Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethyl benzene-1,2,4-tricarboxylate
EC Number:
219-547-3
EC Name:
Trimethyl benzene-1,2,4-tricarboxylate
Cas Number:
2459-10-1
Molecular formula:
C12H12O6
IUPAC Name:
trimethyl benzene-1,2,4-tricarboxylate
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): 1,2,4-Trimethylbenzenetricarboxylate
- Physical state: liquid, viscous, amber
- Analytical purity: 100.00 area-% (233 nm); 99.99 area-% (207 nm)
- Lot/batch No.: 3723340
- Homogeneity: The homogeneity of the test substance is assumed by the analytical laboratory
- Stability under test conditions: the stability of the test substance under storage conditions over the test period was guaranteed until 12 Feb 2015 by the sponsor, and the sponsor holds this responsibility
- Storage condition of test material: room temperature

In vitro test system

Test system:
human skin model
Vehicle:
unchanged (no vehicle)
Details on test system:
Three dimensional human epidermis model:
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Tissue model: Epi-200
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the
extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
irritation test
Value:
99

Any other information on results incl. tables

Test substance

 

tissue 1

tissue 2

tissue 3

mean

SD

NC

mean OD570

2.533

2.719

2.389

2.547

 

viability

[% of NC]

99.4

106.8

93.8

100

6.51

13/0402-1

mean OD570

2.620

2.450

2.531

2.534

 

viability

[% of NC]

102.9

96.2

99.4

99

3.34

PC

mean OD570

0.075

0.070

0.061

0.068

 

viability

[% of NC]

2.9

2.7

2.4

3

0.29

The test substance is not able to reduce MTT directly.

The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 99%.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results and applying the evaluation criteria it was concluded, that 1,2,4-Trimethylbenzenetricarboxylate does not show a skin irritation potential in the EpiDerm™ skin irritation test under the test conditions chosen.