Dl-Proline, 5-oxo-, compd. with N2-coco acyl-l-arginine Et ester

Endpoint summary

Description of key information

Skin Corrosion in vitro: Negative

Skin Irritation in vitro: Under the conditions of this study, the test material is not irritating to skin.

Eye irritation in vitro:

Under the conditions of this study the test material was concluded to be classified as UN GHS eye irritant Category 2 (sub-category 2A).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

06 December 1996 to 21 February 1997

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: A non GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data. The report was redrafted several years after the study period.

GLP compliance:

not specified

Species:

guinea pig

Strain:

Hartley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 375 to 439 g
- Housing: 2 animals per cage (W 360 × D 550 × H 350 mm:)
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 5 °C
- Humidity (%): 55 ± 25 %
- Air changes (per hr): 8 to 10
- Photoperiod (hrs dark / hrs light): 12 hour lighting period from 07.00 to 19.00

Type of coverage:

open

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 ml

Duration of treatment / exposure:

24 hours

Observation period:

14 days

Number of animals:

10 animals, 5 animals in each group

Details on study design:

TEST SITE
- Area of exposure: Dorsal area
- Coverage: 1.5 x 1.5 cm
- Test material re-applied every 24 hours for 14 days

OBSERVATION TIME POINTS:
- Skin was obsereved for dermal reactions every 24 hours before re-applying test material, fur was clipped twice or three times per week.
- The animals were observed for general condition once every day.
- Animals were weighed on the day of application (Day 0) and on days 7 and 14.

Irritant / corrosive response data:

Sample D (the registered substance): At 2.0%, 0.6% and 0.2%, there were no dermal reactions during the observation period.

Other effects:

There were neither abnormalities in general condition nor deaths during the observation period.

TABLE 2: A 14 -day cumulative skin irritation study of sample D (the registered substance) in guinea pigs

Data of cumulatiive skin irritation study

*: Number of days after the start of application

Substance for application	MEAN SCORE
		1*	2	3	4	5	6	7	8	9	10	11	12	13	14
Sample D	2.0%	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Sample D	0.6%	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Sample D	0.2%	0	0	0	0	0	0	0	0	0	0	0	0	0	0

Interpretation of results:

not irritating

Conclusions:

Under the conditions of the study, there were no effects from the test material on the general condition or body weight during the observation period.

Executive summary:

The potential for the test material to cause skin irritation was predicted in an in vivo study using Hartley strain female guinea pigs. The study was conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies which do not affect the quality of the relevant results. The test material was applied open onto the animal skin. 24 hours after application, the skin was observed for dermal reactions and the test material was applied. By repetition of this procedure, 14-day repeated application was conducted. During the application period, fur was clipped twice or 3 times per week.

There were no reactions at 2.0 %, 0.6 % or 0.2 %.

Under the conditions of the study, there were no effects from the test material on the general condition or body weight during the observation period.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 December 2016 to 09 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN™ (SM) three-dimensional human epidermis model.

Justification for test system used:

The EPISKIN™ (SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); 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: A three-dimensional human epidermis model
- Storage: The EPISKIN™(SM) kits were kept in their packaging at 37 °C until the initiation of the test.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: At room temperature (22.9 - 24.2 °C).

PERFORMANCE OF THE STUDY
- Pre-incuation (Day [-1])
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37 °C in an incubator with 5 % CO2 in a >95 % humidified atmosphere.
 - Application (Day 0)
The Assay Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of test material was applied evenly to the epidermal surface of each of two test material treated units and each additional control skin units and then 100 μL physiological saline was added to the test material to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
The plates with the treated epidermis units were incubated for 4 hours (± 10 min) at room temperature (22.9-24.2 °C) covered with the plate lids.
 
REMOVAL OF TEST MATERIAL AND CONTROLS
-Rinsing (Day 0)
After the incubation time (4 hours), all test material treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
-MTT test (Day 0)
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced and the plate incubated at 37 °C in an incubator with 5 % CO2 for 3 hours (±15 minutes), protected from light.

-Formazan extraction (Day 0)
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.
 
-Cell viability measurements (Day 1)
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test material is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test material interference with the viability measurement.

-Check-method for possible direct MTT reduction with test material:
Approximately 20 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours and then any colour change was observed:
-Test materials which do not react with MTT: yellow
-Test materials reacting with MTT: blue or purple
After three hours incubation, yellow colour of the mixture was detected; therefore additional controls were not used in the experiment.

