3,4-dihydroxybenzaldehyde

Endpoint summary

Description of key information

Skin irritation (in vitro): Key study. Test method according to OECD 439, GLP study. The mean percent viability of the treated tissues was 104.7% (considered 100%), versus 1.4% in the positive control (5% SDS). Therefore, the test item is not irritant to the skin. 

Eye irritation (in vitro): Key study.  Test method according to OECD 438, GLP study. The test item results were 3 x II. therefore the test item category is  "no prediction can be made". 

Eye irritation (in vitro): Key study.  Test method according to OECD 492, GLP study. The test item result was 2.7% percent tissue viability versys 47.8% in the positive control. Therefore, the test item category has to be identified as potentially requiring classification and labelling according to UN GHS ÇCategory 2 or Category 1.  

Serious eye damage/eye irritation: Final conclusion on classification of the test substance: Based on the result obtained in the ICE Test (OECD 438) it can be excluded that the test item should be classified as “Category 1”. Based on the result obtained in the EpiOcular™ Test (OECD 492) it can be excluded that the test item should be classified as “No category”, but no prediction can be made between Category 1 and Category 2. Thus, Category 2 (eye irritation) should be the correct classification for the test substance.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 June to 30 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

SkinEthic RHE model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The SkinEthic RHE model has been validated for irritation testing and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be
suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE
- Tissue batch number(s): 22-RHE-089
- Production date: N/A
- Delivery date: 28.06.2022
- Date of initiation of testing: 28.06.2022

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 42 minutes at room temperature.
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 36,8º and 37ºC, 5% CO2
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: there is no direct interaction between the test item and MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDIC
TION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered as non-irritant to skin if the mean percent viability after 60 minutes exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2) if the mean percent tissue viability after 60 minutes exposure and 42 hours of post-treatment incubat ion is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after 60 minutes exposure and 42 hours of posttreatment incubation is ≤ 50% and in absence of information on a skin corrosion test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 at room temperature and

Duration of post-treatment incubation (if applicable):

41 hours and 06 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

104.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(distilled water)

Positive controls validity:

valid

Remarks:

(5% SDS)

	Well ID	OD	Mean OD / disc	Mean OD / product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	SPL 1	1.063 1.088 1.081	1.063	0.989	107.4	100.0	7.7	No Category
	SPL 2	1.012 0.970 1.000	0.994		100.5
	SPL 3	0.877 0.936 0.921	0.911		92.1
Positive control	SPL 4	0.013 0.015 0.014	0.014	0.014	1.4	1.4	0.2	Category 2 'Irritant'
	SPL 5	0.012 0.013 0.013	0.012		1.2
	SPL 6	0.016 0.016 0.017	0.016		1.6
Test item PH-22/0351	SPL 7	0.971 0.990 1.028	0.996	1.036	100.7	104.7	6.4	No Category
	SPL 8	0.994 1.009 1.003	1.002		101.3
	SPL 9	1.085 1.122 1.122	1.109		112.1

# mean of 3 values (triplicate of the same extract).

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The test substance can be considered as not irritant to skin as the mean percent viability of the treated tissues was found 104.7% in the in vitro RhE test.

Executive summary:

An in vitro skin irritation test was conducted for the test item in a reconstructed human epidermis model (Episkin) according to OECD TG 439 (GLP study).

Three epidermis units, previously moistened with 10 μL of distilled water, were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1mL of DPBS. The epidermis units were then incubated for a 41 hours and 06 minutes post-treatment incubation period in fresh medium at 37ºC, 5% CO2. Then placed into 300 μL of an MTT solution at 1.9 mg/mL for 3 hours at 37ºC, 5% CO2.

The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Under the test conditions, the mean percent viability of the treated tissues was 104.7%, versus 1.4% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is considered as not irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09-08-2022 to 25-08-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Description of the cell system used: EpiOcular TM
- RhCE tissue or hCE cell construct used, including batch number: 0.60 cm2 reconstructed human Cornea-like Epithelium. supplied by MatTek Corporation, batch No. 34980.
- Tissues in their shipping container were equilibrated to room temperature for 20 minutes, then were removed from agarose and placed in 6 wells culture plate which had been filled with 1 mL of 37º pre-warmed assay medium and incubated 20 hours and 25 minutes at standard culture conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Positive and negative control dose: 50 μL
- Test item: 50 mg

Duration of treatment / exposure:

- Positive and negative control dose: 50 μL: applied to the surface of 2 RhCE tissue replicates for 5 hours and 55 minutes.
- Test item: 50 mg: applied to the surface of 2 RhCE tissue replicates for 5 hours and 55 minutes.

