Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 September 2018 (Study Initiation) to 22 November 2018 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Fatty acids, C16-18 (even numbered), manganese(II) salts
EC Number:
947-666-7
Molecular formula:
C36H70MnO4, C32H62MnO4 and C28H54MnO4
IUPAC Name:
Fatty acids, C16-18 (even numbered), manganese(II) salts
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities Pvt Ltd.
-Lot/batch No.of test material: MNST9H122A
- Expiration date of the lot/batch: May 2023
- Purity test (release) date: June 2018 (CoA)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), in original container as supplied by the Sponsor. Kept tightly closed in a dry, cool and well ventilated place
- Stability under test conditions: Assumed stable for the duration
- Solubility and stability of the test substance in the solvent/vehicle: Test item administered as received, assumed stable for the duration of the test
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

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: Strain No. PKP.DPQR-2
Justification for test system used:
This study addresses the human health endpoint skin corrosion. It makes use of reconstructed human epidermis (RHE) (human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RHE) is recommended by the OECD and other regulatory authorities. The SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is a recommended model for conducting in vitro skin corrosion studies. The results of the study are believed to be predictive for the potential of inducing skin corrosivity in humans.
Vehicle:
unchanged (no vehicle)
Remarks:
Test item administered as received
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model
- Tissue batch number(s): 18-RHE-111
- Quality Control certified and release date: 11 September 2018
- Shipping date: Not specified
- Delivery date: Not specified
- Date of initiation of testing: 11 September 2018 (Experimental start)
- Date of expiry: 17 September 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes at room temperature and 60 minutes at 37±1 °C in 5±1% CO2 in a 95% humidified atmosphere.
- Temperature of post-treatment incubation (if applicable): None

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
After exposure, tissues were rinsed and dried with cotton buds. Any residual test item was initially removed by knocking the treated insert on a beaker or by holding upside down with forceps. Treated tissues were rinsed 20 times in a constant stream of 1 mL DPBS at a 5-8 cm distance from the insert to remove all residual test item from the epidermal surface. Mesh (applied on negative and positive control treated tissues only) was removed by washing all tissues. The bottom of tissue inserts were dried on sterile absorbent paper (Kim wipes) for 1-2 seconds. The surface of the stratum corneum was gently swept using both ends of a cotton tip (5-6 turns per end). After washing, inserts were transferred to holding plates containing 300 µL maintenance medium.

- Observable damage in the tissue due to washing: None
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: After rinsing and drying, the MTT test was performed. Tissues were placed in 300 µL of MTT (1.0 mg/mL) solution.
- Incubation time: Incubated for 180 minutes at 37±1 °C in 5±1% CO2 in a 95% humidified incubator. At the end of the MTT test, tissues were observed for MTT reduction.
- Spectrophotometer: SynergyHT Microplate Reader. The optical density (OD) of the extracted formazan (200 μL/well of a 96 well plate) was determined in triplicate per tissue using an absorbance microplate reader at 570 nm.
- Wavelength: 570 nm.
- Filter: Not specified
- Filter bandwidth: 570 ±30 nm
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The percent relative viability in tissues treated with the test item was 102.74% after 3 minute exposure and 108.24% after 60 minute exposure. No significant reduction in percent cell viability was observed after 3 minute and 60 minute exposure in treated tissues when compared with that of the concurrent negative control. The difference between viability of treated tissues was less than 2.0%, i.e., % CV.
- Barrier function: The test system used for the in vitro skin corrosion test was the reconstructed human epidermis (SkinEthicTM RHE) as recommended by the OECD 431 guideline. The SkinEthicTM RHE model consists of normal human keratinocytes cultured for 17-days on a 0.5 cm2 polycarbonate filter insert at the air-liquid interface in a chemically defined growth medium. The cells form a multi-layered, highly differentiated and stratified epidermis model of the human epidermis that consists of a main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Morphology: The test system used for the in vitro skin corrosion test was the reconstructed human epidermis (SkinEthicTM RHE) as recommended by the OECD 431 guideline. The SkinEthicTM RHE model consists of normal human keratinocytes cultured for 17-days on a 0.5 cm2 polycarbonate filter insert at the air-liquid interface in a chemically defined growth medium. The cells form a multi-layered, highly differentiated and stratified epidermis model of the human epidermis that consists of a main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Contamination: None
- Reproducibility: Acceptable between triplicate tissues

NUMBER OF REPLICATE TISSUES: hree replicates/time point (3 minutes and 60 minutes)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not produce direct MTT reduction when compared with that of the concurrent negative control (maintenance medium).

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

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): 20 mg ± 3 mg of test item/0.5 cm2. Test item administered as supplied

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL/0.5 cm2 of sterile distilled water40 µL
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL/0.5 cm2 of 8N KOH
- Concentration (if solution): 8N KOH
Duration of treatment / exposure:
Tissues were exposed to Fatty acids, C16-18 (even numbered), manganese(II) salts (test item) and sterile distilled water (negative control) for 3 minutes at room temperature and 60 minutes at 37 ± 1 °C in 5 ± 1% CO2 using three replicates/time point. Positive control tissues were exposed for 60 minutes at 37 ± 1 °C in 5 ± 1% CO2.
Duration of post-treatment incubation (if applicable):
Not applicable
Number of replicates:
Three tissue replicates/time point.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 mins
Run / experiment:
1 (Negative control) Sterile distilled water
Value:
100
Negative controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 mins
Run / experiment:
1 (test item) Fatty acids, C16-18 (even numbered), manganese(II) salts
Value:
102.74
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 (Negative control) Sterile distilled water
Value:
100
Negative controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
60 mins
Run / experiment:
1 (test item) Fatty acids, C16-18 (even numbered), manganese(II) salts
Value:
108.24
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
60 mins
Run / experiment:
1 (Positive control) 8N KOH
Value:
0.39
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: The test item did not produce direct MTT reduction when compared with that of the concurrent negative control (maintenance medium).
- Colour interference with MTT: No difference in absorbance was observed between the negative control (isopropanol) and test item, Fatty acids, C16-18 (even numbered), manganese(II) salts. The colour interference test did not show interference in optical density due to the test item

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test facility is GLP complient and competant in this OECD 431 regulatory method. The laboratory has sufficinet historical data to justify the test item responce within this test system.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control data of mean optical density were within the OECD Guideline 431 range, i.e., ≥ 0.8 and ≤ 3.0 for each exposure time.
- Acceptance criteria met for positive control: Mean viability of tissues exposed for 60 minutes with the positive control (8N KOH), expressed as % of the negative control were < 15%.
- Acceptance criteria met for variability between replicate measurements: In the range of 20-100% viability and for OD’s ≥ 0.3, difference in viability between tissue replicates was not > 30%.
- Range of historical values if different from the ones specified in the test guideline: Not applicable

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
it was concluded that Fatty acids, C16-18 (even numbered), manganese(II) salts is non-corrosive in accordance with the United Nations Globally Harmonised System of Classification and Labelling of Chemicals as indicated in OECD 431 under the specified conditions of this study.