-Check-method to detect the colouring potential of test material:
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol* (simulating a tissue humid environment). As the test material had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSC living %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

CALCULATIONS OF VIABILITY PERCENTAGES
The data calculation using two replicates is shown below. Results were calculated in a similar way when more replicates are used.
Blank:
- The mean of the 6 blank OD values was calculated

Negative control:
– Individual negative control OD values (NCraw) were corrected with the mean blank OD:

OD Negative Control (ODNC) = ODNCraw – ODblank mean

– The corrected mean OD of the 2 negative control values was also calculated: this corresponds to 100% viability 

Positive control:
– Individual positive control OD values (PCraw) were corrected with the mean blank OD:

OD Positive Control (ODPC) = ODPCraw – ODblank mean

– The corrected mean OD of the 2 positive control values was calculated
– The % viability for each positive control replicate was calculated relative to the mean negative control:
Positive Control1 % = (ODPC1 / mean ODNC) ×100
Positive Control2 % = (ODPC2 / mean ODNC) ×100
– The mean value of the 2 individual viability % for positive control was calculated:
Mean PC % = (PC1 % + PC2 %) / 2
 
Test material:
– Individual test material OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 2 test material values was calculated
– The % viability for each test material replicate was calculated relative to the mean negative control:
Treated Tissue1 % = (ODTT1 / mean ODNC) ×100
Treated Tissue2 % = (ODTT2 / mean ODNC) ×100
– The mean value of the 2 individual viability % for test material was calculated:
Mean TT % = (TT1 % + TT2 %) / 2
– The variability for 2 disks was calculated as: (Disk1-Disk2)/((Disk1+Disk2)/2) x 100%
 
-Data calculation for test materials having MTT-interacting potential:
Test materials that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability% as follows:
Non specific MTT reduction calculation (NSMTT%):
NSMTT% = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control treated killed tissues OD
ODKT: test material treated killed tissues OD
ODNC: negative control OD
If NSMTT% is ≤ 50 %, then true MTT metabolic conversion (TODTT) has to be undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test material treated viable tissues
– The % relative viability (RV %) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSMTT% is > 50 % relative to the negative control: additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.
 
- Data calculation for test items having colouring potential:
For test materials detected as able to stain the tissues the non-specific OD was evaluated due to the residual chemical colour (unrelated to mitochondrial activity) and subtracted before calculation of the “true” viability % as detailed below:
Non Specific Colour % (NSCliving %) = (mean ODCTV / mean ODNC) × 100
ODCTV: test material treated viable tissue (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSC living % is ≤ 5 % then the normal calculation mode was used.
If NSC living % is > 5 % and ≤ 50 %, then additional correction (TODTT) has to be undertaken as follows:
TODTT = [ODTV - ODCTV]
ODTT: test material treated viable tissue (incubated with MTT)
ODCTV: test material treated viable tissue (not incubated with MTT)
– The % relative viability (RV% %) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
– The mean value of the 2 individual relative viability % for test material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2
If NSC living % is > 50 % relative to the negative control, additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.
 
Data calculation for test materials having both MTT-interacting and colouring potential
For test materials detected as able to both stain the tissues and interfere with MTT may also require a third set of controls before calculation of the “true” viability %.
Non Specific Colour % with killed tissues (NSCkilled %):
NSCkilled % = (mean ODCTK / mean ODNC) ×100
ODCTK: test material treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
ODTT: test material treated viable tissues (incubated with MTT)
ODKT: test material treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test material treated viable tissues (not incubated with MTT)
ODCTK: test material treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
RV1 % = [TODTT1 / mean ODNC] × 100
RV2 % = [TODTT2 / mean ODNC] × 100
The mean value of the 2 individual relative viability % for test material is calculated:
Mean Relative Viability % = (RV1 % + RV2 %) / 2

PREDICTION MODEL / DECISION CRITERIA
For 2 disks:
If both disks have mean viability of ≥35 % = Non Corrosive
If both disks have mean viability of <35 % = Corrosive (at the corresponding incubation period)
Otherwise:
If the mean value is ≥35 % and the variability is less than 50 % = Non Corrosive
If the mean value is <35 % and the variability is less than 50 % = Corrosive
Otherwise:
If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg (100 µL of physiological saline was added to the test material to ensure good contact with the epidermis)

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

4 hours (± 10 minutes)

Duration of post-treatment incubation (if applicable):

3 hours (± 15 minutes)

Number of replicates:

Two

Irritation / corrosion parameter:

% tissue viability

Value:

96.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS: ADDITIONAL CONTROLS
- Colour interference with MTT: As the test material was coloured, two additional test material-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.005, Non Specific Colour% (NSCliving %) was calculated as 0.6%.This is below the threshold of 5 %, therefore correction due to colouring potential was not necessary.

