Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation/corrosion:

non irritant (According to OECD 439 and OECD 404)

Eye irritation:

non irritant (according to OECD 439 and OECD 405)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Clariant Plastics and Coatings (Deutschland) GmbH, BU Additives
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Justification for test system used:
The assessment of skin irritation has typically involved the use of laboratory animals (OECD TG 404). In relation to animal welfare concerns, TG 404 recommends the use of a tiered testing strategy for the determination of skin corrosion and irritation which includes the use of validated in vitro or ex vivo test methods avoiding pain and suffering of animals.

One of the validated in vitro test methods adopted by the OECD TG 439 makes use of reconstructed human epidermis (RhE) which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
Details on test system:
SKIN DISC PREPARATION
- Procedure used: EpiSkin is an in vitro reconstructed human epidermis (RHE) from normal human keratinocytes cultured on a collagen matrix at the air liquid interface. This is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum. Differentiated and stratified epidermis model comprises the main basal, supra basal, spinous and granular layers and a functional stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.
- Quality control for skin discs: The human keratinocytes come from mammary samples obtained from healthy consenting donors during plastic surgery. HIV 1 & 2, B and C hepatitis tests were carried out on the donor bloods as well as verification of the bacteriological and fungal sterility of the cells and absence of mycoplasma.
The technical data, safety sheet and certificate of analysis of the EpiSkin kit used in this study

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The epidermis units were transferred to 12-well plates containing 2 mL of pre-warmed maintenance medium and incubated in a CO2 incubator for approximately 3 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After the 15 minutes exposure at room temperature, the epidermis units were removed from the wells and thoroughly rinsed with PBS to remove residual matters, if any, from the epidermal surface.

After rinsing each of these epidermal units were gently tapped on an absorbent paper to remove the remaining PBS. The surface gently swabbed with a cotton swab and then placed in a 12-well plate pre-filled with 2 mL pre-warmed assay medium to rinse. After rinsing, all the epidermis units were placed on an absorbent paper to remove excess assay medium and then placed back to the same 12-plate pre-filled with pre-warmed maintenance medium.

DYE BINDING METHOD
- Dye used in the dye-binding assay: [MTT:] After incubation, the epidermis units were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2 mL MTT-solution (0.3 mg/mL) and the plates were incubated for 3-hr at 37°C in a carbon dioxide incubator with 5% CO2
- Spectrophotometer: 200 μL sample from each tube was transferred into the wells of a labeled 96-well flat bottom plate (2 wells/epidermis unit) and the amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Microplate Reader, using acidified isopropanol solution as the blank.
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)

Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Ten microliters (10 μL) of Phosphate Buffered Saline (PBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): Ten milligram (10 mg) of 98.5% pure Sodium Dodecyl Sulfate (SDS) in powder form
Duration of treatment / exposure:
The epidermis units were dried carefully to remove excess medium and were transferred into a 12-well plate prefilled with 2 mL MTT-solution (0.3 mg/mL) and the plates were incubated for 3-hr at 37°C in a carbon dioxide incubator with 5% CO2.
Number of replicates:
Three
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
The relative mean tissue viability obtained after 15 minutes treatment with the test item compared to the negative control tissues was 145.54 %.
Value:
> 145.54
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The test item was checked for possible direct MTT reduction by exposing it in direct contact with the MTT solution.
A quantity of 10 mg of the test item was mixed in a glass tube with 2 mL of MTT solution (0.3 mg/mL) along with water as control and mixed. The resulting mixture was incubated for 3 hours in a carbon dioxide incubator at 37°C protected from light. After the incubation period, the color of the MTT solution was checked.

- Colour interference with MTT: The test item was evaluated for its intrinsic color or ability to become colored in contact with water (simulating a tissue humid environment).
A quantity of 10 mg of the test item was mixed with 90 µL water in a glass tube, kept for 15 minutes at room temperature and then visually observed for color development, if any.


DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: It is considered that the NC meets the acceptance, if the mean OD value of the 3 tissues is > 0.6 and the Standard Deviation value (SD) of the % viability is < 18.
- Acceptance criteria met for positive control: It is considered that the Positive Control (SDS) meets the acceptance if the mean viability expressed as % of the NC, is < 40% and the SD is < 18.
Interpretation of results:
GHS criteria not met
Conclusions:
The mean relative tissue viability for the test item, Licocare RBW 300 FL TP was 145.54 % and hence, in according with UN GHS it is predicted to be non-irritant under the experimental conditions described in this report.
Executive summary:

The test item, Licocare RBW 300 FL TP was tested for its possible skin irritation potential using a three dimensionalReconstructed Human Epidermis model,EpiSkin, through topical application for 15 minutes.

