Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 Oct. 2018 - 10 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, MEDICINES & HEALTHCARE PRODUCTS REGULATORY AGENCY

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
chicken
Strain:
other: Ross 308 Broiler
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Baileys Turkeys Ltd., Cheshire, UK
- Number of animals: multiple, not further specified
- Characteristics of donor animals: approx. 56 days old, approx. 3 kg
- Storage, temperature and transport conditions of ocular tissue: Heads were removed immediately after the chickens were killed, heads were stored individually in a transport box, transport box was lined with paper towel moistened with isotonic saline, heads were transported at ambient temperature
- Time interval prior to initiating testing: eyes were used on day of slaughter
- Indication of any existing defects or lesions in ocular tissue samples: no, eyes were examined for damages and corneal opacity, only acceptable eyes were used for test

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied (test item and positive control): 0.03 g
- Volume applied (negative control): 0.03 mL
Duration of treatment / exposure:
10 s
Number of animals or in vitro replicates:
Test item and positive control: triplicates
Negative control: duplicates
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Eyelids were carefully excised. The integrity of the cornea was measured with 2% (w/v) sodium fluorescein followed by rinsing with isotonic saline after a few seconds. The treated eyes were examined with a Haag-Streit BQ 900 (Switzerland) microscope. An acceptable eye had fluorescein retention and corneal opacity scores of <= 0.5. Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. A portion of the optic nerve remained on the removed eye. Other connective tissue was removed from the eye on an absorbent tray liner.

Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. Once all eyes were placed in the superfusion apparatus, they were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea. Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.

EQUILIBRATION AND BASELINE RECORDINGS
After the approval process, the eyes were incubated for approx. 45 min in an upright position at 30.5 - 33.5 °C for equilibrium purposes, time zero measurements for corneal thickness and opacity were taken to serve as baseline, baseline for the fluorescein measurements were taken at dissection.

OBSERVATION PERIOD
Treated corneas were evaluated prior to treatment, 30, 75, 120, 180 and 240 minutes after removal of test item / positive control.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The test item was rinsed off from the corneas with 20 mL isotonic saline .

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
- Damage to epithelium based on fluorescein retention: The mean fluorescein retention scores for all test eyes are calculated at the 30 min time interval only.
- Swelling: Percentage corneal swelling was assessed from corneal thickness measurements. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea. The calculation is expressed in the following formula: ((corneal thickness at time (t) - corneal thickness at time = 0)/ corneal thickness at time 0) x 100. The mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
- Macroscopic morphological damage to the surface: pitting, sloughing or roughening of the epithelium

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA:
The evaluation and decision criteria as indicated in the TG were used.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
Phase 1
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Phase 1
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
Phase 1
Value:
13.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 240 min
Irritation parameter:
cornea opacity score
Run / experiment:
Phase 2
Value:
0.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Phase 2
Value:
0.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
Phase 2
Value:
-0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 240 min
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Phase 1: No morphological effects were noted in 2/3 (Phase 1) and 0/3 (Phase 2) test item-treated eyes and all negative and positive control-treated eyes (Phase 1 and 2). Adherence of the test item to the cornea was noted in 3/3 treated eyes during the observation period (Phase 1 and 2) and persisted in 1/3 (Phase 1) and 3/3 (Phase 2) treated eyes up to the 240 minute observation.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The Proficiency testing as recommended in OECD TG 438 to show suitability of substances for the ICE test in determining non GHS Category and Category 1 substances was performed at the test facility and is reported.

Any other information on results incl. tables

 Table 1: Summary of Results

Phase 1

Mean Corneal Opacity

(ICE class)

Mean Fluorescein Retention (ICE class)

Mean Corneal Thickness compared to time zero % (ICE class)

Combination of the 3 Endpoints

30 min

75 min

120 min

180 min

240 min

1.0 (II)

0.5 (I)

1.03 (I)

10.31 (II)

8.25 (II)

8.25 (II)

13.40 (II)

 

(II)

1 x I, 2 x II

Classification:

No Category

Phase 2

Mean Corneal Opacity

(ICE class)

Mean Fluorescein Retention (ICE class)

Mean Corneal Thickness compared to time zero % (ICE class)

Combination of the 3 Endpoints

30 min

75 min

120 min

180 min

240 min

 

0.8 (II)

 

0.3 (I)

-3.98

(I)

-1.00

(I)

-0.50

(I)

-2.49

(I)

-0.50

(I)

 

(I)

2 x I, 1 x II

Classification:

No Category

mins: Minutes following treatment

 

Table 2: Individual Scores and Mean Scores for Corneal Effects – Test Item (Phase 1)

End Point

Eye Number

Time (after decontamination) [minutes]

