Benzeneacetaldehyde, cyclic acetal with glycerol

Endpoint summary

Description of key information

Skin corrosion (OECD TG 431): Non-corrosive to skin 
Skin irritation (OECD TG 439): Irritating to skin (Cat 2)
Eye irritation (OECD TG 438): Irritating to eyes (Cat 2).

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:

11 May 2016 to 13 May 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: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

28 July 2015

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.

Cell source:

other: MatTek

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
EpiDerm™ Reconstructed Human Epidermis Model Kit:
Supplier: MatTek
Date received: 10 May 2016
EpiDermTM Tissues (0.63cm2) lot number: 23335
Assay Medium lot number: 050516TMA
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

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

Test for Direct MTT Reduction:
As specified, a test item 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 item was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
If the MTT solution containing the test item turns blue or purple relative to the control, the test item was presumed to have reduced the MTT.
The test item was found to be a possible direct reducer of MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item 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 item like viable tissues.
Freeze-killed tissues were prepared by placing untreated EPIDERM tissues in an empty 12 well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, the MTT reducing test item was applied to two freeze killed tissues per exposure period. In addition, two freeze killed tissues per exposure period remained untreated. The untreated freeze killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Color Interference with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
50 µL of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 oC, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

MAIN TEST
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-labeled 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 Item 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 item 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 item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. 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 labeled 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 stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements:
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-labeled 96-well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 562nm (OD562) of each well was measured using the Anthos 2001 microplate reader.

DATA EVALUATION
Quantitative MTT Assessment (percentage tissue viability):
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 100

Classification of corrosivity potential is based on relative viabilities for both exposure times according to the table presented under Any other information on materials and methods incl. tables.

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control: The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control: Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
Coefficient of Variation: In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

3-Minute exposure period and 60-Minute exposure period

Duration of post-treatment incubation (if applicable):

3 hours MTT incubation period and overnight MTT extraction period

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-Minute exposure period

Value:

87.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Relative mean tissue viability compared to the negative control tissues (viability set at 100%)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-Minute exposure period

Value:

67.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Relative mean tissue viability compared to the negative control tissues (viability set at 100%)

Other effects / acceptance of results:

Direct MTT Reduction:
The test item was found to be a possible direct reducer of MTT. Therefore, an additional procedure using freeze-killed tissues was performed during the determination of skin corrosion potential as a precaution. However, the results obtained showed a negligible degree of interference 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. The results generated from the freeze-killed tissues are presented below.

3 minutes exposure:
Mean of test item treated killed tissues (tkt) = 0.147 OD562
Mean of untreated killed tissues (ukt) = 0.139 OD562
The direct reduction by the test item relative to the negative control value:
(0.147 (tkt) – 0.139 (ukt)) / 1.858 (mean of negative control) = 0.4%

60 minutes exposure:
Mean of test item treated killed tissues (tkt) = 0.143 OD562
Mean of untreated killed tissues (ukt) = 0.136 OD562
The direct reduction by the test item relative to the negative control value:
(0.143 (tkt) – 0.136 (ukt)) / 1.669 (mean of negative control) = 0.4%

Assessment of Color Interference with the MTT endpoint:
The solution (sterile water) containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Test Item, Positive Control Item and Negative Control Item:
Mean OD562 values and viabilities for the negative control, positive control and test item are given under Any other information on results incl. tables.

Quality Criteria
The mean OD562 for the negative control treated tissues was 1.858 for the 3-Minute exposure period and 1.669 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 4.5% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 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.

Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue	Exposure Period	MeanOD562of individual tissues	Mean OD562of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	2.030	1.858	0.243	13.1	100*
		1.686
	60 Minutes	1.676	1.669	0.010	0.6
		1.662
Positive Control	3 Minutes	0.096	0.094	0.003	na	5.1
		0.092
	60 Minutes	0.086	0.075	0.016	na	4.5
		0.063
Test Item	3 Minutes	1.705	1.630	0.106	6.5	87.7
		1.555
	60 Minutes	0.994	1.128	0.189	16.7	67.6
		1.261

OD =Optical density

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

na=  Not applicable

Interpretation of results:

other: Non-corrosive to the skin

Remarks:

in accordance with EU CLP and its amendents (1272/2008 and its amendments)

Conclusions:

The test item was considered to be non-corrosive to the skin, because the viability exceeded 50% after 3 min exposure (87.7%) and 15% after 60 min exposure (67.6%).