ACCEPTANCE OF RESULTS:
After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues was in the recommended range (0.836).
The two positive control treated tissues showed 0.8 % viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 3.3 %.
The difference of viability between the two negative control tissue samples in the MTT assay was 15.0 %.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Viability Results

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The mean OD value for the test item treated skin samples showed a 96.3 % relative viability.

 

Table 1: Optical Density (OD) and the calculated relative viability % of the samples 

Substance	Optical Density (OD)			Viability (% RV)
		Measured	Blank Corrected
Negative control: Physiological saline	1	0.819	0.773	92.5
	2	0.944	0.898	107.5
	Mean	-	0.836	100.0
Positive Control: Glacial acetic acid	1	0.052	0.006	0.8
	2	0.053	0.007	0.8
	Mean	-	0.007	0.8
Test material	1	0.838	0.792	94.8
	2	0.864	0.818	97.9
	Mean	-	0.805	96.3

 

Interpretation of results:

other: Not corrosive in accordance with EU criteria.

Conclusions:

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Executive summary:

An in vitro skin corrosion test was carried out with the test material using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 under GLP conditions.

The model is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Disks of EPISKIN™ (SM) (two units) were treated with the test material and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9 % (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving %) from the test material. For each treated tissue, viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35 % of the negative control, the test material is considered to be corrosive to skin.

Following exposure with the test material, the mean cell viability was 96.3 % compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19 June - 23 June 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A non GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data. Data is however on a 1% solution and is not suitable for assigning C&L.

GLP compliance:

not specified

Specific details on test material used for the study:

Lot No.: 89002

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Diet: 150 g per day of pelleted diet.
- Water: Ad libitum
- Acclimation period: Yes, but time period not stated.

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 3 °C
- Humidity: 55 ± 15 %

Type of coverage:

occlusive

Preparation of test site:

clipped

Remarks:

Before application of the test article, the fur of the rabbits on the right and left trunks was clipped with an electric clipper and healthy animals without island skin were selected.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.3 mL

VEHICLE
- No vehicle

POSITIVE CONTROL
Sodium lauryl sulphate dissolved in distilled water at 5 % concentration.

Duration of treatment / exposure:

24, 48 or 72 hours

Observation period:

72 hours

Details on study design:

TEST SITE
- Area of exposure: Right and left trunks
- Type of wrap if used: Test material was applied to adhesive tape which was applied to the clipped area of the animal and fixed in position using a body cover.

OBSERVATION TIME POINTS
-At 24 hours after the closed application, the body cover and the adhesive tape were removed and the degree of irritation judged according to Draize's evaluation. The judgement was done in the same manner at 48 and 72 hours after the closed application.

SCORING SYSTEM: Draize method
- Erythema and Eschar Formation:
Slight erythema (barely perceptible) = 1
Well defined erythema = 2
Moderate to severe erythema = 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) = 4

- Oedema Formation:
Very slight oedema (barely perceptible) = 1
Slight oedema (edges of the area well defined by definite raising) = 2
Moderate oedema (area raised approximately 1 mm) = 3
Severe oedema (area raised more than 1 mm and extending beyond the area of exposure) = 4

Remarks on result:

not determinable

Slight erythema and oedema were observed at 24 and 48 hours after application, but it was no longer observed thereafter.

After 24 hours		After 48 hours		After 72 hours
Erythema	Oedema	Erythema	Oedema	Erythema	Oedema
6	0	5	0	0	0
1.5		1.3		0.8

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this study the test material was determined to be non-irritating to the skin.

Executive summary:

The study was performed to assess the potential of the test material to cause skin irritation in rabbits.

0.3 mL of the test material was applied directly to the skin under adhesive tape and assessed according to the Draize Method for Erythema and Oedema formation. The sites were assessed at 24, 48 and 72 hours post application.

Slight erythema and oedema were observed at 24 and 48 hours after application, but it was no longer observed thereafter.

Under the conditions of the study the test material is determined to be not irritating to the skin but the results are not suitable for definitive classification and labelling under CLP as the test sample was a 1% solution.

