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]

Endpoint summary

Description of key information

Skin sensitisation (in vitro):
1. Direct Peptide Reactivity Assay (DPRA):The test item solubility was tested in acetonitrile, water, 1:1 (v:v) acetonitrile:water, isopropanol, methanol, ethanol, 1,4-butandiol, N,N-dimethylformamide and tert-butanol. The test item was not soluble in any of these solvents at 100 mM. Acetone and dimethyl sulfoxide were not considered as they influence the degradation of the peptide too much to be valid. Therefore the study was cancelled.

2. ARE-Nrf2 luciferase test assay (KeratinoSens): Negative (OECD 442D/GLP)

3. In vitro human cell line activation test (h-CLAT) assay: Positive (OECD442E/GLP)

The in chemico test (DPRA) was cancelled due to technical infeasibility.The result of in vitro ARE-Nrf2 luciferase test assay ( KeratinoSens) was negative,while the in vitro human cell line activation test (h-CLAT) assay was positive, resulting in no definitve conclusion on the skin sensitisation potential of the substance. As a last resort, an in vivo LLNA (OECD 429) was performed to determine the skin sensitisation potential.

Skin sensitisation (in vivo): Sensitising; Category 1 (OECD 429/GLP)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

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)

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

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

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

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)

Parameter:

SI

Value:

5.6

Test group / Remarks:

12.5%

Remarks on result:

other: ±0.5

Parameter:

SI

Value:

7.3

Test group / Remarks:

25%

Remarks on result:

other: ±2.3

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).

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 July 2020-15 Oct 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)) (migrated information)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: SUQIAN UNITECH CORP., LTD; 190701
- Expiration date of the lot/batch: 24 July 2021
- Purity: 91%

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

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 429 were also performed.

Preparation of the Test Item
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9% NaCl at a final concentration of 500 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical is dissolved or stably dispersed in the chosen solvent and that it does not interfere with the test design. If the test item was not soluble in DMSO or a different organic solvent at 500 mg/mL, the highest soluble concentration was tested by diluting the solution from 500 mg/mL with a constant factor of 2 down to a minimal concentration of 1 mg/mL.
The test item was freshly prepared immediately prior to use. The test item was not soluble in THF at a concentration of 500 mg/mL. Therefore, the test item was dissolved at the highest soluble concentration (250 mg/mL) in THF. Vortex mixing was used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 250 fold with cell culture medium. No precipitation, turbidity or phase separation was observed when diluted 1:250 in cell culture medium. Only in the dose finding assay were 1 precipitates observed at the two highest concentrations. Sonication was used to aid solubilisation. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Controls
A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.

Solvent Controls: Since the test item was solubilized in THF, a THF control served as solvent control for the test item. Since the positive control was solubilized in DMSO, a DMSO control was included and served as solvent control for the positive control. The solvent controls were diluted resulting in a final concentration of 0.2% (v/v) for DMSO and THF.

Positive Control: 2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 μg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted according to the procedure described for the test item in chapter 10.7.1, resulting in a final DMSO concentration of 0.2% (v/v).

Cell line
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 106 cells/mL.

Cells were cultured in 175 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-074) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin; 100 μg/mL streptomycin in a humidified incubator at 37 ± 1°C and 5% CO2.

Dose Groups
1. Medium Control: cell culture medium

2. Solvent Control: 0.2% DMSO (v/v) in cell culture medium; 0.2% THF (v/v) in cell culture medium

3. Positive Control: 4 μg/mL DNCB

4. Test Item: 8 concentrations of the test item
Dose finding assay 1: 500, 250, 125, 62.50, 31.25, 15.63, 7.81, 3.91 μg/mL
Dose finding assay 2 and 3: 20, 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16 μg/mL
Main experiment 1 and 2: 1.36, 1.13, 0.94, 0.79, 0.66, 0.55, 0.46, 0.38 μg/mL

Reactivity Check of the Cells Stock
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. DNCB at a final concentration of 4 μg/mL and nickel sulphate at a final concentration of 100 μg/mL served as positive control while lactic acid at a final concentration of 1000 μg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.

Dose Finding Assay
Starting from 250 mg/mL (dose finding assay 1) and 10 mg/mL (dose finding assay 2 and 3) solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution. The solvent controls and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2. After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 μL FACS buffer. 200 μL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 μg/mL. The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration.
The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation. The CV75 value was used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 Expression (Main tests)
The test item was dissolved using THF as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 500-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.

