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

Endpoint summary

Description of key information

Skin irritation/corrosion :

Non corrosive,

Not irritant to the skin

Eye irritation/eye damage:

Not eye damaging,

Not an eye irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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).

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)

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:

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.

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

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

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

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 %

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.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07.08.2017 - 10.08.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 431, adopted 29. Jul. 2016
“In vitro Skin Corrosion: reconstructed human epidermis (RHE) test method”

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No.440/2008, EU-Method B.40 BIS. “IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST“

Version / remarks:

dated 30. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Evonik Dr. Straetmans GmbH
- Batch No.of test material: NA2253
- Expiration date of the lot/batch: 06-2019

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:

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 were used as source of test system.

Justification for test system used:

The test system (EpiDermTM Skin Corrosivity Test (SCS) (EPI-200) is one of two validated reference methods (VRMs) included in the Test Guideline OECD 431. According to the Guideline the VRM could be used for regulatory purposes for distinguishing corrosive (C) from non-corrosive (NC) substances.

Vehicle:

water

Remarks:

25.8 mg - 26.2 mg test item in 25 µl demineralised water (mean:1.042 g/mL)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
MatTek Corporation’s Reconstructed Human Epidermal Model EpiDermTM (EPI-200-SCT)
- Tissue batch number(s):
25835
- Production date:
not applicable
- Shipping date:
not reported
- Delivery date:
08. Aug. 2017
- Date of initiation of testing:
09. Aug 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during pre-treatment incubation:
37°C ± 1°C (pre-incubation for 1 hour in assay medium and 5.0 ± 0.5% CO2)
- Temperature used during treatment / exposure:
37°C ± 1°C (incubation for 3 minutes and 1 hour at 5.0 ± 0.5% CO2)
- Temperature of post-treatment incubation (if applicable):
not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps:
The tissue was thoroughly rinsed with DPBS buffer, blotted with sterile cellulose tissue and set into wells containing assay medium. After transfer of all tissues, the tissue was immediately blotted at the bottom with cellulose tissue again before tranferring it into wells filled with MTT solution.
- Observable damage in the tissue due to washing:
not reported
- Modifications to validated SOP:
The following deviation from the study plan was documented ans assessed and signed by the study director: The test item was not ground. This can be seen as uncritical, because the test item is a powder-like substance and therefore it was useless to pestle the test item.

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

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
Result: 1.474 ± 0.093
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): 5.93 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:
not applicable
- Contamination:
no contamination
- Reproducibility:
Values for negative control and for positive control were within the range of historical data of the test facility:
Negative control - 3 min incubation time (OD): 1.740 (historical range: 1.197 - 3.077)
Negative control - 1 h incubation time (OD): 1.637 (historical range: 1.377 - 2.571)
Positive control - 3 min incubation time (% Tissue viability): 19.7% (historical range: 9.6 - 57.3%)
Positive control - 1 h incubation time (% Tissue viability): 4.8% (historical range: 4.1 - 24.2%)

NUMBER OF REPLICATE TISSUES:
Two tissue replicates were used for the test chemical and controls for each exposure time (3 minutes and 1 hour).

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 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 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 non-corrosive to skin if the viability after 3 minutes exposure 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 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):
Tissue 1:
26.2 mg (3 minutes)
25.8 mg (1 hour)
Tissue 2:
26.0 mg (3 minutes)
26.2 mg (1 hour)
- Concentration (if solution):
test items were applied to tissue with 25 µl demineralised water (mean conc.: 1.042 g/mL)

VEHICLE
- Amount(s) applied (volume or weight with unit):
test items were applied to tissue with 25 µl demineralised water
- Concentration (if solution):
test items were applied to tissue with 25 µl demineralised water (mean conc.: 1.042 g/mL)
- Lot/batch no. (if required):
20170309
- Purity:
demineralized water (ion-exchanger)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
50 µl demineralised water
- Concentration (if solution):
not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
50 µl KOH (Potassium hydroxide solution, 8M)
- Concentration (if solution):
8M

Duration of treatment / exposure:

"3 minutes" and "1 hour"

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

Two tissue replicates were used for the test chemical and controls for each exposure time (3 minutes and 1 hour).

