bis(2,2,6,6-tetramethyl-1-(phenylthio)piperidin-4-yl)carbonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-28 to 2019-08-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI- National Institute of Pharmacy and Nutrition (22.05.2019)

Test material

Test material information

Constituent 1

Reference substance name:

bis[2,2,6,6-tetramethyl-1-(phenylsulfanyl)piperidin-4-yl] carbonate

EC Number:

827-277-8

Cas Number:

1771689-37-2

Molecular formula:

C31H44N2O3S2

IUPAC Name:

bis[2,2,6,6-tetramethyl-1-(phenylsulfanyl)piperidin-4-yl] carbonate

Test material form:

solid: particulate/powder

Remarks:

White powder

Test animals / tissue source

Species:

human

Strain:

other: Keratinocyte strain 4F1188

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcular™ (OCL-200-EIT) kits are manufactured according to defined quality assurance procedures. All biological components of the EpiOcular™ tissue and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with Triton X-100 (100 µL of 0.3% (v/v) Triton X-100). A certificate of quality as provided by the supplier is available.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The test item was applied in its original form (approximately 50 mg on top of 0.6 cm² tissue; approx. 83.3 mg/cm²), no formulation was required.

Duration of treatment / exposure:

6 h (± 15 min)

Duration of post- treatment incubation (in vitro):

18 h (± 15 min)

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used : The three-dimensional RhCE tissue construct is produced using primary human epidermal keratinocytes i.e., EpiOcular™ OCL-200. The EpiOcular™ OCL-200 RhCE tissues construct is similar to the in vivo corneal epithelium three-dimensional structure. In this assay, the test item is applied to the surface of the cornea epithelial construct for a fixed period, removed, and the tissue allowed to express the resulting damage. Liquids and solids are treated with different exposure and post-exposure incubations. Two construct tissues are used for each treatment and control group. Relative tissue viability is determined against the negative control-treated constructs by the reduction of the vital dye MTT (3-[4,5 -dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide). A concurrent positive control is used with each assay.
- RhCE tissue construct used, including batch number :
EpiOcular™ (OCL-200-EIT)
Supplier: MatTek In Vitro Life Science Laboratories Mlynske Nivy 73, Bratislava, Slovakia
Lot No.: 30608
Expiry date: 30 May 2019
- Doses of test chemical and control substances used :
test item: 50 mg on top of 0.6 cm² tissue (approx. 83.3 mg/cm²)
positive nd negative control: 50 µg on top of 0.6 cm² tissue
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
exposure: 6 h (± 15 min) (37±2°C in an incubator with 5±1% CO2, >95% humidified atmosphere)
post-exposure immersion incubation: 25 ± 2 min at room temperature
post-exposure incubation: 18 hours ± 15 minutes (37±2°C in an incubator with 5±1% CO2, >95% humidified atmosphere)
- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570 nm ±10nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 - 3.1752
- Description of the method used to quantify MTT formazan : The precipitated formazan was quantified spectrophotometrically
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : according to guideline
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : historical OD negative control (range 1.138 - 2.464; mean 1.811); historical OD positive control (range 0.064 - 0.544; mean 0.261)
- Complete supporting information for the specific RhCE tissue construct used :
Biological contaminants: no biological contaminants were detected (HIV-1 virus, Hepatitis B virus, Hepatitis C virus, bacteria, yeast and other fungi)
Tissue viability (MTT QC assay, 1 h, n=3): OD (540-570 nm) = 1.296 ± 0.038
Barrier function (ET-50 assay, 100 µL 0.3 % Triton X-100, 3 time-points, n=2, MTT assay): ET-50 = 23.66 min
- Reference to historical data of the RhCE tissue construct
Tissue viability (MTT QC assay, 1 h, n=3): OD (540-570 nm) = 1.1 - 3.0 -> pass
Barrier function (ET-50 assay, 100 µL 0.3 % Triton X-100, 3 time-points, n=2, MTT assay): ET-50 = 12.2 - 37.5 min -> pass
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.
- Positive and negative control means and acceptance ranges based on historical data :
test positive control (mean OD: 0.233 - mean viability: 11 %), historical OD positive control (range 0.064 - 0.544; mean 0.261)
test negative control (mean OD: 2.050 - mean viability 100 %), historical OD negative control (range 1.138 - 2.464; mean 1.811)
- Acceptable variability between tissue replicates for positive and negative controls : < 20%
- Acceptable variability between tissue replicates for the test chemical: < 20%

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: tissue viability

Run / experiment:

mean [% ]

Value:

90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: tissue viability

Run / experiment:

replicate 1 [%]

Value:

92

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

other: tissue viability

Run / experiment:

replicate 2 [%]

Value:

87

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study using the fifteen Proficiency Chemicals according to OECD Test Guideline No. 492.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, acceptable OD 0.8 - 2.5 (test mean OD of negative control = 2.050)
- Acceptance criteria met for positive control: yes, acceptable percentage viability for positive control < 50 % (test mean viability of positive control: 11 %)
- Range of historical values if different from the ones specified in the test guideline:
historical OD positive control (range 0.064 - 0.544; mean 0.261) - historical viability positive control (range 3 - 41 %; mean 15 %)
historical OD negative control (range 1.138 - 2.464; mean 1.811)

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is, thus, considered as non-irritant to eye (UN GHS No Category).

Executive summary:

The acute ocular irritation potential of the test item was determined in the three-dimensional RhCE tissue in the EpiOcular™ model in vitro according to OECD Guideline 492 following GLP.

Before treatment the tissues were pre-wetted with approximately 20 µL of Ca⁺⁺Mg⁺⁺Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with test item and incubated for 6 hours (± 15 min) at standard culture conditions (37 ± 2°C in an incubator with 5 ± 1% CO₂, >95% humidified atmosphere). Exposure of test material was terminated by rinsing with Ca⁺⁺Mg⁺⁺Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37 ± 2°C in an incubator with 5 ± 1% CO₂, >95% humidified atmosphere and protected from light. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 90 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to eye. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The results obtained from this in vitro eye irritation test, using the EpiOcular™ model, indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, the test item is, thus, considered as non-irritant to eye (UN GHS No Category).