-07-2021
- Purity:91%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8–9 weeks
- Weight at study initiation: 17-22 g
- Housing: The animals were kept in groups of 5 animals in IVC cages, type II L, polys cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least five days
- Indication of any skin lesions: erythema and ear thickness

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 3 °C
- Humidity (%):55 ± 10%
- Air changes (per hr):at least 10 x / hour
- Photoperiod (hrs dark / hrs light):- Artificial light, sequence being 12 hours light, 12 hours dark

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Remarks:

vehicle AOO：(Acetone, VWR, lot no. 20C054005, expiry date: 24/07/2021; olive oil highly refined, Sigma-Aldrich, lot no. BCBW5235, expiry date: 14/12/2020).

Concentration:

Pre-screen test: 25 and 50% (w/v) in AOO
Main test: 12.5%, 25% and 50% (w/v) in AOO

No. of animals per dose:

5 ([pre-screen and main test)

Details on study design:

PRE-SCREEN TESTS:
Four animals were treated by topical application with the test item on three consecutive days at the following concentrations to the entire dorsal surface of each ear:
- animals no. 52 and no. 53 were treated with a test item concentration of 25% (suspended with AOO),
- animals no. 54 and no. 55 were treated with a test item concentration of 50% (suspended with AOO).
- One further animal (no. 51) was treated with 100% AOO and served as negative control.
Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured (see Table 2 in the appendix).
During this period also all clinical signs were recorded. Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).

- Compound solubility:The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AOO
- Irritation & Systemic toxicity: Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal. All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the duration of the prescreen test (see Table 4 in the appendix).
- Ear thickness measurements:On day 6, the ear thicknesses of animal no. 52 were increased up to 28% compared to day 1. As test item residues but no evidence of irritation were apparent, this finding was not considered as irritating effect of the test item. No significant increase in ear thickness was observed for the four remaining animals.

MAIN STUDY
Dose Groups
3 test groups (12.5%, 25% and 50% (w/v), each suspended in AOO (4:1 (v/v) acetone / olive oil) and 1 negative control group (vehicle) were tested.

Test Regime
Topical Application
Each mouse was treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 μCi 3H-methyl thymidine by intravenous injection (tail vein) of 250μL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80μCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Positive control substance(s):

other: 1% phenylenediamine in AOO

Results and discussion

Positive control results:

A positive control (1% phenylenediamine in AOO) was performed concomitantly. The stimulation index was 4.4 which exceeds the stimulation index of 3, confirming the reliability of the test system (Table 8)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

5.6

Test group / Remarks:

12.5%

Remarks on result:

other: ±0.5

Results 2

Parameter:

SI

Value:

7.3

Test group / Remarks:

25%

Remarks on result:

other: ±2.3

Results 3

Parameter:

SI

Value:

4.8

Test group / Remarks:

50%

Remarks on result:

other: ±2.6

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: The proliferative response of lymph node cells was expressed as the number of (DPM/NODE). Before DPM/NODE values were determined, background values were subtracted (Table 6).

DETAILS ON STIMULATION INDEX CALCULATION:The ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals.
The stimulation index at a concentration of 12.5% was 5.6
The stimulation index at a concentration of 25% was 7.3
The stimulation index at a concentration of 50% was 4.8 (Table 6).

EC3 CALCULATION: EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
The EC3 value could not be calculated due to the lack of a dose-response relationship of the data obtained.

CLINICAL OBSERVATIONS: All animals survived throughout the test period without showing any signs of systemic toxicity or local irritation. All animals treated with the test item showed test item residues at the application sites within the days 2–4. Sticky fur was observed at the application sites of all animals treated with the test item at the concentrations 12 .5% or 25% from day 3 or 4 until day 6 (Table 9).

BODY WEIGHTS: All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study (Table 7).

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In an in vivo skin sensitisation LLNA assay, N4,N4’-hexane-1,6-diylbis [N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] is considered to be a dermal sensitiser.

Executive summary:

In a dermal sensitization study (OECD 429/GLP) with N4,N4'-hexane-1,6-diylbis[N-butyl-6-chloro-N,N'-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine]​ (91%), young adult CBA/CaOlaHsd mice (5 females/group) were tested in the Local Lymph Node Assay. The doses tested were 12.5, 25 and 50% (w/v) in AOO (4:1 (v/v) acetone/olive oil). The reliability of the test system was confirmed by a positive control test with 1% phenylenediamine in AOO that was performed concurrently, using 5 animals.

 

In the experiment, the positive control, 1% phenylenediamine, gave the appropriate response (stimulation index 4.4 ± 0.9). All animals survived throughout the test period without showing any signs of systemic toxicity or local irritation. All animals treated with the test item showed test item residues at the application sites within the days 2-4. Sticky fur was observed at the application sites of all animals treated with the test item at the concentrations 12.5% or 25% from day 3 or 4 until day 6. All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study. The SI values were 5.6 ±0.5, 7.3 ±2.3, 4.8 ±2.6 at concentrations of 12.5%, 25% and 50% (w/v), respectively. Each of the tested concentrations exceeded the stimulation index of 3. The EC3 value could not be calculated due to the lack of a dose-response relationship of the data obtained. In this study, N4,N4’-hexane-1,6-diylbis [N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] is considered to be a dermal sensitiser.