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative to the mean of the negative control

Run / experiment:

3 minutes incubation

Value:

80.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive to skin

Irritation / corrosion parameter:

% tissue viability

Remarks:

relative to the mean of the negative control

Run / experiment:

1 hour incubation

Value:

86.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive to skin

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction:
no 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: optical density was 1.7 (3 minutes) resp. 1.6 (1 hour). 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 showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The mean value of relative tissue viability was 4.8%. Values for positive control were within the range of historical data of the test facility.
- Acceptance criteria met for variability between replicate measurements:
Yes; The Coefficient of Variation (CV) between the replicates was below 30% (in the range of 20%-100% viability).
- Range of historical values if different from the ones specified in the test guideline:
Optical Density Negative Control (3 min): 1.197 - 3.077
Optical Density Negative Control (1 hr): 1.377 - 2.571
% Tissue viability Positive Control (3 min): 9.6 - 57.3%
% Tissue viability Positive Control (1 hr): 4.1 - 24.2%

Interpretation of results:

other: non-corrosive to skin

Conclusions:

The test item is considered non-corrosive to skin.

Executive summary:

The study according to OECD 431 was performed to determine the skin corrosion potential of the esters of sodium hydrogen phosphate with myo-Inositol. One valid experiment was performed.

Two tissues of the human skin model EpiDermTM were treated with the test item for 3 minutes and 1 hour, respectively.

The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control and 8M KOH was used as positive control. After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.7 (3 minutes experiment) and 1.6 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 4.8 % for the 1 hour treatment.

After 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 80.6 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, mean value of relative tissue viability was reduced to 86.9%. This value, too, is above the threshold for corrosion potential (15%).

Therefore, the substance is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11. Sep. 2017 - 28.Sep. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Part 492, adopted 28. Jul. 2015, “Reconstructed
Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”/ Protocol provided by MatTek Corporation

Deviations:

no

Principles of method if other than guideline:

Additional information was taken from:
- EpiOcular TM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals” by MatTek Corporation, document no. MK-24-007- 0055, dated 14. Jul. 2014
- Stern M., Klausner M., Alvarado R., Renskers K., Dickens M., 1998. “Evaluation of the EpiOcular Tissue Model as an Alternative to the Draize Eye Irritation Test”. Toxicology in Vitro 12, 455-461

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: Keratinocyte strain 4F1188

Details on test animals or tissues and environmental conditions:

- Commercial Name: EpiOcular™ kit.
- Supplier: MatTek in Vitro Life Science Laboratories, Slovakia
- Designation of the kit: OCL-200-EIT
- Batch: 27006

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
The following amounts were applied to the tissues:
Tissue 1: 50.1 mg test item
Tissue 2: 52.3 mg test item
Controls: 50µl each

Duration of treatment / exposure:

Incubation for 6 hours at 37 ± 1 °C, 5 ± 1.0 % CO2 and 80-100% rel. humidity.

Duration of post- treatment incubation (in vitro):

For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.

Number of animals or in vitro replicates:

The solid test item was applied to two tissue replicates.

Details on study design:

Pre - Tests
1. Assessment of Direct Reduction of MTT by the Test Item.
The test item was tested for the ability of direct MTT reduction. To test for this ability,
47.7 mg of the solid test item were added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2 and 80 – 100 % relative humidity for 3 hours. 1 mL of MTT solution plus 50 μL of H2O demin. was used as negative control.
The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.
2. Assessment of Coloured or Staining Test Items
The test item is colourless. To assess, whether the test item will become coloured after contact with water or isopropanol, 47.8 mg of the solid test item were added to 1 mL of sterile H2O demin. in a 6-well plate and incubated in the dark at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour minutes. For solid test items, moreover, 49.3 mg of test item were added to 2 mL isopropanol and incubated in a 6-well plate for 3 hours at room temperature.
After incubation, no colour development was visible. Therefore, the main test was performed without colourant controls.
MAIN TEST
1. Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use.
The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, negative control and positive control and filled
with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour.
After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.
2. Exposition and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 30 minutes. After that, 50 μL of the controls and a defined amount of the test item were applied in duplicate in 1- min- intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the
plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.
3. MTT Assay and exrtaction
A 24-well-plate was prepared with 300 μL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 3 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
4 Measurement
The inserts were removed from the 6-well plate and discarded. The content of each well
was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate.
Eight wells with 200 μL isopropanol were pipetted also. The plate was read in a plate
spectrophotometer at 570 nm.
DECISION CRITERIA
Viability > 60 % Non eye irritant - No GHS category for eye irritation
Viability ≤ 60 % Eye damage / Eye irritant -GHS category 1 or 2