The test item is in solid form and was applied directly on the top of the skin tissues at 10 mg/tissue, followed by moistening with 5 µL of the negative control, and exposed for 15 minutes.  Ten microliters (10 µL) of PBS and 10 mg of SDS were used as the negative and positive controls, respectively.

After a 42 hour post-incubation period, irritation potential of Licocare RBW 300 FL TP was evaluated by assessing the cytotoxic (irritancy) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the ability of the test item to reduce the cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 minutes treatment with the test item was compared to the negative control tissues.

The absolute mean OD570(optical density at 570 nm) of the negative control tissues was 0.557287. The test item had a mean cell viability of 145.54 % after 15 minutes exposure. The positive control had a mean cell viability of 14.09567 % after 15 minutes exposure, indicating that the test system functioned properly.

 

The study indicated that the test item Licocare RBW 300 FL TP is in accordance with UN GHS predicted to be non-irritant in this In Vitro Skin Irritation Test using Reconstructed Human Epidermis under the conditions of testing employed.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 March 2020 to 27 March 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
Section 4: Health Effects
adopted on 28 July 2015
Deviations:
no
Principles of method if other than guideline:
- Principle of test: to assess the irritation and/or corrosive effects of the test item Licocare RBW 300 FL TP following single dermal application in New Zealand White Rabbits
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Clariant Plastics and Coatings (Deutschland) GmbH


Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Adita Biosys Private Limited
- Age at study initiation: 5 Months
- Weight at study initiation: 2.23421 kg to 2.31546 kg
- Housing: stainless steel wire mesh cage
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: 12 March 2020 to 23 March 2020

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5°C to 22.8°C
- Humidity (%): 46% to 66%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 17 March 2020 To: 27 March 2020
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 mL of distilled water
- Lot/batch no. (if required): 8721 & 8730


Duration of treatment / exposure:
Initial test: 3 min, 1 hour and 4 hours
Confirmatory Test: 4 hours
Observation period:
Initial test: 3 min, 1 hour, 4 hour, 24, 48 and 72 hours
Confirmatory test: 1 hour, 24, 48 and 72 hours
Number of animals:
3
Details on study design:
TEST SITE
- Area of exposure: trunk region
- % coverage: approximately 6 cm2
- Type of wrap if used: crepe bandage

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: Initial test: 3 min, 1 hour and 4 hours
Confirmatory test: 4 hours

OBSERVATION TIME POINTS
Initial test: 3 min, 1 hour, 4 hours, 24, 48 and 72 hours
Confirmatory test: 1 hour, 24, 48 and 72 hours

SCORING SYSTEM:
- Method of calculation: mean score
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: No irritation/corrosive effect
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: no effect observed
Remarks:
no effect observed
Remarks on result:
no indication of irritation
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: no effect observed
Remarks:
no effect observed
Remarks on result:
no indication of irritation

TABLE 1.     INDIVIDUAL ANIMAL CLINICAL SIGNS OF TOXICITY AND MORTALITY RECORD

Phase of the Experiment

Dose

(g/Patch)

Animal

No.

Sex

Clinical Signs of Toxicity and Mortality on Day

1

2

3

4

Initial Test

0.5

Nb1725

Male

N

N

N

N

Confirmatory Test

0.5

Nb1726

Male

N

N

N

N

Nb1727

Male

N

N

N

N

N: Normal


TABLE 2.     INDIVIDUAL ANIMAL SKIN REACTIONS SCORING RECORD

Phase of the

Experiment

Animal

No.