Executive summary:

The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model according to OECD TG 431 and GLP principles. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to be a possible direct reducer of MTT and therefore additional freeze-killed tissues were incorporated into the testing for correction purposes. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT occurred (direct reduction by the test item relative to the negative control value is 0.4%). It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results. The relative mean viability for test item treated tissues after 3-Minute and 60-Minute exposure period was 87.7% and 67.6%, respectively. The test item was considered to be non-corrosive to the skin, because the viability exceeded 50% after 3 min exposure and 15% after 60 min exposure. The mean OD562 for the negative control treated tissues after 3-Minute and 60-Minute exposure period was 1.858 and 1.669, respectively (acceptance criteria ≥ 0.8 and ≤ 2.8). The relative mean tissue viability for the positive control treated tissues was 4.5% relative to the negative control following the 60-Minute exposure period (<15%). In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group ranged between 0.6 and 16.7% (=<30%). The quality criteria required for acceptance of results in the test were satisfied.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2016 to 30 May 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)

Version / remarks:

Adopted 28 July 2015

GLP compliance:

yes (incl. QA statement)

Test system:

other: EPISKIN reconstructed human epidermis model

Source species:

human

Cell type:

other: adult human-derived epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 24 May 2016
EpiSkinTM Tissues (0.38cm2) lot number: 16-EKIN-021
Maintenance Medium lot number: 16-MAIN3-035
Assay Medium lot number: 16-ESSC-021

STUDY DESIGN:

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

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

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

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

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

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

Absorbance/Optical Density Measurements (Day 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

DATA EVALUATION:
Quantitative MTT Assessment (Percentage Tissue Viability):
For the test item the mean tissue viability 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) to generate the relative mean tissue viability. The relative mean viability was calculated in the following way:
Relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 100

A test item is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post-exposure incubation is =<50% of the mean viability of the negative controls.
A test item is considered non-irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post-exposure incubation is >50% of the mean viability of the negative controls.

QUALITY CRITERIA:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is >=0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
Test Item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 µL/cm2)

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): SDS was prepared as a 5% w/v aqueous solution.

Duration of treatment / exposure:

15 minute exposure period

Duration of post-treatment incubation (if applicable):

42 hours post-exposure incubation period

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Relative mean tissue viability compared to the negative control tissues (100%)

Value:

15.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
It was considered unnecessary to perform IL-1alpha analysis as the results of the MTT test were unequivocal.
- Direct-MTT reduction:
The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT:
The solution (10 µL of test item in 90 µL of sterile water) containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.293 and the standard deviation value of the viability was 3.5%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 4.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.9%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for the test item: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 8.8%. The test item acceptance criterion was therefore satisfied.

The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given under Any other information on resutls incl. tables.

Mean OD₅₆₂Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562 of triplicate tissues	±SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	1.265	1.293	0.045	97.8	100*	3.5
	1.345			104.0
	1.269			98.1
Positive Control Item	0.076	0.063	0.011	5.9	4.9	0.9
	0.058			4.5
	0.055			4.3
Test Item	0.280	0.196	0.114	21.7	15.1	8.8
	0.066			5.1
	0.241			18.6

OD = Optical Density

SD = Standard deviation

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

Interpretation of results:

other: Skin irritant, Category 2

Remarks:

according to EU CLP criteria and its amendments (1272/2008)

Conclusions:

The relative mean viability of the test item treated tissues was 15.1% after the 15-minute exposure period and 42-Hour post-exposure incubation period. Since the relative mean tissue viability for the substance treated tissues was below 50%, the substance is considered to be a skin irritant.

Executive summary:

The possible skin irritation potential of the substance was tested in vitro, using a reconstructed human epidermis model, according to OECD TG 439 and GLP principles. Skin tissues were treated by topical application of 10 µL undiluted test substance for 15 minutes. After a 42 hours post-exposure incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean viability of the test item treated tissues was 15.1% after a 15-Minute exposure period and 42-Hour post-exposure incubation period. Since the relative mean tissue viability was below 50% after 15 min exposure, the test item is considered to be irritating to the skin. Reliable negative and positive controls were included. The positive control had a relative mean tissue viability of =< 40% (4.9%) after 15 minutes exposure. The mean OD562 for the negative control treated tissues is >=0.6 and <= 1.5 (1.293). The standard deviation value of the percentage viability of three tissues treated identically was less than 18% for test item (8.8%), positive control (0.9%) and negative control (3.5%), indicating that the test system functioned properly.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-03-2016 to 15-04-2016

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:

Adopted on 26 July 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion, Utrechtseweg 48, 3700 AV, Zeist

Species:

other: eyes of male or female chickens (ROSS, spring chickens)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: approximately 1.5 - 2.5 kg
- Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- The preparation and validation of the eyes prior to the ICE-test were all according to OECD guideline 438.

Vehicle:

unchanged (no vehicle)

Controls:

other: Positive controls: Benzalkonium Chloride. Negative control: Physiological saline.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

3 eyes

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The eyes were rinsed with 20 mL saline
- Time after start of exposure: 10 seconds

SCORING SYSTEM: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes

TOOL USED TO ASSESS SCORE: All examinations were carried out with the slit lamp microscope. Fluorescein retention was only scored at approximately 30 minutes after treatment.

CONTROLS: A negative control (30 µL physiological saline) and 3 positive controls (30 µL Benzalkonium Chloride 5%) were included.