Irritation parameter:

other: Viability

Run / experiment:

Tissue 1

Value:

67.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Viability

Run / experiment:

Tissue 2

Value:

66.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Validity criteria and results are stated below:
Validity
Criterion Demanded Found
OD of negative control > 0.8 and < 2.5 1.6
% mean relative viability < 50% of negative control 42.2%
of positive control
Variation within replicates < 20% 0.2% (negative control)
3.5% (positive control)
1.0% (test item)
Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Interpretation of results:

GHS criteria not met

Conclusions:

After treatment with the test item, the mean value of relative tissue viability was 66.9 %. This value is above the threshold for eye irritation potential (≤ 60%).
Under the conditions of the test the substance is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

Executive summary:

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.

The solid test item was applied to two tissue replicates. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.6. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 42.2 % (< 50%). Variation within tissue replicates was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 66.9 %. This value is above the threshold for eye irritation potential (≤ 60%). Under the conditions of the test, the test item is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

- Justification of the test method and considerations regarding applicability

This in vitro study will be performed in order to evaluate the eye hazard potential of the substance.

The EpiOcular™ Eye Irritation Test (EIT) predicts the acute ocular irritation potential of chemicals by measurement of its irreversible tissue damage caused by cytotoxic effects in the reconstructed human cornea-like tissue model. Within a testing strategy, the EpiOcular™ EIT is used as a replacement of the in vivo Draize Eye Irritation Test. It is utilized for the classification and labelling of chemicals concerning their eye irritation potential. The EpiOcular™ EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B). For these purposes, further testing with other suitable test methods is required

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14.09.2017-28.09.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Adopted 14.02.2017 by Commission Regulation 2017/35

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 160 (Guidance document on The Bovine corneal opacity and permeability (Bcop) an d isolated chicken eye (Ice) test methods: collection of tissues for histological evaluation and co llection of data on non-severe irritants)

Deviations:

no

Principles of method if other than guideline:

Additional information was taken from:
- “Bovine Corneal Opacity and Permeability (BCOP) Assay”, SOP of Microbiological Associates Ltd., UK, Invittox (UK) protocol no. 124, Procedure Details, April 1997, last update Aug. 1999; based on Gautheron et al. (1992), refined by Vanparys et al. (1994)
- “The Bovine Corneal Opacity and Permeability Assay – Method of Gautheron”; Invittox (UK) protocol no. 98, April 1996

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

- Species: Bos primigenius Taurus.
- Source: fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test
- Characteristics of donor animals: the cattle were between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue: The eyes were transported to the test facility in Hank’s balanced salt solution with 1 % Penicillin-Streptomycin solution (Penicillin 100 U/ ml, Streptomycin 100 μg/ml) in a suitable cooled container.
Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for one hour

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µl

Duration of treatment / exposure:

4 h

Duration of post- treatment incubation (in vitro):

90 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe
with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.
NUMBER OF REPLICATES: three for each treatment group (negative control solution, test item and positive control).
NEGATIVE CONTROL USED:
HBSS: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin.
water (1:10), batch no.: 20170914 (1. experiment), 20170921 (2. experiment)
and 2010928 (3. experiment).
POSITIVE CONTROL USED:
Imidazole solution: 20% C3H4N2 (CAS-No. 288-32-4), dissolved in HBSS, batch no.: 20170914 (1. experiment), 20170921 (2. experiment) and 2010928 (3. experiment).
METHOD:
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, 750 μL test item solution and positive control solution were applied to each replicate. According to the characteristics of the test item, the following treatment procedure was performed:
1. Open Chamber Method
In order to apply the test item, the nut was unscrewed to remove the glass disc. The test item could be applied directly on the cornea now.
750 μL of the test item were tested as solution at 20% concentration in HBSS. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with test item. Exposure time on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing with cMEM
with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded
2. Permeability Test
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas. For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/mL was used. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.
EVALUATION:
1. Calculation of Opacity
The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value
was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.
2. Calculation of Permeability
The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.
3. Calculation of IVIS (in - Vitro Irritancy Score)
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference + (15 x corrected OD492 value) The IVIS of each replicate of the positive control and of the test item were calculated from
the following equation: IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]
All calculations are performed with unrounded values.
DECISION CRITERIA: As indicated in OECD 437

