Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 December 2017 - 28 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
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)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3,4,5-trihydroxybenzoic acid
EC Number:
205-749-9
EC Name:
3,4,5-trihydroxybenzoic acid
Cas Number:
149-91-7
Molecular formula:
C7H6O5
IUPAC Name:
3,4,5-trihydroxybenzoic acid
impurity 1
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Oxidane
impurity 2
Reference substance name:
sum of impurities (organic and inorganic) not relevant for classification
Molecular formula:
not available for mixtures
IUPAC Name:
sum of impurities (organic and inorganic) not relevant for classification
Test material form:
solid
Details on test material:
Name: gallic acid
Form: powder
Color: white
Specific details on test material used for the study:
Test item name: Gallic Acid
Chemical name (IUPAC): 3,4,5-Trihydroxybenzoic acid
CAS No.: 149-91-7
Physical appearance: White powder
Batch Produced by: Archroma
Date of Expiry: 13.07.2019
Storage Conditions: Ambient (21 to 29ºC)

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
COLLECTION AND TRANSPORT OF EYES TO THE TEST FACILITY:
Eyes of cattle were collected from a slaughterhouse. To prevent exposure of the eyes to potentially irritant substances, the heads of the animals were not rinsed with detergent. Eyes were enucleated as soon as possible after death and immersed in the Hank’s Balanced Salt Solution (HBSS) with 1% antibiotics (Penicillin and Streptomycin) in a suitable container and were transported to the test facility by placing in cool packs.

SELECTION CRITERIA FOR EYES:
Upon arrival to the test facility, eyes were examined for defects including opacity, scratches and neovascularization. Only corneas free of such defects were used in the experiment.

PREPARATION OF THE EYES:
Before the start of the experiment, opacity of empty cornea holders filled with MEM media were measured and the mean opacity value of the empty corneal holders obtained was considered as I0. Corneas free of defects were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in designated corneal holders by placing the endothelial side of the cornea against the O-ring of the posterior chamber. The anterior chamber was placed over the cornea and both chambers were joined together by tightening the chamber screws then filling the posterior and anterior chambers with MEM without phenol red (Minimum Essential Medium supplemented with 1% Fetal Bovine Serum and 1% Penicillin and Streptomycin). The corneal holders were equilibrated at 32±1ºC for one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity. MEM was replaced with fresh pre-warmed MEM without phenol red in both chambers of cornea holders after completion of equilibrium period. An opacity determination was performed on each of the corneas using an Opacitometer (BASF Opacitometer 2013-19). The opacity of each cornea was read against a MEM filled chamber, and the initial opacity reading thus determined was recorded as baseline opacity.

Test system

Vehicle:
water
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
750 µL of 20% w/v test item.
Duration of treatment / exposure:
4 hours.
Duration of post- treatment incubation (in vitro):
Not specified.
Number of animals or in vitro replicates:
Three replicates per group.
Details on study design:
APPLICATION OF THE TEST ITEM:
The medium from the anterior chamber was removed and the test item was added by following the Close Chamber Method. Distilled water was used as vehicle for test item formulation based on the information provided in the certificate of analysis. 750 µL of 20% w/v test item was introduced into the anterior chamber through the dosing holes of the corneal holder, and the holes were subsequently sealed during the exposure. Incubation was performed horizontally at 32±1ºC. The corneas were exposed to the test item for approximately 4 hours. A similar procedure was followed for negative and positive controls.

POST EXPOSURE:
After the exposure period, the test item, negative and positive controls were removed from the anterior chamber and the epithelium was washed with EMEM containing phenol red until no visual evidence of the test item was observed. Finally the corneas were rinsed with MEM without phenol red. The anterior chamber was then refilled with fresh MEM without phenol red. Post exposure corneas were observed visually for tissue peeling, residual test chemical and non-uniform opacity patterns.

MEASUREMENT OF OPACITY:
Opacity was measured with the aid of an opacitometer. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea and positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item treated and positive control cornea. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

MEASUREMENT OF PERMEABILITY:
Permeability was determined by replacing media with 1 mL of a 5 mg/mL solution of sodium fluorescein in to the anterior chamber of the corneal holder, while posterior chamber was filled with fresh MEM without phenol red. The corneal holders were then incubated in a horizontal position for 90±5 minutes at 32±1ºC. Post incubation, MEM media from the posterior chambers was collected into separate tubes. From each tube, 360 µL of each sample of individual corneas were transferred into each well of a 96 well plate in triplicates. Permeability in terms of optical density was determined by measuring at 490 nm using Spectramax m5e (Make: Molecular devices).
Mean OD490 values were obtained for each corneas and the corrected individual permeability values for each test item treated cornea and positive control was calculated by subtracting the average OD490 value of the negative control corneas. The mean OD490 of each treatment group is then calculated by averaging the corrected opacity values of the treated corneas for each positive control and test item treatment group.
Also, each cornea is observed visually and pertinent observations recorded (e.g. tissue peeling, non-uniform opacity patterns).

