Fatty acids, tallow, compds. with triethanolamine

Endpoint summary

Description of key information

Skin Corrosion

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

Skin Irritation

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

Eye Irritation (in vitro)

Under the conditions of this study, the test material could not be categorised as it had an IVIS score greater than 3 and less than 55, therefore no prediction of eye irritation can be made.

Eye Irritation (in vivo)

Under the condition of this study, the test material has been determined to be a non-irritant in the eyes of New Zealand White Rabbits.

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:

09 December 2015 to 10 December 2015

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EpiDerm™ Reconstructed Human Epidermis

Details on animal used as source of test system:

SOURCE ANIMAL
Model: Three-dimensional reconstructed human epidermis.
- Supplier: MatTek
- Batch number: 23306
- Date recieved: 08 December 2015

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis
- Tissue batch number(s): 23306
- Delivery date: 08 December 2015
- Date of initiation of testing: 09 December 2016
Upon receipt of the EpiDerm™ tissues, the sealed 24-well plate was stored in the refrigerator until use.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
- Temperature of post-treatment incubation: The plates were refrigerated over night.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Observable damage in the tissue due to washing: None specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 60 minutes
- Spectrophotometer: Anthos 2001 microplate reader.
- Wavelength: 562 nm
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks.
Pre-test procedure – Assessment of direct Test Item reduction of MTT
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 dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test material 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.
Test for Direct MTT Reduction:
As specified, a test material may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test material was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test material 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.
If the MTT solution containing the test material turns blue relative to the control, the test material was presumed to have reduced the MTT and the determination of skin corrosion potential would be performed in parallel on viable and freeze-killed tissues for quantitative correction of the results.
The test material was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test material could not be totally rinsed off the tissues, any residual test material present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze-killed tissues.
This step was a functional check which employs freeze-killed tissues that possess no metabolic activity but absorb and bind the test material like viable tissues.
In addition to the normal test procedure, the MTT reducing test material was applied to two freeze-killed tissues. In addition, two freeze-killed tissues remained untreated. The untreated freeze-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

NUMBER OF REPLICATE TISSUES:
Two six-well plates per exposure period.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Freeze-killed tissues
- Procedure used to prepare the killed tissues: Freeze-killed tissues were prepared by placing untreated EpiDerm™ tissues in an empty 12-well plate and storing in the freezer (-14 to -30 °C) for a minimum of 24 hours. Before use, each tissue was thawed by placing in 0.9 mL of maintenance medium for approximately 1 hour at room temperature.
- N. of replicates: Two

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
A test material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed.
50 µL of test material was added to 300 µL of sterile water. The solution was incubated in the dark at 37 °C, 5 % CO2 in air for 60 minutes. A visual assessment of the colour was then made.

PRE-INCUBATION
The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labelled 6-well plates for both the 3-minute and 60-minute exposure periods. EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5 % CO2) for approximately 1 hour before dosing.

APPLICATION OF TEST MATERIAL AND RINSING
Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-minute and 60-minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 µL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-minute exposure period was returned to the incubator, while the other was being dosed for the 60-minute exposure. For the 60-minute exposure period, 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. 50 µL of the test material and 50 µL of 8.0 N potassium hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5 % CO2) for the 60-minute exposure period.
When dosing for the 60-minute exposure period was complete, the same procedure was repeated for the 3-minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test material. 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. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5 % CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3-hour MTT incubation was complete, the inserts were blotted and transferred to labelled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates were placed into a refrigerator overnight, to allow extraction to proceed

PREDICTION MODEL / DECISION CRITERIA
- 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 applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL. The negative control item was used as described.

POSITIVE CONTROL
- Amount applied: 50 µL. The positive control item was used as described.

Duration of treatment / exposure:

3 minute and 60 minute exposure periods.

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Duplicate tissues were treated with the test material, negative and positive control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure period

Value:

103.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 mintute exposure period

Value:

102.4

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:
- Direct-MTT reduction:
An assessment found the test material was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed during the determination of skin irritation potential. However, the results obtained showed a negligible degree of interference (0.9 % relative to the negative control after 3 minutes exposure and 0.0 % relative to the negative control after 60 minutes exposure) due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results.

3 minutes exposure:
Mean of test material treated killed tissues (tkt) = 0.149 OD562
Mean of untreated killed tissues (ukt) = 0.132 OD562
The direct reduction by the test material relative to the negative control value:
(0.149 (tkt) – 0.132 (ukt)) / 1.884 (mean of negative control) = 0.9 %

60 minutes exposure:
Mean of test material treated killed tissues (tkt) = 0.114 OD562
Mean of untreated killed tissues (ukt) = 0.150 OD562
The direct reduction by the test material relative to the negative control value:
(0.144 (tkt) – 0.150 (ukt)) / 1.886 (mean of negative control) = 0.0 %

- Colour interference with MTT:
The solution containing the test material did not become coloured. This was taken to indicate the test material did not have the potential to cause colour interference.

