2-Butenedioic acid, 2-methyl-, 1,4-bis(2-ethylhexyl) ester, (2Z)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP screening OECD 438 study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Principles of method if other than guideline:

The Enucleated Eye Test with isolated eyes of chickens is a well validated and accepted in vitro test system. It has been recognized as a valuable alternative to the Draize eye irritation test, because it represents a test system nearest to the in vivo test, without the need to use live animals. In the Isolated Chicken Eye Test (ICET) the test compound is applied in one single dose onto the cornea of isolated eyes, which are obtained from slaughter animals.
This method can provide detailed information about the effects of test items on the cornea, and is useful to compare products, to classify test items for regulatory use when they are severe irritants or corrosive to the eye, and thus to avoid the need to test severe eye irritants in vivo. The test is described in OECD 438 and is approved by international regulatory agencies as a replacement for the identification of corrosives and severe irritants in the in vivo Rabbit eye assay (OECD 405).

GLP compliance:

no

Remarks:

The laboratory has a GLP certicificate

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(2-ethylhexyl) (2Z)-2-methylbut-2-enedioate

EC Number:

807-715-4

Cas Number:

1354569-12-2

Molecular formula:

C21 H38 O4

IUPAC Name:

1,4-bis(2-ethylhexyl) (2Z)-2-methylbut-2-enedioate

Test material form:

other: liquid

Details on test material:

Stored at controlled room temperature (15-25◦C, below 70 RH%)

Test animals / tissue source

Species:

other: isolated chicken eye

Strain:

other: Ross 308 strain

Details on test animals or tissues and environmental conditions:

Chicken heads collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.

After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (v/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline dripping from a stainless steel tube, at a rate of approximately 3 or 5 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus, were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not have varied by more than ±5% between the -45 and the zero time. Slight changes in thickness (0% to 3%) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

Amount / concentration applied:

After the zero reference measurements, the eye (in its retainer) was removed from the chamber, and placed on a layer of tissue with the cornea facing upwards. The eyes were held in horizontal position, while the test item was applied onto the cornea. The test item was applied in a volume of 30 μL onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance and taking care not to damage or touch the cornea.

The positive control eyes were treated with 30 μL Trichloroacetic acid 30 (w/v) %. The negative control eye was treated with 30μL of Saline (Salsol solution, NaCl 0.9% w/v).

Duration of treatment / exposure:

The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item. The time while the eye was out of the chamber was limited to the minimum.

Observation period (in vivo):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Minor variations within ±5 minutes were considered acceptable. The cornea thickness and cornea opacity were measured at all time points.
Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

5 isolated eyes (1 saline, 2 test item, 2 Triclotoacetic acid TCAA)

Details on study design:

Superfusion chamber apparatus (32 +- 1.5 oC) during the acclimatization and treatment periods.
Examination with a slit lamp microscope.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Maximum corneal swelling

Basis:

mean

Time point:

other: 30, 75, 120, 180 and 240 min after rinse

Score:

0

Max. score:

3

Remarks on result:

other: ICE Class I

Results 2

Irritation parameter:

other: Maximum corneal opacity

Basis:

mean

Time point:

other: 30, 75, 120, 180 and 240 min after rinse

Score:

0.5

Max. score:

1

Remarks on result:

other: ICE Class I

Results 3

Irritation parameter:

other: Fluorescein retention

Basis:

mean

Time point:

other: 0 and 30 min after rinse

Score:

0.25

Max. score:

0.5

Remarks on result:

other: ICE Class I

Irritant / corrosive response data:

Please see the individual results tables, ICE classification and interpretation criteria in the attachments 1, 2 and 3.

Any other information on results incl. tables

Assessments were made at t=0 (base line), 30, 75, 120, 180 and 240 minutes (cornea thickness and opacity).

Fluorescein retention at t=0 and 30 minutes after the post-treatment rinse.

ICE Classes were determined for mean maximum corneal swelling at up to 75 min and 240 min, mean maximum corneal opacity and mean fluorescein retention.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: OECD 438

Conclusions:

In an in vitro screening test on chicken's eye, the test substance was not ocular corrosive or a severe irritant. The test results indicate that the test item is not irritating at all (ICE classification 3 X I).

Executive summary:

In an in vitro screening test on chicken's eye, the test substance was not ocular corrosive or a severe irritant. The test results indicate that the test item is not irritating at all (ICE classification 3 X I). The test was conducted according to OECD 438 but not under GLP. The results of the screening tests are rated as Klimisch 2 and applied for classification and labelling of the substance.