4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with [(dimethylamino)methyl]phenol and piperazine

Endpoint summary

Description of key information

Skin irritation / corrosion:

In Vitro Skin Corrosion, key study:

The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes based on OECD 431. The test item was considered to be non-corrosive to the skin.

In vitro skin irritation, key study:

The skin irritation potential of the test item was evaluated using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours based on OECD 439. The test item was classified as non-irritant.

In vivo skin irritation, supporting study:

AJICURE PN-23, AJICURE MY-24 and AJICURE PN-H were tested in primary skin irritation study accordance with OECD/404 and EU Council Directive. AJICURE PN-23, AJICURE MY-24 and AJICURE PN-H were classified as “No risk phrase (nonirritant to the skin)”.

 

Eye irritation:

In vivo eye irritation, key study:

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit according to OECD 405. The treated eyes appeared normal at the 48 or 72-Hour observation. The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labeling of Chemicals.

BCOP, supporting study:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage based on OECD 437. The In Vitro irritancy score was 3.8 for test item. Thus, no prediction of eye irritation can be made.

Primary eye irritation, supporting study:

AJICURE MY-H was applied to animals at 5 % suspended with Liquid paraffin in the test.

It caused slight redness, minimal swelling and so much discharge in all animals at 1hr after eye application. These irritative findings were disappeared at 24hr after application. From the above results, a 5% suspension of AJICURE MY-H in liquid paraffin was judged as 'Minimal Irritant' to rabbit eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 July 2017 - 14 July 2017

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)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: 161129
Purity: 100%
Physical state/Appearance: Pale brown powder
Expiry Date: 28 November 2017
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. mCorrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:EpiDerm™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s):25829
- Delivery date: 11 July 2017
- Date of initiation of testing: 04 July 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:50 μL of the test item were applied to the tissues in turn. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed.
- Observable damage in the tissue due to washing: None specified.
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
50 μL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.


NUMBER OF REPLICATE TISSUES: 2


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL of the test item


NEGATIVE CONTROL
- Amount(s) applied (volume or weight):50 μL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time.


POSITIVE CONTROL
- Amount(s) applied (volume or weight):50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 μL of sterile water was added for wetting of the test item to increase tissue surface contact.

Duration of treatment / exposure:

3 minutes and 60 minutes.

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

100.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

96

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes in compliance with OECD 431. Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viabilities for each treatment group were as follows:

3 minute the test item was 100.3%

60 minute the test item was 96.0%

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 26 July 2017 and 08 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPIDERM™ Reconstructed Human Epidermis Model and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

EC No. 761/2009 of 23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: THEMIS (EC 500-429-8)
CAS Number: 159034-96-5
EC Number: EC 500-429-8
Batch: 161129
Purity: 100%
Physical state/Appearance: Pale brown powder
Expiry Date: 28 November 2017
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

other: The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.

Details on test system:

Test System
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 25 July 2017
EpiSkinTM Tissues (0.38cm2) lot number : 17-EKIN-030
Maintenance Medium lot number : 17-MAIN3-030
Assay Medium lot number : 17-ESSC-029

Study Design
Pre-Test Procedure

Assessment of Direct Test Item Reduction of MTT

MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory : Yes
Temperature Indicator Color Satisfactory : Yes
Agar Medium Color Satisfactory : Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 3”C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg test item and 10µL controls

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

100.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

91.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

99.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Direct MTT Reduction

The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.  

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was a very pale brown color. This color was attributed to the intrinsic color of the test item itself. It was therefore unnecessary to run color correction tissues.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 97.2% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.

It was considered unnecessary to perform IL-1alpha analysis as the results of the MTT test were unequivocal.

