Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Oct 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

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 Jul 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

National Institute of Pharmacy and Nutrition, Budapest, Hungary

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate

EC Number:

224-580-1

EC Name:

Sodium 1-(3,4-dihydro-6-methyl-2,4-dioxo-2H-pyran-3-ylidene)ethanolate

Cas Number:

4418-26-2

Molecular formula:

C8H7O4.Na

IUPAC Name:

sodium 1-(6-methyl-2,4-dioxo-2H-pyran-3(4H)-ylidene)ethanolate

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., Sárvár, Hungary
- Storage, temperature and transport conditions of ocular tissue: Transport of the chicken heads approximately within 2 h from collection under ambient temperature (19.3 - 20.4 °C). After collection, the heads were inspected for appropriate quality, wrapped with paper moistened with saline and placed in closable box
- Time interval prior to initiating testing: 45 - 60 min

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.03 g

Duration of treatment / exposure:

10 s

Duration of post- treatment incubation (in vitro):

30, 75, 120, 180 and 240 min

Number of animals or in vitro replicates:

Number of eyes for the negative control: 1
Number of eyes for the positive control: 3
Number of eyes for the test substance: 3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Selection:The surface of the cornea was treated with fluorescein solution (2% (w/v)) for a few seconds and examined with slit lamp microscope to ensure that the cornea was not damaged.


EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline for each individual eye. The cornea thickness of the eyes should not change by more than ± 5 - 7% within approximately 45 - 60 min before the start of application. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NEGATIVE CONTROL USED
Saline solution (9 g/L)

POSITIVE CONTROL USED
Imidazole (0.03 g)

APPLICATION DOSE AND EXPOSURE TIME
0.03 g for test item and positive control, 30 uL for negative control; exposure period: 10 s

OBSERVATION PERIOD
30, 75, 120, 180 and 240 min

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The cornea was rinsed thoroughly with 20 mL saline solution at ambient temperature

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity is evaluated by using the area of the cornea that is most densely opacified for scoring. The mean corneal opacity value for all test eyes is calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score is then given for the test substance.
- Damage to epithelium based on fluorescein retention: Fluorescein retention is evaluated at the 30 minute observation time point. The mean fluorescein retention value of all test eyes is then calculated for the 30-minute observation time point, and used for the overall category score given for the test substance.
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: subjective according to the interpretation of the study director

SCORING SYSTEM AND DECISION CRITERIA:
- Mean corneal swelling (%)
Cornea swelling: For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5%) following application are counted as zero. Large negative numbers (> -12%) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5% to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect.

CS at time t : [(CT at time t - CT at t=0)/CT at t=0] x 100
Mean CS at time t = [FECS (at time t) + SECS (at time t) + TECS (at time t)] / 3

CS : Cornea swelling
CT : Cornea thickness
FECS : First eye cornea swelling
SECS : Second eye cornea swelling
TECS : Third eye cornea swelling
Mean CS : The mean percentage of corneal swelling
At time t : Observation time at 30, 75, 120, 180 or 240 min after the post-treatment rinse
At t=0 : Reference value

- Mean maximum opacity score
Score 0: No opacity
Score 0.5: Very faint opacity
Score 1: Scattered or diffuse areas; details of the iris are clearly visible
Score 2: Easily discernible translucent area; details of the iris are slightly obscured
Score 3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
Score 4: Complete corneal opacity; iris invisible

ΔCO at time t : CO at time t – CO at t=0
Mean CO (at time t) : [FEΔCO (at time t) + SEΔCO (at time t) + TEΔCO(at time t)]/3

CO : Cornea opacity
ΔCO : Difference between cornea opacity and cornea opacity reference value
FEΔCO : Difference between first eye cornea opacity and first eye cornea opacity reference value
SEΔCO : Difference between second eye cornea opacity and second eye cornea opacity reference value
TEΔCO : Difference between third eye cornea opacity and third eye cornea opacity reference value
Mean CO : The mean corneal opacity value
At time t : Observation time at 30, 75, 120, 180 or 240 min after the post-treatment rinse
At t=0 : Reference value

