Reaction mass of 2-(cis-4-methylcyclohexyl)propan-2-ol and 2-(trans-4-methylcyclohexyl)propan-2-ol and cis-1-methyl-4-(propan-2-yl)cyclohexanol and trans-1-methyl-4-(propan-2-yl)cyclohexanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12-13 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other 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)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

10 January 2013

Test material

Test material information

Constituent 1

Reference substance name:

cis-α,α,4-trimethylcyclohexanemethanol

EC Number:

230-795-1

EC Name:

cis-α,α,4-trimethylcyclohexanemethanol

Cas Number:

7322-63-6

Molecular formula:

C10H20O

IUPAC Name:

cis-2-(4-methylcyclohexyl)propan-2-ol

Constituent 2

Reference substance name:

trans-α,α,4-trimethylcyclohexanemethanol

EC Number:

225-844-9

EC Name:

trans-α,α,4-trimethylcyclohexanemethanol

Cas Number:

5114-00-1

Molecular formula:

C10H20O

IUPAC Name:

trans-2-(4-methylcyclohexyl)propan-2-ol

Constituent 3

Reference substance name:

cis-4-isopropyl-1-methylcyclohexanol

Cas Number:

3901-95-9

Molecular formula:

C10H20O

IUPAC Name:

cis-4-isopropyl-1-methylcyclohexanol

Constituent 4

Reference substance name:

trans-4-isopropyl-1-methylcyclohexanol

Cas Number:

3901-93-7

Molecular formula:

C10H20O

IUPAC Name:

trans-4-isopropyl-1-methylcyclohexanol

impurity 1

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No. MP8 du 29/10/2013
Purity: 98.8% (sum of the 4 main constituents)
Name of the test item (as cited in the study report): DIHYDROTERPINEOL MULTICONSTITUENT
IUPAC Name of the test item: Reaction mass of cis-2-(4-methylcyclohexyl) propan-2-ol and trans-2-(4-methylcyclohexyl) propan-2-ol and
cis- 4-isopropyl-1-methylcyclohexanol and trans- 4-isopropyl-1-methylcyclohexanol
Synonym: Reaction mass of cis-α,α-4-trimethyl-cyclohexanemethanol and trans-α,α-4-trimethyl-cyclohexanemethanol and cis-1-methyl-4-(1-methylethyl)-cyclohexanol and trans-1-methyl-4-(1-methylethyl)-cyclohexanol
Physical state: colourless – slightly amber liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 28 October 2015

Test animals / tissue source

Species:

other: Bovine eye

Details on test animals or tissues and environmental conditions:

Origin: Bovine eyes were obtained from EVA, Saint-Pierre-sur-Dives, France.
Age: Bovine cattle were up to 12 months old.
Transport from supplier to testing laboratory: The eyes were transported to testing laboratory at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.

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): 750 μL (± 8 μL) of test item was applied on each cornea
- Concentration (if solution): undiluted

CONTROLS:
Negative control: 0.9 % sodium chloride solution
Positive control: 10 % sodium hydroxide solution

Duration of treatment / exposure:

The test item was evaluated by using a treatment time of 10 minutes ± 30 seconds.

Duration of post- treatment incubation (in vitro):

2 hours ± 10 minutes in a water bath at 32 ± 1°C.

Number of animals or in vitro replicates:

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

Details on study design:

Dose formulation application:
As the test item was a non-viscous liquid, the closed-chamber method was used as follows: the test and control items were introduced into the anterior chamber of the corneal holder through the dosing holes, to cover the epithelial side of the cornea. Then the dosing holes were sealed.

Treatment of corneas:
Corneas obtained from freshly slaughtered cattle (from abattoir) were mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber. For pre-incubation, both chambers of the corneal holder were filled with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) and pre-incubated for 1 h and 5 minutes ± 5 minutes at 32 ± 1 °C. Then MEM was removed & then refilled with fresh cMEM and corneas were examined macroscopically for any defects. Then, the opacity of the cornea was measured to obtain OPT0. The medium was removed from anterior chamber and the test item was applied onto the epithelium of the cornea. After application of the dose formulation, the holders were incubated, vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at 32 ± 1 °C, for 10 minutes. At the completion of the treatment period, the test and control items were removed from the front opening of the anterior chamber and the corneas were rinsed. The corneas were then incubated for 2 h ± 10 minutes in a water bath at 32 ± 1 °C and the second opacity measurement (OPT2) was performed.

Permeability determination
- Application of sodium fluorescein: After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution (4 mg/mL). The holders were then incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at 32 ± 1 °C for 90 ± 5 minutes. At the end of the incubation, the optical density at 490 nm (OD490) of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea.

OTHERS:
- Macroscopic examination: After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

93

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

20

Negative controls validity:

valid

Positive controls validity:

valid

In vivo

Irritant / corrosive response data:

- Macroscopic examinations: No notable opaque spots or irregularities were observed on negative control corneas. Fluorescein fixation was observed on all test item-treated corneas. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.
- In Vitro Irritancy Score (IVIS) for test item and positive control were 20 and 187, respectively.

Other effects:

None

Any other information on results incl. tables

Table 7.3.2/1: Eye irritation – results

GROUP		OPACITY				PERMEABILITY		SCORE
	Holder	OPT0	OPT2	OPT2-OPT0	cOPT	OD490 nm	cOD490 nm
Negative control	22	3	2	-1.0	-	0.002	-	-
	35	3	2	-1.0	-	0.004	-	-
	8	3	2	-1.0	-	0.021	-	-
	Mean	-	-	-1.0	-	0.009	-	-
	SD	-	-	0.0	-	0.010	-	-
Test item	45	2	0	-2.0	-1.0	0.106	0.097	0
	5	3	15	12.0	13.0	1.744	1.735	39
	38	3	16	13.0	14.0	0.439	0.430	20
	Mean	-	-	-	8.7	-	0.754	20
	SD	-	-	-	8.4	-	0.866	19.3
Positive control	42	3	129	126.0	127.0	5.248	5.239	206
	18	3	111	108.0	109.0	5.312	5.303	189
	16	4	115	111.0	112.0	3.736	3.727	168
	Mean	-	-	-	116.0	-	4.756	187
	SD	-	-	-	9.6	-	0.892	18.9

 

OD: optical density

cOD: corrected optical density

cOPT: corrected corneal opacity

SD: standard deviation

OPT0: corneal opacity before treatment

OPT2: corneal opacity after the 2 h recovery period

Applicant's summary and conclusion

Interpretation of results:

other: not predictable

Conclusions:

The ocular corrosive or severe irritant potential of test item dihydroterpineol multiconstituent could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (no Category).
According to OECD Guideline 437, further testing should be conducted for classification and labeling purposes.

Executive summary:

In an in vitro eye irritation study performed according to OECD Guideline 437 and in compliance with GLP, 750 μL (± 8 μL) of undiluted test item dihydroterpineol multiconstituent was applied to isolated bovine corneas for 10 minutes followed by an incubation period of 2 h at 32 °C. Three corneas were used for each treated series (undiluted test item; negative control; positive control). 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).

No notable opaque spots or irregularities were observed on negative control corneas. Fluorescein fixation was observed on all test item-treated corneas. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control. In Vitro Irritancy Score (IVIS) for test item and positive control were 20 and 187, respectively. 

 

Therefore, the ocular corrosive or severe irritant potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (no Category).

According to OECD Guideline 437, further testing should be conducted for classification and labeling purposes.