QUALITY CRITERIA
The mean OD562 for the negative control treated tissues was 1.884 for the 3 Minute exposure period and 1.886 for the 60 Minute exposure period. The negative control acceptance criteria were therefore satisfied.

The relative mean tissue viability for the positive control treated tissues was 5.1 % relative to the negative control following the 60 Minute exposure period. The positive control acceptance criterion was therefore satisfied.

In the range 20-100 % viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30 %. The acceptance criterion was therefore satisfied.

Table 1: Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Material

Tissue	Exposure Period	Mean OD562 of individual tissues	Mean OD562 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	2.066	1.884	0.257	13.7	100*
		1.702
	60 Minutes	1.786	1.886	0.141	7.5
		1.985
Positive Control	3 Minutes	0.109	0.107	0.003	N/A	5.2
		0.105
	60 Minutes	0.091	0.096	0.006	N/A	5.1
		0.100
Test Material	3 Minutes	2.025	1.949	0.108	5.6	103.4
		1.872
	60 Minutes	1.879	1.931	0.074	3.8	102.4
		1.983

OD = Optical density

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

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test material was determined to be non-corrosive to the skin

Executive summary:

A study was performed in vitro to assess the corrosive potential of the test material in accordance with the standardised guidelines OECD 431 and EU Method B.40bis under GLP conditions.

Duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. The test employed the use of the EpiDerm™ Human Skin Model. Negative and positive control groups were treated for each exposure period. The test material was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test material 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).

The mean OD562 for the negative control treated tissues was set at 100 for both the 3 minute and 60 minute exposure period. The relative percentage tissue viability for the positive control treated tissues was 5.2 % for a 3 minute exposure and 5.1 for the 60 minute exposure period. 

The relative mean viability of the test material treated tissues was 103.4 for the 3 minute exposure period and 102.4 for the 60 minute exposure period.

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

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 January 2016 to 25 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EPISKIN™ Reconstructed Human Epidermis

Details on animal used as source of test system:

SOURCE ANIMAL
Model: 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.
- Supplier: SkinEthic Laboratories, Lyon, France
- Batch number: 16-EKIN-003

Justification for test system used:

The EPISKINTM 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. A highly differentiated and stratified epidermis model is obtained after a 13 Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Following a full validation study the EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
The procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study.
Test items are applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis
- Tissue batch number(s): 16-EKIN-003
- Delivery date: 19 January 2016
- Date of initiation of testing: 20 January 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

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 Colour Satisfactory: Yes
Agar Medium Colour Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 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 material and each control material. The tissues were incubated at 37 °C, 5 % CO2 in air overnight.

Application of Test Item (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 material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm2) of the test material was applied to the epidermis 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 material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). 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 material). The plates were kept in the biological safety cabinet at room temperature for15 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++.

NUMBER OF REPLICATE TISSUES: Triplicate tissues

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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 incubated at 37 °C, 5 % CO2 in air for 42 hours.
- Observable damage in the tissue due to washing: None specified

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- The test was performed in parallel with viable and water killed tissues.
- Procedure used to prepare the killed tissues: Water-killed tissues were prepared by placing untreated EPISKINTM tissues in a 12 well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5 % CO2 in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, the MTT reducing test material was applied to three water-killed tissues. In addition, three water-killed tissues remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of a 0.3 mg/mL MTT solution was used.
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader.
- Wavelength: 562 nm
- Filter: No reference filter was used.

Test for Direct MTT Reduction
As specified, a test material 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 material is checked for the ability to directly reduce MTT according to the following procedure:
10 µL of the test material 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 material turns blue, the test material 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.
The test material was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test material could not be totally rinsed off the tissues, any residual test material present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues.

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

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-labelled 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 labelled 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.

PREDICTION MODEL / DECISION CRITERIA
For the test material the relative mean tissue viabilities obtained after the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD562 of test material / mean OD562 of negative control) x 100

The test material was shown to directly reduce MTT and water-killed tissues were employed; the results of the MTT assay were therefore corrected as follows:
True viability = mean OD tvt-(OD tkt-OD ukt)
OD = optical density at 562 nm
tvt = treated viable tissues
tkt = treated killed tissues
ukt = untreated killed tissues
If direct reduction by the test material is greater than 30 % of the negative control value, additional steps must be taken into account or the test material may be considered incompatible with this test system.
If direct reduction by the test material is less than 30 % of the negative control value, the mean OD of the test material treated killed control may be subtracted from the mean OD of the test material treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.
- The test substance is considered to be irritating to skin if the viability after 15 minutes exposure is less than or equal to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
10 µL (26.3 µL/cm²) of the test material was applied to the epidermis surface.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

15 minutes

Number of replicates:

Triplicate tissues were treated with the test material. Triplicate tissues treated with DPBS which served as the negative controls and triplicate tissues treated with SDS 5 % w/v which served as the positive control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of triplicate samples

Value:

71.1

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:
- Direct-MTT reduction: The MTT solution containing the test material turned blue which indicated that the test material directly reduced MTT. The direct reduction by the test material relative to the negative control value was 9.9 %. Direct reduction was <30 % relative to the negative control and therefore acceptable.
- Colour interference with MTT: The solution containing the test material was colourless. It was therefore unnecessary to run colour correction tissues.
It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD562 for the negative control treated tissues was 0.858 and the standard deviation value of the viability was 8.6 %. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: Yes. The relative mean tissue viability for the positive control treated tissues was 8.4 % relative to the negative control treated tissues and the standard deviation value of the viability was 1.3 %. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 6.9 %. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

A study was performed in vitro to assess the irritancy potential of the test material. The study was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

The skin irritation potential of the test material was evaluated using the EPISKIN™ reconstructed human epidermis model. Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was found to directly reduce the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt, therefore additional non-viable tissues were incorporated into the testing to counteract false negative results. 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. 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 then measured at 562 nm.

The acceptance criteria was met for negative and positive controls and the standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 6.9 %. The test material acceptance criterion was therefore satisfied.

The relative mean viability of the test material treated tissues was 71.1 % after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 January 2016 to 29 January 2016

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)

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter.
- Characteristics of donor animals: Typically 12 to 60 months old.
- Storage, temperature and transport conditions of ocular tissue: They were transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: The corneas were refrigerated on arrival and used within 24 hours of receipt.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were macroscopically examined before and after dissection. Only corneas free from damage were used.
- Indication of any antibiotics used: The eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with penicillin at 100 IU/mL and streptomycin at 100 µg/mL.

Vehicle:

unchanged (no vehicle)

Remarks:

The test material was used as supplied.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

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 material and three corneas to the positive control.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
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.
The condition of the cornea was visually assessed post treatment and post incubation.

NUMBER OF REPLICATES
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 material and three corneas to the positive control material.

NEGATIVE CONTROL USED
0.75 mL 0.9 % w/v sodium chloride solution

POSITIVE CONTROL USED
0.75 mL ethanol

APPLICATION DOSE AND EXPOSURE TIME
The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes.

TREATMENT METHOD:
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material or control materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test material and control materials 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. The anterior chamber was refilled with fresh complete EMEM. A post-treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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.
- Corneal permeability: Following the final 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 (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes. After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of medium representing each cornea was applied to a designated well on a 96 well plate and the optical density at 492 nm (OD492) was measured. Passage of sodium fluorescein dye measured with the aid of the Anthos 2001 microtiter plate reader (OD492).
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.
- Histopathology: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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.

DECISION CRITERIA:
For an acceptable test the following positive control criterion should be achieved:
Neat ethanol 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 collated during 2014 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 29.6 to 52.0.

For an acceptable test the following negative control criteria should be achieved:
0.9% w/v sodium chloride solution 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 2014 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be =2.9 and for permeability =0.103.

Irritation parameter:

in vitro irritation score

Value:

3.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test material or negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of =2.9 and permeability =0.103. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.

Table 1. In Vitro Irritancy Scores

Treatment	In Vitro Irritancy Score
Test Material	3.5
Negative Control	2.1
Positive Control	39.0

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this study, the test material could not be categorised as it had an IVIS score greater than 3 and less than 55, therefore no prediction of eye irritation can be made.

Executive summary:

The eye irritancy potential of the test material was investigated in vitro in a study conducted through The Bovine Corneal Opacity and Permeability (BCOP) Assay in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions.

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 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 an In Vitro Irritancy Score (IVIS). 

After test material exposure, the corneas treated with the test material or negative control item were clear post treatment and post incubation and the corneas treated with the positive control item were cloudy post treatment and post incubation. The negative and positive control acceptance criteria were satisfied as the negative control gave an opacity reading of =2.9 and permeability =0.103 and the positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The test material gave an opacity reading of 3.3 and permeability of 0.008 (mean corrected values) and had an In Vitro Irritancy Score of 3.5.

Under the conditions of this study, the test material could not be categorised as it had an IVIS score greater than 3 but less than 55, therefore no prediction of eye irritation can be made.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 February 2016 to 10 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Remarks:

Hsdlf:NZW

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12 to 52 weeks old.
- Weight at study initiation: 2.65 or 3.40 kg.
- Housing: The animals were individually housed in suspended cages.
- Diet: Ad libitum
- Water: Ad libitum access to mains drinking water.
- Acclimation period: Acclimatization period of at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): The rate of air exchange was at least fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

Vehicle:

unchanged (no vehicle)

Remarks:

For the purpose of the study the test material was used as supplied.