Table Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570of tissues	Mean OD570of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.928	0.843	0.090	110.1	100**	10.7
	0.749			88.8
	0.852			101.1
Positive Control Item	0.047	0.052	0.010	5.6	6.2	1.2
	0.045			5.3
	0.063			7.5
Test Item	0.844	0.819	0.038	100.1	97.2	4.5
	0.775			91.9
	0.837			99.3

OD= Optical density

SD = Standard Deviation

**=          The mean percentage viability of the negative control tissue is set at 100%

    

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 6.2% relative to the negative control treated tissues and the standard deviation value of the viability was 1.2%.  The positive control acceptance criteria were therefore satisfied.

The mean OD570 for the negative control treated tissues was 0.843 and the standard deviation value of the viability was 0.090%.  The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 4.5%.  The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply:
EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours based on OECD 439.  The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay.  Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.  

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.  At the end of the exposure period each tissue was rinsed before incubating for 42 hours.  At the end of the post-exposure incubation period each tissue was taken for MTT-loading.  The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination.  After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.  

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate.  The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 97.2% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Quality criteria:  The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as non-irritant.  The following classification criteria apply:

EU DSD and CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

This study was conducted in October 2002

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

not specified

Specific details on test material used for the study:

AJICURE PN-23(Lot No. 010217B), AJICURE MY-24(Lot No.020430B) and AJICURE PN-H (Lot No.020412B)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

1) Test Animals: Male New Zealand white rabbits were purchased from SUKA-FLAT. A day before treatment, the hair of both flanks of each rabbit was shaved, using an animal hair trimmer. Rabbits with healthy, intact skin were selected. 3 rabbits were tested for each test article.
2) Husbandry: The animals were accommodated individually in metal bracket cages in an animal room at 23 ± 3°C, 50 ± 20% humidity, 10 to 15 times of all fresh ventilation/hour and 12 hours/day of light (at 200 - 500 lux).
3) Diet and drinking water: Allowed free access to solid food for experimental animals RC-4 (Oriental Yeast Co., Ltd.) and water with chlorine added.

Type of coverage:

occlusive

Preparation of test site:

shaved

Vehicle:

water

Amount / concentration applied:

Treatment concentration and preparation.
The test article was diluted with water for injection to prepare the top concentration paste. And the paste was used for the test.
(1) AJICURE PN-23 (Lot No. 010217B): Conformal
Water for injection (Lot No.K1E78) was added to test article to prepare paste of 50.15%. And the paste was used for the test.
(2) AJICURE MY-24 (Lot No.020430B): Conformal
Water for injection (Lot No.K1E78) was added to test article to prepare paste of 49.90%. And the paste was used for the test.
(3) AJICURE PN-H (Lot No. 020412B): Conformal
Water for injection (Lot No.K1E78) was added to test article to prepare paste of 50.08%. And the paste was used for the test.

Duration of treatment / exposure:

4 hours

Observation period:

1 week

Number of animals:

3 per treatment

Details on study design:

Administration
(1) To prepare the patch, 2.5 x 2.5 cm lint sheet was placed on surgical tape. 0.5mL of test article paste was placed on lint sheet using disposable syringe.
(2) The patch was applied to the shaved areas and covered occlusively with stretchable adhesive tape for 4hours. Maximum 4 patches on each side of flanks was applied, and in order to eliminate bias based on the application site, the patch was applied to different locations for each animal.
(3) 4hours after application, the patch and tape were removed, the test article was removed with the paper towel moistened with water.

Judgement
(1) Observations of the application site were conducted approximately 1 hour after removal of the patch, and erythema and edema of skin were assessed in accordance with following “Judgement criteria”(Judgement 1 hour after removal of the patch).

Judgement criteria
A. Erythema and Eschar formation
0: No erythema
1: Very slight erythema (barely perceptible)
2: Well defined erythema
3: Moderate to severe erythema
4: Severe erythema (beet redness) or eschar formation preventing grading of erythema (damage to deep area)

B. Edema formation
0: No oedema
1: Very slight edema (barely perceptible)
2: Slight edema (edges of area well defined by definite raising)
3: Moderate edema (raised approximately 1 mm)
4: Severe edema (raised more than 1 mm and extending beyond area of exposure)

(2) Simmilar observations of the application site were conducted 24, 48, 72 hour and 1week after removal of the patch, erythema and edema of skin were assessed.

