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 irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08/11//2019-01/04/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes (incl. QA statement)

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

Details on test material:

light yellow powder

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 COPR., 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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle):
The test item was crushed to a fine powder, using a mortar and a pestle.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: normal human kenatinocytes (NHEK)

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200-SIT
- Tissue batch number(s): 30838 (Appendix 1)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 ± 1 min under the sterile flow at room temperature and for the remaining time of 35 ± 1 min at 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C for 24 ± 2 h.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter: filter band pass of maximum ± 30 nm without reference wavelength

NUMBER OF REPLICATE TISSUES: 3 for test item, negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
To check the non-specific MTT-reducing capability of the test item 25 mg of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator. The mixture did not turn blue/purple. Thus, the additional test with freeze-killed tissues and the quantitative corrections were not necessary.

To check the colouring potential of the test item 25 mg of the test item were mixed per 300 μL aqua dest. and per 300 μL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min. The mixture showed no colouring detected by unaided eye-assessment. Thus, the additional test with viable tissues and the quantitative corrections were not necessary. The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The test substance may be considered as non-irritant to skin if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 μL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL 5% SDS solution

Duration of treatment / exposure:

1 hr (25 ± 1 min under the sterile flow at room temperature and for the remaining time of 35 ± 1 min at 37 ± 1 °C)

Duration of post-treatment incubation (if applicable):

The plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation, the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.

Number of replicates:

3 tissues (test item, negative, positive control)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

101.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 101.4± 1.5

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
- Colour interference with MTT: The mixture of 25 mg of the test item per 300 μl aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.
The test item showed no non-specific reduction of MTT and no relevant colouring potential after mixture with aqua dest. and with isopropanol. Therefore, no additional controls for correction of possible false-negative results were necessary.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8.
- Acceptance criteria met for positive control: The mean relative tissue viability (% negative control) of the positive control was ≤ 20% .
- Acceptance criteria met for variability between replicate measurements: Standard deviation of viability of replicate tissues of all dose groups was ≤ 18%.
- Historical control data from 2015 to 2018 was provided.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation test, 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 not a skin irritant.

Executive summary:

In an in vitro skin irritation assay in the human epidermal model EpiDerm (OECD 439/GLP), normal human keratinocytes (moistened with 25µL DPBS) was exposed to 25 mg of 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%) for 1 hr (25 ± 1 min at room temperature and for the remaining time of 35 ± 1 min at 37 ± 1°C). DPBS was used for the negative control and 5% SDS was used for the positive control. After removal of the test substance via washing, tissues were post-incubated for 42 ± 2hrs. Tissues were then incubated with MTT for 3 hours. The OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

The controls confirmed the validity of the study. The mean OD570 for the negative control treated tissues was between ≥0.8 and ≤ 2.8 (1.271). The mean relative tissue viability of the positive control was ≤ 20% (4%). Standard deviation of viability of replicate tissues of all dose/negative control/positive control groups was ≤ 18% (1.5%/0.3%/0.1%). The test item was not directly MTT reducing and had no relevant colouring potential. The average viability of tissues treated by the test item was 101.4 ±1.5% of the negative control average value i.e. viability was > 50%. According to these results, the test item is not irritating.