Registration Dossier

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:
other: GLP guideline study

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
according to
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
according to
other: OECD Guideline 492 (Reconstucted human Cornea-like Epithelium (RHCE) test method for identifiying chemicals not requiring classificaton and labelling for eye irritation or serious eye damage)
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
- Name of test material (as cited in study report): (R)-3-Ammonium-1-hydroxybutyl (S)-mandelat
- Physical state: solid / white
- Analytical purity: 99.8 %
- Lot/batch No.: 15-0010

Test animals / tissue source

other: in vitro test

Test system

Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): The test-substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a magnetic stirrer. After stirring with a magnetic stirrer the test substance was soluble in the vehicle.
-Form of Application:
BCOP: 750 µL of the 20% (w/v) test-substance preparation
EpiOcular: Using a sharp spoon, a bulk volume of ca. 50 µL of the test material was applied covering the whole tissue surface.
Details on study design:
BCOP Test:
Preparation of the bovine corneas and measurement of initial corneal opacity:
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 Eagle’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 pre-warmed 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 < 544 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups. Each corneal holder was uniquely identified with a number on the chambers.
Application of the test substance and washing:
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 20% (w/v) test-substance preparation (non-surfactant) was applied into the anterior chamber using a pipette. For the control tissues 750 µL of de-ionized water (negative control, NC) or 750 µL of 20% (w/v) solution of imidazole in de-ionized water (positive control, PC), were applied into the anterior chamber using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately
4 hours (non-surfactant solids). The test substance, 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).
Measurement of final corneal opacity:
Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
Determination of permeability:
For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (5 mg/mL for solid test substances) 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. An aliquot was diluted 1:5 in Eagle’s MEM (without phenol red) and measured analogously (PC, only).

EpiOcular Test

Direct MTT reduction:
To assess the ability of the test material to directly reduce MTT a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.
The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.
In case where direct MTT reduction occurred, two freeze-killed control tissues each were treated with the test article and the negative control, in the same way as described in the following section.

Basic procedure:
Several test substances were tested in parallel within the present test (test no. 70) using the same control tissues (NC and PC). Two tissues were treated with each, the test substance, the PC and the NC. There are two separate protocols for liquids and solids, differing in exposure time and post-incubation period. Due to the physical state of the test substance the protocol for solids was applied.

Pre-incubation of the tissues:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the pre-incubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours.

Pretreatment of the tissues:
After the pre-incubation, the tissues were pre-treated with 20 µL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

Application of the test substance:
Using a sharp spoon, a bulk volume of ca. 50 µL of the test material was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 µL of sterile de-ionized water (NC) or with 50 µL of methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed.

Removal of the test substance and postincubation period:
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well pre-warmed medium (post-soak immersion) in order to remove residual test substance.
After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period).

MTT incubation:
After the post-incubation 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 at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Results and discussion

Any other information on results incl. tables

Decision criteria of BCOP



< 1.5

no classification for eye irritation1

1.5 – 4.5


> 4.5; < 45

no prediction can be made for eye irritation, further testing with another suitable method is required2

45 – 65


> 65

ocular corrosive or severe irritant

1According to OECD Guideline 437 (adopted July 2013), this prediction is possible, however, not recommended by the test facility. If the IVIS obtained for the substance tested in this study fell within this range.

2The test method according to the OECD Guideline 437 revised and adopted in 2013 does not allow for the evaluation of eye irritation. I.e., the result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study are needed. 

3The borderline“-evaluation (IVIS3.0 ± 1.5 and55.0± 10.0) was determined statistically using historic BASF data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437.

Decision criteria for evaluation of results

Mean tissue viability
(% of negative control)




55 - 65


> 65


4The „borderline“-evaluation (60 ± 5%) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation provided in OECD Guideline 492.

Applicant's summary and conclusion

Interpretation of results:
Migrated information Criteria used for interpretation of results: EU
Based on the results for BCOP and EpiOcular Test and applying the evaluation criteria described in section ‎3.10, (R)-3-Ammonium-1-hydroxybutyl (S)-mandelat shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.
Executive summary:


The potential of (R)-3 -Ammonium-1-hydroxybutyl (S)-mandelat to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 µL of a 20% test-substance preparation to theepithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation foran exposure period of 4 hours.

In addition to the test substance a negative control (NC; de-ionized water) and a positive control (PC; 20% imidazole in de-ionized water) were applied to three corneas each.

Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.


The potential of (R)-3 -Ammonium-1 -hydroxybutyl (S)-mandelat to cause ocular irritation was assessed by a single topical application of ca. 50 µL bulk volume (about 18 mg) of the undiluted test substance to a reconstructed three dimensional human cornea model (EpiOcular™). Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by an 18-hours post-incubation period.

 Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test-substance treated epidermal tissues is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.  

The following results were obtained in the EpiOcular™ eye irritation assay:  

The test substance is not able to reduce MTT directly.  

The mean viability of the test-substance treated tissues was 3.5%.

Summary of individual test results and test strategy evaluation

Test Method

Test Result

Test Evaluation

Evaluation Test Strategy


The mean IVIS of the test-substance treated corneas was 0.3.

Not identified as corrosive or severe irritant.



Ocular irritant




Mean viability of the test-substance treated tissues was 3.5%.