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Description of key information

Skin irritation - Not irritant

Eye irritation - Not irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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 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.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
irritation test
Value:
99

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%.

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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye 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
Qualifier:
according to guideline
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
Vehicle:
unchanged (no vehicle)
Controls:
yes
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.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
0

Test substance

Mean Opacity Value

Mean Permeability Value

Mean In Vitro Irritancy Score

13/0402-1

0.0

0.002

0.0

NC

1.8

0.000

1.8

PC

103.3

2.125

135.2

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 cause ocular corrosion or severe irritation in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin: Based on the results of the available data the substance does not require to be classified and labelled according to Regulation 1272/2008/EC (CLP) or Directive 67/548/EEC (DSD).

Eye: Based on the results of the available data the substance does not require to be classified and labelled according to Regulation 1272/2008/EC (CLP) or Directive 67/548/EEC (DSD).