(3) Risk determination
Based on the above scores, risks were classified as described below in accordance with the European Council Directive2001/59.
Risk phrase: R35 (Causes severe burns)
Full thickness destruction of skin tissue occurs as a result of exposure of up to three minutes.
Risk phrase: R34 (Causes burns)
Full thickness destruction of skin tissue occurs as a result of exposure of up to four hours. .
Risk phrase: R38 (Irritating to skin)
Cases where one of the following occurs at scores of 24, 48, and 72 hours after removal of the patch exposured up to 4 hours, and it continues for at least 24 hours after exposure.
a. The mean value of the scores for either erythema and eschar formation or edema
formation is 2 or more.
b. Two or more animals reveal erythema and eschar formation or edema formation in a score of 2 or more.
c. Skin irritation continues in two or more animals at 72 hour observations.

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Remarks:

AJICURE PN-23

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Remarks:

AJICURE PN-23

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Remarks:

AJICURE MY-24

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Remarks:

AJICURE MY-24

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Remarks:

AJICURE PN-H

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Remarks:

AJICURE PN-H

Total value of erythema and edema score (upper section), the mean value of 1 animal (lower section)

Test article	Animal			Reading time (After removal of the patch)
		1hr		24hrs		48hrs		72hrs		1week
		Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema
AJICURE PN-23 Lot No.010217B	1	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0
	Total	0	0	0	0	0	0	0	0	0	0
	Average	0		0		0		0		0

Test article	Animal			Reading time (After removal of the patch)
		1hr		24hrs		48hrs		72hrs		1week
		Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema
AJICURE MY-24 Lot No.020430B	1	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0
	Total	0	0	0	0	0	0	0	0	0	0
	Average	0		0		0		0		0

Test article	Animal			Reading time (After removal of the patch)
		1hr		24hrs		48hrs		72hrs		1week
		Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema	Edema	Erythema
AJICURE PN-H Lot No.020412B	1	0	0	0	0	0	0	0	0	0	0
	2	0	0	0	0	0	0	0	0	0	0
	3	0	0	0	0	0	0	0	0	0	0
	Total	0	0	0	0	0	0	0	0	0	0
	Average	0		0		0		0		0

Results were summarized in the table above.

 

No abnormal changes were observed in 50.15% paste of AJICURE PN-23 (Lot No. 010217B) at 1hour after removal of the patch, and no change was seen until the next 1 week. Based on the risk judgement, AJICURE PN-23 was classified as “No risk phrase (nonirritant to the skin)”. 

   

No abnormal changes were observed in 49.90% paste of AJICURE MY-24 (Lot No.020430B) at 1hour after removal of the patch, and no change was seen until the next 1 week. Based on the risk judgement, AJICURE MY-24was classified as “No risk phrase (nonirritant to the skin)”.

 

No abnormal changes were observed in 50.08% paste of AJICURE PN-H (Lot No. 020412B) at 1hour after removal of the patch, and no change was seen until the next 1 week. Based on the risk judgement, AJICURE PN-H was classified as “No risk phrase (nonirritant to the skin)”.

Interpretation of results:

GHS criteria not met

Conclusions:

No abnormal changes were observed in 50.15% paste of AJICURE PN-23 (Lot No. 010217B) at 1hour after removal of the patch, and no change was seen until the next 1 week. Based on the risk judgement, AJICURE PN-23 was classified as “No risk phrase (nonirritant to the skin)”.

No abnormal changes were observed in 49.90% paste of AJICURE MY-24 (Lot No.020430B) at 1hour after removal of the patch, and no change was seen until the next 1 week. Based on the risk judgement, AJICURE MY-24was classified as “No risk phrase (nonirritant to the skin)”.

