Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Principles of method if other than guideline:

In vitro eye irritation test using the SkinEthic reconstructed Human Corneal Epithelium (HCE) model

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Test material

Test material information

Constituent 1

Reference substance name:

Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

EC Number:

260-257-1

EC Name:

Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

Cas Number:

56554-53-1

Molecular formula:

C30H56O6

IUPAC Name:

1,3-bis[(3,5,5-trimethylhexanoyl)oxy]propan-2-yl 3,5,5-trimethylhexanoate

Test animals / tissue source

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

TEST TISSUE
- Test model: SkinEthic reconstructed Human Corneal Epithetial model (SkinEthic HCE)
- Source: HCE, SkinEthic Laboratories, Nice, France
- Description: Transformed human corneal epithelial cells of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. The test item is applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.
- Age of tissue at study initiation: 6 days
- Tissue storage/maintenance: Upon arrival, tissues were stored at room temperature prior to transferring into 24-well plates designated “arrival plates” containing 300 µl of maintenance medium. It was ensured that there were no air bubbles present under the tissue inserts.

INCUBATION CONDITIONS
- Temperature (°C): 37
- CO2 (%): 5

PREPARATION OF TISSUE
Prior to treatment, 7-day old tissues were transferred from the “arrival plates” to 6-well plates designated “treatment plates” (for test item, negative and positive controls) containing 1 mL maintenance medium at room temperature.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: yes, negative control solution (Solution A)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 30 µL

NEGATIVE CONTROL
- Identity: Solution A (0.142 g/L Na2HPO4; 1.802 g/L Glucose; 7.149 g/L HEPES; 0.224 g/L KCl; 7.597 g/L NaCl)
- Concentration: as such
- Amount(s) applied in the test: 30 µL

POSITIVE CONTROL SUBSTANCE
- Identity: Sodium Dodecyl Sulphate (SDS)
- Concentration: 2% (w/v)
- Amount(s) applied in the test: 30 µL

Duration of treatment / exposure:

10 min at 37°C

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

3 tissues

Details on study design:

PRE-TEST
ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT
The MTT Assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
The possible interference of the test item with the MTT assay by its potential ability to directly reduce MTT was tested prior to the main test. 30 µL of the test item was added to 1 mL of a 0.5 mg/mL MTT solution and incubated at 37 °C, 5% CO2 in air for 3 h. Untreated MTT solution was used as a control. If the MTT solution turned blue, the test item was presumed to have reduced MTT.

MAIN TEST
POST-EXPOSURE TREATMENT
- Removal of the test item: At the end of the exposure period, tissue inserts were removed from the wells and rinsed with Dulbecco’s Phosphate Buffered Saline without Ca++ and Mg++.
- Post-exposure incubation conditions: Tissues were placed into 24-well plates designated “holding plates” containing 300 µL of maintenance medium (at room temperature) until all tissues had been rinsed.

MTT ASSAY
- Loading: Following rinsing, tissues were transferred to 24-well plates (“MTT Loading plates”), each well containing 300 µL of a 0.5 mg/mL MTT solution freshly prepared in maintenance medium.
- Incubation conditions: 3 h at 37 °C, 5% CO2 in air
- Extraction: At the end of the incubation period, tissues were rinsed twice with phosphate buffered saline, blotted on absorbent paper to remove residual MTT and transferred to 24-well plates (“MTT extraction plates”) containing 750 µL isopropanol per well. Further 750 µL isopropanol were added to each well and plates were sealed to prevent evaporation.
- Extraction conditions: formazan crystals were extracted from the tissues overnight at room temperature protected from light.
- Optical density measurement: At the end of the extraction period, triplicate 200 µL aliquots of the extraction solutions were transferred to a 96-well plate. The optical density was measured at 540 nm (OD540) using the Anthos 2001 microplate reader for determination of tissue viability. Isopropanol served as blank.

TISSUE HISTOLOGY
One tissue per treatment group (test item, negative and positive controls) was retained for possible tissue histopathology.
The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a 1.5 mL Eppendorf tube containing 1 mL of 10% formalin and stored at room temperature.

INTERPRETATION OF RESULTS
- Tissue viability: The mean OD540 values of the duplicate tissues were calculated. Each of these values had already been corrected for blanks by the microplate reader. The relative mean tissue viability (percentage of the negative control) was calculated as follows:

Relative mean tissue viability (%) = [(mean OD540 of test item)/(mean OD540 of negative control)] x 100

SCORING SYSTEM
The mean tissue viability for the test item was compared to the negative control and classified according to the following criteria:

Irritant (I): If the relative mean tissue viability (percentage of negative control) was < 60.
Non-Irritant (NI): If the relative mean tissue viability (percentage of negative control) was ≥ 60.

ASSAY ACCEPTANCE CRITERION
The results of the assay were considered acceptable if the following assay acceptance criterion was achieved:
- Assay acceptance criterion: Positive control
The assay meets the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is < 60% relative to the negative control treated tissues.

Results and discussion

In vitro

Results

Irritation parameter:

other: Relative Mean Viability (%)

Value:

92.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The individual and mean OD540 values and mean viabilities for each treatment group are given in Table 1.

The relative mean viability of the test item treated tissues after a 10 min exposure period was 92.6%

It was considered unnecessary to proceed with tissue histopathology.

The MTT solution containing the test item remained yellow which indicated that the test item did not directly reduce MTT.

Table 1. Assessment of eye irritation potential – Viability of HCE tissues

Item	OD540 of individual tissue	Mean OD540	Relative Mean Viability (%)
Negative control	0.812	0.838	100*
	0.863
Positive control	0.152	0.111	13.2
	0.069
Test item	0.779	0.776	92.6
	0,773

* The mean viability of the negative control tissues was set at 100%

 

Assay Acceptance Criterion

The relative mean viability of the positive control treated tissues after a 10 min exposure period was 13.2%. Thus, the quality criterion required for the acceptance of results in the test was satisfied.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.