1,2-Benzenedicarboxylic acid, tridecyl ester, manuf. of, by-products from

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

Human Cornea Model Test (OECD 492)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2019 - 21 March 2019

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)

Test material

Test material information

1

Reference substance name:

1,2-Benzenedicarboxylic acid, tridecyl ester, manuf. of, by-products from

EC Number:

271-080-4

EC Name:

1,2-Benzenedicarboxylic acid, tridecyl ester, manuf. of, by-products from

Cas Number:

68515-39-9

Molecular formula:

none available

IUPAC Name:

1,​2-​Benzenedicarboxylic acid, tridecyl ester, manuf. of, by-​products from

Specific details on test material used for the study:

Identification: C13 Discard Alcohol (MRD-18-893)
CAS No.: 68515-39-9 / 90193-82-1
Batch: 20181013
Purity: 100% based on UVCB
Appearance: Clear liquid
Expiry Date: 13 October 2021
Storage Conditions: At room temperature
Stability in Solvent: Stable in water (not quantified)

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcular™ tissues, Lot No. 27096, were received from MatTek on 19 March 2019 at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2) overnight (about 17 hours).

RECONSTRUCTED HUMAN CORNEAL-LIKE EPITHELIUM (RHCE) TISSUE
- Model used: EpiOcular (MatTek)
- Tissue batch number(s): 27096
- Delivery date: 19 March 2019
- Date of initiation of testing: 20 March 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1.5°C
- Temperature of post-treatment incubation (if applicable): 37±1.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The study substance was removed by extensively rinsing with PBS. Three clean beakers containing a minimum of 100 mL each of PBS were used per test item. Each test item was rinsed 3 times in each beaker.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: plate reader, Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
-The results are acceptable according to MatTek Protocol, if the difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control. For this experiment, the difference of viability between the two relating tissues of a single item were < 20 p.p. in the same run, indicating viable tissues.
The cells used to produce EpiOcular tissues are screened for potential biological contaminants by the manufacturer. Tests were performed by MatTek for each of the potential biological contaminants below resulting in "no detection": HIV-1 virus (oligonucleotide-directed amplification), Hepatitis-B virus (oligonucleotide-directed amplification), Hepatitis-C virus (oligonucleotide-directed amplification), and Bacteria, yeast and other fungi (long-term antibiotic, antimycotic free culture).
Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness are annexed to the report, including quality control data (determined by MatTek Corporation, 82105 Bratislava, Slovakia) of the respective EpiOcular™ lot.
- Viability: 1.793 ± 0.09 (pass)
- Barrier function: 16.36 minutes (pass)
- Contamination: sterile (pass)

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Control used: Freeze-killed tissues
- Procedure used to prepare the killed tissues: Freeze killed tissues were prepared by placing untreated EpiOcular™ constructs (in a 24-well plate) in the -20°C freezer overnight, thawing to room temperature, and then refreezing (two freeze-thaw cycles).
- No. of replicates: 2
- Method of calculation used: Corrected mean viability = Tissue viabilitytest item −(Tissue viabilityKC test item− Tissue viabilityKC negative control)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
DECISION CRITERIA
- The study substance is considered to be non-irritating (GHS No Category) to eye if the viability
following treatment is greater than 60%
- If the study substance-treated tissue viability is less than or equal to 60% relative to negative control treated tissue viability, no prediction can be made from this result in isolation and requires additional information for classification purposes.
- A single test composed of at least two tissue replicates should be sufficient for a test chemical, when the result is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent tissue viability equal to 60±5%, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 μL (83.3 μL/cm2 according to guideline) of the study substance was dispensed directly atop duplicate EpiOcular™ tissue

POSITIVE CONTROL
- Amount applied: 50 μL
- Concentration: as supplied

NEGATIVE CONTROL
- Amount applied: 50 μL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

2

Details on study design:

Assessment of Colored Substance:
Since the test item was non-coloured, additional tests had to be performed to assess if it becomes coloured after contact with water or isopropanol.
For this purpose, 50 μL of the test item were added to 2.0 mL of isopropanol and shaken for 3 hours at room temperature. 2.0 mL of isopropanol were used as control. Additionally 50 μL of the test item were added to 1.0 mL of deionised water and incubated at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for 1 hour. 1 mL of deionised water was used as control.
According to guideline the absorbance of all samples (200 μL aliquots) were measured in duplicates at 570 nm (OD570) with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.
Since the OD of the test item at 570 nm, after subtraction of the OD for isopropanol or water was < 0.08 (which corresponds to approximately 5% of the mean OD of the negative control) the test item was considered as not interacting with the MTT measurement and an additional test on additional viable tissues (without MTT addition) were not necessary.

Assessment of Direct MTT Reduction by the Test Item:
Test items may have the ability to directly reduce MTT, which could have an impact on the quantitative MTT measurement. For this purpose approximately 50 μl of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for 3 hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was performed concurrently. Since the MTT solution colour turned purple, the test item proved to reduce MTT. A functional check using freeze-killed tissue controls (killed controls = KC) had to be performed in at least one definitive assay to evaluate, whether the test material was not binding to the tissue and leading to a false MTT reduction signal.

Cell Viability Measurement:
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Since the test item was colourless inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2 mL isopropanol in each well so that isopropanol was flowing into the insert. The plates were sealed with parafilm and a standard plate sealer, and were stored 4-72 h at 2-8 °C in the dark. To extract the MTT, the tissues were pierced and the plates were placed on an orbital plate shaker and shaken for 2.5 hours at room temperature. The corresponding negative, positive, and additional viable tissues (without MTT addition) were treated identically with piercing.
The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Results and discussion

In vitro

Results

Irritation parameter:

other: Mean Tissue Viability after Data Correction (%)

Value:

100.45

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The OD of the tissues treated with the negative control was > 0.8 and < 2.5 (2.136 and 2.264).

Positive controls validity:

valid

Remarks:

The tissue viability of the positive control was below 50% of the negative control viability (32.57%).

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Non irritating to the eye according to EU CLP and UN GHS

Conclusions:

Irritating effects were not observed following incubation with the study substance. The study substance does not need to be classified according to UN GHS.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of the study substance by means of the Human Cornea Model Test.

The study substance proved to be an MTT reducer in the MTT pre-test. Therefore, additional tests with freeze-killed tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the normal tests.

Each 50 μL of the study substance, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 30 minutes.

The mean OD of the tissue replicates treated with the negative control was > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of relative viability between the two relating tissues was < 20% in the same run (for study substance tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with the study substance. Compared with the value of the negative control, the mean absorption value corresponding to the tissue viability did not decrease below 60% (determined value for the study substance: 100.45%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the study substance does not need to be classified according UN GHS.