No abnormal changes were observed in 50.08% paste of AJICURE PN-H (Lot No. 020412B) at 1hour after removal of the patch, and no change was seen until the next 1 week. Based on the risk judgement, AJICURE PN-H was classified as “No risk phrase (nonirritant to the skin)”.

Executive summary:

AJICURE PN-23(Lot No. 010217B), AJICURE MY-24(Lot No.020430B) and AJICURE PN-H (Lot No.020412B) were tested in primary skin irritation study accordance with OECD/404 and EU Council Directive. The test items were applied to Male New Zealand white rabbits for 4 hours. All the Edema and Erythema scores after 24, 48, 72 h and 1 week were 0.

No abnormal changes were observed in 50.15% paste of AJICURE PN-23 (Lot No. 010217B) at 1hour after removal of the patch, and no change was seen until the next 1 week. Based on the risk judgement, AJICURE PN-23 was classified as “No risk phrase (nonirritant to the skin)”.

No abnormal changes were observed in 49.90% paste of AJICURE MY-24 (Lot No.020430B) at 1hour after removal of the patch, and no change was seen until the next 1 week. Based on the risk judgement, AJICURE MY-24 was classified as “No risk phrase (nonirritant to the skin)”

No abnormal changes were observed in 50.08% paste of AJICURE PN-H (Lot No. 020412B) at 1hour after removal of the patch, and no change was seen until the next 1 week. Based on the risk judgement, AJICURE PN-H was classified as “No risk phrase (nonirritant 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 vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 08 August 2017 and 01 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

02 October 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: THEMIS (EC 500-429-8)
CAS Number: 159034-96-5
EC Number: EC 500-429-8
Batch: 161129
Purity: 100%
Physical state/Appearance: pale brown powder
Expiry Date: 28 November 2017

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

Details on test animals or tissues and environmental conditions:

Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 3.72 or 4.53 kg and were 12 to 52 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.1 mL of undiluted test material

Observation period (in vivo):

72 h

Number of animals or in vitro replicates:

2

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% proxymetacaine hydrochloride) was applied to each eye.
A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made according to the six point scale shown in Table1.
Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.
After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.
Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977) given in Table 2.
Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Any clinical signs of toxicity, if present, were also recorded.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Data Evaluation
The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:
Score for conjunctivae = (A + B + C) x 2
Score for iris = D x 5
Score for cornea = (E x F) x 5
Using the numerical data obtained a modified version of the system described by Kay J.H. and Calandra J.C. (1962) was used to classify the ocular irritancy potential of the test item. This was achieved by adding together the scores for the cornea, iris and conjunctivae for each time point for each rabbit. The group means of the total scores for each observation were calculated. The highest of these group means (the maximum group mean score) together with the persistence of the reactions enabled classification of the eye irritancy potential of the test item.
If evidence of irreversible ocular damage is noted, the test item will be classified as corrosive to the eye.
The results were also evaluated according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
The results were also evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

probability of mild irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 72 hours

Remarks on result:

probability of mild irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

probability of mild irritation

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

72 h

Score:

0

Max. score:

8

Reversibility:

not specified

Remarks on result:

probability of mild irritation

Irritant / corrosive response data:

Ocular Reactions
Individual and group mean scores for ocular irritation are given in Tables 3 and 4.
No corneal or iridial effects were noted during the study.
Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation in the other treated eye 1 hour after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 24-Hour observation and persisted in one treated eye at the 48-Hour observation.
The treated eyes appeared normal at the 48 or 72-Hour observation.


Body Weight
Both animals showed expected gain in body weight during the study.