Sex

Exposure  

Duration

Application Site

Dose

(g/Patch)

Observations

Skin Reactions Score after Patch

Removal

Mean Score

IAR

1 hr

24 hrs

48 hrs

72 hrs

Initial Test

Nb1725

Male

3 min

Site 2

0.5

ERY

0

-

-

-

-

-

EDE

0

-

-

-

-

-

1 hr

Site 3

0.5

ERY

0

-

-

-

-

-

EDE

0

-

-

-

-

-

4 hrs

Site 1

Untreated

Control

ERY

0

0

0

0

0

0

EDE

0

0

0

0

0

0

4 hrs

Site 4

0.5

ERY

0

0

0

0

0

0

EDE

0

0

0

0

0

0

Confirmatory

Test

Nb1726

Male

4 hrs

Site 1

Untreated

Control

ERY

-

0

0

0

0

0

EDE

-

0

0

0

0

0

Site 2

0.5

ERY

-

0

0

0

0

0

EDE

-

0

0

0

0

0

Nb1727

Male

4 hrs

Site 1

Untreated

Control

ERY

-

0

0

0

0

0

EDE

-

0

0

0

0

0

Site 2

0.5

ERY

-

0

0

0

0

0

EDE

-

0

0

0

0

0

 ERY: Erythema; EDE: Oedema; 0: No Erythema/Oedema; min: Minutes; hr/hrs: Hour/Hours; IAR: Immediately after removal of the test patch; -: Not applicable

 

                                  

 Mean Score For Erythema/ Oedema

:

24 hrs + 48 hrs+ 72 hrs (Observations after patch removal)

3 (Observation time point)

 


TABLE 3.     BODY WEIGHT (kg) AND PERCENT CHANGE IN BODY WEIGHT WITH RESPECT TO DAY 1

Phase of

the

Experiment

Dose

(g/Patch)

Animal No.

 

Sex

Body Weight (kg) on day

Percent Change in Body Weight with Respect to Day

1

4

1 to 4

Initial Test

0.5

Nb1725

Male

2.28069

2.33002

2.16294

Confirmatory

Test

0.5

Nb1726

Male

2.36153

2.41089

2.09017

0.5

Nb1727

Male

2.34631

2.38962

1.84588

 

 

Mean

 

2.35392

2.40026

1.96802

 

 

±SD

 

0.01076

0.01504

0.17274

 

 

n

 

2

2

2

SD: Standard Deviation; n: Number of animals

 

 


TABLE 4.     INDIVIDUAL ANIMAL GROSS PATHOLOGY FINDINGS

Phase of the Experiment

Dose

(g/Patch)

Animal No.

 

Sex

 

Fate

Gross Pathology Findings

External

Internal

Initial Test

0.5

Nb1725

Male

TS

NAD

NAD

Confirmatory Test

0.5

Nb1726

Male

TS

NAD

NAD

Nb1727

Male

TS

NAD

NAD

NAD: No Abnormality Detected; TS: Terminal Sacrifice

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the experiment, it is concluded that single dermal application of Licocare RBW 300 FL TP did not reveal any skin reactions when applied to the skin of New Zealand White Rabbits, hence the test item is considered as non-irritant and did not meet the classification criteria according to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals
Executive summary:

The test item, Licocare RBW 300 FL TPwas evaluated forAcute Dermal Irritation/Corrosion in New Zealand White rabbits as per OECD guideline No. 404, “Acute Dermal Irritation/Corrosion” adopted on 28 July 2015.

The study was performed in two phases i.e. initial test and confirmatory test. Approximately 24 hours before the application of the test item, fur on the dorso-lateral area of the trunk of the animals was removed by clipping closely using an electric hair clipper (approximately 8 × 12 cm). Care was taken to avoid abrasion to the skin and the animals with healthy intact skin were used for experiment.

As there was no information on the test item, in initial test, three patches were applied for 3 minutes, 1 hour and 4 hour exposure period. The application site 1 (anterior left trunk region) of the animal was served as untreated control. On application site 2 (posterior left trunk region), site 3 (posterior right trunk region) and site 4 (anterior right trunk region) of the animal, 0.5 g oftest item moistened with 0.5 mL of distilled water was applied for 3 minutes, 1 hour and 4 hours respectively (semi-occlusive dressing). The first test patch was applied on site 2 and removed after three minutes. No skin reaction was observed after immediate observation, a second test patch was applied at site 3 and removed after one hour. After immediate observation, the observations at this stage indicated that exposure could be humanely allowed to extend to four hours, a third test patch was applied at site 4 and removed after four hours, and the response was graded. The application site 1 (anterior left trunk region) of the animal was covered with blank cotton gauze at the time of site 4 application and was removed along with application site 4. After removal of the test patches, the application sites were washed with distilled water and dried with absorbent cotton without altering the existing response or the integrity of the epidermis. The application sites 1, 2, 3 and 4 were scored immediately after patch removal whereas the application sites 1 and 4 were also scored approximately at 1, 24, 48 and 72 hours after the patch removal.