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item
Value:
ca. 20.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
As the in vitro irritation score is between 3 and 55, the result is considered equivocal and no prediction can be made.
Other effects / acceptance of results:
RESULTS: IN VITRO IRRITANCY SCORE (IVIS)
The opacity and mean permeability values were recorded for all treatment groups and in vitro Irritancy Score (IVIS) for each group was derived. The vehicle control and test item resulted in mean changes in opacity value after treatment of respectively -0.8, 21.2. The mean corrected opacity and mean corrected permeability values of test item are 22.04 and -0.117 respectively. The in vitro Irritancy Score (IVIS) of the test item resulted in 20.3. Positive control resulted in mean change in opacity of 110.4. The mean corrected opacity and mean corrected permeability values of positive control are 111.22 and 1.413 respectively with an in vitro Irritancy Score (IVIS) of 132.4.

STUDY ACCEPTABILITY:
The study was accepted:
• As the negative control response resulted in opacity and permeability values less than the established upper limits of background opacity and permeability values for bovine corneas treated with the respective negative control.
• As the positive control gave an IVIS that falls within two standard deviations of the historical mean.

Any other information on results incl. tables

Group & Treatment

Cornea Holder No.

Initial Opacity Reading (Before treatment)

Opacity Reading (After Treatment)

Change in Opacity Value

Mean Change in Opacity Value

Corrected Opacity Value

Average of Permeability Value

Corrected Permeability Value

Mean of Corrected Opacity Value ±SD

Corrected Permeability Value ±SD

IVIS Value

G1 & Vehicle Control

7

5.2

3.8

-1.4

-0.8

-

0.148

0.239

-

-

-

4

2.6

1.5

-1.0

0.252

10

2.4

2.3

0.0

0.318

G2 & Positive Control

1

3.8

126.8

123.0

110.4

123.8

1.699

1.424

111.22

±14.23

1.413

±0.040

132.4

9

2.8

97.8

95.0

95.8

1.627

1.400

2

1.0

114.3

113.3

114.1

1.631

1.415

G3 & 20% w/v Test Item

8

3.3

19.7

16.3

21.2

17.1

0.144

-0.121

22.04

±4.74

-0.117

±0.022

20.3

5

2.6

24.3

21.6

22.4

0.100

-0.117

3

0.5

26.3

25.8

26.6

0.122

-0.114

IVIS:In VitroIrritancy Score

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Gallic acid was determined to have an in vitro Irritation Score of 20.3. As this result is between the threshold values of 3 and 55, no prediction can be made on the eye irritation potential of Gallic Acid according to UN GHS.
Executive summary:

The test item, Gallic Acidwas evaluated forocular corrosivity or severe irritancyas per the OECD guideline for the testing of chemicals No. 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals not requiring Classification for Eye Irritation or Serious Eye Damage”, adopted on 9thOctober 2017.

Eyes of cattle were collected from a slaughter house by immersing them in the Hank’s Balanced Salt Solution (HBSS) with antibiotics (penicillin and streptomycin) in a suitable container and transported to the test facility by placing on cool packs. Eye balls free of defects were selected for the experiment. Empty cornea holder’s opacity with pre-warmed Eagle’s Minimum Essential Medium was measured and the mean opacity value obtained was determined as I0.Cornea holders with selected Corneas were equilibrated at 32±1ºC for 1 hour with Eagle’s Minimum Essential Medium with1% Fetal Bovine Serum supplemented with 1% antibiotics and baseline opacity was recorded for each cornea. Corneas with opacity units less than 7 were selected and used for the study and distributed for the treatment groups.

A volume of 750 µL of 20% w/v test item, vehicle (distilled water) and positive control (20% w/v imidazole) was introduced into anterior chamber in triplicates to the designated cornea holders and incubated at 32±1ºC for 4 hours. Treated corneas were washedtill no visual evidence of test item observed with EMEM containing phenol redand finally with EMEM without phenol red. The anterior chamber was then refilled with fresh EMEM without phenol red. Opacity was measured with the aid of opacitometer and permeability was determined spectrophotometrically at 490 nm (OD490) using 5 mg/mL sodium fluorescein, post incubation of 90 min at 32±1ºC.

The test item [Gallic Acid] resulted in the mean corrected opacity and mean corrected permeability values of test item are 22.04 and -0.117 respectively. Thein vitroIrritancy Score (IVIS) of test item resulted in 20.3.Whereas the positive control resulted inmean corrected opacity and mean corrected permeability values of positive control are 111.22 and 1.413 respectively where thein vitroIrritancy Score (IVIS) of 132.4, indicatingcorrosivity or severe irritancy to Bovine cornea.

Based on the results obtained in the Bovine Corneal Opacity and Permeability Test, the test item Gallic Acid induced an IVIS of 20.3 after 4 hours of treatment.The results indicated an appreciable increase in the endpoint “opacity” and a small decrease in “permeability”.As the test item induced an IVIS >3 and ≤55, the results are equivocal and no prediction can be made regarding thecorrosivity or severe irritancy to Bovine corneas.