[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-09-01 until 2015-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

EC Number:

267-510-5

EC Name:

[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

Cas Number:

67874-81-1

Molecular formula:

C16H28O

IUPAC Name:

[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

Test material form:

liquid

Test animals / tissue source

Species:

other: eyes of male or female chickens (ROSS, spring chickens)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: approximately 1.5 - 2.5 kg
- Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- The preparation and validation of the eyes prior to the ICE-test were all according to OECD guideline 438.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 µL

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

0, 30, 75, 120, 180, and 240 minutes

Number of animals or in vitro replicates:

3 eyes

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The eyes were rinsed with 20 mL saline
- Time after start of exposure: 10 seconds

SCORING SYSTEM: According to OECD 438 guideline.

TOOL USED TO ASSESS SCORE: All examinations were carried out with the hand-slit lamp microscope. Fluorescein retention was only scored at approximately 30 minutes after treatment. After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

CONTROLS: A negative control (30 µL physiological saline) and 3 positive controls (30 µL Benzalkonium Chloride 5%) were included.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

slit-lamp examination

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

slit-lamp examination

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

slit-lamp examination

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

In vivo

Irritant / corrosive response data:

- Slit-lamp examination:
The test substance caused very slight corneal swelling (1%), very slight or slight opacity (mean score of 0.8) and no fluorescein retention (mean score of 0.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
- Microscopic examination:
Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion (two corneas) and slight vacuolation (one cornea) of the epithelium. In addition, the epithelium was partly detached form the basement membrane in the same cornea. In view of the slit-lamp observations of this cornea, the toxicological significance of these findings was considered dubious and not taken into account for the classification. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities, apart from very slight vacuolation (top region) of the epithelium. The positive control BAC 5% caused moderate or severe erosion, slight vacuolation (one cornea; low region) of the epithelium, and endothelial necrosis.

Applicant's summary and conclusion

Interpretation of results:

other: Not eye irritating

Remarks:

EU CLP 1272/2008 and its amendments

Conclusions:

Under the test conditions (OECD 438 and GLP) the test substance is not an eye irritant using the EU CLP and GHS criteria.

Executive summary:

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused very slight corneal swelling (1%), very slight or slight opacity (mean score of 0.8) and no fluorescein retention (mean score of 0.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion (two corneas) and slight vacuolation (one cornea) of the epithelium. In addition, the epithelium was partly detached form the basement membrane in this cornea. In view of the slit-lamp observations of this cornea, the toxicological significance of these findings was considered dubious and not taken into account for the classification. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities, apart from very slight vacuolation (top region) of the epithelium. The positive control BAC 5% caused moderate or severe erosion, slight vacuolation (one cornea; low region) of the epithelium, and endothelial necrosis. Based on these results the test substance is not considered to be an eye irritant in accordance with EU CLP and GHS criteria.