As there was no corrosive effects observed during initial test, the confirmatory test was performed using two additional rabbits. In the confirmatory test, the patches were applied for 4 hours exposure period. Each animal had two application sites. The clipped area of skin at left trunk region and right trunk region were identified as application sites 1 and 2 respectively.

In the confirmatory test, the application site 1 (left trunk region) of each animal was served as untreated control covered with blank cotton gauze. On application site 2 (right trunk region) of each animal, 0.5 g oftest item moistened with 0.5 mL of distilled water was applied for 4 hours exposure period and removed after 4 hours. After removal of the test patches, the application sites were washed with distilled water and dried with absorbent cotton without altering the existing response or the integrity of the epidermis. The application sites 1 and 2 were scored approximately at 1, 24, 48 and 72 hours after patch removal.

The mean score across 3 scoring times (24, 48 and 72 hours after patch removal) for both initial test animal and confirmatory test animals was ‘0’ for erythema and oedema grades. No erythema and oedema was observed at the test item application site in both initial and confirmatory test animals.

All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality during the observation period. No clinical signs of toxicity and mortality was observed in both initial and confirmatory test animals.

Body weight was recorded on the day of receipt and prior to application of test item (day 1) and at termination. No changes were observed in body weight andpercent change in body weight with respect to day 1in both initial and confirmatory test animals. All the animals revealednormal increase in the body weightduring the observation period.

At termination, all the animals were sacrificed by intravenous administration of sodium thiopentone. Nogross pathological findings were observed in any of the animals in both initial and confirmatory test.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-04 to 2017-12-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Clariant Plastics and Coatings (Deutschland) GmbH, BU Additives

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Local abattoir (Slaughter house), Near Frazer town, Bengaluru.

IN-LIFE DATES: From: 04 July 2017 To: 06 July 2017
Vehicle:
other: 0.9% w/v Sodium chloride
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The neat test item Licocare RBW 300 FL TP (enough quantity to completely cover the cornea) grounded to fine powder was applied.

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL of negative control (i.e. 0.9% NaCl w/v) was applied to each designated cornea.
- Batch no. (if required): C263586
Duration of treatment / exposure:
4 hours exposure
Duration of post- treatment incubation (in vitro):
After final opacity measurement, EMEM medium was removed and replaced by fluorescein solution. After 91 minutes, the fluid from posterior chamber was transferred into labeled glasstubes for further investigations.
Number of animals or in vitro replicates:
3 replicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- Procurement of corneas: The bovine eyes were procured from local abattoir and transported in a box containing Hank’s Balanced Salt Solution (HBSS) and penicillin-streptomycin (100 IU & 100 µg/mL) in an ice box. On day 1 of experiment 20 numbers and on day 2 of experiment 30 numbers of corneas were procured.

- Dissection: On both days of experiment, post pretest examination, eyes were dissected to isolate the corneas from surrounding tissue and then placed in a container of fresh HBSS (Hank Balanced Salt Solution).

- Mounting: The isolated corneas were mounted in the cornea holder, which separated holder into anterior and posterior chambers. During mounting, it was ensured that the epithelium of the cornea projected into the anterior chamber. The EMEM medium was added in to both the chambers and kept in an incubator (32°C) for 1 to 2 hours.

QUALITY CHECK OF THE ISOLATED CORNEAS
- Pretest examination: On both days of experiment, immediately after receiving the eyes in the lab, all eyes were observed for any evidence of vascularization, pigmentation, opacity or scratches.
- Initial opacity: Corneas exhibited initial opacity < 7 were considered for experiment

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% w/v Sodium Chloride (NaCl)

POSITIVE CONTROL USED: Dibenzoyl-L-tartaric acid

APPLICATION DOSE AND EXPOSURE TIME: Enough of the neat test item (grounded to powder) to cover the cornea; 0.75 mL of the positive control

