methyl 2-hydroxybut-3-enoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-04-12 to 2021-05-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2017-02-14

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018-06-25

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

methyl 2-hydroxybut-3-enoate

EC Number:

831-109-9

Cas Number:

5837-73-0

Molecular formula:

C5H8O3

IUPAC Name:

methyl 2-hydroxybut-3-enoate

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni utca 129. Hungary
- Number of animals: Not indicated, but all eyes used in the assay were from the same groups of eyes collected on one specific day.
- Storage, temperature and transport conditions of ocular tissue: After collection, the heads were wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box). The heads were then transported to the laboratory approx. within 2 hours from collection. The temperature was between 19.3 ºC and 20.2 ºC during the transport.
- Indication of any existing defects or lesions in ocular tissue samples: One small drop of fluorescein solution 2 (w/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 slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged.
- Selection and preparation of corneas: Eyelids were carefully cut away with scissors, avoiding damaging the cornea. If the cornea was in good condition (as determined by fluorescein examination), the eyeball was carefully removed from the orbit. The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye 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.
- Quality check of the isolated corneas: After being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected. 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 μL

Number of animals or in vitro replicates:

Three eyes were used for the test substance and positive control treatment and one eye was used for the negative control treatment.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES: If the corneas were in good condition (as determined by fluorescein staining), the eyeball was carefully removed from the orbit. The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. 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). 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 saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes.

EQUILIBRATION AND BASELINE RECORDINGS: If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers 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 baseline (t=0) for each individual eye.

NUMBER OF REPLICATES: Three eyes were used for the test substance and positive control treatment and one eye was used for the negative control treatment.

NEGATIVE CONTROL USED: NaCl (9 g/L saline)

POSITIVE CONTROL USED: Benzalkonium chloride solution (5%)

APPLICATION DOSE AND EXPOSURE TIME: The test item was applied unchanged at a volume of 30 µL for 10 seconds.

OBSERVATION PERIOD: Eyes were evaluated at approx. 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE:
- Volume and washing procedure after exposure period: After treatment, the eyes were immediately rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The cornea opacity was measured at all time points. Eyes were examined with a slit lamp microscope.
- Damage to epithelium based on fluorescein retention: Fluorescein retention was determined at baseline (t = 0) and 30 minutes after the post-treatment rinse. Eyes were examined with a slit lamp microscope.
- Swelling: The cornea thickness was measured at all time points. Eyes were examined with a depth measuring device on the slit lamp microscope (slit-width setting: 9½, equaling 0.095 mm).
- Macroscopic morphological damage to the surface: If morphological effects were observed, the classification of these findings were done by study director (e.g. pitting or loosening of the epithelium.
- Other: Corneas were stored in preservative fluid (4% formaldehyde) for potential histopathology after all measurements were done. Histopathology of the corneas was not performed as a clear result was obtained.

SCORING SYSTEM:
- Mean corneal swelling (%): See "Any other information on materials and methods incl. tables"
- Mean maximum opacity score: See "Any other information on materials and methods incl. tables"
- Mean fluorescein retention score at 30 minutes post-treatment: See "Any other information on materials and methods incl. tables"

DECISION CRITERIA: Decision criteria as indicated in the TG were used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean of three eyes

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Fluorescein retention of 0.7

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Mean of three eyes

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Corneal opacity of 3.7

Results 3

Irritation parameter:

corneal swelling 

Run / experiment:

Mean of three eyes

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Corneal swelling of 35% within 240 minutes

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method, the laboratory demonstrated the technical proficiency in a separate study using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In an Isolated Chicken Eye Test according to OECD guideline 438, the test item is categorized as corrosive to the eyes (Category 1).

Executive summary:

The Isolated Chicken Eye Test (ICET) was conducted according to OECD guideline 438 and GLP and aimed to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 μL/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS/CLP)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test item exposure) were determined at each of the above time points. The test item, the positive control ( 5% solution of Benzalkonium chloride), and the negative control (NaCl, 9 g/L saline) were applied in a volume of 30 μL/eye, in such a way that the test items evenly cover the whole cornea surface of each tested eye. Three test item treated eyes, three positive control eyes and one negative control eye were used in this study. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, the overall ICE classes of the test item were once II (based on the fluorescein retention of 0.7) and twice IV (based on the cornea swelling of 35 % within 240 minutes and corneal opacity score of 3.7). The positive control was classed as corrosive/severely irritating, UN GHS/CLP Classification: Category 1. The positive and negative controls showed the expected results and were within the respective historical control ranges. The negative control had not significant effects on the eye and the positive control showed the expected effects for a Category 1 substance (three times ICE class IV) (The experiment was therefore considered to be valid.

 

In this ICET, the overall ICE classes were once II (based on the fluorescein retention of 0.7) and twice IV (based on corneal swelling of 35% within 240 minutes and the opacity score of 3.7). According to the guideline OECD 438, the test item is categorized as corrosive (Category 1).