Reference 4

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2017 to 18 February 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:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Version / remarks:

Version 1.8 (February 2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN™ (SM) three-dimensional human skin model.

Justification for test system used:

The EPISKIN™ (SM) model has been validated for irritation testing in an international validation study 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: EPISKIN™ (SM)
- Tissue batch number(s): 17-EKIN-007
- Expiry Date: 20 February 2017
- Source: SkinEthic, France
- The model is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
For killed epidermis, living epidermis units (Manufacturer: SkinEthic, France, Batch No.:16-EKIN-037, Expiry Date: 19 September 2016) were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37 °C in an incubator with 5 % CO2, in a >95 % humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen ( frozen units can be used up to 6 months). Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Assay Medium). Further use of killed tissues was similar to living tissues.

KIT RECEPTION
- The pH of the agar medium used for transport was checked by checking the colour of the medium: orange colour = good and yellow or violet colour = not acceptable.
- The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C (the colour change is irreversible, independent of the length of the period above 40 °C): White colour = good and grey or black colour = not acceptable.
- The kits were found to be in good order at reception.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (22.8 to 26.7 °C).
- Temperature of post-treatment incubation: 37 °C

CHECK-METHOD FOR DIRECT MTT REDUCTION
- Approximately 10 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in a shaking water bath for 3 hours (± 5 minutes) protected from light, and then any colour change was recorded: Test materials which do not react with MTT turn yellow and test materials reacting with MTT turn blue or purple.
- After three hours incubation, purple colour (purple precipitate) of the mixture was detected in the test tube. Thus, the test material reacted with MTT and therefore the use of additional controls was necessary.

CHECK-METHOD FOR COLOURING POTENTIAL OF THE TEST MATERIAL
- Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or extracting solution (e.g. acidified isopropanol) (simulating a tissue humid environment). As the test material had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non-Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
- The test material was showed being an MTT-interacting substance and the test material had an intrinsic colour a third set of controls were necessary. The test material may bind to both living and killed tissues and therefore the NSMTT control may not only correct for potential direct MTT reduction by the test chemical, but also for colour interference arising from the binding of the test chemical to killed tissues. This could lead to a double correction for colour interference since the NSCliving control already corrects for colour interference arising from the binding of the test chemical to living tissues. To avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled) was needed.
- Therefore, in addition to the normal procedure, two additional test material-treated living tissues and two additional test material-treated killed tissues were used for the non-specific OD evaluation. These tissues followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

PROCESS FOR TEST MATERIALS WITH BOTH MTT REDUCTION AND COLOUR INTERFERENCE
The test material was identified as producing both direct MTT reduction and colour interference was also require a third set of controls. This is usually the case with darkly coloured test chemicals interfering with the MTT assay (e.g., blue, purple, black) because their intrinsic colour impedes the assessment of their capacity to directly reduce MTT. These test chemicals may bind to both living and killed tissues and therefore the NSMTT control may not only correct for potential direct MTT reduction by the test chemical, but also for colour interference arising from the binding of the test chemical to killed tissues. This could lead to a double correction for colour interference since the NSCliving control already corrects for colour interference arising from the binding of the test chemical to living tissues. To avoid a possible double correction for colour interference, a third set of control for non-specific colour in killed tissues (NSCkilled) was performed.

APPLICATION/ TREATMENT
- Pre-incubation (Day [-1]): The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere.
- Test Material (Day 0): As the test material was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 20 mg of the test material was applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a pipette tip without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
- Negative and positive controls: 50 μL of negative control (PBS) or positive control (5 % (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
- The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (22.8 to 26.7 °C).
- As the test material was showed being an MTT-interacting substance, in addition to the normal procedure, two test material treated killed epidermis and two negative control treated killed epidermis was used in the study (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues. The same treatment steps were followed in case of the killed tissues as in case of the living tissues.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The test material stuck on surface of the epidermis so additional rinsing was used. The test material was totally washed off from the skin. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
- After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1 h) at 37 °C in an incubator with 5 % CO2 in a > 95 % humidified atmosphere.

MTT TEST (Day 2)
- After the 42 hours incubation, all EPISKINTM (SM) units (except for two colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKINTM (SM) units were incubated for 3 hours at 37 °C in an incubator with 5 % CO2 in a >95 % RH % protected from light.
- Formazan extraction (Day 2): After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
- Cell viability measurements (Day 2): Following the formazan extraction, 2 × 200 μL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank. The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800)) at the required wavelength on each day before use.

