4-amino-5-(ethylsulphonyl)-o-anisic acid

Endpoint summary

Description of key information

Skin irritation

Key study: Test method OECD 430. GLP study. Test item do not lead to skin corrosion/severe irritation.

Eye irritation

Key study: Test method OECD 438. GLP study. No prediction can be made, since the ICE Class combination of the 3 endpoints were 2xII and 1xI (the first run) and 2xIII and 1xI (the second run). The test item did not cause eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 2014 - 26 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): 4-amino-5-(ethylsulphonyl)-o-anisic acid
- Physical state: Solid
- Analytical purity: 99.81%
- Lot/batch No.: MP1032.31

Test system:

isolated skin discs

Source species:

rat

Source strain:

Wistar

Details on animal used as source of test system:

SOURCE OF THE BIOLOGICAL MATERIAL:
TEST ANIMALS:
- Source: Centre for Experimental Medicine at the Medical University in Katowice
- Age at study initiation: 21 days old (hair follicles in dormant phase)
- Housing: Plastic cage covered with a wire bar lid. Dimensions: 58 x 37 x 21 cm. Bedding: UV-sterilized wood shavings.
- Diet (e.g. ad libitum): ad libitum, "Murigran” standard granulated laboratory fodder
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 ºC
- Humidity (%): 55-70%
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Vehicle:

water

Details on test system:

SKIN DISC PREPARATION
- Procedure used:The animals were euthanized by intraperitoneal administration of morbital at a dose of 200 mg/kg b.w.
After euthanasia, the dorso-lateral skin of each animal was removed and stripped of excess subcutaneous fat by carefully peeling it away from the skin using a paper towel. The skin discs were cut out using a scalpel. Each skin disc was placed over one of the ends of a PTFE (polytetrafluoroethylene) tube, ensuring that the epidermal surface was in contact with the tube. A rubber ‘O’ ring was press-fitted over the end of the tube to hold the skin in place and excess tissue is trimmed away. The rubber ‘O’ ring was then carefully sealed to the end of the PTFE tube with petroleum jelly. The tube was supported by a spring clip inside a receptor chamber containing MgSO4 solution (154 mM). The skin disc should be fully submerged in the MgSO4 solution. As many as 11 skin discs with a diameter of 20-mm each were obtained from a single rat skin. Two of them were used to control the quality of the procedure, whereas the remaining nine were used for the purpose of the experiment.
The age of the animals was particularly important. The age of 29 days ensures that the hair follicles are in the dormant phase before adult hair growth begins [SOP/T/57].

- Quality control for skin discs:
Before the test, the electrical resistance of two skin discs obtained from each rat was measured as a quality control procedure for each animal skin. Both discs should give resistance values greater than 10 kΩ for the remainder of the discs to be used for the test.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:21-22ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1 time washing with jet of tap water at up to 30ºC.
- Observable damage in the tissue due to washing:No

DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:] : Not necessary,since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

NUMBER OF INDEPENDENT TESTING RUNS: 3 replicates

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the mean TER value obtained for the test item is less than or equal to 5 kΩ and the skin disc is obviously damaged.
- The test substance is considered to be non-corrosive to skin if the mean TER value obtained for the test item is greater than 5 kΩ, or the mean TER value is less than or equal to 5 kΩ, and the skin disc shows no obvious damage.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

VEHICLE
- Amount(s) applied (volume or weight with unit):test item moistened with 150 μL of destiled water.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):150 μL.

POSITIVE CONTROL
- Amount(s) applied (volume or weight):150 μL.

Duration of treatment / exposure:

24 hours

Number of replicates:

3

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

MEAN TER value, TEST 1

Value:

ca. 15.75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

SD+-041

Irritation / corrosion parameter:

transcutaneous electrical resistance (in kΩ)

Run / experiment:

MEAN TER value TEST 2

Value:

ca. 19.57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

SD+- 0.43

Irritation / corrosion parameter:

other: Morphologics effects

Remarks on result:

not measured/tested

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system:No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES - the mean TER values were higher than 5 kΩ
- Acceptance criteria met for positive control: YES - the mean TER value is less than 5 kΩ.

