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Toxicological information

Eye irritation

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Administrative data

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

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethyl benzene-1,2,4-tricarboxylate
EC Number:
EC Name:
Trimethyl benzene-1,2,4-tricarboxylate
Cas Number:
Molecular formula:
1,2,4-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

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Isolated bovine cornea: The test system (target tissue) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months). Schlachthof Bensheim, Germany

Test system

unchanged (no vehicle)
other: Negative control (NC): De-ionized water Positive control (PC): 100% dimethylformamide for liquid test substances and surfactants
Details on study design:
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 530 opacity units2 were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number on the chambers.
Each treatment group (test substance, NC and PC) consisted of 3 corneas.
Before application the medium in the anterior chamber was removed using a syringe.
750 μL of the undiluted liquid, viscous test substance was applied directly to the epithelial surface of the cornea using a pipette (open chamber method).
Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (negative control, NC) or with 750 μL of 100% dimethylformamide (positive control, PC) using a pipette.
The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes. The NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red). Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.
The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.
Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:

Any other information on results incl. tables

Test substance

Mean Opacity Value

Mean Permeability Value

Mean In Vitro Irritancy Score













Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Based on the observed results and applying the evaluation criteria it was concluded, that 1,2,4-Trimethylbenzenetricarboxylate does not cause ocular corrosion or severe irritation in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.