Duration of post- treatment incubation (in vitro):

17 hour and 45 minutes post exposure incubation

Number of animals or in vitro replicates:

4 RhCE tissue replicates

Details on study design:

TREATMENT, POST-IMMERSION AND POST-INCUBATION:
- Pre-treatment: tissues were pre-wetted with 20 μL of Ca2+, Mg2+ Free-DPBS and incubated for 30 minutes.
- Treatment: Test item (dose 50 mg) was applied to 2 RhCE tissue (5 hours, 55 minutes); positive and negative control were applied (50 μL) to 2 RhCE tissue (5 hous and 55 minutes).
- Post exposure incubation period: substances were washed from tissues and checked for coloration (noted to be yellowish). Immersion of 25 minutes at room temperature in 5 mL fresh medium and incubated for 17 hour and 45 minutes in 1 mL fresh mediu, 37ºC, 5% CO2.

NUMBER OF REPLICATES: 4

NEGATIVE CONTROL USED:
distilled water - ADL Prochillab - Batch No. 211021

POSITIVE CONTROL USED:
Methyl acetate - Sigma Aldriche, batch No. BCBX8836.

APLICATION DOSE AND EXPOSURE TIME:
Positive and negative control dose: 50 μL: applied to the surface of 2 RhCE tissue replicates for 5 hours and 55 minutes.
Test item: 50 mg: applied to the surface of 2 RhCE tissue replicates for 5 hours and 55 minutes.

POST TREATMENT DURATION
17 hour and 45 minutes post exposure incubation

CELL VIABILITY MEASUREMENTS:
Measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
RhCE constructs were placed in 300 μL of a MTT solution at 1.0 mg/mL (3h and 3 minutes). The precipitated blue formazan product was then extracted form the lower layer of the tissues by placing each insert in 2 mL of isopropanol (1h and 57 minutes).
Concentration of formazan was measured by determining the OD at 570 nm.

OD MEASURES:
OD at 570 nm was measured in triplicate samples of formazan extracts.
The measured OD are proportional to the number of living cells.
The measurement of OD was performed using ELx800 absorbance microplate reader supplied by BioTek and the validated software Gen5 ELISA

EVALUATION AND INTERPRETATION OF RESULTS:

OD values were used to calculate the mean percent tissue viability normalized to the negative control, which was set to 100%. The percentage tissue viability cut-off value distinguishing classified from non-classified test items is 60%. Results interpretation:
- Test item is identified as not requiring classification and labelling according to UN GHS No Category: if mean percent tissue viability after exposure and post-exposur incubation is >60%. In this case, no futher testing in other test methods required.
- The item is identified as potentially requiring classification and labelling accoding to UN GHS (Category 2 or Category 1): if mean percent tissue viability after exposure and post-exposure incubation is ≤60%. futher testing with other methods will be required because the RhCE tets method shows a certain number of false positive and cannot revolve between UN GHS Categories 1 and 2.

Irritation parameter:

percent tissue viability 

Run / experiment:

Mean corrected percent tissue

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- The difference of viability between two tissue replicates should be less than 20%
- Acceptance criteria met for negative control: OD values of the two replicates should be in the range > 0.8 and < 2.8. As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range < 0.4 and < 1.4 for the negative control.
- Acceptance criteria met for positive control: Tissues trated with the positive control substance should show a mean tissue viability <50%.

The difference between the replicates was higher than 20% for positive control. As the mean tissue viability of positive control is less than 50%, this deviation is considered as without impact on the conclusion of the study.

