Trihexyl benzene-1,2,4-tricarboxylate

Skin irritation / corrosion

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018 February - 2018 April

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, 28 July 2015.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

“In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. 06-Jul-2012.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: L’Oréal Standard Operating Procedure: “EpiSkin™ Skin Irritation Test Method"

Version / remarks:

ECVAM Skin Irritation Validation Study - Validation of the EpiSkin™ Test Method15 min - 42 hours for the Prediction of Acute Skin Irritation of Chemicals. Version 1.8, Feb-2009.

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ECVAM DB-ALM Protocol No. 131

Version / remarks:

“EpiSkin™ Skin Irritation Test15 min – 42 hours. 09-Jun-2012.

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:

The test substance (Batch 0000140180) was provided by the study sponsor, Vertellus Holdings, and stored at room temperature. The test substance appeared as a clear oily liquid with no odour.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS “Category 2”. Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic)
- Tissue batch number(s): 18 EKIN 011
- Date of initiation of testing: 09 February 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Once with DPBS. Excess DPBS was removed by blotting bottom with blotting paper.
- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: Stock: 3 mg/mL MTT (Sigma, Lot No.: MKBZ5197V; VWR, Lot No.: 1017C385) in PBS (Gibco; Lot No.: 1909266). MTT medium: MTT stock solution was diluted 1 + 9 with DMEM-based medium (final concentration 0.3 mg/mL).
- Incubation time: 3 h
- Spectrophotometer: Plate spectrophotometer (acidic isopropanol as blank)
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Mean 11.28%
- Barrier function: 1.9 mg/mL
- Morphology: 7.2%
- Contamination: None
- Reproducibility: Fully reproducible

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : The mixture of 10 µL test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixtures of 10 µL of the test item per 90 µL aqua dest. (distilled water) and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSCliving equalled 0%.
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : 3
- Method of calculation used: NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritating to skin if the viability after 3 minutes exposure is less than or equal to 50% relative to negative control (Irritant (I): UN GHS “Category 2”).
- The test substance is considered to be non-irritating to skin if the viability after 3 minutes exposure is greater than 50% (Non-Irritant (NI): UN GHS “No Category”).
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: N/A

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control (mean)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control (mean)

Value:

5.6

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item (mean)

Value:

115.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was evaluated for skin irritation in vitro by assessing its effect on a reconstituted three-dimensional human epidermis model according to OECD guideline 439. The test substance was observed to be non-irritating at doses up to 10 µL.