NUMBER OF REPLICATE TISSUES: In this assay, three replicates were used for the test material. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test material was coloured, two additional test material-treated living tissues were used for the nonspecific OD evaluation. Furthermore, as the two test materials had an MTT interacting potential, two additional test material-treated killed epidermis and two negative control treated killed epidermis were used. To avoid a possible double correction for colour interference, for non-specific colour in killed tissues, two additional disks were used.

CALCULATIONS OF VIABILITY PERCENTAGES
- Data calculation for normal test materials
Blank: The mean of the six blank OD values was calculated

Negative control: Individual negative control OD values (NCraw) were corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean.
The mean corrected OD of the 3 negative control samples were calculated: this value corresponds to 100 % viability

Positive control: Individual positive control OD values (PCraw) were corrected with the mean blank OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean.
The mean corrected OD of the 3 positive control samples were calculated. The % viability for each positive control replicate was calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
The mean value of the 3 individual relative viability % for positive control was calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test material: Individual test material OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean.
The mean corrected OD of the 3 test material samples were calculated. The % viability for each test material replicate was calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
The mean value of the 3 individual relative viability % for test material was calculated:
Mean TT % = (%TT1 +%TT2 +%TT3) / 3

- Data calculation for test materials having MTT-interacting potential
Test materials that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability % as detailed below:
Non-specific MTT reduction calculation (NSMTT):
NSMTT (%) = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control killed tissues OD
ODKT: test material treated killed tissues OD
ODNC: negative control OD
If NSMTT is ≤ 30 %, then true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test material treated viable tissues
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test material is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3.
If NSMTT is > 30 % relative to the negative control, then additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

- Data calculation for test materials having colouring potential
For test materials detected as able to stain the tissues the non-specific OD due to the residual chemical colour (unrelated to mitochondrial activity) is evaluated and subtracted before calculation of the “true” viability % as detailed below:
Non Specific Colour % with viable tissues (NSCliving %) = (mean ODCTV / mean ODNC)×100
ODCTV: test substance treated viable tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)
If NSCliving % is ≤ 5 % then the normal calculation mode is used.
If NSC % is > 5 % a nd ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken as follows.
TODTT = [ODTT – mean ODCTV]
ODTT: test substance treated viable tissue (incubated with MTT)
ODCTV: test substance treated viable tissue (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test material is calculated:
Mean Relative Viability % = (% RV 1 +% RV 2 +% RV 3) / 3
If NSC living % is > 30 % relative to the negative control, additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

- Data calculation for test materials having both MTT interacting potential and colouring potential
For test substances detected as able to stain the tissues and interfere with MTT also require a third set of controls before calculation of the “true” viability %.
Non Specific Colour % with killed tissues (NSCkilled%) = (mean ODCTK / mean ODNC)×100
ODCTK: test material treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)

TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
ODTT: test material treated viable tissues (incubated with MTT)
ODKT: test material treated killed tissues OD
ODKNC: negative control killed tissues OD
ODCTV: test material treated viable tissues (not incubated with MTT)
ODCTK: test material treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test material is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3

INTERPRETATION OF TEST RESULTS
- In case duplicates are spread over different categories, the test will be repeated.
- In the present study, the irritancy potential of test materials is predicted by the mean tissue viability of tissues exposed to the test material.
- The test material is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 15 minutes exposure to the test material and 42 hours post incubation is less or equal (≤) to 50 % of the mean viability of the negative controls.
- In case the test material is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2.
-The test material considered to be non-irritant to skin (No Category), if the mean relative viability of three individual tissues after 15 minutes exposure to the test material and 42 hours post incubation is more than (˃) to 50 % of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg (with 10 µL distilled water to improve contact with the eye)

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 5 % (w/v)

Duration of treatment / exposure:

15 minutes (± 0.5 minutes)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

77.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
ADDITIONAL CONTROLS
- Colour interference with MTT: As the test material was coloured, two additional test material-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of tissues were 0.016, Non Specific Colour % was calculated as 1.7 %. This value was below 5 %, therefore additional data calculation was not necessary.
- Direct-MTT reduction: As colour change (purple precipitate) was observed after three hours of incubation of the test material in MTT working solution, thus the test material might interact with MTT. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Based on these observed mean OD (0.012), the calculated NSMTT is 1.3 %. This was considered to be significant, thus correction with NSMTT were made.
- As the test material was showed being an MTT-interacting substance and the test material had an intrinsic colour, two additional test material-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.022, Non Specific Colour % (NSCkilled %) was calculated as 2.4%. NSCliving % was not used, therefore correction with NSCkilled% was not necessary.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The OD values for the test material treated skin samples showed 77.4 % relative viability compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.