The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

On the grounds of the study, the test item belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Table No.1: Results of the control transcutaneous electrical resistance test (TER):

Animal number	Skin disc number	TER value (kΩ)
1	1	17.14
	2	16.57
2	1	14.72
	2	13.98

The skin discs gave the resistance values greater than 10 kΩ; therefore, the remainder of the skin discs of the animals could have been used in the experiment.

Table No.2: Results of the transcutaneous electrical resistance test (TER):

Animal number	Tested substance	Skin disc number	TER value (kΩ)	Mean TER value ± SD (kΩ)
1	Positive control – 36% HCl	1	0.90	0.90 ± 0.01
		2	0.89
		3	0.90
	Negative control – distilled water	1	18.75	18.63 ± 0.46
		2	18.13
		3	19.02
	Test item	1	15.28	15.75 ± 0.41
		2	16.03
		3	15.95
2	Positive control – 36% HCl	1	0.88	0.89 ± 0.01
		2	0.88
		3	0.90
	Negative control – distilled water	1	19.78	19.29 ± 1.11
		2	19.01
		3	19.09
	Test item	1	19.74	19.57 ± 0.52
		2	19.08
		3	19.88

The concurrent mean values for the positive and negative controls were within the acceptable ranges for the method:

Positive control: 0.5-1.0 kΩ

Negative control: 10 -25 kΩ

The mean TER results for the skin discs treated with the test item were equal to 15.75 kΩ (animal no. 1) and 19.57 kΩ (animal no. 2).

Table No.3: Gross changes on the surface of the treated skin discs:

Animal number	Tested substance	Skin disc number	Gross changes
1	Positive control – 36% HCl	1	perforation
		2	perforation
		3	perforation
	Negative control – distilled water	1	no changes
		2	no changes
		3	no changes
	Test item	1	no changes
		2	no changes
		3	no changes
2	Positive control – 36% HCl	1	perforation
		2	perforation
		3	perforation
	Negative control – distilled water	1	no changes
		2	no changes
		3	no changes
	Test item	1	no changes
		2	no changes
		3	no changes

The gross examination showed that the positive control skin discs exhibited skin perforation, whereas the negative control skin discs and the ones treated with the test item did not reveal any changes.

Interpretation of results:

GHS criteria not met

Remarks:

non-corrosive/severe irritant.

Conclusions:

The substance do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Executive summary:

The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD Guideline 430 Guideline and EU Method B.40. Skin discs used in the experiment were obtained from two 29-day-old rats. The test item (ground to a powder) was uniformly applied to the epidermal surface of the skin disc placed inside a tube. Positive (36% hydrochloric acid) and negative (distilled water) controls were conducted concurrently. Three skin discs obtained from each animal were used for the test item and three for each control item. The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. Then, they were removed by washing with a jet of tap water and the surface tension of the skin was reduced by adding 70% ethanol. After removing the ethanol the tissue was hydrated by the addition of 3 mL of a solution of MgSO4 (154 mM). A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩ by placing the databridge electrodes on either side of the skin disc. The skin discs were subjected to a gross examination. The mean TER results were equal to 15.75 kΩ (animal no. 1) and 19.57 kΩ (animal no. 2). The concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations of the skin discs did not reveal any pathological changes. On the grounds of the study, it may be stated that the test item do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 October 2014 - 24 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): 4-amino-5-(ethylsulphonyl)-o-anisic acid
- Physical state: Solid
- Analytical purity: 99.81%
- Lot/batch No.: MP1032.31

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice
- Age at study initiation: 7-week-old
- Weight at study initiation: 1.5 - 2.5 kg

BIOLOGICAL MATERIAL
After sedation of the chickens by electric shock and incision of the neck for bleeding, their heads were transported to the laboratory in a plastic container at
ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container.
The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 30 minutes.