0

30

75

120

180

240

Corneal Opacity

3A

0

0.5

0.5

1

1

1

6A

0

0.5

1

1

1

1

8A

0

0.5

0.5

0.5

0.5

1

Mean

0.0

0.5

0.7

0.8

0.8

1.0

ICE Class

II

Fluorescein Retention

3A

 

0.5

 

 

 

 

6A

 

0.5

 

 

 

 

8A

 

0.5

 

 

 

 

Mean

 

0.5

 

 

 

 

ICE Class

I

Corneal Thickness

3A

0.63

0.62

0.72

0.70

0.68

0.74

6A

0.65

0.70

0.70

0.68

0.70

0.74

8A

0.66

0.64

0.72

0.72

0.72

0.72

Mean

0.65

0.65

0.71

0.70

0.70

0.73

Mean Corneal Swelling (%)

 

1.03

10.31

8.25

8.25

13.40

ICE Class

II

Epithelium Condition

3A

 

TA

TA

TA

TA

N

6A

 

TA

TA

TA

TA

TA

8A

 

TA

N

N

N

N

ICE Classes Combined:

1x I, 2 x II

Classification:

No Category

TA: Test Item Adherence; N: Normal

Table 3: Individual Scores and Mean Scores for Corneal Effects – Test Item (Phase 2)

End Point

Eye Number

Time (after decontamination) [minutes]

0

30

75

120

180

240

Corneal Opacity

3A

0.5

0.5

0.5

0.5

1

1

6A

0.5

0.5

0.5

1

1

1

8A

0.5

0.5

0.5

0.5

0.5

0.5

Mean

0.5

0.5

0.5

0.7

0.8

0.8

ICE Class

II

Fluorescein Retention

3A

 

0.5

 

 

 

 

6A

 

0.5

 

 

 

 

8A

 

0

 

 

 

 

Mean

 

0.3

 

 

 

 

ICE Class

I

Corneal Thickness

3A

0.68

0.65

0.72

0.75

0.70

0.68

6A

0.67

0.63

0.650

0.64

0.66

0.65

8A

0.66

0.65

0.62

0.61

0.60

0.67

Mean

0.67

0.64

0.66

0.67

0.65

0.67

Mean Corneal Swelling (%)

 

-3.98

-1.00

-0.50

-2.49

-0.50

ICE Class

I

Epithelium Condition

3A

 

TA

TA

TA

TA

TA

6A

 

TA

TA

TA

TA

TA

8A

 

TA

TA

TA

TA

TA

ICE Classes Combined:

2x I, 1 x II

Classification:

No Category

TA: Test Item Adherence

 

Table 4: Individual Scores and Mean Scores for Corneal Effects – Positive Control (Phase 1)

End Point

Eye Number

Time (after decontamination) [minutes]

0

30

75

120

180

240

Corneal Opacity

2A

0

4

4

4

4

4

5A

0

4

4

4

4

4

7A

0

4

4

4

4

4

Mean

0.0

4.0

4.0

4.0

4.0

4.0

ICE Class

IV

Fluorescein Retention

2A

 

3

 

 

 

 

5A

 

3

 

 

 

 

7A

 

3

 

 

 

 

Mean

 

3.0

 

 

 

 

ICE Class

IV

Corneal Thickness

2A

0.67

0.70

0.82

0.82

0.90

0.86

5A

0.64

0.78

0.86

0.86

0.90

0.88

7A

0.63

0.76

0.83

0.88

0.86

0.90

Mean

0.65

0.75

0.84

0.85

0.89

0.88

Mean Corneal Swelling (%)

 

15.46

29.38

31.96

37.11

36.08

ICE Class

IV

Epithelium Condition

2A

 

N

N

N

N

N

5A

 

N

N

N

N

N

7A

 

N

N

N

N

N

ICE Classes Combined:

3 x IV

Classification:

Category 1

N: Normal

 

Table 5: Individual Scores and Mean Scores for Corneal Effects – Positive Control (Phase 2)

End Point

Eye Number

Time (after decontamination) [minutes]

0

30

75

120

180

240

Corneal Opacity

2A

0

4

4

4

4

4

5A

0

4

4

4

4

4

7A

0.5

4

4

4

4

4

Mean

0.2

4.0

4.0

4.0

4.0

4.0

ICE Class

IV

Fluorescein Retention

2A

 

3

 

 

 

 

5A

 

3

 

 

 

 

7A

 

3

 

 

 

 

Mean

 

3.0

 

 

 

 

ICE Class

IV

Corneal Thickness

2A

0.65

0.83

0.91

0.95

0.91

0.94

5A

0.61

0.78

0.77

0.84

0.82

0.87

7A

0.68

0.79

0.83

0.91

0.94

0.96

Mean

0.65

0.80

0.84

0.90

0.89

0.92

Mean Corneal Swelling (%)

 

23.71

29.38

39.18

37.63

42.78

ICE Class

IV

Epithelium Condition

2A

 

