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Diss Factsheets

Administrative data

eye irritation: in vitro / ex vivo
Type of information:
other: read across from similar substance
Adequacy of study:
key study
Study period:
From the 20th to the 20th June, 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
The test was conducted according to an internationally accepted test guideline.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
according to guideline
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Principles of method if other than guideline:
SOP 118 00 850
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate
EC Number:
EC Name:
Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate
Cas Number:
Molecular formula:
Disodium 3-[[ethyl[4-[[4-[(3-sulphonatophenyl)azo]-1-naphthyl]azo]phenyl]amino]methyl]benzenesulphonate
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Tested directly, without dilution or preparation of a solution.

Test animals / tissue source

other: Bos primigenius Taurus
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 Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin).
Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 hour.

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Tissue 1: 182.2 mg
Tissue 2: 198.4 mg
Tissue 3: 183.1 mg
Duration of treatment / exposure:
4 hours
Number of animals or in vitro replicates:
Three reprlicates
Details on study design:
HBSS- solution: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10), added Penicillin 100 IU/mL-Streptomycin 100µg/mL- solution (1%)
Imidazole C3H4N2, CAS-No. 288-32-4, dissolved in HBSS solution, 20% solution (200 g/L)
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.
For each treatment group (negative control solution, test item and positive control solution), three replicates were used.
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. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution resp. positive control solution were applied to each replicate.
Open Chamber Method The “open chamber-method” is used for solid substances. In order to apply the test item, the nut was unscrewed to remove the glass disc. The test item could be applied directly on the cornea now. The following amounts of the test item were tested neat and applied directly on the cornea using a weighing funnel.
The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with test item. Exposure time on the corneas was 4 hours at 32 ± 1 °C. 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. The cMEM without phenol red was then removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 5 mg/mL) was added to the front chamber. The chambers were then closed again and incubated for 90 min at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid at 492 nm.
The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.
The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group
The IVIS of each replicate of the negative control was calculated from the following equation: IVIS = opacity difference + (15 x corrected OD492±2 value)
The IVIS of each replicate of the positive control and of the test item were calculated from the following equation: IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492±2 – mean OD492±2 of the negative control)]
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean. The negative control has to show an IVIS ≤ 3.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
The calculated IVIS (in vitro irritancy score) is 389.21.
Under the conditions of this test, the test item induced serious eye damage on the cornea of the bovine eye.
Executive summary:


An vitro study was performed to assess the corneal damage potential of the substance, according to the OECD Guideline for the Testing of Chemicals, Method No. 437 and the EU Method B. 47 of the Regulation (EU) No. 1152/2010 amending Regulation (EC) No. 440/2008, in GLP.

One valid experiment was performed. Bovine corneas were collected from slaughtered cattle which were between 12 and 60 months old. The test item was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 4 hours at 32 °C.

After removal ofthe test item, opacity and permeability values were measured.


The test item could not be completely rinsed off the cornea. Therefore the opacity meas-urement after removal of the test item was not completely correct. The opacity values were very high and probably false positive due to the remaining test item on the cornea. But the permeability measurement was that high, even if the opacity measurement would have been completely negative, the substance is classified in category I, due to the high permeability of the cornea caused by the test item.

HBSS solution was used as negative control. The negative control showed no irritating effect and the positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean.


Under the conditions of this study, the test item induced serious eye damage on the cornea of the bovine eye.The calculated IVIS (in vitro irritancy score) is 389.21.