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Eye irritation

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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:

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethyl citrate
EC Number:
EC Name:
Trimethyl citrate
Cas Number:
Molecular formula:
1,2,3-trimethyl 2-hydroxypropane-1,2,3-tricarboxylate
Specific details on test material used for the study:
Designation in Test Facility: 17100206G
Date of Receipt: 02. Oct. 2017
Condition at Receipt: Room Temperature, in proper conditions
6.1.1 Specification
The following information concerning identity and composition of the test item was provid-ed by the sponsor.
Name Trimethyl citrate
Batch no. 20170901
Appearance white crystalline powder
Composition Trimethyl citrate
Purity 99.4%
Homogeneity homogeneous
Expiry date 01. Sep. 2019
Storage Room Temperature: (20 ± 5°C)

The following additional information, provided by the sponsor too, was relevant to the
conduct of the study, according to OECD 437:
CAS No. 1587-20-8
EINECS-No. 216-449-2
Chemical Class not stated
Volatility unknown
pH-value not stated
Stability H2O: unknown; EtOH: unknown; Acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility H2O: unknown; EtOH: unknown; Acetone: unknown; CH3CN: unknown; DMSO: unknown

6.1.2 Storage
The test item was stored in the test facility in a closed vessel at room temperature (20 ± 5°C).
6.1.3 Preparation
In a non-GLP pre-test, the solubility of the test item was determined in olive oil and in Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10). The test item was not sufficiently soluble in a concentration of 20% in HBSS or olive oil. Moreover neither a homogeneous nor a pipettable suspension could be prepared.
Therefore, the test item was tested directly, without dilution or preparation of a solution.

Test animals / tissue source

other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The following amounts of the test item were tested neat and applied directly on the cornea using a weighing funnel:
Amounts of Test Item
Replicate Amount
1 500.3 mg
2 501.7 mg
3 498.4 mg
The test item was given on the epithelium so that the cornea was completely covered with test item.
Duration of treatment / exposure:
Exposure time on the corneas was 4 hours at 32 ± 1 °C.
Duration of post- treatment incubation (in vitro):
After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded.
Number of animals or in vitro replicates:
Details on study design:
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, a defined amount of test item and positive control solution were applied to each replicate.
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas.
For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
ca. 4.77
Negative controls validity:
Positive controls validity:
Remarks on result:
not determinable
Other effects / acceptance of results:
In the negative control, no signs of eye irritation were observed.
The positive control induced serious eye damage, which would be classified as GHS category I.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
The test item Trimethyl citrate showed effects on the cornea of the bovine eye. The calcu-lated IVIS (In Vitro Irritancy Score) is 4.77. According to OECD Guideline no. 437 (Oct: 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category.