Table 3    Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex		75742 Female				75769 Female
		IPR = 0				IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	1	1	0	0	2	1	1	0
B = Chemosis	1	0	0	0	1	1	0	0
C = Discharge	1	0	0	0	1	0	0	0
Score (A + B + C) x 2	6	2	0	0	8	4	2	0
Total Score	6	2	0	0	8	4	2	0

IPR=Initial pain reaction

Table 4   Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex		Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
75742 Female	6	2	0	0
75769 Female	8	4	2	0
Group Total	14	6	2	0
Group Mean Score	7.0	3.0	1.0	0.0

Interpretation of results:

GHS criteria not met

Conclusions:

The test item produced a maximum group mean score of 7.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Executive summary:

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit according to OECD 405.

 

A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation in one treated eye and minimal conjunctival irritation in the other treated eye 1 hour after treatment. Minimal conjunctival irritation was noted in both treated eyes at the 24-Hour observation and persisted in one treated eye at the 48-Hours observation.

The treated eyes appeared normal at the 48 or 72-Hour observation.

The test item produced a maximum group mean score of 7.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 27 July 2017 and 31 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:

26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: THEMIS (EC 500-429-8)
CAS Number: 159034-96-5
EC Number: EC 500-429-8
Batch: 161129
Purity: 100%
Physical state/Appearance: pale brown powder
Expiry Date: 28 November 2017
Storage Conditions: room temperature in the dark

Species:

other: Eyes from adult cattle

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of the test item or control items were applied to the cornea.

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 Minutes

Number of animals or in vitro replicates:

3 corneas per treatment

Details on study design:

Study Design
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Data Evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

Visual Observation
The condition of the cornea was visually assessed post treatment.

Data Interpretation
The test item was classified according to the following prediction model:

IVIS Classification
≤ 3 No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55 No prediction of eye irritation can be made
> 55 Category 1. UN GHS or EU CLP Causes serious eye damage

Criteria for an Acceptable Test
For an acceptable test the following positive control criterion should be achieved:
20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2015 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 50.8 to 100.4.

For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2015 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤5.4 and for permeability ≤0.070.

Irritation parameter:

in vitro irritation score

Value:

ca. 3.8

Negative controls validity:

valid

Remarks:

1.4

Positive controls validity:

valid

Remarks:

101.9

Remarks on result:

other: No prediction of eye irritation can be made.

Other effects / acceptance of results:

The corneas treated with the test item or negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

Criteria for an acceptable test

The positive controlIn VitroIrritancy Score was not within the range of 50.8 to 100.4. The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation.

The negative control gave opacity of ≤5.4 and permeability ≤0.070. The negative control acceptance criteria were therefore satisfied

Table 1 Individual and Mean Corneal Opacity and Permeability Measurements when needed in this detail (delete when editing):

Treatment	Cornea Number		Opacity			Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment Pre-Treatment	Corrected Value		Corrected Value
Negative Control	7	3	5	2		0.004
	8	3	4	1		0.010
	9	3	4	1		0.003
				1.3*		0.006**		1.4
Positive  Control	1	2	77	75	73.7	1.193	1.187
	2	2	77	75	73.7	1.740°	1.734
	3	2	86	84	82.7	2.140°	2.134
					76.7§		1.685§	101.9
Test Item	4	5	8	3	1.7	0.089	0.083
	5	4	9	5	3.7	0.052	0.046
	6	2	7	5	3.7	0.035	0.029
					3.0§		0.053§0	3.8

OD= Optical density            

* = Mean of the post-incubation -pre‑treatment values          

**= Mean permeability

§ = Mean corrected value

° = 1 in 5 dilution performed

Table 2 Corneal Epithelium Condition Post Treatment and Post Incubation:

Treatment	Cornea Number	Observation Post Treatment
Negative Control	7	Clear
	8	Clear
	9	Clear
Positive Control	1	Cloudy
	2	Cloudy
	3	Cloudy
Test Item	4	Clear
	5	Clear
	6	Clear

Interpretation of results:

GHS criteria not met

Conclusions:

No prediction of eye irritation can be made.

Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage based on OECD 437.

Method

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate anIn VitroIrritancy Score (IVIS).  

