Soybean meal, protein extn. residue

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Soybean meal, protein extn. residue

EC Number:

293-693-6

EC Name:

Soybean meal, protein extn. residue

Cas Number:

91081-84-4

Molecular formula:

UVCB substance

IUPAC Name:

Not determined

Test material form:

solid: particulate/powder

Details on test material:

- Physical state/Appearance: Beige-yellow/tan powder
- Storage Conditions: Room temperature in the dark

In vitro test system

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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.