	Well ID	OD	Mean OD / disc	Mean OD / product	Viability %	Mean viability %	SD viability %	Viability difference between replicates %	Conclusion
Negative Control	SPL1	0.804 0.776 0.810	0.797	0.784	101.7	100.0	2.3	3.3	No Category
	SPL2	0.711 0.826 0.775	0.771		98.3
Positive control	SPL 3	0.514 0.513 0.486	0.504	0.375	64.3	47.8	23.4	33.0	UN GHS Category 2 or 1
	SPL 4	0.237 0.255 0.244	0.245		31.3
Test item PH-ZZ/0351	SPL 9	0.068 0.070 0.073	0.070	0.068	8.9	8.7	0.4	0.5	UN GHS Category 2 or 1
	SPL 10	0.066 0.065 0.066	0.066		8.4
Test item PH-22/0351 NSMTT	SPL 7	0.047 0.047 0.047	0.047	0.047	6.0	5.9	0.1	0.1
	SPL 8	0.046 0.045 0.046	0.046		5.9
Test item PH-22/0351 corrected						2.7

Interpretation of results:

other: No prediction can be made (CLP Regulation EC no. 1272/2008)

Conclusions:

Under the experimental conditions adopted and inaccordance with the Regulation EC No. 1272/2008, the test item has to be identified as potentially requiring classification and labelling according to UN GHS Category 2.

Executive summary:

An in vitro RhCE study according to OECD 492 was conducted under GLP conditions to assess the irritation potential of the test item. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Based on the preliminary tests, two tissues (0.6 cm2) were pre-wetted with 20 µL Ca2+ Mg2+Free-DPBS, incubated for 30 min, then dosed topically with 50 mg test item and exposed for 5 hours and 55 minutes at standard culture conditions.After exposure, the tissues were rinsed with DPBS, transferred to fresh assay medium, and incubated for 25 min. Then, they were transferred to fresh medium again and incubated for 17 hours and 45 min at 37ºC, 5% CO2. The MTT assay for cell viability evaluation was performed on the tissues, by incubating them for 3 hours and 3 minutes at standard culture conditions. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically (OD at 570 nm) in order to determine cell viability.

Concurrent positive and negative controls were run in parallel. All validity criteria were met. The relative mean viability of the tissues treated with the test item was 2.7%. This value is below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage. Thus, no prediction can be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption.
- Number of animals: Not specified.
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old. 1.5 - 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):30 mg of test item

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

No post-treatment incubation is performed.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit
by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and t he nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.0ºC and 32.1ºC.
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure using sodium fluor
escein. Corneal thickness was also measured at this time at the corneal apex using the depth measur ing device on the slit-lamp microscope.
Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that were not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes were rejected. (see table appendix No.4 on "Any other information on results incl. tables")
Once all eyes had been examined and approved, the eyes were incubated between 62 and 77 minutes to equilibrate them to the test system prior to dosing.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED
30 μL physiological saline - Dutscher Batch No. 3014011 (one eye)
SOLVENT CONTROL USED: not applicable.
POSITIVE CONTROL USED
Sodium hydroxide – Fisher Scientific, Batch No. 1550248 - 30 mg (three eyes)

APPLICATION DOSE AND EXPOSURE TIME
30 mg of the test item was applied for 10 seconds.

OBSERVATION PERIOD: Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: NO

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points (see table 4)
- Damage to epithelium based on fluorescein retention: Fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item (see table 5)
- Swelling: optical pachymeter on a slit-lamp microscope ((HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I). The slit-width was set at 9 1/2 equalling 0.095 mm. The mean perc
entage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score
was then given for each test item (see table 3).
- Macroscopic morphological damage to the surface: The aim of this evaluation was to determine whe ther any “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea were visible.These findings can vary in severity and may occur simultaneously.

SCORING SYSTEM:
- Mean corneal swelling: It was expressed as a percentage and was calculated from corneal thicknes measurements according to the following formula:
(corneal thickness measurement at time t - corneal thickness at time=0 / corneal thickness at ime=0 )*100
- Mean maximum opacity score:
0 - No opacity,
0.5 -Very faint opacity
1- Scattered or diffuse areas; details of the iris clearly visible
2- Easily discernible translucent area; details of the ris are slightly obscured,
3-Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely
discernible
4-Complete corneal opacity; iris invisible

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

Irritation parameter:

morphological effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No effects

Irritation parameter:

percent corneal swelling

Run / experiment:

Highest mean

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean at 30 minutes post-treatment

Value:

1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Irritation parameter:

cornea opacity score

Run / experiment:

Highest mean

Value:

0.7

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: NO, no morphological effects were noted, whatever the examination time.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES, the combination of the three endpoints for the negative control, physiological saline, was 3xI classified as “No Category”
- Acceptance criteria met for positive control: YES, the combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV,classified as “Corrosive/Severe Irritant ”