TREATMENT METHOD: open chamber; 4 hour exposure

POST-INCUBATION PERIOD: yes. If YES please specify duration: 91 minutes with 1 mL of fluorescein solution (500 mg of Sodium fluorescein was weighed and added to 100
mL of Dulbecco’s Phosphate-Buffered Saline to obtain 5mg/mL concentration)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Using OP3.0 Opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [microtiter plate reader] (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS): IVIS = Mean Opacity Score + (15 × Mean Optical Density Score)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1
Test item of IVIS score, ≤ 3 would be considered as UN GHS No Category.
Test item of IVIS score, >3 and ≤ 55 would be considered as “No prediction can be made”, subsequently testing with any other adequate method remains at the discretion of the sponsor.
Test item of IVIS score, > 55 would be considered as severe irritant causing serious eye damage and classified as Category 1.
Irritation parameter:
in vitro irritation score
Run / experiment:
The corneas treated with Licocare RBW 300 FL TP exhibited IVIS score as 0.1 which is classified as “No Catagory”.
Value:
<= 3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Negative control/mean
Value:
2.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
positive control/ mean
Value:
187.5
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item /mean
Value:
0.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
Interpretation of results:
GHS criteria not met
Conclusions:
The test item Licocare RBW 300 FL TP, negative control 0.9% w/v NaCl and positive control 20% w/v Dibenzoyl-L-Tartaric acid were tested for their occular irritancy score using bovine corneas as test system in Bovine Corneal Opacity and Permeability assay (3 corneas per compound) using Opacitometer and Microplate reader/Spectrophotometer to measure opacity and permeability respectively.
All the test groups were tested in triplicates. The mean of all three corneas treated with test item Licocare RBW 300 FL TP exhibited mean opacity -1.0 and mean permeability 0.0716. Under similar conditions, the positive control (20% w/v Dibenzoyl-L-Tartaric acid) exhibited a mean opacity score of 179.6 and mean permeability 0.5279. Whereas negative control (0.9% w/v NaCl) exhibited a mean opacity score 0.7 and mean permeability 0.1423, confirming the sensitivity of the test system to occular irritancy.
The IVIS for Licocare RBW 300 FL TP (Solid) was found to be 0.1 while the positive control 20% w/v Dibenzoyl-L-Tartaric acid scored 187.5. The negative control, 0.9% w/v NaCl scored 2.8.
Based on the In Vitro Irritancy Score (IVIS), it is concluded that the test item Licocare RBW 300 FL TP (Solid) is an occular non-irritant classified under ‘No Category’ as per UN GHS. Whereas the positive control proved to be severe/corossive eye irritant classified under ‘Category I’. The negative control is classified as ‘No Category’ based on its IVIS.
Executive summary:

The occular irritancy of the test item “Licocare RBW 300 FL TP (Solid)” was carried out using Bovine Corneal Opacity and Permeability assay. The Licocare RBW 300 FL TP was applied directly (neat) to the corneas by means of open chamber method. The positive control i.e. Dibenzoyl-L-Tartaric Acid was applied to the corneas as 20% w/v in 0.9% NaCl solution by open chamber method whereas negative control i.e. 0.9% w/v Sodium Chloride solution was applied to the corneas by means of closed chamber method. The control and treated corneas (n=3)were subjected to the opacity and permeability testing. 

All the test groups were tested in triplicates. The IVIS for Licocare RBW 300 FL TP (Solid) was found to be 0.1 while the positive control 20% w/v Dibenzoyl-L-Tartaric acid scored 187.5. The negative control, 0.9% w/v NaCl scored 2.8, confirming the sensitivity of the test system to occular irritancy.

Based on theIn VitroIrritancy Score (IVIS), it is concluded that the test item Licocare RBW 300 FL TP (Solid) is an occular non-irritant classified under ‘No Category’ as per UN GHS. Whereas the positive control proved to be severe/corossive eye irritant classified under ‘Category I’. Similarly, ‘No Category’ for negative control based on its IVIS.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Clariant Plastics & Coatings (Deutschland) GmbH., BU Additives
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: In Vivo Bio Sciences, Shed No.23, Khatha No. 3169, Assessment No.154, Kodigehalli Village, Magadi Road, Bangalore - 560 091, Karnataka, India

- Age at study initiation: 7 months
- Weight at study initiation: 2.2710 to 2.3047 kg
- Housing: The rabbits were housed individually in rabbit cages (approx. size: L 65 x B 65 x H 45 cm) with noryl shallow cage body and facilities for pelleted food (Stainless steel feed hopper) and drinking water (750 mL markrolon bottle fitted with sipper tube). The perforated noryl raised shelf for enrichment was provided. The litter collection trays (noryl waste tray) were changed daily (except on Sunday). The water bottles and feed hoppers were changed once a week.

- Diet (e.g. ad libitum): The animals were offered rabbit feed manufactured Krishna Valley Agrotech LLP, MIDC Kupwad block, Sangli, Maharashtra.

