Soybean meal, protein extn. residue

Endpoint summary

Description of key information

Skin Corrosion (Warren, 2018)

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

Skin Irritation (Warren, 2018)

Under the conditions of this study, the test material is not classified as a skin irritant. 

Eye Irritation (Henzell, 2018)

Under the conditions of this study, the test material induced an in vitro irritancy score of 0.4 and therefore does not require classification as an eye irritant.

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:

25 October 2017 to 27 October 2017

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:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit.
-Supplier: MatTek
- Tissue lot number: 25850
- Delivery date: 24 October 2017

TEST FOR DIRECT MTT REDUCTION
- 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: 25 mg 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/purple relative to the control, the test material was presumed to have reduced the MTT.
- 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 item like viable tissues.
Freeze-killed tissues were prepared prior to the study 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 material 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 COLOUR INTERFERENCE WITH THE MTT ENDPOINT
A test material may interfere with the MTT endpoint if it is coloured or if it becomes coloured when in wet or aqueous conditions. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed. 25 mg 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.


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-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 THE TEST MATERIAL
- 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. 25 mg of the test material and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 μL of sterile water was added for wetting of the test material to increase tissue surface contact. 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.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (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.

MTT EXTRACTION
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 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-labelled 96-well plate. 200 μL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader.

NUMBER OF REPLICATE TISSUES: 2 per exposure time

DATA EVALUATION
- Quantitative MTT Assessment (percentage tissue viability):
The corrosivity potential of the test material 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 OD570 of test material/ mean OD570 of negative control) x 100

Classification of corrosivity potential is based on relative viabilities for both exposure times according to the following:
STEP 1
< 50 % viability after 3 min exposure= Corrosive
≥ 50 % viability after 3 min exposure AND < 15 % viability after 60 min exposure = Corrosive
≥ 50 % viability after 3 min exposure AND ≥ 15 % after 60 min exposure = Non-corrosive

STEP 2 for test materials identified as corrosive in step 1
< 25 % viability after 3 min exposure = H314 Sub-category 1A
≥ 25 % viability after 3 min exposure = H314 Combination of sub-categories 1B-and-1C

- Quality Criteria:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
- The absolute OD570 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 OD570 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.
- 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 %.
-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:

TEST MATERIAL
- Amount(s) applied: 25 mg

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8.0 N

Duration of treatment / exposure:

3 minutes of exposure and 60 minutes of exposure

Duration of post-treatment incubation (if applicable):

Incubated for 3 hours with MTT

Number of replicates:

2 per exposure time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure period

Value:

102.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

98.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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. However, the results obtained showed that negligible 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 or for reporting purposes.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- 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.

TEST MATERIAL, POSITIVE AND NEGATIVE CONTROLS
- Mean OD570 values and viabilities for the negative control, positive control and test material are given in Table 1.

QUALITY CRITERIA
- The mean OD570 for the negative control treated tissues was 1.620 for the 3-Minute exposure period and 1.704 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 3.8 % 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.

Table 1: Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material

Tissues	Exposure Period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control	3 Minutes	1.633	1.620	0.019	1.2	100*
		1.606
	60 Minutes	1.692	1.704	0.017	1.0
		1.716
Positive Control	3 Minutes	0.095	0.086	0.013	N/A	5.3
		0.077
	60 Minutes	0.069	0.066	0.005	N/A	3.8
		0.062
Test Material	3 Minutes	1.674	1.656	0.026	1.6	102.2
		1.637
	60 Minutes	1.684	1.683	0.002	0.1	98.8
		1.681

 OD = Optical Density

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

N/A = not applicable

Interpretation of results:

other: Not corrosive in accordance with EU criteria

Conclusions:

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

Executive summary:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. The corrosivity potential was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40bis, under GLP conditions.

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 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-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

The relative mean viabilities for the test material treated tissues were 102.2 and 98.8 % after 3 and 60 minute exposure times, respectively. The quality criteria required for acceptance of results in the test were satisfied.

