Reaction mass of 3,5,6,6-tetramethyl-4-methyleneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one

EC Number:

946-245-5

Molecular formula:

C12H22O

IUPAC Name:

Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one

Test material form:

liquid

Details on test material:

According to ECHA communication with reference to Annotation number: SUB-C-2114629169-42-01/F, the substance ID for Koavone has been changed with EC# from 939-627-8 to 946-245-5, and the IUPAC name from Reaction mass of (3R,5R)-3,5,6,6-tetramethyl-4-methylideneheptan-2-one and (3R,5S)-3,5,6,6-tetramethyl-4-methylideneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one to Reaction mass of (E)-3,4,5,6,6-pentamethylhept-3-en-2-one and 3,5,6,6-tetramethyl-4-methyleneheptan-2-one.

Test animals / tissue source

Species:

other: chicken eyes (in vitro)

Strain:

other: ROSS (spring chickens)

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

Duration of post- treatment incubation (in vitro):

Ca. 0, 30, 75, 120, 180 and 240 minutes after treatment

Number of animals or in vitro replicates:

3 eyes for the substance, 3 eyes for positive control and 1 eye for negative control

Details on study design:

Approximately 7 weeks old, male or female chickens were used as eye donors. Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus. The chambers of the superfusion apparatus were temperature controlled at approximately 32 ºC. The eyes were examined with the slit-lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. 1. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that showed opacity (score > 0.5), or were unacceptably scored with fluorescein (score higher than 0.5), or eyes showing any other signs of damage were rejected. The eyes were treated with 30 μLof the test substance for 10 seconds and rinsed with 20 mL saline. The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment using the following criteria:

Corneal swelling: expressed as a percentage (max. 100%): calculated as corneal thickness at time t minus corneal thickness at time 0, divided by corneal thickness at time 0 and multiplied by 100.

Corneal opacity:
0 = no opacity
0.5 = very faint opacity (very slight)
1 = scattered or diffuse areas, details of iris clearly visible (= slight)
2 = easily discernible translucent area, details of iris slightly obscured ( = moderate)
3 = severe corneal opacity, no specific details of iris visible, size of pupil barely discernible (= severe)
4 = complete corneal opacity, iris invisible (= very severe)

Fluorescein retention:
0 = no fluorescein retention
0.5 = very minor single cell staining ( = very slight)
1 = single cell staining cattered throughout the treated area of the cornea ( = slight)
2 = focal or confluent dense single cell staining (moderate)
3 = confluent large area of the cornea retaining fluorescein (= severe)

Fluorescein retention was only scored at approximately 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.

The scores were converted into the irritation categories according to the following scheme:
I = no effect; II = slight effect; III = moderate effect; IV = severe effect

Regulatory GHS Classification:

A: not irritating: 3 x Category I; 2 x Category I, 1 x Category II
B: slightly irritating: 3 x Category II; 2 x Category II, 1 x Category III; 1 x Category 1, 1 x Category II, 1 x Category III
C: moderately irritating (GHS Category 2A): 3 x Category III; 2 x Category III, 1 x Category II; 2 x Category III, 1 x Category I; 2 x Category I, 1 x Category IV; 2 x Category II, 1 x Category IV; 2 x Category III, 1 x Category IV; 1 x Category II, 1 x Category III, 1 x Category IV
D: severely irritating (GHS Category 1): 3 x Category IV; 2 x Category IV, 1 x Category III; 2 x Category IV, 1 x Category II; 2 x Category IV, 1 x Category I; immediate corneal opacity score 3 in at least 2 corneas; corneal opacity score 4 in at least 2 corneas; severe loosening of epithelium in at least one cornea

Irritation index was calculated as follows: maximum corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

240 minutes after treatment

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Irritancy category II

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

mean

Run / experiment:

240 minutes after treatment

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Irritancy category I

Results 3

Irritation parameter:

fluorescein retention score

Remarks:

mean

Run / experiment:

240 minutes after treatment

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Irritancy category II

Other effects / acceptance of results:

The following irritation categories were reported for the substance: I (swelling), II (opacity), II (fluorescin retention); irritation index 40
For saline (negative control): corneal swelling 0, corneal opacity 0, fluorescein retention 0
For 5% BAC (positive control): 29% corneal swelling, corneal opacity 3.0, fluorescein retention 3.0; irritation categories III, IV and IV, respectively; irritation index 149.

Applicant's summary and conclusion

Interpretation of results:

other: Not irritating

Remarks:

in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance caused no swelling of the cornea, slight corneal opacity and slight fluorescein retention in the in vitro Isolated Chicken Eye test (OECD TG 438). The substance is considered to be not irritating to the eyes.

Executive summary:

The eye irritating properties of the substance were tested in vitro, in an Isolated Chicken Eye (ICE) test according to OECD TG 438 and in compliance with GLP. The test included a negative control (saline) and a positive control (5% benzalkonium chloride). The isolated chicken eyes were treated with of 30 μL of the substance for 10 seconds and then rinsed with 20 mL saline. Corneal swelling, corneal opacity and fluorescein retention were measured to disclose possible adverse effects. The substance caused no swelling of the cornea (0%), slight corneal opacity (score 1 out of max. 4) and slight fluorescein retention (score 1 out of max.3). The corresponding irritation categories were I, II and II, respectively. The irritation index (calculated as maximum corneal swelling + maximum mean opacity (x 20) + mean fluorescein score (x 20)) was 40 (out of max. 200). The positive control produced moderate corneal swelling (29%), severe opacity (score 3.0) and severe fluorescein retention (score 3.0). The calculated irritation index was 149. Based on the results of this in vitro study, the substance is considered to be not irritating to the eyes.