Reaction Mass of Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, (alpha.R,1R,6S)- and Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, (alpha.S,1R,6S)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 11, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 437 without any deviation.

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:

2013

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)

Test material

Test material information

Constituent 1

Reference substance name:

(3R)-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]-3-hexanol

Cas Number:

253454-12-5

Molecular formula:

C15H30O

IUPAC Name:

(3R)-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]-3-hexanol

Constituent 2

Reference substance name:

(3S)-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]-3-hexanol

Cas Number:

253454-10-3

Molecular formula:

C15H30O

IUPAC Name:

(3S)-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]-3-hexanol

Test material form:

liquid

Details on test material:

- Physical state: Colourless liquid
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature, protected from light, in the original container (aluminium bottle)

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir as a by-product from freshly slaughtered animals
- Number of animals: not mentioned
- Characteristics of donor animals (e.g. age, sex, weight): adult (12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported to the test facility over ice packs on the same day of slaughter
- Time interval prior to initiating testing: The corneas were prepared immediately on arrival
- indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: None

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 was applied on each cornea
- Concentration: Undiluted

Duration of treatment / exposure:

10 minutes at 32 ± 1 °C

Observation period (in vivo):

- Corneal opacity was measured pre-treatment, post-treatment and post-incubation (after 120 minutes of incubation).
- Application of Sodium Fluorescein (4 mg/mL) and corneal permeability was measured after 90 min of incubation at 32 ± 1 °C.

Duration of post- treatment incubation (in vitro):

120 minutes at 32 ± 1 °C.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes.
The medium from both chambers of each holder was replaced with fresh complete MEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitomete. The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls

NEGATIVE CONTROL USED
0.9% w/v sodium chloride solution. Batch 300999 104. Purity: 0.9%. Expiry: 01 January 2015.

POSITIVE CONTROL USED
Ethanol. Batch SZBA0290. Expiry: 06 August 2014.

APPLICATION DOSE AND EXPOSURE TIME
- Application:0.75 mL of the test item or control items
- Exposure time: 10 minutes at 32 ± 1 ºC

TREATMENT METHOD: not reported

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

POST-EXPOSURE INCUBATION: 120 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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 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.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of Anthos 2001 microplate reader (OD492)
- Others: The corneas were retained after testing for possible conduct of histopathology

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean 3 corneas

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS = 2.3

Positive controls validity:

valid

Remarks:

IVIS = 46.7

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: not included

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤4.7 and permeability ≤0.080. The negative control acceptance criteria were therefore satisfied
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 27.8 to 51.0. The positive control acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: not reported

Any other information on results incl. tables

Table 7.3.2/1: 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	7	2	3	5	3		0.052
	18	2	2	2	0		0.035
	9	2	2	4	2		0.047
					1.7*		0.045¨		2.3
Positive Control	4	2	24	25	23	21.3	1.822	1.777
	5	3	23	24	21	19.3	1.516	1.471
	6	1	24	29	28	26.3	1.664	1.619
						22.3·		1.623·	46.7
Test Item	1	2	3	3	1	0.0	0.018	0.000
	2	1	1	1	0	0.0	0.019	0.000
	3	1	1	1	0	0.0	0.038	0.000
						0.0·		0.000·	0.0

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

Table 7.3.2/2: Corneal Epithelium Condition Post Treatment and Post Incubation

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, with an IVIS < 3, test item does not require classification for eye irritation or serious eye damage according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In an in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, 0.75 mL of undiluted test item was applied to isolated bovine corneas for 10 minutes followed by an incubation period of 120 minutes.Three corneas were used for each treated series (undiluted test item; negative control; positive control: ethanol). Before the treatment, a first opacity measurement was performed using an opacitometer. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). 

The test item, negative and positive control induced an IVIS of 0.0, 2.3 and 46.7, respectively.

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

The positive control In Vitro Irritancy Score was within the range of 27.8 to 51.0, therefore the acceptance criterion was satisfied. The negative control gave opacity of ≤4.7 and permeability ≤0.080, therefore the acceptance criterion was satisfied.

Under the test conditions, with an IVIS < 3, test item does not require classification for eye irritation or serious eye damage according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.