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

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 January 2018 to 15 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, supplied by SkinEthic Laboratories, Lyon, France.
- Tissue lot number: 18-EKIN-002
- Delivery date: 09 January 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation: 37 °C

TEST FOR DIRECT MTT REDUCTION
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 mg 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 or purple, 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.
This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test material like viable tissues.
Water-killed tissues were prepared prior to the study 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 a minimum of 48 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.

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 mg 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.

MAIN TEST
PRE-INCUBATION: DAY 0
Before removal from the transport plate each tissue was inspected for: any air bubbles between the agarose gel and the insert, if tissues are satisfactory, if the temperature indicator colour is satisfactory and if the agar medium colour is satisfactory. 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 MATERIAL: 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. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test material and the epidermis. Approximately 10 mg (26.3 mg/cm²) of the test material was then applied to the epidermal surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5 % w/v served as the positive controls. To ensure satisfactory contact with the positive control material 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 material). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.

REMOVAL OF TEST MATERIAL AND CONTROLS
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 Ca²+ and Mg²+. 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 material. 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 °C, 5 % CO2 in air for 42 hours.

MTT LOADING/ FORMAZAN EXTRACTION: DAY 3
Following the 42 hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise 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 °C 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™ 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.

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 coloured solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre labelled 96 well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

NUMBER OF REPLICATE TISSUES: 3

DATA EVALUATION
Quantitative MTT Assessment (Percentage Tissue Viability)
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 OD570 of test material / mean OD570 of negative control) x100

Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42-Hour post-exposure incubation period as follows:
Relative mean tissue viability is ≤ 50 %: Irritant,
Relative mean tissue viability is > 50 %: Non-irritant.
If the relative mean tissue viability is ≤50%, differentiation between EU CLP/UN GHS Category 1 and Category 2 will not be possible based on the results of this study.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 mg (26.3 mg/cm²)

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % w/v aqueous solution and was used within 2 hours of preparation.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

91.4

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:

DIRECT MTT REDUCTION
- The MTT solution containing the test material turned black which indicated the test material was a strong direct MTT reducer.
- An assessment found the test material was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test material was a yellow colour. This colour was attributed to the intrinsic colour of the test material itself. It was therefore unnecessary to run colour correction tissues.

TEST MATERIAL , POSITIVE CONTROL AND NEGATIVE CONTROL
- A summary of the results can be seen in Table 1.
- The relative mean viability of the test material treated tissues was 91.4 % after a 15-minute exposure period and 42-Hour post-exposure incubation period.
- It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Table 1: Summary of Results

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control	0.578	0.638	0.053	90.6	100*	8.3
	0.658			103.1
	0.679			106.4
Positive Control	0.032	0.033	0.001	5.0	5.2	0.2
	0.033			5.2
	0.034			5.3
Test Material	0.603	0.583	0.019	94.5	91.4	3.0
	0.580			90.9
	0.565			88.6

OD = Optical density

SD = Standard deviation

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

Quality Criteria

- The relative mean tissue viability for the positive control treated tissues was 5.2 % relative to the negative control treated tissues and the standard deviation value of the viability was 0.2 %. The positive control acceptance criteria were therefore satisfied.

- The mean OD570 for the negative control treated tissues was 0.638 and the standard deviation value of the viability was 8.3 %. The negative control acceptance criteria were therefore satisfied.

- The standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 3.0 %. The test material acceptance criterion was therefore satisfied.

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material is not classified as a skin irritant.

Executive summary:

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

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

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

The relative mean viability of the test material treated tissues was 91.4 % after the 15-minute exposure period and 42-Hours post-exposure incubation period.

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

Under the conditions of this study, the test material is not classified as a skin irritant. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- The test material was prepared as a 20 % w/v solution in sodium chloride 0.9 % w/v.
- The test material was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
- No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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

Vehicle:

other: sodium chloride 0.9 % w/v

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 20 % w/v

VEHICLE
- Amount(s) applied: 0.75 mL
- Concentration: 0.9 % w/v
- Lot No.: 3012487

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes with sodium fluorescein

Number of animals or in vitro replicates:

Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test material and three corneas to the positive control item.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 75 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.

