Registration Dossier

Administrative data

Description of key information

Based on the results of the study the test substance is not classified as an irritant. The results from this study show that the test substance is not corrosive to skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 September 2018 (Study Initiation) to 19 November 2018 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceuticals Ltd.
- Lot/batch 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 origanal container as supplied by the Sponsor. Container kept tightly closed in a dry, and well ventillated 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
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 specified
- 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 at a 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 specifieed

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The mean cell viability in tissues treated with the test item was 97.09% 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], into a blue formazan salt that is quantitatively measured after extraction from tissues.
- 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),manganese(II) salts, a negative and a positive control for 42 minutes.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : Fatty acids, C16-C18 (even numbered), manganese(II) 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 (volume or weight with unit): 16.4,16.4 and 16.0 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 applied16 ± 2 mg

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 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 hr
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 (Negative control) Dulbecco’s Phosphate Buffered Saline (DPBS)
Value:
100
Vehicle controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 (test item) Fatty acids, C16-18 (even numbered), manganese (II) salts
Value:
97.09
Remarks on result:
no indication of irritation
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-C18 (even numbered), manganese(II) 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, Fatty acids, C16-C18 (even numbered), manganese(II) 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
Interpretation of results:
GHS criteria not met
Conclusions:
Based on results of this study, the classification for Fatty acids, C16-C18 (even numbered), manganese(II) salts is as follows:

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study Initiation (4 October 2018) to 16 January 2019 (Study Completion)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Nitika Pharmaceutical Specialities Pvt. Ltd.
- Lot/batch No.of test material: MNST9H122A
- Expiration date of the lot/batch: May 2023
- Purity test date: 12 June 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. Containers were kept tightly closed in a dry, cool and well ventilated place
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: The test item formed a homogenous suspension in corn oil and was tested at a concentration of 20% (w/v) in corn oil. The test item was insoluble in normal (physiological) saline.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulated as a homogenous suspension in corn oil at 20% (w/v).

FORM AS APPLIED IN THE TEST (if different from that of starting material) : The test item was formulated as a homogenous suspension in corn oil and tested at a concentration of 20% (w/v) in corn oil.

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: At the end of exposure period, the test item, positive, negative and vehcile controls were removed from the anterior chamber and the corneal epithelium washed until no visual evidence of test item was observed using EMEM (containing phenol red). pH and osmolality are not specified.
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra, India
- Age at study initiation: Boving age was between 1 to 5 years (animal age was determined based on the teeth count and horn ring count in addition to the Horizontal Diameter of corneas and central corneal thickness)
- Weight at study initiation: Not specified
- Housing: Transported (in a sealed plastic container) under cold conditions in Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL]. Eyes were used within 24 hours of slaughter after confirmation each was free from defects following pre-test procedures. Corneas that had an opacity of less than seven opacity units or equivalent for the opacitometer were used in the study.

- Acclimation period: As part of the pre-test procedure, the corneal holder was equilibrated at 32 ± 1°C for at least one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible.
Vehicle:
other: Corn oil
Remarks:
750 μL Fatty acids, C16-18 (even numbered), manganese(II) salts (in suspension) at 20% (w/v) in corn oil
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume with unit): 750 μL
- Concentration (if solution): In suspension at 20% (w/v) in corn oil.

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL Corn oil
- Lot/batch no. (if required): MKCC0462
- Purity: Not specified
Duration of treatment / exposure:
Corneas were exposed for 4 h ±5 minutes at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
The holders were incubated in a horizontal position for 90 ±5 min at 32 ± 1 ºC. After incubation the medium in the posterior chamber was transferred into labelled tubes.
Number of animals or in vitro replicates:
Four sets, of three corneas were tested.
Set 1: Control: normal (physiological) saline.
Set 2: Positive control: 20% (w/v) imidazole in normal (physiological) saline.
Set 3: Vehicle: Corn oil
Set 4: Test item: Fatty acids, C16-18 (even numbered), manganese(II) salts (in suspension) at 20% (w/v) in corn oil.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS : Eyes were used within 24 hours of slaughter. Eyes were examined prior to use and only corneas free from any visible defects were used. Corneas that had an opacity of less than seven opacity units or equivalent for the opacitometer were used.

QUALITY CHECK OF THE ISOLATED CORNEAS : A baseline reading for each cornea holder was taken with medium prior to loading the cornea onto the cornea holder. Corneas, free from defects, were dissected so they had a 2 to 3 mm rim of sclera and were transferred to a container containing Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL].

