Trihexyl benzene-1,2,4-tricarboxylate

Eye irritation

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Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018 January 25 - 2018 May 31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT)

Version / remarks:

For the prediction of acute ocular irritation of chemicals For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Clear oily liquid, molecular weight 462.61, relative density 1.025

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Vertellus, 0000140180
- Expiration date of the lot/batch: 15 June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: assumed to be stable

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinised surface, showing a cornea-like structure analogous to that found in vivo.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

6 hr

Duration of post- treatment incubation (in vitro):

18 hr

Number of animals or in vitro replicates:

2/dose

Details on study design:

- Details of the test procedure used : RhCE tissue was treated with test substance for 6 hrs. After an 18 hr post-treatment incubation, tissue samples were evaluated for viability using an MTT reduction assay.
- RhCE tissue construct used, including batch number : EpiOcular™ tissues on agarose (Lot No.: 27019 for NSMTT control, 27039 for main experiment)
- Doses of test chemical and control substances used : Negative control: 50 µL. Positive control: 50 µL. Test item: 50 mg. The test item was ground into a fine powder using a mortar and pestle. Approximately 50 mg (83.3 mg/cm^2) of the test item were applied directly to the EpiOcular TM tissue using an application spoon, avoiding compression of the test item. The test item was spread to match size of the tissue.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : Exposure: 6 hr, 37 °C. Post-exposure immersion: 25 min, room temperature. Post-exposure incubation: 18 hr, 37 °C.
- Justification for the use of a different negative control than ultrapure H2O (if applicable) : N/A.
- Justification for the use of a different positive control than neat methyl acetate (if applicable) : N/A
- Description of any modifications to the test procedure : N/A
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : The mixture of 50 mg test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : 570
- Description of the method used to quantify MTT formazan : Absorbance (OD570)
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable) : N/A
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities obtained after treatment compared to the negative control tissues concurrently treated with Aqua dest. (distilled water). The test item is considered to be irritant to the eye but it cannot be differentiated between UN GHS “Category 1” or “Category 2”, if the relative tissue viability is less or equal to 60%. The test item is considered to be non-irritant in accordance with UN GHS “No Category” if relative tissue viability is higher than 60%.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : Yes
- Complete supporting information for the specific RhCE tissue construct used : Yes
- Reference to historical data of the RhCE tissue construct: Yes
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : Yes
- Positive and negative control means and acceptance ranges based on historical data : Yes
- Acceptable variability between tissue replicates for positive and negative controls: Yes
- Acceptable variability between tissue replicates for the test chemical: Yes

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Tissue Viability [%]

Run / experiment:

Negative Control

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other: Tissue Viability [%]

Run / experiment:

Positive Control

Value:

20.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

other: Tissue Viability [%]

Remarks:

NSMTT corrected

Run / experiment:

Test item

Value:

98.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Observed values in range of historical values

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was evaluated for eye irritation potential according to OECD Guideline 492. In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”.