N

N

N

N

N

5A

 

N

N

N

N

N

7A

 

N

N

N

N

N

ICE Classes Combined:

3 x IV

Classification:

Category 1

N: Normal

 

Table 6: Individual Scores and Mean Scores for Corneal Effects – Negative Control (Phase 1)

End Point

Eye Number

Time (after decontamination) [minutes]

0

30

75

120

180

240

Corneal Opacity

1A

0

0

0

0

0

0

4A

0

0

0

0

0

0

Mean

0.0

0.0

0.0

0.0

0.0

0.0

ICE Class

I

Fluorescein Retention

1A

 

0

 

 

 

 

4A

 

0

 

 

 

 

Mean

 

0.0

 

 

 

 

ICE Class

I

Corneal Thickness

1A

0.64

0.64

0.66

0.64

0.66

0.64

4A

0.66

0.64

0.67

0.66

0.68

0.66

Mean

0.65

0.64

0.67

0.65

0.67

0.65

Mean Corneal Swelling (%)

 

-1.54

2.31

0.00

3.08

0.00

ICE Class

I

Epithelium Condition

1A

 

N

N

N

N

N

4A

 

N

N

N

N

N

ICE Classes Combined:

3 x I

Classification:

No Category

N: Normal

 

Table 7: Individual Scores and Mean Scores for Corneal Effects – Negative Control (Phase 2)

End Point

Eye Number

Time (after decontamination) [minutes]

0

30

75

120

180

240

 

Corneal Opacity

1A

0

0.5

0.5

0.5

0.5

0.5

4A

0.5

0.5

0.5

0.5

0.5

0.5

Mean

0.3

0.5

0.5

0.5

0.5

0.5

ICE Class

I

Fluorescein Retention

1A

 

0.5

 

 

 

 

4A

 

0.5

 

 

 

 

Mean

 

0.5

 

 

 

 

ICE Class

I

 

Corneal Thickness

1A

0.61

0.62

0.62

0.59

0.59

0.58

4A

0.64

0.61

0.62

0.61

0.61

0.65

Mean

0.63

0.62

0.62

0.60

0.60

0.62

Mean Corneal Swelling (%)

 

-1.60

-0.80

-4.00

-4.00

-1.60

ICE Class

I

Epithelium Condition

1A

 

N

N

N

N

N

4A

 

N

N

N

N

N

ICE Classes Combined:

3 x I

Classification:

No Category

N: Normal

 

Table 8: Historical control data, Negative Controls

Study Number

Negative control

Date

Maximum Mean CO

Maximum Mean % CT

Maximum Mean FR

ME

CR76PS

saline

30/01/2017

0.3

5.19

0

None

VQ32JY

saline

30/01/2017

0.5

2.11

0

None

HK86QX

saline

03/10/2016

0.3

2.16

0.3

None

PJ73HL

saline

05/12/2016

0.3

2.86

0

None

KX84DP

saline

28/11/2016

0.3

2.9

0

None

KK41FP

saline

22/08/2016

0.5

3.57

0

None

41502580

saline

11/01/2016

0.5

7.04

0.5

None

41502299

saline

09/11/2015

0.5

‐0.7

0

None

41502380

saline

12/10/2015

0.5

1.42

0.3

None

ME: Morphological effects; FR: Fluorescein Retention; CT: Corneal Thickness; CO: Corneal Opacity

 

Table 9: Historical control data, Positive Controls

Study Number

Positive control

Date

Maximum Mean CO

Maximum Mean % CT

Maximum Mean FR

ME

CR76PS

5% Benzalkonium chloride

30/01/2017

4

54.23

3

S (two eyes)

VQ32JY

5% Benzalkonium chloride

30/01/2017

4

53.77

3

S (three eyes)

HK86QX

Imidazole

03/10/2016

4

46.92

3

S (one eye)

PJ73HL

5% Benzalkonium chloride

05/12/2016

4

44.5

3

S (two eyes)

KX84DP

5% Benzalkonium chloride

28/11/2016

2.7

35.71

2.7

S (one eye)

KK41FP

5% Benzalkonium chloride

22/08/2016

3.7

38.54

3

S (three eyes)

41502580

5% Benzalkonium chloride

11/01/2016

4

45.41

3

S (three eyes)

41502299

5% Benzalkonium chloride

09/11/2015

4

47.89

3

S (three eyes)

41502380

5% Benzalkonium chloride

12/10/2015

4

34.12

3

S (one eye)

ME: Morphological effects; FR: Fluorescein Retention; CT: Corneal Thickness; S: Sloughing (loosening); CO: Corneal Opacity

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
Conclusions:
In two independent experiments, the test material was assessed to have no potential to cause eye irritation or serious eye damage in the Isolated Chicken Eye test. No classification is warranted.