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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 April 2017 to 12 September 2017
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Sulfates of potassium, sodium and calcium, byproduct from fermentation
Molecular formula:
SO42-, K+, Na+, Ca2+
IUPAC Name:
Sulfates of potassium, sodium and calcium, byproduct from fermentation
Test material form:
solid
Remarks:
light to beige solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the Sponsor, EXP LIberica Lote: AI16503047
- Expiration date of the lot/batch: 30 November 2019
- Purity test date: 1 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%), protected from light and humidity
- Stability under test conditions: not applicable
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle was used
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: the test item did not react with MTT or Medium Assay

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was used as supplied

In vitro test system

Test system:
artificial membrane barrier model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: reconstructed epiderdermis
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin
- Tissue batch number(s): 17-EKIN-017
- Production date: 25 April 2017
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 26 April 2017

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


REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: It was rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL(stock solution) ; 0.3 mg/mL per well
- Incubation time: 3 hours (± 5 min)
- Spectrophotometer: Plate reader Thermo Fisher Scientific, Catalogue Number: 240 72800
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 0.805
- Barrier function: Yes
- Morphology: Well-differentiated epidermis consisting of a basal monolayer, several spinous and granular layers and thick stratum corneum
- Contamination: Absence of HIV1 and 2, Hepatitic C, Hepatitis B and absence of bacteria, fungus and mycoplasma
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: triplicates were used

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
killed tissues
- Procedure used to prepare the killed tissues (if applicable): not specified
- N. of replicates : triplicates
- Method of calculation used:
Non Specific Colour % with killed tissues (NSCkilled%):
NSCkilled % = (mean ODCTK / mean ODNC)×100
ODCTK: test substance treated killed tissues (not incubated with MTT)
ODNC: negative control OD (incubated with MTT)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3 test sequences : Pre incubation / Application and Rinsing / MTT Test

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after after 15 minutes exposure to the test item and 42 hours post incubation is less than 50%
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure to the test item and 42 hours post incubation is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 439: not specified
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
As the test item was solid, first an appropriate amount (10 µL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis and then 20 mg of powdered the test item was applied evenly to the epidermal surface. If necessary, the test item was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

CONTROLS
50 µL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test Item
Value:
103
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative Control
Value:
100
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive Control
Value:
3.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No effect
- Direct-MTT reduction: No effect
- Colour interference with MTT: No effect

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control treated tissues showed 3.4% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.5.

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: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.7.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

 

 

 

Substance

Optical Density (OD)

Viability

SD

 

Measured

Blank corrected

(% RV)

Negative Control:

1

0.780

0.734

94.5

 

-

Phosphate buffered saline

2

0.846

0.799

103.0

 

3

0.842

0.796

102.5

 

mean

--

0.776

100.0

4.7

Positive Control:

1

0.067

0.021

2.6

 

-

5%(w/v)SDSsolution

 

2

0.065

0.019

2.4

 

3

0.086

0.040

5.1

 

mean

--

0.026

3.4

1.5

Test Item:

1

0.804

0.758

97.6

 

-

Sulfates of potassium, sodium and calcium,

2

0.859

0.813

104.7

by-product from fermentation

3

0.874

0.828

106.6

 

mean

--

0.799

103.0

4.7

Notes:

1.         Mean blank value was0.047.

2.         Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3.         SD: Standard deviation

 

Table 2 : HISTORICAL CONTROL DATA

(updated11 August 2016)

 

 

Negative control (PBS)

Positive control (5% (w/v) SDS

solution)

Mean optical density (OD)

0.802

0.094

Standard deviation

0.157

0.048

Minimum optical density (OD)

0.573

0.032

Maximum optical density (OD)

1.362

0.354

Number of cases

111

102

PBS: Phosphate buffered saline SDS: Sodium dodecyl sulphate OD: Optical density (absorbance)

Note: All OD values (measured at 570 ±30 nm) are background corrected values.

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in this in vitro EPISKINTM (SM) model test with Sulfates of potassium, sodium and calcium, by-product from fermentation (Batch number: EXP LIberica Lote: AI16503047), the results indicated that the test item was non-irritant to skin. Hence, the test item was not classified for Skin Irritation according to CLP regulation.
Executive summary:

A GLP compliant in vitro skin irritation test of Sulfates of potassium, sodium and calcium, by-product from fermentation test item was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKINTM (SM) (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Following exposure with Sulfates of potassium, sodium and calcium, by-product from fermentation, the mean cell viability was 103.0% compared to  the negative  control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In  conclusion,   in   this   in   vitro   EPISKINTM   (SM)   model   test   with   Sulfates of potassium, sodium and calcium, by-product from fermentation (Batch number: EXP LIberica Lote: AI16503047), the results indicated that the test item was non-irritant to skin. Hence, the test item was not classified for Skin Irritation according to CLP regulation.