For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 106 cells/mL. Then, 500 μL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 106 cells/well). The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.

After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 μL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 μg/mL).
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. A total of 10,000 living (PI negative) cells were acquired and based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated. The cell viability was calculated.

Data Analysis
FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 (EC150) and Effective Concentration 200 (EC200) values were performed using the software Microsoft Excel 2010. The mean values and standard deviations of the single replicates were determined using the respective excel commands.

Run / experiment:

other: Experiment 1

Parameter:

other: %RFI (CD54)

Value:

234

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

>200% at 1.36 μg/mL

Run / experiment:

other: Experiment 2

Parameter:

other: %RFI (CD54)

Value:

240

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

>200% at 0.94 μg/mL

Other effects / acceptance of results:

OTHER EFFECTS:
Reactivity Check of the Cell Stock
Doubling time of the cells was monitored and found to be 47.9 h which is within the doubling time range specified by the manufacturer (35 - 55 h). The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86. The cell batch was accepted for further testing.

Dose Finding Assay
The dose finding assay was performed using stock solutions with a concentration of 250 mg/mL (applied concentration 500 μg/mL, dose finding assay 1) and 10 mg/mL (applied concentration 20 μg/mL, dose finding assay 2 and 3). The mean CV75 was derived from three independent runs and was found to be 1.14 ± 0.38 μg/mL. Based on the mean CV75, the main experiment was performed covering a concentration range from 1.36 – 0.38 μg/mL (0.68 – 0.19 mg/mL stock solution).

ACCEPTANCE OF RESULTS: The controls confirmed the validity of the study for all experiments as shown in Table 6. Historical control data generated from 2016 to 2019 was provided (Table 7).

Interpretation of results:

other: WoE classification with OECD 442D and OECD 429 studies.

Conclusions:

In the in vitro human cell line activation test (h-CLAT), 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] did upregulate the cell surface marker CD54 in at least two independent experiment runs.

Executive summary:

In an in vitro skin sensitisation: human Cell Line Activation Test (h-CLAT; OECD 442E/GLP), 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] was evaluated by the ability to induce an increase in cell surface marker expression (CD54, CD86) in THP-1 cells. 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] (in 0.2% THF in RPMI culture medium) was applied to THP-1 cells at concentrations from 0.38, 0.46, 0.55, 0.66, 0.79, 0.94, 1.13 and 1.36 μg/mL for 24 hrs in 2 independent runs. Flow cytometry analysis was carried out on IgG1-FITC, CD86-FITC and CD54-FITC antibody labelled cells. The % Relative Fluorescence Index (%RFI) for CD86 and CD54 expression for each tested concentration was then calculated. The positive control was 2,4-Dinitrochlorobenzene (DNCB).

All acceptance criteria for controls and test item were reached in each run. The study was therefore considered to be valid. Relative cell viability at the highest test item concentration was reduced to 80.6% (CD86), 80.9% (CD54) and 80.2% (isotype IgG1 control) in the first experiment and to 85.22% (CD86), 83.99% (CD54) and 85.02% (isotype IgG1 control) in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 (threshold 200%) was upregulated to 234% at a concentration of 1.36 μg/mL in the first experiment and to 240% - 297% (0.94 μg/mL - 1.36 μg/mL) in the second experiment.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 429 were also performed. The data generated with this test will be considered in the context of integrated approached such as IATA, combining the result with other complementary information from the other 2 tests.

Reference 3

Endpoint:

skin sensitisation: in chemico

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

The test item solubility was tested in acetonitrile, water, 1:1 (v:v) acetonitrile:water, isopropanol, methanol, ethanol, 1,4-butandiol, N,N-dimethylformamide and tert-butanol. The test item was not soluble in any of these solvents at 100 mM. Acetone and dimethyl sulfoxide were not considered as they influence the degradation of the peptide too much to be valid. Therefore the study was stopped and cancelled.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 March 2020- 22 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material::SUQIAN UNITECH CORP., LTD; 190701
- Expiration date of the lot/batch: 24 July 2021
- Purity:91%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability of the test material during storage: stable

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442E and OECD 429 were also performed.

