Fatty acids, C16-18 (even numbered), iron(III) salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 September 2018 (Study Initiation) to 4 December 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), iron(III) salts

EC Number:

947-665-1

Molecular formula:

C54H105FeO6, C48H93FeO6 and C42H81FeO6.

IUPAC Name:

Fatty acids, C16-18 (even numbered), iron(III) 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: IRST7H112A
- 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). Container kept tightly closed and in a dry, cool and well ventilated place
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: Test item administered as received
- 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: PKP.DPQR-2

Justification for test system used:

This study addresses the human health endpoint skin irritation. It makes use of reconstructed human epidermis (RhE) (obtained from 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 a recommended model for conducting in vitro skin irritation studies. The results of the study are believed to be predictive for the potential of inducing skin irritation in humans.

Vehicle:

unchanged (no vehicle)

Remarks:

Test item administered as received

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE model
- Tissue batch number(s): 18-RHE-111
- Certified and release date: 11 September 2018
- Shipping date: not specifed
- Delivery date: not specified
- Date of initiation of testing: 11 September 2018
- Date of expiry: 17 September 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All treated tissues were incubated at room temperature for 42 minute exposure.
- Temperature of post-treatment incubation (if applicable): After washing and drying, tissues were incubated in 6-well plates containing 2 mL growth medium at 37± 1°C in 5± 1% CO2 in a humidified incubator for 42 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After exposure, tissues were rinsed and then dried with cotton buds. The test item was removed by rinsing 25 times in a constant stream of 1 mL DPBS from 5-8 cm distance from the insert to remove all test item from the epidermal surface. Mesh (applied on negative and positive control tissues) was removed during washing. 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: Tissues were placed in MTT (1.0 mg/mL) solution and incubated for 180 minutes at 37 ± 1°C in 5 ± 1% CO2 in a 95% humidified incubator. At the end tissues were observed for MTT reduction.
- Incubation time: 180 minutes
- Spectrophotometer: SynergyHT Microplate Reader
- Wavelength: Absorbance (OD) was measured at 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 mean cell viability in tissues treated with the test item was 94.33% after 42 minutes exposure. No significant reduction in percent cell viability was observed in treated tissues when compared with that of the concurrent negative control.
- Barrier function: The RhE test method is based on the premise that irritant chemicals are able to penetrate the stratum corneum and damage the underlying layers of keratinocytes and other skin cells. Cell viability is measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide], into a blue formazan salt that is quantitatively measured after extraction from tissues.
Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels.
- Morphology: 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: Three replicates for each, were exposed to Fatty acids, C16-18 (even numbered), iron(III) salts, a negative and a positive control for 42 minutes.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : None. Fatty acids, C16-18 (even numbered), iron(III) salts did not cause 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

DECISION CRITERIA
- The test substance is considered to be irritant to skin in accordance with UN GHS Category 2, if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
- The test substance is considered to be non-irritant to skin in accordance with UN GHS No Category, if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (weight with unit): 16.7mg , 15.8 mg and 16.5 mg for tissues 1, 2 and 3.
For test item treatment, the epidermal surface of the tissue was wetted using 10 µL of sterile distilled water followed by application of 16 ± 2 mg of test item/0.5 cm2 and gently spread across the surface using a blunt spatula. All treated tissues were incubated at room temperature for 42 minute exposure.

NEGATIVE CONTROL
- Amount(s) applied (volume): 16 µL/0.5 cm2 sterile dulbecco’s phosphate buffered saline (DPBS) was applied.

POSITIVE CONTROL
- Amount(s) applied (volume): 16 µL/0.5 cm2 of 5% sodium dodecyl sulphate (5% aq.) was applied
- Concentration (if solution): 5%

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 (Negative control) Dulbecco’s Phosphate Buffered Saline (DPBS)

Value:

100

Negative controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 (test item) Fatty acids, C16-18 (even numbered), iron(III) salts

Value:

94.33

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 (Positive control) Sodium dodecyl sulphate (5% aq.)

Value:

1.52

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: Fatty acids, C16-18 (even numbered), iron(III) salts did not cause direct MTT reduction when compared with that of the concurrent negative control (Maintenance Medium).
- Colour interference with MTT: No significant difference in absorbance was observed between the negative control (isopropanol) and test item, i.e., Fatty acids, C16-18 (even numbered), iron(III) salts. Therefore results did not show interference in optical density due to the test item.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The test facility is GLP compliant and technically profiocient in this OECD 439 regulatory method. It has suffient historical data to justify the test item classification based on the results of this study.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- 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:

Based on the results of this study, the classification for Fatty acids, C16-18 (even numbered), iron(III) salts is as mentioned below:

Globally Harmonized System of Classification and Labelling of Chemicals: No Category (Non Skin Irritant)