Irritation parameter:

percent corneal swelling

Run / experiment:

Slit-lamp examination

Value:

13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Maximum mean score

Irritation parameter:

cornea opacity score

Run / experiment:

Slit-lamp examination

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Maximum mean score

Irritation parameter:

fluorescein retention score

Run / experiment:

Slit-lamp examination

Value:

2.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Slit-lamp examination: The test substance caused corneal effects consisting of slight corneal swelling (mean of 13%), moderate opacity (mean score of 2.0) and moderate-to-severe fluorescein retention (mean score of 2.5). In addition, loosening (veil-like) of epithelium was observed in one cornea. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination: Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion (two corneas), moderate necrosis (two corneas) and moderate vacuolation of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas).

In order to translate the eye irritancy scores from the ICE test to an EU-CLP regulatory classification, four irritancy classes from the ICE study need to be reconciled into three classes within the EU-CLP regulatory classification scheme. This was achieved through application of the prediction model defined in Annex 1 of the study report which is based on scientific judgment and which is supported by several years of experience with conduct of the ICE test. This has resulted in 3 classes of the EU CLP: non eye irritant; serious eye irritation and causes serious eye damage. Despite the serious eye irritation class not considered fully validated in the OECD TG guideline, the categorisation as outlined in the report serious eye irritation is sufficiently scientifically valid, the substance is in the applicability domain of the method and the result is adequately and reliably documented.

Interpretation of results:

other: Irritating to eyes, Cat 2

Remarks:

according to EU CLP (1272/2008 and its amendments)

Conclusions:

Under the test conditions (OECD 438) the substance has to be classified as Cat. 2 and 2A in accordance with EU CLP and GHS, respectively.

Executive summary:

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused corneal effects consisting of slight corneal swelling (mean of 13%), moderate opacity (mean score of 2.0) and moderate-to-severe fluorescein retention (mean score of 2.5). In addition, loosening (veil-like) of epithelium was observed in one cornea. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion (two corneas), moderate necrosis (two corneas) and moderate vacuolation of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas). Based on these results, the substance is considered an eye irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

In vitro tests

Skin corrosion: The corrosivity potential of the test item was evaluated using the EpiDerm™ Human Skin Model according to OECD TG 431 and GLP principles. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to be a possible direct reducer of MTT and therefore additional freeze-killed tissues were incorporated into the testing for correction purposes. However, the results obtained showed a negligible degree of interference due to direct reduction of MTT occurred (direct reduction by the test item relative to the negative control value is 0.4%). It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results. The relative mean viability for test item treated tissues after 3-Minute and 60-Minute exposure period was 87.7% and 67.6%, respectively. The test item was considered to be non-corrosive to the skin, because the viability exceeded 50% after 3 min exposure and 15% after 60 min exposure. The mean OD562 for the negative control treated tissues after 3-Minute and 60-Minute exposure period was 1.858 and 1.669, respectively (acceptance criteria ≥ 0.8 and ≤ 2.8). The relative mean tissue viability for the positive control treated tissues was 4.5% relative to the negative control following the 60-Minute exposure period (<15%). In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group ranged between 0.6 and 16.7% (=<30%). The quality criteria required for acceptance of results in the test were satisfied.

Skin irritation: The possible skin irritation potential of the substance was tested in vitro, using a reconstructed human epidermis model, according to OECD TG 439 and GLP principles. Skin tissues were treated by topical application of 10 µL undiluted test substance for 15 minutes. After a 42 hours post-exposure incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean viability of the test item treated tissues was 15.1% after a 15-Minute exposure period and 42-Hour post-exposure incubation period. Since the relative mean tissue viability was below 50% after 15 min exposure, the test item is considered to be irritating to the skin. Reliable negative and positive controls were included. The positive control had a relative mean tissue viability of =< 40% (4.9%) after 15 minutes exposure. The mean OD562 for the negative control treated tissues is >=0.6 and <= 1.5 (1.293). The standard deviation value of the percentage viability of three tissues treated identically was less than 18% for test item (8.8%), positive control (0.9%) and negative control (3.5%), indicating that the test system functioned properly.

In vitro eye irritation

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused corneal effects consisting of slight corneal swelling (mean of 13%), moderate opacity (mean score of 2.0) and moderate-to-severe fluorescein retention (mean score of 2.5). In addition, loosening (veil-like) of epithelium was observed in one cornea. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion (two corneas), moderate necrosis (two corneas) and moderate vacuolation of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas).Based on these results, the substance is considered an eye irritant.

Justification for classification or non-classification

Based on the negative result in the skin corrosion test (OECDTG 431) the substance does not need to be classified for skin corrosion.

Based on the positive results in the skin irritation test (OECDTG 439) the substance needs to be classified as a skin irritant. According to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 this results in skin irritation, Category 2, H315: Causes skin irritation.

Eye irritation: The substance needs to be classified "Irritating to eyes" based on the positive results in the OECDTG 438, resulting in Cat. 2, H319: Causes serious eye irritation, in accordance with EU CLP (1272/2008 and its amendments).