Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted on 07 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
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:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
Identification: Diammonium phthalate
Batch: 7172002
Purity: 99.3%
Appearance: White crystalline powder
Expiry Date: August 2017
Storage Conditions: At room temperature, protected from moisture
Stability in Solvent: Not indicated by the Sponsor

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Rationale: OECD 437
Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Collection of Bovine 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 supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes.

Test system

Vehicle:
other: The test item was tested as a 20% solution (w/v) in saline.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% solution (w/v) in saline

VEHICLE Saline
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9% NaCl in deionised water


POSITIVE CONTROL Benzalkonium chloride
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 10% (w/v)
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
For permeability determination corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS.
Number of animals or in vitro replicates:
3 for test item, positive and negative control
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
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 (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 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.

NUMBER OF REPLICATES
3

Exposure of the Corneae to the Test Groups:
The anterior compartment received the test item solution or negative or positive control at a volume of 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 and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

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).


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Depending on the score obtained, the test item is classified according to OECD guideline 437

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
2.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

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

1

0.33

0.074

0.073

2.11

1.43

Not categorized

0

0.073

1.10

0

0.072

1.08

Positive Control

118.67*

0.043*

119.31

122.17

Category 1

124.67*

0.098*

126.14

119.67*

0.092*

121.05

Diammo-nium phthalate

2.67*

0.050*

3.42

2.10

Not categorized

1.67*

-0.019*

1.38

1.67*

-0.012*

1.49

*corrected values

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, Diammonium phthalate is not categorized (GHS)).
Executive summary:

This in vitro study was performed to assess the corneal damage potential of Diammonium phthalate 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)solution in saline (0.9% (w/v) NaCl in deionised water) of the test item Diammonium phthalate, 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.

T

he 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 Diammonium phthalate did not cause an increase of the corneal opacity or permeability compared with the values caused by the negative control. The calculated mean in vitro irritancy score was 2.1. According to OECD 437 the test item is not categorized (GHS).