ACCEPTANCE OF RESULTS:
- After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.
- The mean OD value of the three negative control tissues was in the recommended range (0.897). Standard deviation of the viability results for negative control samples was 3.3.
- The positive control treated tissues showed 4.3 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.5.
- The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 4.6.
- The mean OD value of the blank samples (acidified isopropanol) was 0.047.
- All these parameters met the acceptability criteria, therefore the study was considered to be valid.

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Treatment	Optical Density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative control: PBS	1	0.931	0.884	98.6
	2	0.922	0.875	97.6
	3	0.978	0.931	103.8
	Mean	-	0.897	100.0
Positive control: 5 % (w/v) SDS solution	1	0.086	0.039	4.4
	2	0.089	0.042	4.7
	3	0.081	0.034	3.8
	Mean	-	0.039	4.3
Test material	1	0.742	0.695	76.2
	2	0.717	0.670	73.5
	3	0.798	0.751	82.5
	Mean	-	0.705	77.4

Mean blank value was 0.047.

Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is not irritating to skin.

Executive summary:

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions.

The in vitro skin irritation test was performed in a reconstructed human epidermis model. EPISKIN™ (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay.

Disks of EPISKIN™ (SM) (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2 in a > 95 % RH %. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 in a > 95 RH % protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control, the test item is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 77.4 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of this study, the test material is not irritating to 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:

supporting study

Study period:

2 July - 6 July 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: A non GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data. Data is however on a 1% solution and is not suitable for assigning C&L.

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Housing: individually housed
- Diet: 150 g per day
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3 °C
- Humidity (%): 55 ± 15 %

Vehicle:

water

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 ml

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

groups of 4 animals.

Interpretation of results:

study cannot be used for classification

Conclusions:

Application of the test material caused reddening of the conjunctiva, slight edema and adhesion of discharge, but the effects were slight. The results are not suitable for definitive classification and labelling under CLP as the test sample was a 1% solution.

Executive summary:

The potential for the test material to cause eye irritation was assessed in an in vivo irritation study in rabbits. The study was a non GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data. The test material was administered as a 1% solution to 4 male New Zealand white rabbits. At 24, 48 and 72 hours after the ocular application, the degree of irritation reactions was jusdged according to Draize's evaluation standards. Application of the test material caused reddening of the conjunctiva, slight edema and adhesion of discharge but the effects were slight. The results are not suitable for definitive classification and labelling under CLP as the test sample was a 1% solution.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2016

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Number of animals: Not specified. Seven eyes were used in the experiment.
- Characteristics of donor animals: Approximately 7 weeks old. Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and transported to the testing laboratory at ambient temperature at the earliest convenience.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box).
- Time interval prior to initiating testing: The heads were received at the testing laboratory and processed within approximately 2 hours of collection.
- Indication of any existing defects or lesions in ocular tissue samples: After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2 % (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
- Indication of any antibiotics used: None specified.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from theorbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
- The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
- The appropriate number of eyes was selected and placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e.,> 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5 °C) during the acclimatisation and treatment periods.
- At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5 % within the -45 min and the zero time. No corneal thickness changes (0.0 %) were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test material related effect after treatment. All eyes were considered to be suitable for the assay.
-After the zero reference measurements, the eye (in its retainer) was removed from the chamber, held in a horizontal position and the test item was applied onto the centre of the cornea, taking care not to damage or touch the cornea.

NUMBER OF REPLICATES: Three test material treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

NEGATIVE CONTROL USED: 30 μL of physiological saline.

POSITIVE CONTROL USED: 30 mg of powdered Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 30 mg of test material was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test material for 10 seconds.

REMOVAL OF TEST SUBSTANCE:
- The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test material if possible.
- Additional gentle rinsing with 20 mL saline was performed at each time point when the test material or control material remaining on the cornea was observed. The test material treated eyes were rinsed additional gentle rinsing with 4x20 mL saline after treatment in each experiment.

OBSERVATION PERIOD:
- The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ± 5 minutes were considered acceptable.
- Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. A slit-lamp microscope was used for the measurements.
- Morphological effects: Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test material to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator.

