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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-06 - 2016-12-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Remarks:
follow-up skin irritation study
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-02-13 - 2017-02-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Remarks:
previous skin corrosion study
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
B.46 BIS. “IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST”, dated 24. Aug. 2009
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
OECD Guideline for the Testing of Chemicals, Version 439, 26. July 2013, “In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method”
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in the test facility in a closed vessel at room temperature (20±5°C), protected from humidity.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: multiple donors, not specified
Source strain:
other: not applicable
Details on animal used as source of test system:
- Source: The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.
Justification for test system used:
specified in the guideline
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: commercially available EpiDermTM-Kit, procured by MatTek In Vitro Life Science Laboratories, Bratislava., EPI-218-SIT
- Tissue batch number(s): 23393
- Delivery date: 2017-02-14
- Date of initiation of testing: 2017-02-14

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
- Observable damage in the tissue due to washing: none stated
- Modifications to validated SOP: none stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h
- Spectrophotometer: plate spectrophotometer: 96-well-plate photometer, Anthos Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3 replicates for each test item, positive and negative control

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 experiment, 3 replicates

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-corrosive to skin if the viability after 1h exposure is greater than or equal to 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 26.0, 25.9 and 25.6 mg
- Concentration (if solution): The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size.

NEGATIVE CONTROL
Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and without MgCl2).
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
Sodium dodecyl sulphate (SDS), CAS No. 151-21-3, solution in demineralised water containing 5% SDS. Procured from MatTek In Vitro Life Science Laboratories, batch no.: 020217ZSC.
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
1h
Duration of post-treatment incubation (if applicable):
23 hours and 30 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1st replicate test item
Value:
14.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2nd replicate test item
Value:
13.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3rd replicate test item
Value:
14.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value test item
Value:
14
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean positive control
Value:
2.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none stated
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Testing was performed via a GLP OECD 439 guideline study on the registered substance itself. The present in vitro method is recommended in a tiered testing approach, it was used as subsequent testing to a in vitro skin corrosion test, which revealed that the test item was non-corrosive to the skin.
The validity criteria are met, making the results sufficiently reliable to assess the irritating potential of the test item to the skin. The present in vitro method allows the identification of irritating chemical substances and mixtures.
The relative absorbance value was reduced to 14.0% after the treatment. This value is below the threshold for skin irritation potential (50%). Therefore, the test item is considered as irritant to skin in the Reconstructed Human Epidermis (RhE) Test Method.
Executive summary:

In an OECD 439 (in vitro skin irritation) study under GLP, three tissues of the human skin model EpiDermTM were treated with 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control, 5% SDS solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.7. The positive control showed clear irritating effects. Relative absorbance was reduced to 2.9% (required: < 20%).

Variation within the tissue replicates was acceptable; 5.9% for the negative control, 0.1% for the positive control and 0.4% for the test item (required: < 18%).

 

After the treatment with the test item, the relative absorbance values were reduced to 14.0 %. This value is below the threshold for skin irritation potential (50%).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 430 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test Method (TER))
Version / remarks:
OECD Guideline for the Testing of Chemicals No. 430 (2015): “In vitro skin corrosion: transcutaneous electrical resistance test (TER)”
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
EU Method B.40.: “In vitro skin corrosion: transcutaneous electrical resistance test (TER)”. Commission Regulation (EU) No. 1152/2010 of December 08, 2010 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No. 440/2008 of May 30, 2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the registration, evaluation, authorisation, and restriction of chemicals (REACH).
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item should be stored at room temperature (20 ± 5ºC). It should be protected from exposure to humidity and water.

In vitro test system

Test system:
isolated skin discs
Source species:
rat
Cell type:
other: skin cells, various
Cell source:
other: Skin obtained from two rats
Source strain:
Wistar
Details on animal used as source of test system:
SOURCE ANIMAL
- Source: Rats were obtained from the conventional husbandry of laboratory animals of the Centre for Experimental Medicine at the Medical University in Katowice (number in the register of units entitled to the husbandry of laboratory animals: 0053). The Rat Birth Certificates issued by the Centre for Experimental Medicine at the Medical University in Katowice confirmed the animals’ good health.
- Sex: female
- Age at study initiation (in days): 19 days
- Housing: Rats were kept in a plastic cage covered with a wire bar lid. The dimensions of the cage were 58 x 37 x 21 cm. UV-sterilized, autoclaved, dust-free wood shavings were used as bedding. The environment of the animals was enriched by placing wooden blocks and nesting materials for laboratory animals in the cages.
- Diet (e.g. ad libitum): The animals had ad libitum access to the "Labofeed H Standard" standard laboratory fodder produced by Zofia Połczyńska Wytwórnia Pasz “Morawski”, Kcynia
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 5 days
Justification for test system used:
As indicated by the tonnage-driven data requirements under REACH and as set out in the guideline.
Vehicle:
other: substance was applied as powder (neat) and wetted with 150µl deionized water
Details on test system:
SKIN DISC PREPARATION
- Procedure used: The animals were euthanized by intraperitoneal administration of morbital at a dose of 200 mg/kg b.w.. After euthanasia, the dorso-lateral skin of each animal was removed and stripped of excess subcutaneous fat by carefully peeling it away from the skin using a paper towel. The skin discs were cut out using a scalpel. Each skin disc was placed over one of the ends of a PTFE (polytetrafluoroethylene) tube, ensuring that the epidermal surface was in contact with the tube. A rubber ‘O’ ring was press-fitted over the end of the tube to hold the skin in place and excess tissue is trimmed away. The rubber ‘O’ ring was then carefully sealed to the end of the PTFE tube with petroleum jelly. The tube was supported by a spring clip inside a receptor chamber containing MgSO4 solution (154 mM). The skin disc should be fully submerged in the MgSO4 solution. As many as 11 skin discs with a diameter of 20-mm each were obtained from a single rat skin. Two of them were used to control the quality of the procedure, whereas the remaining nine were used for the purpose of the experiment.
The age of the animals was particularly important. The age of 30 days ensures that the hair follicles are in the dormant phase before adult hair growth begins.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was > 10 kΩ.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 21-22°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: removed by washing with a jet of tap water at up to 30°C
- Observable damage in the tissue due to washing: none stated
- Modifications to validated SOP: none stated

