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]

Eye irritation

Administrative data

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

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 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)

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 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.

Test animals / tissue source

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.

Test system

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

Results and discussion

In vitro

Results

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.

Applicant's summary and conclusion

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.