4-acetyl-2-methylbenzoic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start: 30 July 2019, experimental end: 30 July 2019

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-acetyl-2-methylbenzoic acid

EC Number:

856-079-4

Cas Number:

55860-35-0

Molecular formula:

C10H10O3

IUPAC Name:

4-acetyl-2-methylbenzoic acid

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

chicken

Strain:

other: Ross 308

Details on test animals or tissues and environmental conditions:

Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni u 129., Hungary
Chicken heads were collected from a commercial abattoir after chickens (approximately 7 weeks old) had been slaughtered for human consumption. Heads were collected by a slaughter house technician. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a sealed plastic box (4-5 heads/box).
The heads were immediately transported to Charles River Laboratories Hungary Kft. at ambient temperature. The heads were received at Charles River Laboratories Hungary Kft. and processed within 2 hours of collection.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg of test substance, 30 mg of powdered imidazole (positive control), 30 μL of physiological saline (0.9% w/v NaCl)

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

The treated eyes were evaluated after 30, 75, 120, 180 and 240 minutes after the rinses following the exposure period.

Number of animals or in vitro replicates:

Negative control: single treated eye
Test substance and positive control: 3 replicates

Details on study design:

Preparation of eyes: The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting 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.
Examination and acclimatisation: The prepared eye was placed in a steel retainer. The cornea was positioned vertically with the eye in the correct relative position (same position as in the chicken head), taking care to avoid putting too much pressure on the eye by the retainer. Due to 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 retainer holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes (approximately nine to twelve) were selected, after being placed in the superfusion apparatus they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to clearly see the physiological 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 with an optical pachymeter on a slit-lamp microscope, which was set at a 0.095 mm slit-width. Any eye with cornea thickness deviating by 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 was started and 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.
Base line assessment: At the end of the acclimatization period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a base line (t=0) for each individual eye. The corneal thickness of the eyes should not change by more than 5% between the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes. Following the equilibration period, the fluorescein retention was also 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.
Treatment: The eye was held in horizontal position, while the test item was applied onto the centre of the cornea. The powdered test or positive control substance was applied in an amount of 30 mg onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, taking care not to damage or touch the cornea. The negative control eye was treated with 30 μL of physiological saline.
Removal: 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 at least 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item if possible. The eye was returned to the chamber after rinsing. The time while the eye was out of the chamber was limited to the minimum required. Additional gentle rinsing with 20 mL saline (3 or 4 times) was performed at each time point when the test item or the positive control material was observed to remain on the cornea.
Observation and assessment of corneal effects: Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse. A Haag-Streit BP 900 slit-lamp microscope was used for the measurements and was set at a 0.095 mm slit-width.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

corneal swelling 

Run / experiment:

Up to 75 minutes

Value:

2.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0% corneal swelling

Positive controls validity:

valid

Remarks:

10.2% corneal swelling

Results 2

Irritation parameter:

corneal swelling 

Run / experiment:

Up to 240 minutes

Value:

2.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0% corneal swelling

Positive controls validity:

valid

Remarks:

27.3% corneal swelling

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

Up to 240 minutes

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Score of 0.0 for corneal opacity

Positive controls validity:

valid

Remarks:

Score of 4.0 for corneal opacity

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

Up to 240 minutes

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Score of 0.0 for fluorescein retention

Positive controls validity:

valid

Remarks:

Score of 3.0 for fluorescein retention

Applicant's summary and conclusion

Interpretation of results:

other: Additional information is required for definitive classification.

Conclusions:

The test substance is not classified as a severe irritant and not classified as non-irritant.

Executive summary:

An in vitro eye irritation study of the test substance was performed under GLP on isolated chicken’s eyes to OECD TG 438 (25 June 2018). The study used 3 eyes for test substance treatment, alongside 1 eye treated with the negative control physiological saline and 3 positive control eyes treated with powdered imidazole. After the baseline measurements, each eye in the treatment group was held in a horizontal position and 30 mg of powdered test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. In the experiment the positive control eyes were treated in a similar way with 30 mg of powdered imidazole and the negative control eye was treated with 30 μL of physiological saline. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (pitting or loosening of the epithelium) were evaluated over a four-hour observation period. No significant corneal swelling (mean 2.7%) was observed during the four-hour observation period on the eyes treated with the substance. Slight corneal opacity change (severity 1.0) and moderate fluorescein retention change (severity 2.0) was noted on three eyes treated with the substance. Test item was stuck on all corneal surfaces (3/3) after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 30 minutes after the post-treatment rinse. The substance was considered to be not severely irritating, but could not be identified as non-irritating. The negative and positive control group results demonstrated that the study was valid and demonstrated the sensitivity of the assay, identifying physiological saline as non-irritating and imidazole as severly irritating.