PREPARATION OF TEST SOLUTIONS:
All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in Tetrahydrofuran (THF, Sigma, Lot No.: STBJ0170; Merck, Lot No.: I0984207848). A stock solution of 50 mM (experiment 1) and of 1 mM (experiment 2 and 3) was prepared by pre-weighing the test material into a suitable tube (this was the highest concentration which leads to a soluble pre dilution in medium after a 1:25 dilution). A factor of 1.1 to correct for the purity of the test item was used. Vortex mixing was used to aid solubilisation

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. Since the test item was dissolved in THF, THF was added at a final concentration of 4% (v/v).
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

CONTROLS
A blank, a negative control, a solvent control and a positive control were set up in parallel in order to confirm the validity of the test.
Negative Control: DMSO (AppliChem; Lot No.: 0001446873, 0001755581) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate.
Solvent Control: THF (Sigma, Lot No.: STBJ0170; Merck, Lot No.: I0984207848) at a final concentration of 1% (v/v) in test item exposure medium was used as solvent control. Six wells were included in every testing plate.
Positive Control: Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Alfa Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001446873, 0001755581) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

CELL LINE
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 06 in experiment 1; P 08 in experiment 2; P 04 in experiment 3) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.

DOSE GROUPS
1. Negative/Solvent Controls: 1% (v/v) DMSO in test item exposure medium and 1% (v/v) THF
2. Positive Control: CA: 4 μM, 8 μM, 16 μM; 32 μM; 64 μM
3. Test Item: 12 concentrations of the test item as follows: 500, 250, 125, 62.50, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, 0.24 μM (experiment 1) and 10, 5, 2.50, 1.25, 0.63, 0.31, 0.16, 0.08, 0.04, 0.02, 0.01, 0.00 μM (experiment 2 and 3)

Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per 96-well plate in every independent run.

EXPERIMENTAL PROCEDURE
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase Activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 μL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 μL test item exposure medium. 27 μL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 μL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 over the weekend (experiment 1, 2 and 3). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

- Description on study acceptance criteria
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.
- Other:

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density)
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
Only cells at a low passage number <25 (P 06 in experiment 1; P 08 in experiment 2; P 04 in experiment 3) were used.

Positive control results:

The luciferase activity induction obtained with the positive control, Cinnamic aldehyde, was statistically significant above the threshold of 1.5-fold in at least one concentration (Tables 3 and 4).

The EC1.5 of the positive control was within two standard deviations of the historical mean (23.24 μM and 15.97 μM in experiment 2 and 3, respectively). A dose response was observed and the induction at 64 μM was higher than 2-fold (2.56-fold and 2.63-fold in experiment 2 and 3, respectively).

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

0.91

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EC1.5 [μM] = N/A; IC30 [μM] = 0.24; IC50 [μM]=0.32

Run / experiment:

other: Experiment 3

Parameter:

other: Imax

Value:

0.79

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EC1.5 [μM] = N/A; IC30 [μM] = .28; IC50 [μM]=0.41

Run / experiment:

mean

Parameter:

other: Imax

Value:

0.85

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EC1.5 [μM] = N/A; IC30 [μM] = 0.26; IC50 [μM]=0.37

Other effects / acceptance of results:

OTHER EFFECTS:
- In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range (See Table 2 and Figure 1). Therefore, no EC1.5 value could be calculated. Because in the first experiment toxic effects were seen in all concentrations tested this test cannot be evaluated and two further experiments with an adjusted concentration range were performed

ACCEPTANCE OF RESULTS: The controls confirmed the validity of the study (see Table 7). Historical data from November 2015 and March 2020 was provided (Table 8).

Interpretation of results:

other: WoE classification with OECD 442E and OECD 429 studies.

Conclusions:

In this study under the given conditions the test item ,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],did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs.

Executive summary:

In an in vitro skin sensitisation: ARE-Nrf2 luciferase test method assay (KeratinoSens; OECD 44D/GLP), 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%) was evaluated for its potential to activate the Nrf2 transcription factor in KeratinoSens cells. The test item (in 1% (v/v) THF in DMEM culture medium) was applied to KeratinoSens cells at concentrations from 0.24 to 500 µM (first experiment) and 0.00 – 10 μM (second and third experiment; top dose was limit of solubility) for 48 h at 37°C. Luciferase production was measured by flash luminescence. Cytotoxicity was measured using an MTT assay. The positive control was Cinnamic aldehyde and the negative controls were 1% THF and 1% DMSO.