- Mean fluorescein retention score at 30 minutes post-treatment
Scores for fluorescein retention:
Score 0: No fluorescein retention
Score 0.5: Very minor single cell staining
Score 1: Single cell staining scattered throughout the treated area of the cornea
Score 2: Focal or confluent dense single cell staining
Score 3: Confluent large areas of the cornea retaining fluorescein

ΔFR at time t : FR at time t – FR at t=0
Mean FR (at time t) : [FEΔFR (at time t) + SEΔFR (at time t) + TEΔFR(at time t)]/3

FR : Fluorescein retention
ΔFR : Difference between fuorescein retention and fuorescein retention reference value
FEΔFR : Difference between first eye fuorescein retention and first eye fuorescein retention reference value
SEΔFR : Difference between second eye fuorescein retention and second eye fuorescein retention reference value
TEΔFR : Difference between third eye fuorescein retention and third eye fuorescein retention reference value
Mean FR : The mean fuorescein retention value
At time t : Observation time at 30 min after the post-treatment rinse
At t=0 : Reference value

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

75 min

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2%

Positive controls validity:

valid

Remarks:

24%

Results 2

Irritation parameter:

percent corneal swelling

Run / experiment:

240 min

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2%

Positive controls validity:

valid

Remarks:

29%

Results 3

Irritation parameter:

cornea opacity score

Run / experiment:

mean maximum o 3 eyes

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0

Positive controls validity:

valid

Remarks:

4.0

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

mean of 3 eyes

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0

Positive controls validity:

valid

Remarks:

3.0

Other effects / acceptance of results:

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

Any other information on results incl. tables

Table 1 Results of ICE Assay

Na-Dehydroacetic acid (0.03 g/eye)
Relative Observation time (min)	Corneal thickness (instrument units)								Corneal opacity score						Fluorescein retention score
	-45-60	0	Δ	30	75	120	180	240	0	30	75	120	180	240	0	30
5	59	59	0%	60	60	61	61	61	0	0	0	0.5	0.5	0.5	0	0.5
6	60	60	0%	60	60	60	60	60	0	0	0	0.5	0.5	0.5	0	0
7	60	60	0%	61	61	61	61	61	0	0	0	0	0	0	0	0
	Mean Swelling			1%	1%	2%	2%	2%	MOS	0.0	0.0	0.3	0.3	0.3	MFRS	0.2
Imidazole (0.03 g/eye)
Relative Observation time (min)	Corneal thickness (instrument units)								Corneal opacity score						Fluorescein retention score
	-45-60	0	Δ	30	75	120	180	240	0	30	75	120	180	240	0	30
2	62	62	0%	70	74	74	75	77	0	4	4	4	4	4	0	3
3	60	60	0%	72	75	75	77	79	0	4	4	4	4	4	0	3
4	60	60	0%	73	76	78	79	79	0	3	4	4	4	4	0	3
	Mean Swelling			18%	24%	25%	27%	29%	MOS	3.7	4.0	4.0	4.0	4.0	MFRS	3.0
NaCl (9g/L saline solution: 30 uL/eye)
Relative Observation time (min)	Corneal thickness (instrument units)								Corneal opacity score						Fluorescein retention score
	-45-60	0	Δ	30	75	120	180	240	0	30	75	120	180	240	0	30
2	60	60	0%	61	61	61	61	61	0	0	0	0	0	0	0	0.5
	Swelling			2%	2%	2%	2%	2%	MOS	0	0	0	0	0	FRS	0.0

 Δ = cornea thickness change between the start of the acclimatization period (t = -60 to -45 min) and the baseline (t = 0)

MOS =  Mean opacity score

OS = Opacity score

MFRS = Mean fluorescein retention score

FRS = Fluorescein retention score

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

Under the conditions of this study the test material was shown not to be an eye irritant.

Executive summary:

In an isolated chicken eye test method conducted to assess potential eye irritation the test material was shown not to be irritating to the eye.