Selected corneas were mounted on corneal holders with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then placed on the top of the cornea and fixed in place. Both chambers were then filled to excess with pre-warmed phenol red free Eagle's Minimum Essential Medium (EMEM) (the posterior chamber filled first to allow the cornea to return to its natural concave position), ensuring no bubbles were present. The device was then equilibrated at 32 ± 1°C for at least one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible.

Following equilibration, the medium was removed from both chambers and fresh pre-warmed phenol red free EMEM added to both chambers. A baseline opacity reading was then taken for each cornea.

NUMBER OF REPLICATES : Four sets, of three corneas were tested.

NEGATIVE CONTROL USED : 750 μL normal (physiological) saline

VEHICLE CONTROL USED: 750 μL corn oil

POSITIVE CONTROL USED : 750 μL 20% (w/v) imidazole in normal (physiological) saline.

APPLICATION DOSE AND EXPOSURE TIME :
750 μL Fatty acids, C16-18 (even numbered), manganese(II) salts (in suspension) at 20% (w/v) in corn oil. Corneas were exposed for 4 h ±5 minutes at 32 ± 1 °C.
The same volume was used for corneal administration for the control (normal (physiological) saline), positive control (20% (w/v) imidazole in normal (physiological) saline), and the vehicle (corn oil).

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: yes, 90 ± 5 min at 32 ± 1 ºC.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of exposure period, the test item, positive and negative controls were removed from the anterior chamber and the corneal epithelium washed until no visual evidence of test item was observed using EMEM (containing phenol red). Once the medium was free of test item, the corneas were given a final rinse with EMEM (without phenol red)

- POST-EXPOSURE INCUBATION: The holders were incubated in a horizontal position for 90 ±5 min at 32 ± 1 ºC. After incubation the medium in the posterior chamber was transferred into labelled tubes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity is measured quantitatively, as the amount of light transmission through the cornea.
Mean Opacity value =
Step 1. Step 1 value of section 2.11.1 – post treatment opacity reading (calculated for each cornea)
Step 2. Calculate the mean of the control group
Step 3. {[Step 1 value of each cornea (except control) – Step 2 value] – b}/a
Step 4. Calculate Mean of Step 3 value
Where b = 0.9894 and a = 0.0251 (a and b values are empirically derived for the instrument)

- Corneal permeability: Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The passage of sodium fluorescein dye is measured with the aid of a plate reader set to read at OD490.

- Others (e.g, pertinent visual observations, histopathology): None

SCORING SYSTEM: In vitro irritancy score (IVIS)
In vitro irritancy score (IVIS) was calculated for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The in vitro irritancy score was calculated for each individual treatment.

DECISION CRITERIA: The decision criteria as indicated in the OECD 437 TG was used.
The IVIS cut-off values for identifying a test item as inducing serious eye damage (UN GHS Category 1) and a test item not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given below:

IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Irritation parameter:
in vitro irritation score
Remarks:
mean value
Run / experiment:
1: (Negative control) Normal (physiological) Saline, 0.75 mL
Value:
1.91
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Remarks:
mean value
Run / experiment:
1 (Positive control) Imidazole at 20% (w/v) in normal (physiological) saline, 0.75 mL
Value:
135.92
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Remarks:
mean value
Run / experiment:
1: (Vehicle control) Corn Oil, 0.75 mL
Value:
2.73
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Remarks:
mean value
Run / experiment:
1: Test item (Fatty acids, C16-18 (even numbered), manganese(II) salts (in suspension) at 20% (w/v) in Corn Oil, 0.75 mL
Value:
0.52
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: Perfomed at a GLP proficient laboratory with well established methodology for perfoming the OECD 437 test guidline. Historical data was provided for the negative, vehicle and positive control to sup[port the IIVS vlaues obtained for the test item.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Valid, the mean IVIS of the negative control (Saline) and vehicle control (Corn oil) were within acceptable min - max values for histroical control data, confirming the reliability of the test procedure.
- Acceptance criteria met for positive control: Valid, the mean IVIS of imidazole (20% w/v) treated corneas was 135.92, this was within the range of two standard deviations of the mean of the historical control data, confirming the reliability of the test procedure.

This met the OECD 437 criteria for a valid test as acceptable historical data ranges were observed for the concurrent positive, negative and vehicle control.
Interpretation of results:
GHS criteria not met
Conclusions:
The IVIS score for corneas treated with 750 μL Fatty acids, C16-18 (even numbered), manganese(II) salts (in suspension) at 20% (w/v) in corn oil was 0.52.

Based on results of this study, the classification for Fatty acids, C16-18 (even numbered), manganese(II) salts is as follows: Classification (OECD 437 UN GHS): No Category
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the results of the studies, the test substance is not an irritant or corrosive to skin or eyes and will not be classified as such.