Appendix No. 4: Selected eyes for the performance of the ICE test

Chamber	Fluorescein retention	Corneal opacity	Morphological	Corneal thickness (e)
nº1	0.5	0	N.t.R.	0.46
nº2	0.5	0	N.t.R.	0.44
nº3	0.5	0	N.t.R.	0.48
nº4	0.5	0	N.t.R.	0.46
nº5	0.5	0	N.t.R.	0.48
nº6	0.5	0	N.t.R.	0.48
nº7	0.5	0	N.t.R.	0.46
nº8	0.5	0	N.t.R.	0.46
nº9	0.5	0	N.t.R.	0.46
nº10	0.5	0	N.t.R.	0.48
nº11	0.5	0	N.t.R.	0.48
nº12	0.5	0	N.t.R.	0.46
nº13	0.5	0	N.t.R.	0.48
nº14	0.5	0	N.t.R.	0.48
nº15	0.5	0	N.t.R.	0.46
nº16	0.5	0	N.t.R.	0.48
Mean corneal thickness value=				0.47
Range of accepted thickness:				0.42 /≤e≤ / 0.52

N.t.R: Nothing to Report 

Table 9: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESION AFTER TREATMENT 

Endpoint measured	Eye No.	-45	30	75	120	180	240
Corneal opacity	4	0	0.5	0.5	0.5	0.5	0.5
	5	0	0.5	0.5	0.5	0.5	0.5
	6	0	1	1	1	1	1
Mean		0	0.7	0.7	0.7	0.7	0.7
ICE class		II
Fluorescein retention	4	0.5	1
	5	0.5	1
	6	0.5	2
Mean		0.5	1.3
ICE class			II
Corneal thickness	4	0.46	0.52	0.52	0.54	0.54	0.54
	5	0.48	0.48	0.48	0.50	0.50	0.50
	6	0.48	0.50	0.50	0.50	0.52	0.52
Corneal swelling (%)	4	0	13	13	17	17	17
	5	0	0	0	4	4	4
	6	0	4	4	4	8	8
Mean			6	6	9	10	10
ICE class					II
Combination of the 3 Endpoints		3 X II
CLASSIFICATION		No prediction can be made

Note: No morphological effects were noted, whatever the examination time 

Table 8: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT 

Endpoint measured	Eye No.	-45	30	75	120	180	240
Corneal opacity	4	0	0.5	0.5	0.5	0.5	0.5
	5	0	0.5	0.5	0.5	0.5	0.5
	6	0	1	1	1	1	1
Mean		0	0.7	0.7	0.7	0.7	0.7
ICE class		II
Fluorescein retention	4	0.5	1
	5	0.5	1
	6	0.5	2
Mean		0.5	1.3
ICE class			II
Corneal thickness	4	0.46	0.52	0.52	0.54	0.54	0.54
	5	0.48	0.48	0.48	0.50	0.50	0.50
	6	0.48	0.50	0.50	0.50	0.52	0.52
Corneal swelling (%)	4	0	13	13	17	17	17
	5	0	0	0	4	4	4
	6	0	4	4	4	8	8
Mean			6	6	9	10	10
ICE class					II
Combination of the 3 Endpoints		3 X II
CLASSIFICATION		No prediction can be made

Note:
( - ): evaluation of corneal swelling not possible (Corneal opacity = 4 at each examination time)
Severe loosening of the corneal epithelium noted from 30 minutes post-dose in eyes No. 1, No. 2 and
No. 3 

Table 7: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT. 

Endpoint measured	Eye No.	-45	30	75	120	180	240
Corneal opacity	16	0	0	0	0	0	0
Mean		0.0	0.0	0.0	0.0	0.0	0.0
ICE class					I
Fluorescein retention	16	0.5	0.5
Mean		0.5	0.5
ICE class		0.5	0.5
Corneal thickness	16	0.48	0.48	0.48	0.48	0.48	0.48
Corneal swelling (%)	16	0	0	0	0	0	0
Mean		0.0	0	0	0	0	0
ICE class					I
combination of the 3 Endpoints		3 x I
CLASSIFICATION		No category

Interpretation of results:

other: other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The test item result was determined as 'no prediction can be made'.

Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hidroxide (positive control) or 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results
of each endpoint were assigned to ICE classes according to OECD guideline 438. According to CLP Regulation EC no. 1272/2008 the test item result was 'no prediction can be made' for eye irritation and serious eye damage, since the combination of the 3 endpoints for the test item was 3 x II.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available