8,9,10-trinorborna-2,5-diene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2017 - January 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 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)

Test material

Test material information

Constituent 1

Reference substance name:

8,9,10-trinorborna-2,5-diene

EC Number:

204-472-0

EC Name:

8,9,10-trinorborna-2,5-diene

Cas Number:

121-46-0

Molecular formula:

C7H8

IUPAC Name:

bicyclo[2.2.1]hepta-2,5-diene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch number of test material: N° ELI 70700450
- Production date: 31 August 2017
- Expiry date February 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material under test conditions and during storage: stable

Test animals / tissue source

Species:

chicken

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:
Eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France)


- Characteristics of donor animals:
The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, I .5 - 2.5 kg).

- Storage, temperature and transport conditions of ocular tissue:
Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Phycher on 04 December 2017 at 10:20 am.

- Time interval prior to initiating testing:
Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus, placed in a horizontal position, and 30 µL of the test item was applied, as supplied, to the cornea such that the entire surface of the cornea was evenly covered with the test item.

- Indication of any existing defects or lesions in ocular tissue samples:
None

- Selection and preparation of corneas:
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.7°C and 32.4°C
Once all eyes had been examined and approved, the eyes were incubated between 45 and 64 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

- Quality check of the isolated corneas:
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

30 µL

Duration of treatment / exposure:

10s

Duration of post- treatment incubation (in vitro):

30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse

Number of animals or in vitro replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

3

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

3

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

corneal swelling 

Run / experiment:

Mean

Value:

17

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Endpoint measured	Eye No	Time (min)
		0	30	75	120	180	240
Corneal opacity	7	0	3	3	3	3	3
	8	0	3	3	3	3	3
	9	0	3	3	3	3	3
Mean		0.0	3.0	3.0	3.0	3.0	3.0
ICE class		IV
Fluorescein retention	7	0.5	3	-	-	-	-
	8	0.5	3	-	-	-	-
	9	0.5	3	-	-	-	-
Mean		0.5	3.0	-	-	-	-
ICE class		IV
Corneal thickness	7	0.58	0.61	0.64	0.65	0.66	0.67
	8	0.60	0.63	0.66	0.67	0.69	0.70
	9	0.57	0.60	0.63	0.64	0.67	0.67
Corneal swelling	7	-	5	10	12	14	16
	8	-	5	10	12	15	17
	9	-	5	11	12	18	18
Mean		-	5	10	12	15	17
ICE class		II
Combination of the 3 Endpoints		2 x IV, 1 x II
Classification		Category 1 : Corrosive / Severe irritant

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the test item has to be classified in Category 1 "Irreversible effects on the eye"
The signal word "Danger" and hazard statement H318 "Causes serious eye damage" are required.

Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe imitating effects of the test item after administration on enucleated chicken eyes.

The test item was applied, as supplied, at the dose of 30 µL, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 438 adopted 26 July 2013 and the test method B.48 — Commission Regulation (EU) No. 1152/2010 dated 08 December 2010 (EU Journal L324) - ATP Council regulation No. 440/2008 of 30 May 2008 (E. U. Journal L142).

The ocular reactions observed in eyes treated with the test item were:

• maximal mean score of corneal opacity: 3.0, corresponding to ICE class IV;


• mean score of fluorescein retention: 3.0, corresponding to ICE class IV;


• maximal mean corneal swelling: 17%, corresponding to ICE class II.

The combination of the three endpoints for the test item was 2 x IV, 1 x II.

The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as "No Category", as expected.

In accordance with the Regulation (EC) No. 1272/2008, the test item has to be classified in Category 1 "Irreversible effects on the eye".

The signal word "Danger" and hazard statement H318 "Causes serious eye damage" are required.