6,8-Dioxabicyclo[3.2.1]octan-4-one, (1S,5R)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1 Oct. 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

(1S,5R)-6,8-dioxabicyclo[3.2.1]octan-4-one

EC Number:

807-130-4

Cas Number:

53716-82-8

Molecular formula:

C6H8O3

IUPAC Name:

(1S,5R)-6,8-dioxabicyclo[3.2.1]octan-4-one

Details on test material:

- Name of test material (as cited in study report): Cyrene™- Analytical purity: 99%- Expiration date of the lot/batch: 30-Sep-2015- Physical state: clear, pale yellow liquid- Storage condition of test material: at room temperature

Test animals / tissue source

Species:

other: cattle

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

TEST METHOD: The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye damage. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.
IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Schlachthof Bensheim, Bensheim, Germany- Donor animals: at least 9 month old donor cattle
- Date and time of eye collection: 18. Sep. 2014
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) at ambient temperature supplemented with penicillin/streptomycin
PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (vascularization, pigmentation, scratches, opacity): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Type of cornea holder used: cornea holder according to the description given in OEDC guideline 437
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea.
- Test medium and temperature conditions used in the cornea holder: Minimum Essential Medium (MEM) with sodium bicarbonate and L-glutamine, supplemented immediately before use with 1% [v/v] fetal calf serum- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: free of macroscopical defects, each cornea with an basal opacity > 7 was discarded.
DETERMINATION OF THE BASAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.
- Specification of the device: OP_KiT opacitometer (Electro Design, 63-Riom France)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: number of corneas for the negative control: 3; number of corneae for the positive control: 3

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied in the test: 750 µL
POSITIVE CONTROL
- Substance: 2-Ethoxyethanol- Amount(s) applied in the test: 750 µL
NEGATIVE CONTROL
- Substance: (0.9% (w/v) NaCl solution (Saline)- Amount(s) applied in the test: 750 µ

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

The test substance were rinsed off from the anterior compartment with saline. The corneas were incubated at 32 ± 1 °C for further two hours in a vertical position.

Number of animals or in vitro replicates:

Number of corneas for the test item: 3

Details on study design:

TEST CONDITIONS
- Short description of the method used: The anterior compartment received the test item or negative or positive control at a volume of 750 µL on the surface of the corneas. The corneas were incubated in a horizontal position at 32 ± 1 °C in a water-bath. The incubation time lasted 10 min.
POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance were rinsed off from the anterior compartment with saline. The corneas were incubated at 32 ± 1 °C for further two hours in a vertical position.
DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer
- Time of determination: After further incubation of the corneae in medium for two hours at 32 ± 1 °C in a water-bath, the opacity value was determined (t130).
- Specification of the device: OP_KiT opacitometer (Electro Design, 63-Riom France)
DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1°C. Complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
- Amount and concentration of the dye: 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS.
- Incubation time: 90 min at 32 ± 1°C

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean / out of 3 corneas / 10 min

Value:

29.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: permeability

Run / experiment:

mean / out of 3 corneas / 10 min

Value:

0.469

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

mean / out of 3 corneas / 10 min

Value:

36.37

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

With the negative control neither an increase of opacity nor permeability of the corneas could be observed. The measured IVIS value of 1.10 lies within the historical range of the IVIS negative control (0.00 - 2.84). The positive control showed clear opacity and distinctive permeability of the corneas (mean IVIS = 68.22). Relative to the negative control, the test item caused an increase of the corneal opacity and the permeability. The calculated mean IVIS was 36.37 (threshold for serious eye damage: IVIS ≥ 55). According to OECD Test Guideline 437 no conclusion can be made on the eye irritation potential of the test substance Cyrene™.

Any other information on results incl. tables

Results after 10 Minutes Incubation 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	0.33	0.051	0.051	0.77	1.10	Not categorized
	1		0.053		1.80
	0		0.049		0.74
Positive Control	53.676		0.718*		64.44*	68.22	Category 1
	61.67		0.906*		75.26*
	54.67		0.687*		64.97*
Test Substance	27.67		0.582*		36.40*	36.37	No prediction can be made
	29.67		0.365*		35.14*
	30.67		0.461*		37.58*

  *corrected values

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In the in vitro eye irritation study, conducted according to OECD Test Guideline 437 and in compliance with GLP, no conclusion on the eye irritation potential of Cyrene™ can be made.