Hexyl hexanoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-May-2018 to 15-May-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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)

Version / remarks:

09 October 2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

09 December 2010

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Hexyl hexanoate

EC Number:

228-952-4

EC Name:

Hexyl hexanoate

Cas Number:

6378-65-0

Molecular formula:

C12H24O2

IUPAC Name:

hexyl hexanoate

Test material form:

liquid

Specific details on test material used for the study:

The test article, a clear colourless liquid, was identified as Hexyl Caproate and was received as follows:

Test Article
Hexyl Caproate

CAS Number
6378-65-0

Storage
Room temperature,

Batch Number
202462

Expiration Date
19 November 2019

Purity
99.29%, assumed 100% for testing

A certificate of analysis for the test article was provided by the Sponsor and is presented in the Attachments.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System and Study Design

Specification
Corneas from bovine eyes were obtained from a local abattoir. The eyes were removed after slaughter, completely immersed in physiological saline in a suitably sized container and transported on the same day to the testing facility.

Assessment on Arrival
On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

Excision and Preparation of Corneas
The isolated corneas were stored in a petri dish with cMEM (Earle’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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The negative control substance was physiological saline, 750 µL, supplied by Eurovet Animal Health, Bladel, The Netherlands
The positive control substance was ethanol, 750 µL
The test article was administered without dilution.

Duration of treatment / exposure:

120 ± 10 minutes at 32±1°C.

Duration of post- treatment incubation (in vitro):

Corneas were incubated in a horizontal position for 10±1 minutes at 32±1°C

Number of animals or in vitro replicates:

3

Details on study design:

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 (Earle’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.

Treatment of Corneas and Opacity Measurements:
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.

The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity=[(I0/I)-0.9894]/(0.0251)

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.

The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean value

Value:

ca. -0.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

opacity reading of 1.8

Positive controls validity:

valid

Remarks:

opacity reading of 16

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other:

Run / experiment:

Corneal Permeability, mean value

Value:

ca. 0.002

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

The mean group corrected optical density for the negative control was -0.016

Positive controls validity:

valid

Remarks:

The mean group corrected optical density for the positive control was 1.625

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

RESULTS:
Hexyl Caproate was tested neat.

Table 1 of Appendix 1 summarizes the opacity, permeability and in vitro irritancy scores of Hexyl Caproate and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Table 2 - 5. The individual in vitro irritancy scores for the negative controls ranged from 0.8 to 2.7. The individual positive control in vitro irritancy scores ranged from 31 to 48 (Appendix 2, Table 5). The corneas treated with the negative and positive control items were translucent and
turbid, respectively, after the 10 minutes of treatment. The corneas treated with Hexyl Caproate showed opacity values ranging from -1.2 to 1.5 and permeability values ranging from 0.002 to 0.003. The corneas were translucent after the 10 minutes of treatment with Hexyl Caproate. No pH effect of the test item was observed on
the rinsing medium. Hence, the in vitro irritancy scores ranged from -1.2 to 1.5 after 10 minutes of treatment with Hexyl Caproate.

DISCUSSION:
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 40 and within two standard deviations of the current historical positive control mean (Appendix 3). It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Hexyl Caproate did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.0 after 10 minutes of treatment.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, since Hexyl Caproate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Hexyl Caproate as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of Hexyl Caproate was tested through topical application for 10 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 202462 of Hexyl Caproate was a clear colourless liquid with a purity of 99.29%. The test item was applied as it is (750 µL) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive control (Ethanol) was 40 and was 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.

Hexyl Caproate did notinduce ocular irritation through both endpoints, resulting in a meanin vitroirritancy score of 0.0 after 10 minutes of treatment. In conclusion, since Hexyl Caproate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.