Myo-Inositol, hexakis(dihydrogen phosphate), sodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11. Sep. 2017 - 15. Sep. 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the Testing of Chemicals, Version 439, adopted 28. July 2015,
“In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”

Deviations:

yes

Remarks:

The pre-incubation time was 1 hour, instead of 18- 24 hours. This can be seen as uncritical, because the pre-incubation time must be at least 1 hour (in consultation with the tissue supplier; MatTek In Vitro Life Science Laboratories).

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending Regulation (EC) No.440/2008,
Annex III, EU method B.46 “IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN
EPIDERMIS MODEL TEST”, adopted 06. Jul. 2012

Deviations:

yes

Remarks:

The pre-incubation time was 1 hour, instead of 18- 24 hours. This can be seen as uncritical, because the pre-incubation time must be at least 1 hour (in consultation with the tissue supplier; MatTek In Vitro Life Science Laboratories).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Myo-Inositol, hexakis(dihydrogen phosphate), sodium salt

EC Number:

238-242-6

EC Name:

Myo-Inositol, hexakis(dihydrogen phosphate), sodium salt

Cas Number:

14306-25-3

Molecular formula:

C6H18O24P6.xNa

IUPAC Name:

Esters of sodium hydrogen phosphate with myo-Inositol, plant-derived (old name: myo-Inositol, hexakis(dihydrogen phosphate), sodium salt)

Test material form:

solid: bulk

Details on test material:

- Appearance: white solid powder
- Homogeneity: homogenous
- Density: 2.0401 ± 0.0009 g/cm3 at 20 ± 0.2 °C
- Moisture content: 0.1 - 10% (water is an inherent constituent of the UVCB substance)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Evonik Dr. Straetmans GmbH
- Expiration date of the lot/batch:
NA2253
- Purity test date:
June 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
The test item was stored in the test facility away from light and humidity in a closed vessel at room temperature (20 ± 5°C).
- Stability under test conditions:
H2O: unknown; EtOH: 96h; acetone: 96h; CH3CN: 96h;
DMSO: 96h
- Solubility and stability of the test substance in the solvent/vehicle:
H2O: >1 g/L; EtOH: unknown; acetone: unknown; CH3CN:
unknown; DMSO: unknown
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
unknown

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
none
- Preliminary purification step (if any):
none
- Final dilution of a dissolved solid, stock liquid or gel:
none
- Final preparation of a solid:
none

FORM AS APPLIED IN THE TEST
white solid powder (not different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
not applicable

OTHER SPECIFICS:

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

other: Keratinocyte strain 00267

Details on animal used as source of test system:

no animals used as source of test system

Justification for test system used:

The test system (EpiDerm Skin Irritation Test (EPI-200-SIT) is one of the validated reference methods (VRMs) included in Annex 2 the Test Guideline OECD 439. For this VRMs, pre-validation, optimisation and validation studies have been completed and the VRMs have been used to develop the present Guideline OECD 439 and the Performance Standards.

Vehicle:

other: Dulbecco’s Phosphate Buffered Saline solution

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
MatTek Corporation’s Reconstructed Human Epidermal Model EpiDermTM (EPI-200-SIT)
- Tissue batch number(s):
25841
- Production date:
not available
- Shipping date:
not available
- Delivery date:
13. Sep. 2017
- Date of initiation of testing:
13. Sep. 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during pre-treatment incubation:
37°C ± 1°C
- Temperature used during treatment / exposure:
37°C ± 1°C
- Temperature of post-treatment incubation (if applicable):
37°C ± 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 with DPBS in 1-minute-intervals.
After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 22 hours at 37 ± 1°C and 5.0 ± 0.5% CO2. After post-incubation the tissues were removed from the incubator and shaken for
5 minutes (120 rpm). Then, the inserts were transferred into fresh assay medium (0.9 mL) and were incubated for 16 hours for post-incubation at 37 ± 1°C and 5.0 ± 0.5 % CO2.
- Observable damage in the tissue due to washing:
not reported
- Modifications to validated SOP:
No deviations from the study plan were observed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
1 mg/mL
- Incubation time:
3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
- Spectrophotometer:
Microtiter plate photometer "Anthos Reader 2010 Flexi" (Anthos Microsysteme GmbH)
- Wavelength:
570 nm
- Filter:
570 nm
- Filter bandwidth:
not reported
- Linear OD range of spectrophotometer:
not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
Result: 1.27 ± 0.071 (OD 540-570 nm)
Method: MTT QC assay (4 hrs, n=3)
Acceptance criteria tissue viability: 1.0-3.0 (OD 540-570 nm) (Pass)
- Barrier function:
Result (ET-50): 6.28 hours
Method: ET-50 assay (100µl 1% Triton x-100, 4 time-points, n=3, MMT assay)
Acceptance criteria for ET-50: 4.77-8.72 hours (Pass)
- Morphology:
Tissue viability and the barrier function test are within the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination:
No contamination (sterile) (Pass)
- Reproducibility:
Values for negative control and for positive control were within the range of historical data of the test facility:
Negative control (OD): 1.965 (historical range: 0.476 - 2.471)
Positive control (% OD compared to Negative Control): 2.5% (historical range: 1.8 - 17.1%)

