3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

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:

other: Guideline study, to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2016

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

EC Number:

276-650-6

EC Name:

3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

Cas Number:

72403-67-9

Molecular formula:

C15H24O2

IUPAC Name:

[3-(4-methylpent-3-en-1-yl)cyclohex-3-en-1-yl]methyl acetate; [4-(4-methylpent-3-en-1-yl)cyclohex-3-en-1-yl]methyl acetate

Details on test material:

Identification: Myraldyl Acetate
Appearance: Colourless to pale yellow liquid
Batch: SC00014327
Purity/Composition: See Certificate of Analyses
Test item storage: At room temperature protected from light
Stable under storage conditions until: 28 April 2017 (expiry date)

Test animals / tissue source

Species:

other: Bovine eyes were used as soon as possible after slaughter.

Strain:

not specified

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 minimise 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.

Preparation of corneas
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32  1C. The corneas were incubated for the minimum of 1 hour at 32+/- 1 °C.

Cornea selection and Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a
cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Results and discussion

In vivo

Results

Irritation parameter:

other: IVIS

Basis:

mean

Score:

>= -1.7 - <= -0.3

Reversibility:

fully reversible within: 10 minutes

Remarks on result:

other: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Irritant / corrosive response data:

The corneas treated with Myraldyl Acetate showed opacity values ranging from -1.7 to -0.3 and permeability values ranging from 0.000 to 0.006. The corneas were clear after the 10 minutes of treatment with Myraldyl Acetate. No pH effect of the test item was observed on the rinsing medium.
Hence, the in vitro irritancy scores ranged from -1.7 to -0.3 after 10 minutes of treatment with Myraldyl Acetate.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Migrated information

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 (10% (w/v) Benzalkonium Chloride) was 141 and was within two standard deviations of the current historical positive control mean. (APPENDIX 3, Table 6). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Myraldyl Acetate did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.9 after 10 minutes of treatment. Since Myraldyl Acetate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.