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): 0.03 g

Application and removal of the test item and the control items:
There were three groups, i.e. one group treated with the test item and two control groups, including a positive one (imidazole) and a negative one (physiological
salt) used concurrently in the study to ensure its quality. The test item and the control items were tested on three eyeballs each. Immediately following the zero
reference measurements, the eyeballs in their holders were removed from the superfusion apparatus and placed in a horizontal position in order to apply the test
item and the control items. The test item (ground to a powder) and the item used in the positive control (imidazole) were applied in the amount of 0.03 g,
whereas the item used in the negative control (physiological salt) was applied in a volume of 0.03 mL. The test item and the control items were uniformly applied
to the corneal surface for 10 seconds. Then, they were rinsed from the eye with 20 mL of physiological salt at ambient temperature.

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

4 hours after post-treatment rinse

Number of animals or in vitro replicates:

9 eyebalss (3 eyeballs per treatment: test item, negative control, positive control)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes (physiological salt)
- Time after start of exposure: After 10 seconds of exposure.

MEASURED PARAMETERS:
Pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.
At all observation times points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The
quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure.

Scoring system
Florescein retention:
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

Corneal opacity:
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent areas; details of the iris 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

Corneal swelling:
The degree of corneal swelling was determined by measuring corneal thickness with a SP-100 pachymeter.

Gross evaluation:
To determine whether any morphological effects, e.g. pitting of corneal epithelial cells, roughening of the corneal surface, and sticking of the test item to the
cornea were visible.

Histopathological evaluation of the treated corneas:
Following the final evaluation of the treated eyeballs (240 minutes after the application of the test item and the control items), the eyeballs were fixed in a 4%
solution of formaldehyde. Next, specimens were collected (one specimen in the plane including the cornea, lens, and optic nerve). The tissue material was
dehydrated and prepared using a paraffin technique. Paraffin blocks were cut into smaller parts, whose thickness was 5 μm, with a microtome and stained using
Hematoxylin and Eosin. The following layers of the cornea were evaluated: anterior epithelium, anterior elastic lamina (Bowman’s membrane), corneal stroma,
posterior elastic lamina (Descemet’s membrane), and posterior epithelium. All treated eyeballs were subject to this evaluation.

Irritation parameter:

fluorescein retention score

Run / experiment:

MEAN, 1 run

Value:

ca. 1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE class II

Irritation parameter:

fluorescein retention score

Run / experiment:

MEAN, RUN 2

Value:

ca. 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE class III

Irritation parameter:

cornea opacity score

Run / experiment:

MEAN, RUN 1

Value:

ca. 1.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE class II

Irritation parameter:

cornea opacity score

Run / experiment:

MEAN, RUN 2

Value:

ca. 2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE class III

Irritation parameter:

percent corneal swelling

Run / experiment:

MEAN, RUN 1

Value:

ca. 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE class I

Irritation parameter:

percent corneal swelling

Run / experiment:

MEAN, RUN 2

Value:

ca. 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

ICE class I

Irritation parameter:

morphological effects

Remarks on result:

not measured/tested

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:yes (SEE BELOW)
- Acceptance criteria met for positive control:yes (SEE BELOW)

Fluorescein retention :RUN 1 and RUN 2 - The mean fluorescein retention values for the concurrent positive and negative controls were 3.0 (ICE class IV) for imidazole and 0.0 (ICE class I) for physiological salt
Corneal opacity :RUN 1 and RUN 2 - The mean corneal opacity values for the concurrent positive and negative controls were 4.0 (ICE class IV) for imidazole and 0.0 (ICE class I) for physiological salt
Corneal swelling : FIRST RUN:The mean corneal swelling values for the positive control (imidazole) were from 56.7 to 99.2 (ICE class IV). As for the concurrent negative control samples (physiological salt), no swelling was observed (ICE class I).
: SECOND RUN: The mean corneal swelling values for the positive control (imidazole) were from 45.6 to 78.9 (ICE class IV). As for the concurrent negative control samples (physiological salt), no swelling was observed (ICE class I)

These values fell within the acceptable ranges.

Irritant / corrosive response data:

On the grounds of the study and the overall in vitro Irritancy Classification, it may be stated that the test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints were 2xII and 1xI (the first run) and 2xIII and 1xI (the second run).

Other effects:

- Lesions and clinical observations: NO
- Ophthalmoscopic findings:NO
- Gross evaluation The negative control eyeballs and the ones treated with the test item did not exhibit any changes of the corneal surface (the first and the second run).

