[No public or meaningful name is available]

Eye irritation

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

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. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, MEDICINES & HEALTHCARE PRODUCTS REGULATORY AGENCY

Test material

Test material information

Constituent 1

Reference substance name:

Vapour grown graphitic carbon fibre

Molecular formula:

C

IUPAC Name:

Vapour grown graphitic carbon fibre

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

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Phase 1

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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.