Irritation parameter:

in vitro irritation score

Run / experiment:

Experiment 1

Value:

5.39

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The first experiment was considered as insufficient for assessment, because two of the three replicates gave discordant predictions from the mean value.

Irritation parameter:

in vitro irritation score

Run / experiment:

Experiment 2

Value:

2.33

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Experiment 3

Value:

2.91

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ASSESSMENT:
The first experiment was considered as insufficient for assessment, because two of the three replicates give discordant predictions from the mean value.
Therefore, the experiment was repeated; and the second experiment was sufficient for assessment, but the results showed a non-concordant prediction from the results of the first experiment.
Based on these results, a third experiment was performed; this experiment confirmed the results of the second experiment
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: IVIS of HBSS <= 3 and results are within the range of historical data of the test facility.
- Acceptance criteria met for positive control: IVIS of 20% imidazole solution 70.37-159.05 and results are within the range of historical data of the test facility.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, based on all three experiments, the test item, tested as 20% solution in HBSS, showed no effects on the cornea of the bovine eye.
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Executive summary:

The corneal damage potential of the substance was assessed by quantitative measurements of changes in opacity and permeability in a bovine cornea, according to the OECD Guideline 437. The test item, tested as 20% solution in HBSS, was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. The negative control (HBSS solution) and the positive control (20% imidazole solution) have met the validity criteria.

Under the conditions of this study, the test item shows no effects on the cornea of the bovine eye.

The calculated IVIS (in vitro irritancy score) based on two experiments was ≤ 3.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion/irritation:

Results obtained by OECD 431 (skin corrosion)

After 3 minutes treatment with the test item, the mean value of relative tissue viability was reduced to 80.6 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, mean value of relative tissue viability was reduced to 86.9%. This value, too, is above the threshold for corrosion potential (15%). Therefore, the substance is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Result obtained by OECD 439 (Skin irritation):

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, the substance is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Eye damage/irritation:

Results obtained by OECD 492

After treatment with the test item, the mean value of relative tissue viability was 66.9 %. This value is above the threshold for eye irritation potential (≤ 60%). Under the conditions of the test, the test item is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

Results obtained by OECD 437:

Under the conditions of this study, the test item shows no effects on the cornea of the bovine eye.

The calculated IVIS (in vitro irritancy score) based on two experiments was ≤ 3. A substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Justification for classification or non-classification

Skin irritation:

According to the Guidance on the application of CLP criteria (v.5.0), the OECD 439 can reliably distinguish non-classified from classified substances but cannot distinguish between corrosives and irritants when used alone.  Therefor two in - vitro tests have been performed following the OECD guideline 439 and 431.

The available data on skin performed with "Esters of sodium hydrogen phosphate with myo-Inositol, plant-derived" (old name: myo-Inositol, hexakis(dihydrogen phosphate), sodium salt) following OECD guidelines OECD 439 and OECD 431 do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.

Eye irritation:
According to the Guidance on the application of CLP criteria (v.5.0), A substance can be considered as causing serious eye damage (Category 1) based on positive results the BCOP test. Four adopted OECD TGs can be used for identifying substances not causing serious eye damage/eye irritation which are the ICE test, BCOP test, STE test and Reconstructed human Cornea-like Epithelium (RhCE) (OECD TG 492). Negative results from the ICE, BCOP, STE, RhCE and CM test methods can be used for classification purposes,

The available results obtained from studies performed with"Esters of sodium hydrogen phosphate with myo-Inositol, plant-derived" (old name: myo-Inositol, hexakis(dihydrogen phosphate), sodium salt) following OECD 437 and - 492 (eye damage/irritation) do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.