DYE BINDING METHOD
The dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: triplicates of each two animals

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged.
- The test substance is considered to be non-corrosive to skin if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A sufficient amount of the test item (ground to a powder) was applied evenly to the disc to ensure that the whole surface of the epidermis was covered.

VEHICLE
- Amount(s) applied (volume or weight with unit): 150 μL deionized water were added on top of the solid

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 150 µl distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 150 µl of 10M hydrochloric acid
Duration of treatment / exposure:
24h
Duration of post-treatment incubation (if applicable):
n/a
Number of replicates:
triplicates of each two animals

Test animals

Species:
rat
Strain:
Wistar

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
TER result for the skin discs treated with the test item, animal No. 1
Value:
5.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
vehicle control
Positive controls validity:
valid
Irritation / corrosion parameter:
transcutaneous electrical resistance (in kΩ)
Run / experiment:
TER result for the skin discs treated with the test item, animal No. 2
Value:
6.11
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
vehicle control
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Yes, see attachment

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The concurrent mean values for the negative control (distilled water) were 15.12 kΩ (animal no. 1) and 13.27 kΩ (animal no. 2). These values fell within the acceptable ranges for the method (10 - 25 kΩ).
- Acceptance criteria met for positive control: The concurrent mean values for the positive control (10M HCl) were 0.97 kΩ (animal no. 1) and 0.99 kΩ (animal no. 2). These values fell within the acceptable ranges for the method (0.5 – 1.0 kΩ).
- Acceptance criteria met for variability between replicate measurements: The mean TER results for the skin discs treated with the test item were equal to 5.90 kΩ (animal no. 1) and 6.11 kΩ (animal no. 2).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The study was conducted under GLP according to OECD guideline 430 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and only one minor deviation to the study plan concerning humidity (see "Overall remarks, attachments"). The validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the corrosive potential of the test substance to the skin in vitro.
On the grounds of the study, it may be stated that the test item, i.e. 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.
In a tiered in vitro testing strategy, this result (non-corrosive) may however only give an indication of the skin-damaging potential, but does not allow a definitive classification according to Regulation 1272/2008. So, an additional skin irritation test should be conducted.
Executive summary:

The in vitro skin corrosion: transcutaneous electrical resistance test (TER) was performed according to OECD TG 430 (GLP) in order to obtain information on health hazards resulting from skin contact with the test item, i.e. 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS).

Skin discs used in the experiment were obtained from two 30-day-old WISTAR female rats (outbred).

In order to control the procedure quality, the electrical resistance of two skin discs obtained from each test animal was measured before the start of the experiment. In each case, the skin disc resistance values were greater than 10 kΩ; therefore, the remainder of the animals’ skin discs could have been used in the experiment.

The test item (ground to a powder) was uniformly applied to the epidermal surface of the skin disc placed inside a tube and wetted. Concurrent positive (10M hydrochloric acid) and negative (distilled water) controls were used. Three skin discs obtained from each animal were used for the test item and three for each control item.

The test item and the control items were evenly applied to the discs for 24 hours and kept at 21-22°C. Then, they were removed by washing with a jet of tap water. Prior to measuring the electrical resistance, the surface tension of the skin was reduced by adding a sufficient volume of 70% ethanol to cover the epidermis. After a few seconds, the ethanol was removed from the tube, whereas the tissue was then hydrated by the addition of 3 mL of a solution of MgSO4 (154 mM). A LCR 6401 low-voltage, alternating current databridge was used to measure the electrical resistance of the skin in kΩ by placing the databridge electrodes on either side of the skin disc.

After the transcutaneous electrical resistance test (TER), the MgSO4 solution was removed from the tube, whereas the skin discs were subjected to a gross examination in order to reveal possible damage.

The dye binding procedure was not necessary in this case since all TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.

The experiment was performed in duplicate, because the difference of TER means of both skin discs treated with the test item were below 5 ± 0,5 kΩ.

The mean TER results for the skin discs treated with the test item were equal to 5.90 kΩ (animal no. 1) and 6.11 kΩ (animal no. 2). They can be accepted because the concurrent positive and negative control values fell within the acceptable ranges for the method.

Gross examinations of the skin discs treated with the test item did not reveal any pathological changes.

On the grounds of the study, it may be stated that the test item, i.e. 3-(2-Benzothiazolylthio)-propanesulfonic acid, sodium salt (ZPS) belongs to a group of substances which do not lead to skin corrosion/severe irritation. The mean TER values for the test item were higher than 5 kΩ and there were not any visible changes on the skin discs.