NUMBER OF REPLICATE TISSUES:
Three tissue replicates were used for the test item and for the controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The two pre-tests (testing the ability of direct MTT reduction and the testing the ability of color formation without MTT addition) both resulted in a colourless solution. Therefore, the test item is not presumed to directly reduce the MTT and further not to develop a colour without MTT addition.
As a consequence, additional tests ("Direct Reduction of MTT with freeze killed Tissues" and "Binding capacity) had not to be performed and no data correction was necessary.
- Fresh tissues / killed tissues:
not applicable
- Procedure used to prepare the killed tissues (if applicable):
not applicable
- N. of replicates:
not applicable
- Method of calculation used:
not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
One valid experiment was performed.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritant to skin if the tissue viability is less or equal 50%
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:
not applicable

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
26.1 mg (Tissue 1)
26.3 mg (Tissue 2)
25.3 mg (Tissue 3)
- 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 (mean conc. 1.012 g/mL).

VEHICLE
- Amount(s) applied (volume or weight with unit):
The tissues were wetted with 25 μL DPBS buffer before applying the test item and spreading
it to match the tissue size
- Concentration (if solution):
not applicable
- Lot/batch no. (if required):
not applicable
- Purity:
not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
30 μL DPBS buffer
- Concentration (if solution):
not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
30 μL 5% SDS solution
- Concentration (if solution):
SDS solution (5%)

Duration of treatment / exposure:

1 hour (25 minutes at RT and 35 minutes at 37 ± 1°C)

Duration of post-treatment incubation (if applicable):

- 22 hours at 37 ± 1°C and 5.0 ± 0.5% CO2 in 0.9 mL fresh assay medium
- shaken for 5 minutes (120 rpm)
- 16 hours at 37 ± 1°C and 5.0 ± 0.5% CO2 in 0.9 mL fresh assay medium
- after post-incubation followed the MTT assay

Number of replicates:

Three tissue replicates were used for the test item and for the controls.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1

Value:

84.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

87.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

82.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

84.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

± standard deviation: 2.4%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system:
not reported
- Direct-MTT reduction:
not direct-MTT reduction observed
- Colour interference with MTT:
no colour interference with MTT observed

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
Yes; The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: The mean optical density of the three tissues was 1.965. Variation
within replicates (standard deviataion) was 5.2% and therefore within the accepted range (required: ≤ 18%). Values for negative control were within the range of historical data of the test facility.
- Acceptance criteria met for positive control:
Yes; The positive control reduced absorbance to 2.5% compared to the negative control (required ≤ 20%). Variation within replicates (standard deviataion) was 0.2% and therefore within the accepted range (required: ≤ 18%). Values for positive control were within the range of historical data of the test facility.
- Acceptance criteria met for variability between replicate measurements:
Yes; Variation within replicates (standard deviataion) was within the accepted range for negative control, positive control and test item (required: ≤ 18%).
- Range of historical values if different from the ones specified in the test guideline:
Negative control (DPBS buffer) (OD): 0.476 - 2.471
Positive control (5% SDS) (% OD compared to Negative control): 1.8 - 17.1 %

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

not irritating to skin

Conclusions:

The results in this in-vitro skin irritation test with the EpiDerm model indicates that the test item dermofeel PA-3 (dried) is not irritant.

Executive summary:

One valid experiment was performed.

Three tissues of the human skin model EpiDerm^TM were treated with the test item dermofeel PA-3 (dried) 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 and 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 2.0. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.5% (required: ≤ 20%). The variation within the tissue replicates of negative, control, positive control and test item was acceptable (required: ≤ 18%). For these reasons, the experiment was considered to be valid.

After the treatment with the test item, the mean value of relative tissue viability was reduced

to 84.7 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

Therefore, dermofeel PA-3 (dried) is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.