NUMBER OF REPLICATES
- Three corneas were randomly 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: Sodium chloride 0.9 % w/v

POSITIVE CONTROL USED: Imidazole, used as a 20 % w/v solution in sodium chloride 0.9 % w/v.

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL for 240 minutes

TREATMENT METHOD:
- The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material preparation 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 °C for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- At the end of the exposure period the test material and control materials were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-treatment opacity reading was taken after removal of the test material and each cornea was visually observed. 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 opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes. After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labelled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. A 1:5 dilution was performed on two of the positive controls. 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.
- Others: 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. No histopathology was required for this study.

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 material induced a response through only one of the two endpoints.
- The condition of the cornea was visually assessed post treatment.

DECISION CRITERIA:
The test material was classified according to the following prediction model:
IVIS ≤ 3: No Category
IVIS >3; ≤ 55: No prediction can be made
IVIS > 55: Category 1

ACCEPTABILITY OF THE TEST
- The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2017 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 71.2 to 132.9.
- 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 2017 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.3 and for permeability ≤0.044.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

0.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL EPITHELIUM CONDITION
- The corneas treated with the test material were clear post treatment. The corneas treated with the negative control material were clear post treatment. The corneas treated with the positive control material were cloudy post treatment.

ACCEPTABILITY OF THE TEST
- The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied.
- The negative control gave opacity of ≤2.3 and permeability ≤0.044. The negative control acceptance criteria were therefore satisfied.

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD492)		IVIS
		Pre-treatment	Post-treatment	Post-treatment – Pre-treatment	Corrected Value		Corrected Value
Negative Control	1	6	8	2		0.015
	2	4	6	2		0.000
	3	3	4	1		0.000
				1.7*		0.005♦		1.7
Positive Control	4	7	83	76	74.3	1.540	1.535
	5	4	76	72	70.3	1.805	1.800
	6	4	66	62	60.3	1.201	1.196
					68.3•		1.510•	91.0
Test Material	7	4	6	2	0.3	0.040	0.035
	9	5	6	1	0.0	0.007	0.002
	10	4	6	2	0.3	0.003	0.000
					0.2•		0.012•	0.4

OD = Optical density

* = Mean of the post-treatment − pre-treatment values

♦ = Mean permeability

• = Mean corrected value

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material induced an in vitro irritancy score of 0.4 and therefore does not require classification as an eye irritant.

Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47, under GLP conditions.

The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test material is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test material was applied at a concentration of 20 % w/v in sodium chloride 0.9 % w/v for 240 minutes. Negative and positive control materials 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).

The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤2.3 and permeability ≤0.044. The negative control acceptance criteria were therefore satisfied.

Under the conditions of this study, the test material induced an in vitro irritancy score of 0.4 and therefore does not require classification as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion (Warren, 2018)

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. The corrosivity potential was investigated in accordance with the standardised guidelines OECD 431 and EU Method B.40bis, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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 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-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

The relative mean viabilities for the test material treated tissues were 102.2 and 98.8 % after 3 and 60 minute exposure times, respectively. The quality criteria required for acceptance of results in the test were satisfied.

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

Skin Irritation (Warren, 2018)

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

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

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

The relative mean viability of the test material treated tissues was 91.4 % after the 15-minute exposure period and 42-Hours post-exposure incubation period.

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

Under the conditions of this study, the test material is not classified as a skin irritant. 

Eye Irritation (Henzell, 2018)

The eye irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test material is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test material was applied at a concentration of 20 % w/v in sodium chloride 0.9 % w/v for 240 minutes. Negative and positive control materials 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).

The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤2.3 and permeability ≤0.044. The negative control acceptance criteria were therefore satisfied.

Under the conditions of this study, the test material induced an in vitro irritancy score of 0.4 and therefore does not require classification as an eye irritant.

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 corrosion or irritation.