4-isopropyl-1-methylcyclohexene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-isopropyl-1-methylcyclohexene

EC Number:

226-841-5

EC Name:

4-isopropyl-1-methylcyclohexene

Cas Number:

5502-88-5

Molecular formula:

C10H18

IUPAC Name:

1-methyl-4-(propan-2-yl)cyclohex-1-ene

impurity 1

Reference substance name:

rel-(1R,4R)-1-isopropyl-4-methylcyclohexane

Cas Number:

1678-82-6

Molecular formula:

C10H20

IUPAC Name:

rel-(1R,4R)-1-isopropyl-4-methylcyclohexane

impurity 2

Reference substance name:

rel-(1S,4S)-1-isopropyl-4-methylcyclohexane

Cas Number:

6069-98-3

Molecular formula:

C10H20

IUPAC Name:

rel-(1S,4S)-1-isopropyl-4-methylcyclohexane

Test material form:

liquid

Specific details on test material used for the study:

Identification: Para-Menthene
Appearance: Colourless to pale yellow liquid
Batch: VE00460490
Purity/Composition: See Certificate of Analysis
Test item storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 16 December 2018 (expiry date)
Additional information
Test Facility test item number: 208591/A
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum-foil

Test animals / tissue source

Species:

cattle

Strain:

other: Cornea only used.

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:

physiological saline

Controls:

yes

Amount / concentration applied:

The medium from the anterior compartment was removed and 750 l of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 10  1 minutes at 32  1C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.

Duration of post- treatment incubation (in vitro):

Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120  10 minutes at 32  1C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

The corneas treated with the test item showed opacity values ranging from -1.7 to 2.8 and permeability values ranging from -0.006 to 0.005.

Value:

1.1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

In vivo

Irritant / corrosive response data:

The individual in vitro irritancy scores for the negative controls ranged from -0.4 to 1.2. The individual positive control in vitro irritancy scores ranged from 28 to 48. The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

The corneas treated with the test item showed opacity values ranging from -1.7 to 2.8 and permeability values ranging from -0.006 to 0.005. Two corneas were translucent and one slightly translucent after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.8 to 2.8 after 10 minutes of treatment with the test item.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 35 and within two standard deviations of the current historical positive control mean.
It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.0 after 10 minutes of treatment.

Executive summary:

The test item did not induce ocular irritation after 10 minutes of treatment.