- Histopathological findings:
The first run - The negative control corneas had a normal histological structure. Histopathological examinations of the positive control corneas revealed defects and dissection of the anterior corneal epithelium in all eyeballs (eyeballs no. 4, 5, and 6). These changes confirmed corrosive properties of imidazole.Histopathological examinations of the corneas treated with the test item showed exfoliation of the anterior corneal epithelium (eyeballs no. 2). The corneas had a normal histological structure in eyeball no 1 and no. 3.
The second run -The negative control corneas had a normal histological structure.Histopathological examinations of the positive control corneas revealed defects and dissection of the anterior corneal epithelium (eyeball no. 4, no. 5 and no. 6.); cell vacuolation and detachment of the posterior corneal epithelium (eyeball no. 4). These changes confirmed corrosive properties of imidazole. Histopathological examinations of the corneas treated with the test item showed exfoliation of the anterior corneal epithelium in all eyeballs (eyeballs no. 1, no. 2, and no. 3).
- Effects of rinsing or washing: NO

RESULTS:

Evaluation of fluorescein retention– the first run.

Observation after (minutes)	Test item		Positive control Imidazole		Negative control Physiological saline
	Average	ICE class	Average	ICE class	Average	ICE class
30	1.3	II	3.0	IV	0.0	I

 

Evaluation of fluorescein retention – the second run.

Observation after (minutes)	Test item		Positive control Imidazole		Negative control Physiological saline
	Average	ICE class	Average	ICE class	Average	ICE class
30	2.0	III	3.0	IV	0.0	I

 

Evaluation of corneal opacity– the first run.

Observation after (minutes)	Test item		Positive control Imidazole		Negative control Physiological saline
	Average	ICE class	Average	ICE class	Average	ICE class
30	1.3	II	4.0	IV	0.0	I
75	1.3	II	4.0	IV	0.0	I
120	1.3	II	4.0	IV	0.0	I
180	1.3	II	4.0	IV	0.0	I
240	1.3	II	4.0	IV	0.0	I

 

Evaluation of corneal opacity – the second run.

Observation after (minutes)	Test item		Positive control Imidazole		Negative control Physiological saline
	Average	ICE class	Average	ICE class	Average	ICE class
30	2.0	III	4.0	IV	0.0	I
75	2.0	III	4.0	IV	0.0	I
120	2.0	III	4.0	IV	0.0	I
180	2.0	III	4.0	IV	0.0	I
240	2.0	III	4.0	IV	0.0	I

 

Evaluation of corneal swelling (%) – the first run.

Observation after (minutes)	Test item		Positive control Imidazole		Negative control Physiological saline
	Average	ICE class	Average	ICE class	Average	ICE class
30	0.9*	I	56.7	IV	1.5*	I
75	2.5*	I	66.8	IV	2.5*	I
120	4.5*	I	84.4	IV	4.3*	I
180	5.8*	I	93.1	IV	5.9*	I
240	7.8*	I	99.2	IV	6.8*	I

* - percentage of corneal thickness decrease, no swelling

 

Evaluation of corneal swelling (%) – the second run.

Observation after (minutes)	Test item		Positive control Imidazole		Negative control Physiological saline
	Average	ICE class	Average	ICE class	Average	ICE class
30	0.3*	I	45.6	IV	1.4*	I
75	1.0*	I	53.4	IV	2.4*	I
120	3.1*	I	64.5	IV	4.5*	I
180	5.7*	I	75.0	IV	5.3*	I
240	6.6*	I	78.9	IV	6.5*	I

* - percentage of corneal thickness decrease, no swelling

 Fluorescein retention- the first run.

observation after time t (minutes)	test item     eyeball no.			positive control imidazole   eyeball no.			negative control physiological saline   eyeball no.
	1	2	3	4	5	6	7	8	9
0	0	0	0	0	0	0	0	0	0
30	1	2	1	3	3	3	0	0	0

 Fluorescein retention- the second run.

