Carboxylic acids, di-, C5-9

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jul 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

adopted June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI, National Institute of Pharmacy and Nutrition, Budapest, Hungary

Test material

Test material information

Constituent 1

Reference substance name:

Carboxylic acids, di-, C5-9

EC Number:

701-329-9

Molecular formula:

C5di: C5H8O4 C6di: C6H10O4 C7di: C7H12O4 C8di: C8H14O4 C9di: C9H16O4

IUPAC Name:

Carboxylic acids, di-, C5-9

Test animals / tissue source

Species:

chicken

Strain:

other: COBB 500

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 mg

Duration of treatment / exposure:

10 sec

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

Test and positive control item: 3 eyes each
Negative control item: 1 eye

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and transported to the laboratory at ambient temperature as soon as possible. After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a closed plastic box. The heads were received at the laboratory and processed within 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 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a 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 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 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.

EQUILIBRATION AND BASELINE RECORDINGS
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 physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/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. 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 see clearly 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, 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 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 baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 minute and the zero time. No significant changes in thickness (-1.6% or 1.6%) were observed in two eyes and no changes in thickness were observed in other eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
Test and positive control item: 3 eyes each
Negative control item: 1 eye

NEGATIVE CONTROL USED
0.9% sodium chloride

POSITIVE CONTROL USED
30 mg powdered imidazole

APPLICATION DOSE AND EXPOSURE TIME
Test item and positive control item: 30 mg for 10 sec
Negative control item: 30 µL

OBSERVATION PERIOD
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 min after the post-treatment rinse. Minor variations within approximately ± 5 min were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible. Additional gentle rinsing with at least 3 x 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was still observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: not reported
- Damage to epithelium based on fluorescein retention: not reported
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: not reported
- Macroscopic morphological damage to the surface: not reported

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: The evaluation and decision criteria as indicated in the TG were used (see Table

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class IV

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class IV

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 75 min

Value:

17.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class III

Results 4

Irritation parameter:

percent corneal swelling

Run / experiment:

up to 240 min

Value:

17.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse. The positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. No other morphological effect was observed in the study.

DEMONSTRATION OF TECHNICAL PROFICIENCY: not reported

ACCEPTANCE OF RESULTS:
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range. This study was considered to be valid.

Any other information on results incl. tables

Table 1. Summary of results of test item treated eyes

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	17.1%	III
Mean maximum corneal swelling at up to 240 min	17.1%	II
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

Table 2. Table of individual data - test item

Study Code:	19/192-038C S															Strain:		COBB 500
Date of Exposure:	18 July 2019															Test Item:		Carboxylic Acids, di-, C4-11
Chamber number ↓	Corneal thickness (instrument units)															Corneal opacility score							Fluorescein retention
Relative observation time (min) →	-45	0	C h a n g e	30	change at 30	75	change at 75	Max change up to 75	120	change at 120	180	change at 180	240	change at 240	Max change up to 240	0	30	75	120	180	240	Max Δ Opac	0	30	Δ Flu ret
14	62	63	1.6%	68	7.9%	72	14.3%	14.3%	72	14.3%	72	14.3%	72	14.3%	14.3%	0	4	4	4	4	4	4.0	0	3	3.0
15	63	62	-1.6%	70	12.9%	74	19.4%	19.4%	74	19.4%	74	19.4%	74	19.4%	19.4%	0	4	4	4	4	4	4.0	0	3	3.0
16	62	62	0.0%	69	11.3%	73	17.7%	17.7%	73	17.7%	73	17.7%	73	17.7%	17.7%	0	4	4	4	4	4	4.0	0	3	3.0
Mean values:					10.7%		17.1%	17.1%		17.1%		17.1%		17.1%	17.1%							4.00			3.00

Table 3. Individual data - imidazole

Study Code:	19/192-038CS															Strain:		COBB 500
Date of Exposure:	18 July 2019															Positive Control:		Imidazole
Chamber number  ↓	Corneal thickness (instrument units)															Corneal opacity score							Fluorescein retention
Relative observation time (min)  →	-45	0	C h a n g e	30	change at 30	75	change at 75	Max change up to 75	120	change at 120	180	change at 180	240	change at 240	Max change up to 240	0	30	75	120	180	240	Max Δ Opac	0	30	Δ Flu ret
17	63	63	0.0%	66	4.8%	71	12.7%	12.7%	73	15.9%	78	23.8%	82	30.2%	30.2%	0	4	4	4	4	4	4.0	0	3	3.0
18	62	62	0.0%	67	8.1%	70	12.9%	12.9%	73	17.7%	77	24.2%	80	29.0%	29.0%	0	4	4	4	4	4	4.0	0	3	3.0
19	63	63	0.0%	67	6.3%	70	11.1%	11.1%	74	17.5%	79	25.4%	82	30.2%	30.2%	0	4	4	4	4	4	4.0	0	3	3.0
Mean values:					6.4%		12.2%	12.2%		17.0%		24.5%		29.8%	29.8%							4.00			3.00

Table 4. Individual data - physiological saline

Study Code:	19/192-038CS															Strain:		COBB 500
Date of Exposure:	18 July 2019															Negative Control:		Physiological saline (NaCl 0.9% w/v)
Chamber number  ↓	Corneal thickness (instrument units)															Corneal opacity score							Fluorescein retention
Relative observation time (min)  →	-45	0	C h a n g e	30	change at 30	75	change at 75	Max change up to 75	120	change at 120	180	change at 180	240	change at 240	Max change up to 240	0	30	75	120	180	240	Max Δ Opac	0	30	Δ Flu ret
20	63	63	0.0%	63	0.0%	63	0.0%	0.0%	63	0.0%	63	0.0%	63	0.0%	0.0%	0	0	0	0	0	0	0.00	0	0	0.00

5. Summary table for UN GHS classification

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II:	False
No severe corneal morphological changes:	True
Test item was not stuck to the cornea at 240 minutes after the post- treatment rinse:	True
Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	True
Corneal opacity = 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	True
Severe loosening of epithelium (in at least 1 eye):	False
Criteria for “No prediction can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study:	False

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria met, classified as Eye Irrit. 1 according to Regulation (EC) No 1272/2008

Conclusions:

Eye Irrit. 1

Executive summary:

In this reliable and GLP-conform Isolated Chicken Eye (ICE) study according to OECD 438, exposure of chicken eyes with the test item for 10 sec, resulted in corneal swelling (both at 75 and 240 min after treatment) as well as increased corneal opacity and fluorescein retention, compared to the negative control. The test item is therefore considered as severly irritating to the eye.

The experiment met the validity criteria, therefore the study was considered to be valid.