EVALUATION:
- Corneal swelling was calculated according to the following formulae:
CS at time t = [(CT at time t – CT at t=0)/ CT at t=0] x 100
Mean CS at time t = (FECS(at time t)+ SECS(at time t) + TECS(at time t)) / 3
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point
Small negative numbers for swelling (0 to -5 %) following application are evaluated as class I. Large negative numbers (>12 % below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

- Cornea opacity was calculated according to the following formulae:
ΔCO at time t = CO at time t – CO at t=0
Mean ΔCOmax = [FECOmax(30min to 240min)+ SECOmax(30min to 240min) + TECOmax(30min to 240min)] / 3
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t=0 = baseline cornea opacity
ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

- Fluorescein retention was calculated according to the following formulae:
ΔFR at time t = FR at time t – FR at t=0
Mean ΔFR = [FEFR (30min) + SEFR(30min) + TEFR(30min)] / 3
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Irritation parameter:

other: maximum corneal swelling at up to 75 min (%)

Run / experiment:

mean

Value:

3.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Irritation parameter:

other: maximum corneal swelling at up to 240 min (%)

Run / experiment:

mean

Value:

13.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Other effects / acceptance of results:

- The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change are given below. The conclusion on eye irritancy was based on the OECD guideline quantitative assessments. The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for EC and GHS classification.
- Based on this in vitro eye irritation in the isolated chicken eyes test, the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

HISTOPATHOLOGY
- Semi-quantitative microscopic evaluation was performed on the cornea in the assay.
- The negative control Physiological saline (Salsol solution 0.9 %) cornea showed no abnormalities.
- Positive control, 30 mg powdered Imidazole associated with moderate epithelial erosion without partly detached layer from basal membrane in 3/3 cases. No compromised basement membrane, Bowman’s and Descemet’s membrane, or as well as no stromal and endothelial changes were recorded.
- The test material produced from very slight to slight erosion of the epithelium in 6/6 cases. Slight vacuolation of the top/mid or low layer of the corneal epithelium in 3/6 sections were also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 2A.

VALIDITY OF THE TEST
- The positive control Imidazole was classified as severely irritating and the negative control Physiological saline was classified as non-irritating.
- The results from all eyes used met the quality control standards. The negative control and positive control results were in line with historic data. This experiment was considered to be valid.

DISCUSSION
- To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test material (6 sections).
- Microscopic evaluation showed from very slight to slight erosion of the epithelium in 6/6 cases. Slight vacuolation of the top/mid or low layer of the corneal epithelium in 3/6 sections were also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 2A.
Taking into account the OECD guideline data with the supplemental histopathology data, the weight of evidence indicates an overall conclusion classification of UN GHS eye irritant Category 2 (sub-category 2A).

Table 1: Summary of results

Observation	Test material		Positive control		Negative control
	Value	ICE Class	Value	ICE Class	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	3.8 %	I	10.3 %	II	0.0 %	I
Mean maximum corneal swelling at up to 240 min	13.1 %	II	27.7 %	III	0.0 %	I
Mean maximum corneal opacity	2.00	III	4.00	IV	0.0	I
Mean fluorescein retention	3.00	IV	3.00	IV	0.0	I
Other Observations	Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were cleared at 30 minutes after the post-treatment rinse.		Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.		None
Overall ICE Class	1xII 1xIII 1xIV		1xIII 2xIV		3xI

 

Table 2: Summary table for UN GHS classification

Criteria for no category:	True/false
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoints classed as II	False
Test item were not stuck to the cornea at 240 minutes after post-treatment rinse	True
Criteria for Category 1:	True/false
2 or more endpoints classed as IV	False
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes)	False
Corneal opacity = 4 at any time point (in at least 2 eyes)	False
Severe loosening of epithelium (in at least 1 eye)	False
Criteria for No prediction can be made:	True/false
Based on the endpoints not classifiable for No Category and for Category 1	True
Particles of test item were stuck to the cornea and could not be washed off during the study.	False

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of this study the test material was concluded to be classified as UN GHS eye irritant Category 2 (sub-category 2A).

Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions with an in vitro eye irritation study performed in isolated chicken’s eyes.

After the zero reference measurements, the eye was held in horizontal position and 30 mg of test material was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 mg of powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test material treated eyes, three positive control treated eyes and one negative control treated eye were examined. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

Slight corneal swelling was observed during the four-hour observation period on test material treated eyes. Moderate corneal opacity change (severity 2.0) was noted on all three eyes. Severe fluorescein retention change (severity 3) was noted on all three eyes. No other corneal effect was observed.

