Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
Reconstructed Human Epidermis Test Method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 1, 2017 - February 16, 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cytidine 3'-(dihydrogen phosphate)
EC Number:
200-556-6
EC Name:
Cytidine 3'-(dihydrogen phosphate)
Cas Number:
63-37-6
Molecular formula:
C9H14N3O8P
IUPAC Name:
4-amino-1-(5-O-phosphonopentofuranosyl)pyrimidin-2(1H)-one
Test material form:
solid
Details on test material:
Synonyms: 5-CMP acid, 5'-Cytidylic acid, Cytidine 5'-monophosphate
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

In vitro test system

Test system:
human skin model
Remarks:
SkinEthic™ RHE model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Justification for test system used:
The SkinEthic™ RHE model has been validated for irritation testing (Validation study bsed on the original ECVAM Performace Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439), therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Remarks:
The test item was applied as supplied
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE model
- Tissue batch number(s): 17-RHE-017
- Delivery date: 14 February 2017
- Expiration date: 20 February 2017
- Date of initiation of testing: 14 February 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- 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: 300 µl of a MTT solution at 1.0 mg/mL
- Incubation time: 2 h 58 min at 37ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.3 (CV=4.1%) specification OD > 0.7. Historical negative control mean OD range = 0.834 - 1.574
- Barrier function: 4.7 h (Specification 4 h < ET50 < 10 h)
- Morphology: 5.5 cell layers, absence of significant histological abnormalities, well differentiated epidermis (Specification > 4)
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE. no interference

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32 mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours and 05 minutes.
Number of replicates:
3

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
55.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.5% tissue viability
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
77.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.5% tissue viability
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
45.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.5% tissue viability
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
44.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.5% tissue viability
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The mean corrected percent viability of the treated tissues was 55.9%, versus 1.5% in the positive control (5% Sodium Dodecyl sulfate). However, the results obtained on the three epidermises treated with the test item were heterogeneous: 77.6%, 45.3% and 44.9% (two epidermises classified as irritant and one epidermis classified as non irritant, leading to a mean viavility at 55.9% with a standard deviation at 18.8%).

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: A cloudy yellow solution was observed after 3 hours of incubation between 36.3ºC and 37.8ºC, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT:
In water: A colourless solution at the bottom of the well was obtained after 3 hours of incubation between 36.3ºC and 37.8ºC, 5% CO2
In isopropanol: A colourless solution was obtained after 2 hours of incubation at room temperature. Therefore, the test item will not interfere with the MTT assay and there is no need to add nonspecific coloration controls to the study.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes,a full demonstration of proficiency was performed with Episkin-SM model, plus a reduced validation with SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals. Summary of proficiency chemicals tested according to OECD 439 criteria included in the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the standard deviation of the cell viability for the epidermises treated with the test item was 18.8%, instead of 18% as initially scheduled. This deviation is considered as without impact on the conclusion of the test. The mean OD of the negative control is 0.819 and the acceptability criteria is OD>0.6x<1.5
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: the results are heterogeneous on the three epidermises (two classified as irritant and one classified as non-irritant). A second run should be performed to infirm or confirm the results.

Any other information on results incl. tables

The results were expressed as a viability percentage compared with the negative control:

 

Viability %= ODtest item/ ODnegative controlx 100

 

The OD values obtained for each test simple were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%.

 

Table 1. Individual and average values of OD after 42 minutes exposure

 

 

Skin

OD

Mean OD / disc (#)

Mean OD / product

Viability %

Mean viability %

SD

Conclusion

Negative control

1

0.827

0.811

0.819

99.0

100.0

1.1

 

0.800

0.806

2

0.789

0.829

101.2

0.843

0.857

3

0.812

0.818

99.8

0.823

0.820

Positive control

4

0.015

0.015

0.013

1.8

1.5

0.3

Irritant

0.015

0.015

5

0.012

0.011

1.3

0.012

0.011

6

0.012

0.012

1.5

0.013

0.012

Test item PH-16/0670

25

0.612

0.636

0.458

77.6

55.9

18.8

Non irritant

0.677

0.621

26

0.373

0.371

45.3

0.378

0.364

27

0.366

0.368

44.9

0.376

0.364

# mean of 3 values (triplicate of the same extract)

OD: optival density

Acceptability criteria: SD18%

 

Notes:

           

- If the viability obtained for the test item is greater than 50%, the test item has to be considered as non irritant.

- If the viability obtained for the test item is less than or equal to 50%, the test item has to be considered as irritant.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The mean corrected percent viability of the treated tissues was 55.9% versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item has to be considered as non-irritant to skin.
Executive summary:

The evaluation of the possible irritating effects of the test item has been tested after topical application on in vitro human reconstructed epidermis (SkinEthic RHE model) in accordance with OECD 439 and EU method B.46, following GLP. The test item was applied, as supplied, at a dose of 16 mg to 3 living reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 42 h and 5 min post-incubation period at 37ºC, 5% CO2. Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Positive and negative controls were run in parallel. All acceptability criteria were met. The mean corrected percent viability of the treated tissues was 55.9%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). However, the results obtained on the three epidermises treated with the test item were heterogeneous: 77.6%, 45.3% and 44.9% (two epidermises classified as irritant and one epidermis classified as non irritant, leading to a mean viability at 55.9% with a standard deviation at 18.8%).