Results

The In Vitroirritancy scores are summarized as follows:

Treatment	In VitroIrritancy Score
Test Item	3.8
Negative Control	1.4
Positive Control	101.9

Conclusion

No prediction of eye irritation can be made.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

This study was conducted in August 1999

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

GLP compliance:

not specified

Specific details on test material used for the study:

AJICURE MY-H Lot No. 990116

Species:

rabbit

Strain:

New Zealand White

Vehicle:

other: Liquid Paraffin

Controls:

no

Amount / concentration applied:

AJICURE MY-H (Lot No. 990116) was applied to animals at 5 % suspended with Liquid paraffin (Lot No. CKP4951) in the test.

Observation period (in vivo):

1 week

Details on study design:

Test animal; 3 Male New Zealand white rabbits
Evaluation method; Abnormality and discharge of cornea, iris and conjunctivae were assessed and scored in accordance with “Draize scale”, showing below, at 1, 24, 48, 72, 96 hours and 1 week after the application of the test article. Individual score for each animal was caluculated based on the “Formulation”, showing below. Furtheremore, the means were caluculated from the individual score at each observation time point. The preliminal irritancy of the test compound was classified according to “Criteria of Kay and Calandra No.1”, showing below, with the highest value during the observation period per group. The final irritancy was judged according to “Criteria of Kay and Calandra No.2”.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 1 hour

Score:

10

Max. score:

10

Reversibility:

fully reversible within: 24 hours

Remarks on result:

probability of mild irritation

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 24/48/72/96 hours and 1 week

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

< Mean scores for eye irritation>

Test article	1hr	24hr	48hr	72hr	96hr	1 week
AJICURE MY-H Lot No. 990116	10.0	0	0	0	0	0

Results were shown in the table above. 

AJICURE MY-H (Lot No. 990116) at 5 % suspended in liquid paraffin caused slight redness, minimal swelling and so much discharge in all animals at 1hr after eye application. These irritative findings were dissappered at 24hr after application. 

From the above results, a 5% susupension of AJICURE MY-H (Lot No. 990116) in liquid paraffin was judged as 'Minimal Irritant' to rabbit eye.  

Interpretation of results:

other: Minimal Irritant

Conclusions:

AJICURE MY-H (Lot No. 990116) at 5 % suspended in liquid paraffin caused slight redness, minimal swelling and so much discharge in all animals at 1hr after eye application. These irritativefindings were dissappered at 24hr after application.
From the above results, a 5% susupension of AJICURE MY-H (Lot No. 990116) in liquid paraffin was judged as 'Minimal Irritant' to rabbit eye

Executive summary:

AJICURE MY-H was applied to 3 Male New Zealand white rabbits at 5 % suspended with Liquid paraffin in the test.

AJICURE MY-H at 5 % suspended in liquid paraffin caused slight redness, minimal swelling and so much discharge in all animals at 1hr after eye application. These irritative findings were disappeared at 24hr after application. 

From the above results, a 5% suspension of AJICURE MY-H in liquid paraffin was judged as 'Minimal Irritant' to rabbit eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation / corrosion:

In Vitro Skin Corrosion, key study, OECD 431:  non-corrosive

In vitro skin irritation, key study, OECD 439: non-irritant

In vivo skin irritation, supporting study, OECD 404: non irritant

According to Regulation (EC) No 1272/2008, section 3.2.2.1, this substance should not be classified for this endpoint.

 

Eye irritation:

In vivo eye irritation, key study, OECD 405:

Mean scores for cornea opacity, iris, conjunctivae and chemosis at 24, 48, and 72h:

cornea opacity: 0, 0;

iris: 0, 0;

conjunctivae: 0.33, 0.67;

chemosis: 0, 0.33.

BCOP, supporting study, OECD 437: no prediction of eye irritation can be made.

Primary eye irritation, supporting study: Minimal Irritant

According to Regulation (EC) No 1272/2008, table 3.3.1 and 3.3.2, this substance should not be classified for this endpoint.