Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-09-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 2011-07-19, 2011-07-20 & 2011-07-21; Date of signature 2011-08-31

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

EC Number:

939-227-3

Cas Number:

84238-39-1

Molecular formula:

Not applicable (a generic Molecular formula cannot be provided for this substance)

IUPAC Name:

Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

Test animals / tissue source

Species:

other: Excised Bovine Cornea

Strain:

other: Not Applicable

Details on test animals or tissues and environmental conditions:

Not applicable

Test system

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
-Amounts(s) applied (volume or weight with unit): 0.75 mL of the test material was applied to triplicate corneas.
-Concentration (if solution): the test material was used as supplied.

VEHICLE
- No vehicle used

Duration of treatment / exposure:

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes.

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Details on study design:

TEST SITE
- Area of exposure: 0.75 mL of the test material was applied to triplicate corneas.
- Percentage coverage: the test material was topically applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea.
-Type of wrap used: none.

REMOVAL OF TEST SUBSTANCE
-Washing (if done): at the end of the exposure period the test material was removed from the anterior chamber and each cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM.
-Time after start of exposure: 10 minutes post exposure

SCORING SYSTEM
- Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
- Opacity Measurement: the change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Permeability Measurement: the corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
- In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value).

DATA INTERPRETATION
- A test material that induces an in vitro irritancy score > than or equal to 55.1 is defined as an ocular corrosive or severe irritant.
- Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.
- Visual Observation: the condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

120 minutes post rinsing

Value:

0.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The neat test material induced an in vitro irritancy score of 0.8 and was therefore considered not to be an ocular corrosive or severe irritant.
The corneas treated with the test item were clear post treatment and post incubation.

Any other information on results incl. tables

RESULTS

CORNEAL OPACITY AND PERMEABILITY MEASUREMENT

- Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Table 1.

CORNEAL EPITHELIUM CONDITION

- The condition of the cornea immediately after rinsing and at the final opacity measurement is given in Table 2.

- The corneas treated with the test item or negative control item were clear post treatment and post incubation. 

- The corneas treated with the positive control item were cloudy post treatment and post incubation.

IN VITRO IRRITANCY SCORE

- The results are summarised as follows:

Treatment	In Vitro Irritancy Score
Test Material	0.8
Negative Control	1.3
Positive Control	36.1

CRITERIA FOR AN ACCEPTABLE TEST

- The positive control In Vitro irritancy Score was within the range of 30.9 to 67.7. The positive control acceptance criterion was therefore satisfied.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD)		In vitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post-Incubation	Post-Incubation-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	1	2	3	3	1		0.085
	2	4	4	5	1		0.028
	3	2	2	2	0		0.020
					0.7*		0.044+		1.3
Positive Control	4	4	32	30	26	25.3	1.173	1.129
	5	4	32	29	25	24.3	0.753	0.709
	6	2	24	21	19	18.3	0.891	0.847
						22.7·		0.895·	36.1
Test Item	7	4	5	5	1	0.3	0.048	0.004
	8	3	4	4	1	0.3	0.046	0.002
	9	3	4	5	2	1.3	0.058	0.014
						0.7·		0.006·	0.8

OD= Optical density                 * = Mean of the post treatment-pre‑treatment values      += Mean permeability              ·= Mean corrected value

Table 2: Corneal epithelium condition

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control	1	clear	clear
	2	clear	clear
	3	clear	clear
Positive Control	4	cloudy	cloudy
	5	cloudy	cloudy
	6	cloudy	cloudy
Test Item	7	clear	clear
	8	clear	clear
	9	clear	clear

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The neat test item was considered not to be an ocular corrosive or severe irritant and as the IVIS was below 3, the test item should not be classified for eye irritancy.