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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:
01 August 2018 - 17 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
yes
Remarks:
deviations were considered to have not affected the integrity or validity of the study.
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
yes
Remarks:
deviations were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Details on test material:
Although the test item used within this test contains different identifiers as compared to the subtance that is being registered, the tested substance is within the boundary composition of the substance being registered. Therefore, this particular batch of the tested substance, can be considered as the same substance as (Z,Z)-di-9-octadecenyl phosphonate. It can be considered that all tests conducted on Reaction products of triphenyl phosphite, oleyl alcohol and alcohols, C10-16, Batch 2955, are considered relevant and reliable for (Z,Z)-di-9-octadecenyl phosphonate.
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: applied as supplied
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: n/a

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: Epider RHE model supplied by MatTek Slovakia
Cell source:
other: The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200- SIT Kit
- Tissue batch number(s): 28644
- Production date: Not reported
- Shipping date: Not reported
- Delivery date: August 14, 2018
- Date of initiation of testing: August 1, 2018


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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1) with a 570 nm filter.
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤20% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%. The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.8 and ≤ 2.8 and the SD value of the percentage viability is ≤18%.
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: Not reported
- Reproducibility: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- n/a, test item did not reduce MTT.
- Procedure used to prepare the killed tissues (if applicable): n/a
- N. of replicates : n/a
- Method of calculation used: n/a

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure is equal to or less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes 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): 30 µL
- Concentration (if solution): unchanged- applied as supplied.

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % w/v
Duration of treatment / exposure:
15 min test item exposure
Duration of post-treatment incubation (if applicable):
42 h followed by 3 h MTT incubation
Number of replicates:
3 per test group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 min exposure
Value:
97.78
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is ≥ 0.8 and ≤ 2.8, and the SD value of the percentage viability is ≤18%.
- Acceptance criteria met for positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤20% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
- Acceptance criteria met for variability between replicate measurements: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.
- Range of historical values if different from the ones specified in the test guideline: Laboratory historical control data was performed to verify the functioning of the test system

Any other information on results incl. tables

Table 1. Results after treatment with AD-464 and the controls

Treatment Group

Tissue No.

OD 570 nm

Mean OD of
3 Wells

Mean OD of 3 Wells blank corrected

Mean OD
of 3 tissues blank corrected

 

Rel. Viability [%] Tissue 1, 2 + 3*

Standard Deviation

Mean Rel. Viability [%]*

Blank

 

0.036

 

0.036

0.036

0.036

 

Negative Control

1

1.883

 

1.996

 

2.032

 

1.971

 

1.935

 

1.885

 

102.634

 

3.9

 

100.0

 

2

1.931

 

1.961

 

1.978

 

1.956

 

1.920

 

101.877

 

3

1.820

 

1.806

 

1.882

 

1.836

 

1.800

 

95.488

 

Positive Control

 

1

0.095

 

0.092

 

0.093

0.093

0.057

 

0.060

 

3.036

 

0.1

 

3.20

 

2

0.097

 

0.098

0.097

0.097

0.061

 

3.245

 

3

0.098

 

0.100

0.098

0.098

0.062

 

3.312

 

Test Item

 

1

1.769

 

1.842

 

1.805

1.805

1.769

 

1.843

 

93.868

 

4.6

 

97.78

 

2

1.965

 

1.991

 

1.971

 

1.976

 

1.940

 

102.901

 

3

1.868

 

1.867

 

1.834

 

1.856

 

1.820

 

96.565

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study the test item was classified as non-irritant (UN GHS and Regulation (EC) 1272/2008 classification criteria not met).
Executive summary:

OECD 439 (2018) - The skin irritation potential of the test item was assessed using a Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

 

Triplicate tissues were exposed to the test item for 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a microplate reader (Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1).

Each three tissues of the human skin model EpiDermTM were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item AD-464 the mean relative viability value decreased to 97.78% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, AD-464 is non-irritant to skin.