Based on this in vitro eye irritation assay in the isolated chicken eyes test with, the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test material (6 sections). Microscopic evaluation showed from very slight to slight erosion of the epithelium in 6/6 cases. Slight vacuolation of the top/mid or low layer of the corneal epithelium in 3/6 sections were also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 2A.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation/Corrosion

Skin Corrosion In Vitro (Varga-Kanizsai, 2017)

An in vitro skin corrosion test was carried out with the test material using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 under GLP conditions.The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The model is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay.

Disks of EPISKIN™ (SM) (two units) were treated with the test material and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9 % (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving %) from the test material. For each treated tissue, viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35 % of the negative control, the test material is considered to be corrosive to skin.

Following exposure with the test material, the mean cell viability was 96.3 % compared to the negative control. This is above the threshold of 35 %, therefore the test material was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Skin Irritation In Vitro (Varga-Kanizsai, 2017)

The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The in vitro skin irritation test was performed in a reconstructed human epidermis model. EPISKIN™ (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay.

Disks of EPISKIN™ (SM) (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2 in a > 95 % RH %. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5 % CO2 in a > 95 RH % protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore to avoid a possible double correction for colour interference, a third set of controls for non-specific colour in killed tissues (NSCkilled), two additional disks were used. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control, the test item is considered to be irritant to skin.

Following exposure with the test material, the mean cell viability was 77.4 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

Under the conditions of this study, the test material is not irritating to skin.

Skin Irritation In Vivo (Shibata, 1997)

The potential for the test material to cause skin irritation was predicted in an in vivo study using Hartley strain female guinea pigs. The study was conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies which do not affect the quality of the relevant results. The test material was applied open onto the animal skin. 24 hours after application, the skin was observed for dermal reactions and the test material was applied. By repetition of this procedure, 14-day repeated application was conducted. During the application period, fur was clipped twice or 3 times per week.

There were no reactions at 2.0 %, 0.6 % or 0.2 %.

Under the conditions of the study, there were no effects from the test material on the general condition or body weight during the observation period.

Skin Irritation In Vivo (Life Sciences Laboratories, 1990)

The study was performed to assess the potential of the test material to cause skin irritation.

0.3 mL of the test material was applied directly to the skin under adhesive tape and assessed according to the Draize Method for Erythema and Oedema formation. The sites were assessed at 24, 48 and 72 hours post application.

Slight erythema and oedema were observed at 24 and 48 hours after application, but it was no longer observed thereafter.

Under the conditions of the study the test material is determined to be not irritating to the skin.

Eye Irritation

Eye Irritation In Vitro (Varga-Kanizsai, 2017)

The eye irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions with an in vitro eye irritation study performed in isolated chicken’s eyes. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

After the zero reference measurements, the eye was held in horizontal position and 30 mg of test material was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 mg of powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test material treated eyes, three positive control treated eyes and one negative control treated eye were examined. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) evaluated.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

Slight corneal swelling was observed during the four-hour observation period on test material treated eyes. Moderate corneal opacity change (severity 2.0) was noted on all three eyes. Severe fluorescein retention change (severity 3) was noted on all three eyes. No other corneal effect was observed.

Based on this in vitro eye irritation assay in the isolated chicken eyes test with, the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

To further establish the classification, histopathological observations were made on two sections of each of the 3 corneas treated with test material (6 sections). Microscopic evaluation showed from very slight to slight erosion of the epithelium in 6/6 cases. Slight vacuolation of the top/mid or low layer of the corneal epithelium in 3/6 sections were also seen. No stromal or endothelial changes were observed as well as no effects on integrity of basement, Bowman’s and Descemet’s membranes. Based on the published criteria for histopathological changes, the test material was classified as Category 2A.

Eye Irritation In Vivo (Life Sciences Laboratories, 1990)

The potential for the test material to cause eye irritation was assessed in an in vivo irritation study in rabbits. The study was a non GLP study performed to sound scientific principles with a sufficient level of detail to assess the quality of the submitted data. The test material was administered as a 1% solution to 4 male New Zealand white rabbits. At 24, 48 and 72 hours after the ocular application, the degree of irritation reactions was jusdged according to Draize's evaluation standards. Application of the test material caused reddening of the conjunctiva, slight edema and adhesion of discharge but the effects were slight. The results are not suitable for definitive classification and labelling under CLP as the test sample was a 1% solution

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin corrosion. The test material is classified for eye irritation, Category 2A.