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 irritation/corrosion: Not irritating (OECD439/GLP)

Serious eye damage/eye irritation: Category 1 (OECD437/GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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.

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)

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31st March 2020 - 14th May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

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: : July 24, 2021
- Purity:91%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At receipt, the test item was stored, in its original packaging, in premises fitted out to that effect, at room temperature and away from light.Stable under the storage and test conditions.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Calves eyes were collected in the slaughterhouse of Sobeval Boulazac 24759 - France

- Characteristics of donor animals (e.g. age, sex, weight): Corneas of calves aged less than 8 months.

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported in a Hanks’s buffered saline solution with antibiotic to the laboratory.

- Selection and preparation of corneas:For each selected eye, an incision with a scalpel was practiced at the level of the scleral ring by means of scissors.

- Quality check of the isolated corneas: At reception, the eyes were carefully examined under lighting and these showing a visible (scratches, pigmentation, neo-vascularization) defect were eliminated.

Vehicle:

other: paraffin oil (validated solvent)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 20%

Test item appearance after dilution: thick granular yellowish (liquid which then became a paste). The dilution of the test item was tested at room temperature and was mixed by vortex just before contact with the cornea, in order to aid the solubilisation process.

VEHICLE: Paraffin oil (Sigma, CAS: 8012-95-1; Batch: BCBT6467)

Duration of treatment / exposure:

4 hours.

Number of animals or in vitro replicates:

3 corneas per group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas were deposited, endothelial side upwards, on the posterior part of cornea holders. Then the anterior part was firmly clamped in place with 3 screws. The anterior (epithelial side) and posterior (endothelial side) compartments were then filled (posterior chamber first), with pre-warmed Eagle’s Minimum Essential Medium (EMEM) without phenol, with a pipette, taking care to eliminate air bubbles. The watertightness was insured by toric caps.

As soon as the corneas were mounted, the holders were maintained at 32 ± 1°C for 1 hour (pre-incubation) in a water bath, in horizontal position, immersed at the three-quarters of their height. After pre-incubation, compartments were emptied with the specific sucking up system and fresh EMEM pre-warmed at 32 ± 1°C was added in both compartments.

QUALITY CHECK OF THE ISOLATED CORNEAS
The opacity at t=0 (OPT0) was then determined. Corneas showing a value of opacity greater than seven opacity units were discarded. 3 corneas were selected as negative control. The remaining corneas were then distributed into treatment and positive control groups.

NUMBER OF REPLICATES：3

NEGATIVE CONTROL USED：Paraffin oil

POSITIVE CONTROL USED：Imidazole 20% in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME：The test item was tested diluted at 20 % with paraffin oil. The exposure time was 4 hours in a bain-marie (32 ± 1°C).

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: No.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least three times.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity was determined by the amount of light transmission thought the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (OP-KIT), resulting in opacity values measured on a scale. The opacitometer was calibrated and the opacity of each cornea was measured at 2 times, just before treatment with the test item (measurement called OPT0) and immediately after the end of the exposure period. (measurement called OPT2).
- Corneal permeability: The permeability was determined by the amount of sodium fluorescein solution (1 ml of a sodium fluorescein solution 5 mg/mL at 32 ±1°C for 90 ± 5 mins) that penetrated all corneal cell layers. The Optic Density (OD) of the different media was then measured with a spectrophotometer (Labsystems) at 490 nm (software VISION liteTM version 2.2). For each cornea a value was obtained and therefore 3 values for the control and 3 values for the test item.
- Others: The aspect of the cornea was observed and visible modifications of the cornea were noted.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
An In Vitro Irritancy Score (IVIS) was then calculated with the mean adjusted values according to the formula:
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA: As per guideline

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item

Value:

24.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: 24.4 ± 10.3

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The aspect of the cornea was observed and visible modifications of the cornea were noted: High epithelium detachment (test item); Oedema and High epithelium detachment (positive control).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The test was considered acceptable if the negative control gives the opacity OP < 8 and the optical density OD < 0.053. They were respectively: OP=1.0 and OD= 0.008.
- Acceptance criteria met for positive control: The test was considered acceptable if the positive control gives an IVIS that fall within two standard deviations of the current historical mean (87.3< IVIS < 148.3). It was 123.3 ± 38.6.
Historical database: The historical (10 years of data) averages and difference were calculated every time an acceptable test was realized.

Interpretation of results:

other: As there is evidence of epithelial detachment, the substance will be classified as Category 1.

Conclusions:

In the in vitro BCOP assay, no prediction can be made for the classification of the test item N4, N4’hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetrametylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] concerning eye irritation or serious eye damage.

Executive summary:

In the in vitro eye irritation Bovine Corneal Opacity and Permeability (BCOP) assay (OECD 437/GLP), isolated bovine corneas were exposed to N4, N4’hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetrametylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] (91%) in a paste (20 % in paraffin oil) for 4 hours using the open-chamber method. Paraffin oil was used for the negative control and 20% imidazole in 0.9% sodium chloride was used for the positive control. The corneas were rinsed and then the opacity and permeability of each cornea was recorded.

The positive control gave the appropriate response (IVIS = 123.3). High epithelial detachment was noted in test item-treated corneas. The mean opacity value for the test substance was 22.7±10.1. The mean permeability OD490 for the test substance was 0.116±0.068. The IVIS for the test substance was 24.4±10.3. The IVIS for N4, N4’hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetrametylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] is > 3 and simultaneously ≤ 55, therefore the classification according to CLP classification criteria for eye irritation or serious eye damage is: no prediction can be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

There is one in vitro skin irritation study available.

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.

Eye irritation

There is one in vitro eye irritation study available.

In the in vitro eye irritation Bovine Corneal Opacity and Permeability (BCOP) assay (OECD 437/GLP), isolated bovine corneas were exposed to N4, N4’hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetrametylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] (91%) in a paste (20 % in paraffin oil) for 4 hours using the open-chamber method. Paraffin oil was used for the negative control and 20% imidazole in 0.9% sodium chloride was used for the positive control. The corneas were rinsed and then the opacity and permeability of each cornea was recorded. The positive control gave the appropriate response (IVIS = 123.3). High epithelial detachment was noted in test item-treated corneas. The mean opacity value for the test substance was 22.7±10.1. The mean permeability OD490 for the test substance was 0.116±0.068. The IVIS for the test substance was 24.4±10.3. The IVIS for N4, N4’hexane-1,6-diylbis[N-butyl-6-chloro-N,N’-bis(2,2,6,6-tetrametylpiperidin-4-yl)-1,3,5-triazine-2,4-diamine] is > 3 and simultaneously ≤ 55, therefore the classification according to CLP classification criteria for eye irritation or serious eye damage is: no prediction can be made. However, as there is evidence of epithelial detachment, the substance will be classified as Category 1.

Justification for classification or non-classification

Based on the available information in the dossier, the substance 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) does not need to be classified for skin irritation/corrosion when considering the criteria outlined in the CLP Regulation (Annex I of 1272/2008/EC).

 

Based on the available information in the dossier, the substance 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 Category 1 for serious eye damage/eye irritation when considering the criteria outlined in the CLP Regulation (Annex I of 1272/2008/EC).