Bis-O-(benzylidene)-D-glucitol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 17 February 2017. Experimental completion date 17 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Bis-O-(benzylidene)-D-glucitol

EC Number:

251-136-4

EC Name:

Bis-O-(benzylidene)-D-glucitol

Cas Number:

32647-67-9

Molecular formula:

C20H22O6

IUPAC Name:

1-{2,6-diphenyl-hexahydro-[1,3]dioxino[5,4-d][1,3]dioxin-4-yl}ethane-1,2-diol

Test material form:

solid: particulate/powder

Details on test material:

Identification: Geniset D
CAS number: 32647-67-9
Appearance/physical state: white powder
Batch: 5434
Purity: 99.4%
Expiry date: 31 October 2018
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

Identification: Geniset D
Batch: 5434
Purity: 99.4%
Appearance: White powder
Expiry Date: 31 October 2018
Storage Conditions: Room temperature

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin). The corneae were isolated on the same day after delivery of the eyes.

Test system

Vehicle:

other: saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was tested as a 20% suspension (w/v) in saline. The test item absorbed the saline completely and formed a pulp, which was spread to match the surface of the corneae.
Due to the suspension’s consistency, it could not be pipetted exactly. Therefore, each approximately 0.75 ml were distributed to the cornea surface.

Volume of app. 0.75 mL of positive and negative control.

Duration of post- treatment incubation (in vitro):

The incubation time lasted 240 minutes.

Number of animals or in vitro replicates:

Triplicate

Details on study design:

Preparation of Corneae:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was
carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were
directly used in the BCOP test on the same day.

Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior
compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea
and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex
position. Care was taken to assure no air bubbles were present within the compartments.

For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).

The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the
negative and positive controls.

Exposure of the Corneae to the Test Groups:
The corneae were distributed as follows:
Groups Number of Corneae
1 Negative Control 3
2 Positive Control 3
3 Test Item 3
The anterior compartment received the test item suspension or negative or positive control at a volume of app. 0.75 mL each on the surface of the corneae. The corneae were incubated in
a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.
Afterwards, the test item or control items, respectively, were rinsed off from the application side with saline, and fresh incubation medium was added into the anterior compartment and
opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined.

Opacity Measurement:
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer
(OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed
in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the test groups and after rinsing the opacity value was determined again (t240).

Permeability Determination:
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the
incubation medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro
Enterprise (version 4.7.1).

IVIS Calculation:
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following Category
according to OECD guideline 437:
IVIS In vitro Irritancy Score (GHS)
≤3 No Category
>3; ≤55 No prediction can be made
>55 Serious eye damage according to CLP/EPA/GHS (cat 1)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

(test item)

Run / experiment:

mean of triplicate runs

Value:

0.82

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.22).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 105.46) corresponding to a
classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

The test item Geniset D was tested as suspension. Relative to the negative control, the test item Geniset D did not cause an increase of the corneal opacity or permeability. The
calculated mean IVIS was 0.82 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437, the test item is not categorized.

Any other information on results incl. tables

Results after 240 minutes incubation time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0	0	0.89	0.081	1.34	1.22	No category
	0		0.083		1.25
	0		0.071		1.07
Positive Control	95.00*		0.077*		96.16	105.46	Category 1
	117.00*		0.059*		117.89
	102.00*		0.023*		102.35
Geniset D	0.00*		-0.005*		0.00**	0.82	No category
	1.00*		0.038*		1.57
	1.00*		-0.002*		0.97

*corrected values

**negative values are set to zero

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Geniset D was tested as suspension. Relative to the negative control, the test item Geniset D did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.82 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437, the test item is not categorized.

In conclusion, according to the current study and under the experimental conditions reported, Geniset D is not categorized (GHS).

Executive summary:

This in vitro study was performed to assess the corneal damage potential of Geniset D by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item Geniset D, the positive and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t₂₄₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item Geniset D did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.82. According to OECD 437 the test item is not categorized (GHS).