Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

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)

Version / remarks:

July 2013

Deviations:

yes

Remarks:

The OECD guideline 437 recommends the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details

Version / remarks:

April 1997

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

EC Number:

273-453-7

EC Name:

Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

Cas Number:

68966-86-9

Molecular formula:

C14H22O2

IUPAC Name:

Methyl 4(or 1)-isopropyl-1(or 4)-methylbicyclo[2.2.2]oct-5-ene-2-carboxylate

Test animals / tissue source

Species:

other: Bovine eyes

Strain:

other: Cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH Aschaffenburg
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): At least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):Isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled abattoir until transportation on the same morning to the laboratory.
- Time interval prior to initiating testing: The corneae were isolated on the same day as delivery
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects with those presenting defects discarded.
- Indication of any antibiotics used: Eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): Neat test item

VEHICLE: not applicable

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: All eyes were carefully examined macroscopically for defects with those presenting defects discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue was left for stability and handling of the isolated cornea. Each isolated cornea was mounted in a specially designed cornea holder according to the OECD 437 guideline 437. The endothelial side of the cornea was positioned against the sealing ring of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium with the posterior compartment filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

QUALITY CHECK OF THE ISOLATED CORNEAS: At the end of the incubation period, the basal opacity was determined (t0). Cornea with a score of > 7 was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Saline (0.9% NaCl in deionised water, using ultrasonic technique)

POSITIVE CONTROL USED: 2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of neat test item

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:1
- POST-EXPOSURE INCUBATION:2 hours at 32 ± 1 ºC

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry(OD490)
- Others (e.g, pertinent visual observations, histopathology): None reported

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: According to guideline

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 1

Value:

0.05

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 2

Value:

-0.22

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Cornea 3

Value:

1.27

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

0.37

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: Validity criteria met

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: Not applicable

Any other information on results incl. tables

Results after 10 minute treatment time:

Test Group	Opacity value = Difference (t130 - t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy score
		Mean		Mean
Negative control	0	0.00	0.085	0.072	1.28	1.09	Not categorised
	0		0.067		1.01
	0		0.065		0.98
Positive control	96.00*		1.152*		113.28	103.28	Category 1
	83.00*		1.501*		105.51
	77.00*		0.938*		91.07
Test item	0.00*		0.004*		0.05	0.37	Not categorised
	0.00*		-0.014*		-0.22
	1.00*		0.018*		1.27

 *corrected values

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 0.37.
According to OECD 437 (see table in chapter 3.8.3) the test item is not categorized (GHS).

Executive summary:

The test item was assessed for eye irritation in a Bovine Corneal Opacity and Permeability Assay (BCOP) conducted following OECD

437, and EU B.47 in undiluted form alongside a postive and negative control. The results of the test indicates that the test item does not possess an eye irritating potential according to the UN GHS regulations.

The study is a GLP compliant guideline experiment study available as an unpublished study report. The study has no restrictions and is fully adequate for assessment.