Registration Dossier

Toxicological information

Eye irritation

Currently viewing:

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2016 - 25 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test method according to OECD 438. GLP 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 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Theobromine.- Physical state: White powder.- Analytical purity: 99.8 %- Purity test date: 12/01/2015- Lot/batch No.: M15015C- Expiration date of the lot/batch: 01/2020- Storage condition of test material: Room temperature
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material:M15015C
- Expiration date of the lot/batch: 01/2020
- Purity test date:12/01/2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test animals / tissue source

Species:
other: Eyeballs isolated from chikens killed for human consumption
Details on test animals or tissues and environmental conditions:
ANIMALS
The eyeballs used in the experiment were obtained from chickens killed for human consumption (Ex vivo).
- Source: Slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice.
- Age at study initiation: approximately 7 weeks.- Weight at study initiation: 1.5 - 2.5 kg.
- Other: After electric shock and incision of the neck for bleeding, the chicken heads were transported to the laboratory in a plastic container at ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container. The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 30 minutes. All eyeballs used in the tests came from the same group of eyes collected on a specific day.Till the moment of their transfer, the eyeballs were kept in special containers at temperature of -18°C.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.03 g
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
240 minutes
Number of animals or in vitro replicates:
The test item and controls (positive and negative) were tested on three eyeballs each.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): With 20 mL physiological saline.
- Time after start of exposure: 10 seconds

SCORING SYSTEM:
Combination of fluoresceing retention, corneal opacity and corneal swelling.
Fluoresceing scores: Score Observations
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

Corneal opacity scores: Score Observations
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent areas; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible
Corneal swelling was calculated as % as follows:corneal swelling = [corneal thickness at time t – corneal thickness at time t = 0/ corneal thickness at time t = 0] x100

TOOL USED TO ASSESS SCORE:
- Fluoresceing retention (30 minutes after end of exposure)
- Corneal opacity by assigning appropiate values to opaque areas, the mean corneal opacity value was calculated for all test eyeballs for all observation time points. Based on the highest mean score corneal opacity the final score was given.
- The degree of corneal swelling was determined by measuring corneal thickness using a slit-lamp microscope and an SP-100 pachymeter.The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal swelling, at any time point, the final category score was given.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
fluorescein retention score
Run / experiment:
Fist run
Value:
2.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Irritation parameter:
fluorescein retention score
Run / experiment:
Second run
Value:
2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Irritation parameter:
cornea opacity score
Run / experiment:
First run
Value:
1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Irritation parameter:
cornea opacity score
Run / experiment:
Second run
Value:
1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Irritation parameter:
percent corneal swelling
Run / experiment:
First run
Value:
-8.1
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Irritation parameter:
percent corneal swelling
Value:
-8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of 3 eyeballs
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:
In the first run histopathological examinations of the corneas treated with the test item revealed: karyorrhexis (eyeball No. 2), slight vacuolation of the anterior corneal epithelium (eyeballs No. 1, No. 2, No. 3), slight coagulation (eyeball No. 1), exfoliation (eyeball No. 2), necrosis (eyeballs No. 2, No. 3), slight erosions of the superficial layer of the anterior corneal epithelium (eyeballs No. 1, No. 2, No. 3), deposits of the test item on the corneal surface (eyeballs No. 2, No. 3), dissection of the corneal stroma (eyeballs No. 1, No. 2, No. 3). In the second run histopathological examinations of the corneas treated with the test item revealed: slight vacuolation and local coagulation of the anterior corneal epithelium (eyeballs No. 1, No. 2, No. 3), slight exfoliation (eyeballs No. 1, No. 2), necrosis and slight erosions of the superficial layer of the anterior corneal epithelium (eyeballs No. 1, No. 2, No. 3).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.
- Range of historical values if different from the ones specified in the test guideline: No.

Any other information on results incl. tables

Evaluation of fluorescein retention– the first run.

observation after time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

2.3

III

3.0

IV

0.0

I

 

 Evaluation of fluorescein retention – the second run.

observation after time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

2.0

III

3.0

IV

0.0

I

 

Evaluation of corneal opacity– the first run.

observation after

time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

0.8

II

4.0

IV

0.0

I

75

1.0

II

4.0

IV

0.0

I

120

1.0

II

4.0

IV

0.0

I

180

1.0

II

4.0

IV

0.0

I

240

1.0

II

4.0

IV

0.0

I

 

Evaluation of corneal opacity – the second run.

observation after

time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

0.8

II

4.0

IV

0.0

I

75

1.0

II

4.0

IV

0.0

I

120

1.0

II

4.0

IV

0.0

I

180

1.0

II

4.0

IV

0.0

I

240

1.0

II

4.0

IV

0.0

I

 Evaluation of corneal swelling (%)– the first run.

observation after time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

-0.8

I

53.8

IV

-6.5

I

75

-0.5

I

63.2

IV

-5.0

I

120

-1.6

I

78.1

IV

-7.9

I

180

-4.5

I

94.7

IV

-7.9

I

240

-8.1

I

104.8

IV

-5.4

I

“-“          - percentage of corneal thickness decrease, no swelling

 

Evaluation of corneal swelling (%) – the second run.

observation after time t

(minutes)

test item

positive control

imidazole

negative control

physiological saline

average

ICE class

average

ICE class

average

ICE class

30

-0.1

I

52.3

IV

-4.8

I

75

-2.4

I

64.1

IV

-4.8

I

120

-4.8

I

69.3

IV

-6.6

I

180

-8.0

I

79.3

IV

-9.9

I

240

-8.0

I

74.2

IV

-9.4

I

“-“          - percentage of corneal thickness decrease, no swelling

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Based on EU criteria
Conclusions:
The substance can not be classified as severe irritant or corrosive to the eyes and it is not possible to state that the test item is not irritant to the eyes according to the UN GHS classification which is in accordance with the CLP criteria. According to the histopathological findings the test can be regarded as moderately irritating to the eyes.
Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the substance theobromine according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to 0.03 g of the test item, the same amount of imidazole and 0.03 mL of physiological saline were applied to other eyeballs as positive and negative controls. Three eyeballs were used for the test item and for each control. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438 recomendations, histopathological evaluation of the corneal layers was conducted. Because the test item was a solid material and the first run lead to a no-classification outcome, a second run of three eyes for the test item was performed as recommended by the guideline, concurrent controls were included. According to the overall in vitro classification (UN GHS), no prediction can be made since the combinations of the 3 endpoints were 2xII and 1xIII in both runs, however taking into account the histopathological findings, the test can be considered as moderately irritating.