Controls:

other: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.1 mL

Duration of treatment / exposure:

The test material was not removed.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

One male and one female rabbit.

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 % tetracaine 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 a six point scale.
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.

REMOVAL OF TEST SUBSTANCE
- Washing: The rabbit eyes were not irrigated.

SCORING SYSTEM: The eyes were examined and scored for ocular reactions at the following intervals after instillation: 1, 24, 48 and 72 hours after treatment. Effects were scored in accordance with the Draize scale. Any other ocular effects were also noted. 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.

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

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:

4

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.667

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.667

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.333

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.333

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No corneal effects were noted during the study.
Irridial inflammation was noted in one treated eye 1 hour after treatment.
Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24- and 48-hour observations.
Both treated eyes appeared normal at the 72-Hour observation period.

Other effects:

- Other observations: Both animals showed expected gain in body weight during the study.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the condition of this study, the test material has been determined to be not irritating to the eyes of New Zealand White Rabbits. The test material does not meet the criteria for classification according to the EU Classification and Labelling of Chemicals.

Executive summary:

A study was conducted to investigate the potential of the test material to cause eye irritation in accordance with the standardised guidelines OECD 405 and EU Test Method B.5 under GLP conditions.

One male and one female rabbits (New Zealand White strain) were treated with the test material over a 72 hour period. A volume of 0.1 mL of the test material 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 material, and then released. The left eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the Draize numerical evaluation. Any other ocular effects were also noted. Examination of the eyes 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.

Both animals tested survived the test period and showed expected gain in body weight during the study.The single application of the test material to the non-irrigated eyes of two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 72-Hour observation period.

Under the condition of this study, the test material has been determined to be not irritating to the eyes of New Zealand White Rabbits. The test material does not meet the criteria for classification according to the EU Classification and Labelling of Chemicals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin Corrosion

A study was performed in vitro to assess the corrosive potential of the test material in accordance with the standardised guidelines OECD 431 and EU Method B.40bis under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

Duplicate tissues were treated with the test material for exposure periods of 3 and 60 minutes. The test employed the use of the EpiDerm™ Human Skin Model. Negative and positive control groups were treated for each exposure period. The test material was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test material 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).

The mean OD562 for the negative control treated tissues was set at 100 for both the 3 minute and 60 minute exposure period. The relative percentage tissue viability for the positive control treated tissues was 5.2 % for a 3 minute exposure and 5.1 for the 60 minute exposure period. 

The relative mean viability of the test material treated tissues was 103.4 for the 3 minute exposure period and 102.4 for the 60 minute exposure period.

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

Skin Irritation

A study was performed in vitro to assess the irritancy potential of the test material. The study was conducted in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The skin irritation potential of the test material was evaluated using the EPISKIN™ reconstructed human epidermis model. Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was found to directly reduce the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt, therefore additional non-viable tissues were incorporated into the testing to counteract false negative results. 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. 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 then measured at 562 nm.

The acceptance criteria was met for negative and positive controls and the standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 6.9 %. The test material acceptance criterion was therefore satisfied.

The relative mean viability of the test material treated tissues was 71.1 % after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

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

Eye Irritation (in vitro)

The eye irritancy potential of the test material was investigated in vitro in a study conducted through The Bovine Corneal Opacity and Permeability (BCOP) Assay in accordance with the standardised guidelines OECD 437 and EU Method B.47 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 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 an In Vitro Irritancy Score (IVIS). 

After test material exposure, the corneas treated with the test material or negative control item were clear post treatment and post incubation and the corneas treated with the positive control item were cloudy post treatment and post incubation. The negative and positive control acceptance criteria were satisfied as the negative control gave an opacity reading of =2.9 and permeability =0.103 and the positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The test material gave an opacity reading of 3.3 and permeability of 0.008 (mean corrected values) and had an In Vitro Irritancy Score of 3.5.

Under the conditions of this study, the test material could not be categorised as it had an IVIS score greater than 3 but less than 55, therefore no prediction of eye irritation can be made from this study.

Eye Irritation (in vivo)

An in vivo study was conducted to investigate the potential of the test material to cause eye irritation in accordance with the standardised guidelines OECD 405 and EU Test Method B.5 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

One male and one female rabbits (New Zealand White strain) were treated with the test material over a 72 hour period. A volume of 0.1 mL of the test material 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 material, and then released. The left eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the Draize numerical evaluation. Any other ocular effects were also noted. Examination of the eyes 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.

Both animals tested survived the test period and showed expected gain in body weight during the study.The single application of the test material to the non-irrigated eyes of two rabbits produced iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 72-Hour observation period.

Under the condition of this study, the test material has been determined to be not irritating to the eyes of New Zealand White Rabbits. The test material does not meet the criteria for classification according to the EU Classification and Labelling of Chemicals.

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 and eye irritation or corrosion.