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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 23, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD test Guideline No. 437 without any deviation.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
other: Validated draft study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on July 13-16, 2015 / signed on September 14, 2015

Test material

Constituent 1
Chemical structure
Reference substance name:
Diethyl tartrate
EC Number:
201-783-3
EC Name:
Diethyl tartrate
Cas Number:
87-91-2
Molecular formula:
C8H14O6
IUPAC Name:
diethyl tartrate
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Tartrate diethyle
- Physical state: Incolor liquid
- Storage condition of test material: Stored at room temperature, in the dark

Test animals / tissue source

Species:
other: Bovine eye
Details on test animals or tissues and environmental conditions:
Test System: Freshly isolated bovine cornea (at least 9 to 60 month old donor cattle)
Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany.

Collection of Bovine Eyes: Freshly isolated bovine eyes of at least 9 month old donor cattle (at least 9 month to 60 month) were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in HBSS at cooled temperature. The corneae were isolated on the same day after delivery of the eyes. The corneae were directly used in the BCOP test on the same day.
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.

Incubation Medium: The incubation medium consisted of MEM, supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin (500 units penicillin, and 500 µg streptomycin per 5mL) per 500 mL medium. Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS).
The OECD recommends the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control: 0.9 % w/v Sodium chloride. Positive control: 2-Ethoxyethanol (purity: 99%)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of test item was applied on each cornea.
- Concentration (if solution): The test item was tested undiluted.
Duration of treatment / exposure:
Test item was applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C, then followed by an incubation period of 120 minutes at 32 ± 1 °C in a vertical position.
Observation period (in vivo):
- Corneal opacity was measured pre-treatment, post-treatment and post-incubation (after 120 minutes of incubation).
- 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.
Number of animals or in vitro replicates:
3 corneas per treatment group (undiluted test item, negative control and positive control)
Details on study design:
EXPERIMENTAL PERFORMANCE:
Preparation of cornea:
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 (O-ring) 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 corneae with a value of the basal opacity > 7 was discarded. Each cornea was examined for defects, such as lacerations, scratches or wrinkling. Corneas with any observed defects were discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

Treatment and incubation:
The anterior compartment received the test item or negative or positive control at a volume of 0.75 mL 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 ten minutes.
After the test item or control items, respectively, were rinsed off from the application side with saline, the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). The opacity measurement is described in chapter 3.7.
In the second step of the assay, permeability of the corneae 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, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).

Epithelium Condition:
Also, after the 2-hour post-exposure incubation period each cornea was observed visually and pertinent observations recorded (e.g., tissue peeling, residual test item, non-uniform opacity patterns). These observations are important as they may be reflected by variations in the opacitometer readings.

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 and from the posterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution (Sigma-Aldrich) in HBSS (anterior compartment) and by fresh cMEM (posterior compartment). 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 was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

CONTROLS:
Negative Control: Saline (0.9% NaCl in deionised water, Envigo CRS GmbH) was included as negative control in each experiment in order to detect non-specific changes in the test system, as well as to provide a baseline for the assay endpoints.
Positive Control: A known ocular irritant was included in each experiment to verify that an appropriate response is induced. 2-Ethoxyethanol (purity: 99%, Sigma-Aldrich) served as positive control.

Results and discussion

In vivo

Results
Irritation parameter:
other: In Vitro Irritancy Score (IVIS)
Basis:
mean
Remarks:
3 corneas
Time point:
other: 120 minutes
Score:
45.04
Irritant / corrosive response data:
Relative to the negative control, the test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 45.04.
Negative control:The opacity mean change value was 0.33 and the permeability mean of the negative control was 0.067. The negative control acceptance criterion were therefore satisfied.
Positive control: IVIS = 77.72. The positive control IVIS was within the range of 56.25 to 97.96. The positive control acceptance criterion was therefore satisfied.
Other effects:
Visual observation: Shortly after application of the test item the corneae became murky.

Any other information on results incl. tables

Table 7.3.2/1: Summary of results


Test Group

Opacity value = Difference (t130-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

Individual values

 

Mean

Individual values

Mean

Negative Control

0

0.33

(SD: 0.58)

0.064

0.067

(SD: 0.010)

0.96

1.34

Not categorized

1

0.059

1.89

0

0.078

1.17

Positive Control

58.67*

1.586*

82.46

77.72

Category 1

54.67*

1.680*

79.87

53.67*

1.145*

70.84

Test Item

31.67*

0.678*

41.84

45.04

No hazard prediction can be made

30.67*

1.062*

46.60

22.67*

1.601*

46.68

 

*corrected values

 

Visual observation: Shortly after application of the test item the corneae became murky.

Negative control: With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase ofopacity nor permeability of the corneae could be observed (mean IVIS=1.34).

Positive control:The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS =77.72) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Applicant's summary and conclusion

Interpretation of results:
other: No prediction can be made based on the decision criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions reported, the calculated mean in vitro irritancy score was 45.04 for the substance Tartrate diethyle and therefore no prediction for ocular irritation can be made as the result is outside the decision criteria (i.e. ≤3 or >55) according to the EU CLP.
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to assess the corneal damage potential of Tartrate diethyle by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 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. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130). 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 (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS=1.34). The opacity mean change value was 0.33, which was equal to the maximum acceptance value of 0.33. The permeability mean of the negative control was 0.067, which was below the the maximum acceptance value of 0.091.

The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The mean IVIS was of 77.72, within the historical range (56.25-97.96) for the 30 assays performed from February 2015 to September 2015.

Relative to the negative control, the test item Tartrate diethyle caused an increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 45.04.

 

Under the experimental conditions reported, the calculated mean in vitro irritancy score was 45.04 for the substance Tartrate diethyle and therefore no prediction for ocular irritation can be made as the result is outside the decision criteria (i.e. ≤3 or >55) according to the EU CLP.