Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-09 to 2017-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
28 July 2015
Deviations:
yes
Remarks:
inter-tissue variability in viability for 3-minutes exposure to test item > 30% (i.e. 31.8%), however no influence on classification (relative mean viability >50% for this group)
Qualifier:
according to
Guideline:
other: EU Method B.40 BIS:"In vitro Skin Corrosion: Human Skin Model Test"
Version / remarks:
30 May 2008
Deviations:
yes
Remarks:
inter-tissue variability in viability for 3-minutes exposure to test item > 30% (i.e. 31.8%), however no influence on classification (relative mean viability >50% for this group)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Appearance: clear liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Identification: Jeffcat LE 30
- CAS number: 65286-55-7
- Physical state/appearance: clear colorless liquid
- Source and lot/batch No.of test material: DR74271215
- Expiration date of the lot/batch: 2018-12-30
- Purity: 98.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was used as supplied.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
Cell type: epithelial, derived from human skin, and formed into a stratified, cornified epithelium
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Tissue batch number(s): 25835
- Production date: no data
- Delivery date: 08 August 2017 (Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator (2 to 10 °C) until use)
- Date of initiation of testing: experimental start date: 09 August 2017
- Assay Medium lot number: 080317TMA

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

REMOVAL OF TEST MATERIAL AND CONTROLS
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.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- MTT loading: The plate was incubated (37 °C, 5% CO2) for 3 hours.
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 570nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL of test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): for both 60-Minute and 3-Minute exposure period: 50 μL of sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): for both 60-Minute and 3-Minute exposure period: 50 μL of 8.0 N Potassium Hydroxide
Duration of treatment / exposure:
3 minutes and one hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
duplicates: 4 tissues per test item, negative control and positive control: 2 for the 3-minute exposure period and 2 for the 1 hour-exposure

Test system

Details on study design:
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 t ested concurrently to act as a control. If the MTT solution containing the test item turns blue/purple relative to the control, the test item was presumed to have reduced the MTT. The test item was shown to directly reduce 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 act ivity but absorb and bind the test item like viable tissues. Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM 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 peri od, 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 an d 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 570nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader.


Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute application
Value:
75.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour application
Value:
12.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: coefficient of variation
Run / experiment:
3-minute application
Value:
31.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation / corrosion parameter:
other: Coefficient of Variation
Run / experiment:
1-hour application
Value:
5.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation / corrosion parameter:
other: optical density
Run / experiment:
3-minute application
Value:
1.078
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
other: optical density
Run / experiment:
1-hour application
Value:
0.193
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
3-minute application:
- relative mean viability:
negative control: The mean % viability of the negative control tissue is set at 100%
positive control: 6.4
- coefficient of variation between tissue replicates
negative control: 15.2
positive control: not applicable
- mean optical density:
negative control: 1.423
positive control: 0.091

1-hour application:
- relative mean viability:
negative control: The mean % viability of the negative control tissue is set at 100%
positive control: 0.9
- coefficient of variation between tissue replicates
negative control: 3.7
positive control: not applicable
- mean optical density:
negative control: 1.567
positive control: 0.014

Results: An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that no 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. The solution 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. The mean viability of the negative control tissues is set at 100%

- OTHER EFFECTS:
- Direct-MTT reduction:
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that no 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.
- Colour interference with MTT:
The solution 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The mean OD570 for the negative control treated tissues was 1.423 for the 3-Minute exposure period and 1.567 for the 60-Minute exposure period. 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 0.9% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements:
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30% with the exception of the test item treated 3 minute exposure group which was 31.8% However, as the relative tissue viability of this group was considerably higher than 50% this failure would not affect the overall classification of corrosivity. This acceptance criterion was therefore not fully satisfied and is reported as a deviation (see above in text field Any other information on materials and methods incl. tables).

Any other information on results incl. tables

Direct MTT Reduction

An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze‑killed tissues was performed. However, the results obtained showed that no 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 color interference with the MTT

The solution 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 below:

Tissue

Exposure Period

MeanOD570of individual tissues

Mean OD570of duplicate tissues

Standard Deviation

Coefficient of Variation

(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.575

1.423

0.216

15.2

100*

1.270

60 Minutes

1.526

1.567

0.057

3.7

1.607

Positive Control

3 Minutes

0.076

0.091

0.021

na

6.4

0.105

60 Minutes

0.013

0.014

0.001

na

0.9

0.015

Test Item

3 Minutes

1.320

1.078

0.343

31.8

75.7

0.835

60 Minutes

0.186

0.193

0.010

5.1

12.3

0.200


OD: Optical density

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

na: Not applicable

The relative mean viabilities of the test item treated tissues were as follows:

 3 minutes exposure: 75.7%

60 minutes exposure: 12.3%

     

Quality Criteria

The mean OD570 for the negative control treated tissues was 1.423 for the 3‑Minute exposure period and 1.567 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 0.9% 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% with the exception of the test item treated 3 minute exposure group which was 31.8% However, as the relative tissue viability of this group was considerably higher than 50% this failure would not affect the overall classification of corrosivity. This acceptance criterion was therefore not fully satisfied and is reported as a deviation.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Conclusions:
The test item was considered to be corrosive: UN GHS H314 Combination of sub-categories 1B and 1C. Category 1B has been indicated in interpretation of results and section 2.1 GHS classification in a precautionary approach.