observation after time t (minutes)	test item     eyeball no.			positive control imidazole   eyeball no.			negative control physiological saline   eyeball no.
	1	2	3	4	5	6	7	8	9
0	0	0	0	0	0	0	0	0	0
30	2	2	2	3	3	3	0	0	0

Corneal capacity:first run

observation after time t (minutes)	test item     eyeball no.			positive control imidazole   eyeball no.			negative control physiological saline   eyeball no.
	1	2	3	4	5	6	7	8	9
0	0	0	0	0	0	0	0	0	0
30	1	2	1	4	4	4	0	0	0
75	1	2	1	4	4	4	0	0	0
120	1	2	1	4	4	4	0	0	0
180	1	2	1	4	4	4	0	0	0
240	1	2	1	4	4	4	0	0	0

Corneal capacity:second run

observation after time t (minutes)	test item     eyeball no.			positive control imidazole   eyeball no.			negative control physiological saline   eyeball no.
	1	2	3	4	5	6	7	8	9
0	0	0	0	0	0	0	0	0	0
30	2	2	2	4	4	4	0	0	0
75	2	2	2	4	4	4	0	0	0
120	2	2	2	4	4	4	0	0	0
180	2	2	2	4	4	4	0	0	0
240	2	2	2	4	4	4	0	0	0

Corneal swelling (%) – the first run

observation after time t (minutes)	test item     eyeball no.			positive control imidazole   eyeball no.			negative control physiological saline   eyeball no.
	1	2	3	4	5	6	7	8	9
0	-	-	-	-	-	-	-	-	-
30	-1.0	-0.7	-1.0	50.8	56.6	62.7	-0.7	-1.0	-2.78
75	-2.0	-2.1	-3.4	60.9	69.2	70.2	-1.0	-1.7	-4.7
120	-4.4	-4.1	-5.1	79.9	83.4	89.8	-2.0	-5.3	-5.7
180	-5.1	-5.8	-6.4	87.3	92.7	99.3	-4.0	-6.6	7.0
240	-6.8	-7.9	-8.8	93.6	99.0	105.1	-5.1	-7.3	-8.1

Corneal swelling (%) – the second run

observation after time t (minutes)	test item     eyeball no.			positive control imidazole   eyeball no.			negative control physiological saline   eyeball no.
	1	2	3	4	5	6	7	8	9
0	-	-	-	-	-	-	-	-	-
30	-0.9	0.3	-0.3	45.4	46.6	44.8	-1.2	-0.6	-2.3
75	-1.5	-0.6	-0.9	56.3	52.3	51.5	-2.4	-1.4	-3.4
120	-5.9	-1.8	-1.5	64.1	60.9	68.6	-6.5	-2.8	-4.3
180	-6.2	-5.9	-5.0	73.3	76.6	75.0	-7.1	-3.4	-5.4
240	-7.1	-6.2	-6.4	78.2	80.9	77.6	-8.2	-4.3	-6.9

Gross evaluation of the treated corneas - the first run.

Observation after (minutes)	Test item			Positive control Imidazole			Negative control Physiological saline
	Eyeball no.			Eyeball no.			Eyeball no.
	1	2	3	4	5	6	7	8	9
30	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
75	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
120	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
180	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
240	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC

NC = no changes

SIGNS = roughening of the corneal surface

 

Gross evaluation of the treated corneas - the second run.

Observation after (minutes)	Test item			Positive control Imidazole			Negative control Physiological saline
	Eyeball no.			Eyeball no.			Eyeball no.
	1	2	3	4	5	6	7	8	9
30	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
75	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
120	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
180	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC
240	NC	NC	NC	SIGNS	SIGNS	SIGNS	NC	NC	NC

NC = no changes

SIGNS = roughening of the corneal Surface

Histological examination of the corneas treated with the test item - the first run.

The negative control corneas had a normal histological structure.

Histopathological examinations of the positive control corneas revealed defects and dissection of the anterior corneal

epithelium in all eyeballs (eyeballs no. 4, 5, and 6). These changes confirmed corrosive properties of imidazole.

Histopathological examinations of the corneas treated with the test item showed exfoliation of the anterior corneal

epithelium (eyeballs no. 2). The corneas had a normal histological structure in eyeball no 1 and no. 3.