The controls confirmed the validity of the study. In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the first experiment, toxic effects were seen at all concentrations tested, therefore this test cannot be evaluated and two further experiments with an adjusted concentration range were performed. In the second and third experiments, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated (Imax = 0.85; IC30 [μM] = 0.26; IC50 [μM]=0.37; mean of 2 experiments). No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as a non-sensitiser.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442E and OECD 429 were also performed. The data generated with this test will be considered in the context of integrated approached such as IATA, combining the result with other complementary information from the other 2 tests.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation:

A tiered strategy for skin sensitization assessment was used. OECD 442D, OECD 442E and OECD 429 were performed. The registrant attempted to perform OECD 442C but there were no suitable solvents. The data generated with these tests was considered in the context of an integrated approach, and the final result was decided by combining the results from all tests.  

Skin sensitisation (in vitro):

The registrant attempted to perform OECD 442C. Test item solubility was tested in acetonitrile, water, 1:1 (v:v) acetonitrile:water, isopropanol, methanol, ethanol, 1,4-butandiol, N,N-dimethylformamide and tert-butanol. The test item was not soluble in any of these solvents at 100 mM. Acetone and dimethyl sulfoxide were not considered as they influence the degradation of the peptide too much to be valid. Therefore the study was stopped and cancelled.

Two in vitro skin sensitisation assays are available:  ARE-Nrf2 luciferase test assay (KeratinoSens) and human cell line activation test (h-CLAT).

ARE-Nrf2 luciferase test assay (KeratinoSens)

In an in vitro skin sensitisation: ARE-Nrf2 luciferase test method assay (KeratinoSens; OECD 44D/GLP), 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%) was evaluated for its potential to activate the Nrf2 transcription factor in KeratinoSens cells. The test item (in 1% (v/v) THF in DMEM culture medium) was applied to KeratinoSens cells at concentrations from 0.24 to 500 µM (first experiment) and 0.00 – 10 μM (second and third experiment; top dose was limit of solubility) for 48 h at 37°C. Luciferase production was measured by flash luminescence. Cytotoxicity was measured using an MTT assay. The positive control was Cinnamic aldehyde and the negative controls were 1% THF and 1% DMSO. The controls confirmed the validity of the study. In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the first experiment, toxic effects were seen at all concentrations tested, therefore this test cannot be evaluated and two further experiments with an adjusted concentration range were performed. In the second and third experiments, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated (Imax = 0.85; IC30 [μM] = 0.26; IC50 [μM]=0.37; mean of 2 experiments). No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as a non-sensitiser.

In vitro human cell line activation test (h-CLAT)

In an in vitro skin sensitisation: human Cell Line Activation Test (h-CLAT; OECD 442E/GLP), 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] was evaluated by the ability to induce an increase in cell surface marker expression (CD54, CD86) in THP-1 cells. 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] (in 0.2% THF in RPMI culture medium) was applied to THP-1 cells at concentrations from 0.38, 0.46, 0.55, 0.66, 0.79, 0.94, 1.13 and 1.36 μg/mL for 24 hrs in 2 independent runs. Flow cytometry analysis was carried out on IgG1-FITC, CD86-FITC and CD54-FITC antibody labelled cells. The % Relative Fluorescence Index (%RFI) for CD86 and CD54 expression for each tested concentration was then calculated. The positive control was 2,4-Dinitrochlorobenzene (DNCB). All acceptance criteria for controls and test item were reached in each run. The study was therefore considered to be valid. Relative cell viability at the highest test item concentration was reduced to 80.6% (CD86), 80.9% (CD54) and 80.2% (isotype IgG1 control) in the first experiment and to 85.22% (CD86), 83.99% (CD54) and 85.02% (isotype IgG1 control) in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 (threshold 200%) was upregulated to 234% at a concentration of 1.36 μg/mL in the first experiment and to 240% - 297% (0.94 μg/mL - 1.36 μg/mL) in the second experiment.

Conclusion: The result of in vitro ARE-Nrf2 luciferase test assay ( KeratinoSens) was negative,while the in vitro human cell line activation test (h-CLAT) assay was positive, resulting in no definitve conclusion on the skin sensitisation potential of the substance. As a last resort, an in vivo LLNA (OECD 429) was performed to determine the skin sensitisation potential.

Skin sensitisation (in vivo):

There is one in vivo test (LLNA) available in mice.

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. 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.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information in the dossier, the substance 4,6-dichloro-N-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]​ (CAS No. 83420-16-0) is classified as a skin sensitiser Category 1 when the criteria outlined in Annex I of 1272/2008/EC and Annex I of 286/2011/EC are applied.