Histopathological examination of the corneas treated with the test item - the second run.

The negative control corneas had a normal histological structure.

Histopathological examinations of the positive control corneas revealed defects and dissection of the anterior corneal

epithelium (eyeball no. 4, no. 5 and no. 6.); cell vacuolation and detachment of the posterior corneal epithelium

(eyeball no. 4). These changes confirmed corrosive properties of imidazole.

Histopathological examinations of the corneas treated with the test item showed exfoliation of the anterior corneal

epithelium in all eyeballs (eyeballs no. 1, no. 2, and no. 3).

Interpretation of results:

study cannot be used for classification

Remarks:

ICE Class combination of the 3 endpoints were 2xII and 1xI (the first run) and 2xIII and 1xI (the second run).

Conclusions:

The test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made.

Executive summary:

The isolated chicken eye test (in vitro) was performed according to OECD Guideline 438 and EU Method B.48. The study was conducted in two runs. The first study led to a GHS NC outcome, so a second run of nine eyeballs was conducted to confirm or discard the negative outcome. The test item (ground to a powder) and the item used in the positive control (imidazole) were applied in the amount of 0.03 g, whereas the item used in the negative control (physiological salt) was applied in a volume of 0.03 mL. Three eyeballs were used for the test item and three for each control item. Every time, the test item and the control items were applied to the corneal surface for 10 seconds and kept at temperature between 20 – 23º C. Then, they were rinsed from the eye with 20 mL of physiological salt at ambient temperature. The corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse. At all observation time points, corneal opacity and swelling were evaluated, whereas morphological changes of the corneal surface were recorded. The quantitative determination of fluorescein retention was performed only once, i.e. 30 minutes after the end of the exposure. Following the final evaluation of the treated eyeballs, i.e. 240 minutes after the application of the test item and the control items, the eyeballs were fixed in a 4% solution of formaldehyde in order to allow histopathological examinations to be conducted. The test item did not cause eye damage. The mean fluorescein retention scores for the eyeball treated with test item were 1.3 (ICE class II) and 2.0 (ICE class III) in the first and second round respectively. The mean corneal opacity scores were 1.3 (ICE class II) and 2.0 (ICE class III). No swelling was observed in both rounds (ICE class I). Gross examination did not reveal any changes of the corneal surface. The histopathological examinations of the corneas showed exfoliation of the anterior corneal epithelium in one eye in the first run and in all three eyes in the second one. These results were accepted since the concurrent positive and negative control values fell within the acceptable ranges for the method. On the grounds of the study results and the overall in vitro Irritancy Classification, it may be stated that the test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints were 2xII and 1xI (the first run) and 2xIII and 1xI (the second run).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion: Key study: The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD Guideline 430 Guideline and EU Method B.40. The mean TER results were equal to 15.75 kΩ (animal no. 1) and 19.57 kΩ (animal no. 2). Gross examinations of the skin discs did not reveal any pathological changes. It was determined that the test item do not lead to skin corrosion/severe irritation, since the mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

Eye irritation: Key study: The isolated chicken eye test (in vitro) was performed according to OECD Guideline 438 and EU Method B.48. The test item did not cause eye damage. The mean fluorescein retention scores for the eyeball treated with test item were 1.3 (ICE class II) and 2.0 (ICE class III) in the first and second round respectively. The mean corneal opacity scores were 1.3 (ICE class II) and 2.0 (ICE class III). No swelling was observed in both rounds (ICE class I). Gross examination did not reveal any changes of the corneal surface. The histopathological examinations of the corneas showed exfoliation of the anterior corneal epithelium in one eye in the first run and in all three eyes in the second one. These results were accepted since the concurrent positive and negative control values fell within the acceptable ranges for the method. On the grounds of the study results and the overall in vitro Irritancy Classification, it may be stated that the test item did not cause eye damage. According to UN GHS classification criteria, no prediction can be made, since the ICE Class combination of the 3 endpoints were 2xII and 1xI (the first run) and 2xIII and 1xI (the second run).

Justification for classification or non-classification

Based on the available data, the substance is not classified according to CLP Regulation (EC) no. 1272/2008.