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India, was provided to animals in markrolon bottles with stainless steel sipper tubes.

- Acclimation period: Start 23 August 2018 End: 28 August 2018 (single animal); 29 August (additional animals)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 23°C
- Humidity (%): 65 to 66%
- Air changes (per hr): 13.3 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark cycle.

IN-LIFE DATES: From: 29 August (single animal); 30 August (additional animals)
To: 02 September 2018
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): On test day one (treatment day), a quantity of 100 mg of the finely ground test item was instilled into the conjunctival sac of the left eye of the animal after gently pulling the lower lid away from the eyeball. The lids were then held together gently for about one second to prevent loss of test item. The right eye remained untreated as the reference control (RBa4451). At 24 hours, the treated eye irrigated with normal saline for one minute to remove the residual test item.
The score at 24 hours indicated that the test item was not corrosive / severe irritant to the eye.

Following the initial test, the confirmatory test was performed using two additional animals (RBa4452 & RBa4453) in the same manner.


Duration of treatment / exposure:
On test day one (treatment day), a quantity of 100 mg of the finely ground test item was instilled into the conjunctival sac of the left eye of the animal after gently pulling the lower lid away from the eyeball. The lids were held together gently for about one second prevents loss of test item. The right eye remained untreated and served as the reference control. All the rabbits were treated in a similar manner.

Observation period (in vivo):
The rabbit used for the single animal test was observed for a period of 5 days and the additional two rabbits were observed for a period of 4 days post-instillation. The eyes of each rabbit were examined for ocular reaction at 1, 24, 48 and 72 hours post-instillation and scored.
Number of animals or in vitro replicates:
3 male rabbits
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes,
- Time after start of exposure: At 24 hours, the treated eye irrigated with normal saline for one minute to remove the residual test item.

SCORING SYSTEM: The eyes of each rabbit were examined by using an opthalmoscope (OECD guideline 405) at 1, 24, 48 and 72 hours post-instillation and scored.
In addition, all the treated eyes were examined using ophthalmic fluorescein sodium at 24 hours post-instillation for all the three rabbits


TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
cornea opacity score
Basis:
other: 1 + 2 rabbits
Time point:
24/48/72 h
Score:
0
Irritation parameter:
iris score
Basis:
other: 1 + 2 rabbits
Time point:
24/48/72 h
Score:
0
Irritation parameter:
conjunctivae score
Basis:
other: 1 + 2 rabbits
Time point:
24/48/72 h
Score:
0
Irritation parameter:
chemosis score
Basis:
other: 1 + 2 rabbits
Time point:
24/48/72 h
Score:
0
Irritation parameter:
overall irritation score
Basis:
other: 1 + 2 rabbits
Time point:
24/48/72 h
Score:
0
Irritant / corrosive response data:
Non irritant

 

TABLE 1.   Individual Body Weight, Clinical Signs and Necropsy Findings

Rabbit No.

Body weight (kg)

Clinical signs

 

Day 1

 

Day 2

 

Day 3

 

Day 4

 

Day 5

 

Necropsy findings

At Start of Acclimatization

At

Treatment

At

Termination

1 h

2 h

3 h

4 h

AM

PM

AM

PM

AM

PM

AM

PM

RBa4451

2.1800

2.2710

2.3557

N

N

N

N

N

N

N

N

N

N

N

N

NAD

RBa4452

 

2.1976

2.2851

2.4103

N

N

N

N

N

N

N

N

N

N

N

-

NAD

RBa4453

2.2244

2.3047

2.4172

N

N

N

N

N

N

N

N

N

N

N

-

NAD

NAD: No Abnormality Detected   h: hour  AM: antemeridian    PM: post meridian    N: Normal

Note :There were no pre-terminal deaths

 


TABLE 2.           Individual Animals Eye Irritation Scores (Treated Eyes) 

 Rabbit Number

RBa4451

RBa4452

 

              RBa4453

Eye Reactions

Hour

Hour

Hour

1

24

48

72

1

24

48

72

1

24

48

72

Corneal Opacity

0

0

0

0

0

0

0

0

0

0

0

0

Area of Opacity

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

NA

Iris

0

0

0

0

0

0

0

0

0

0

0

0

Redness

0

0

0

0

0

0

0

0

0

0

0

0

Chemosis

0

0

0

0

0

0

0

0

0

0

0

0

 NA: Not applicable

 

 

 

                                                                                                                                                                                                                     

 


TABLE 3.           Eye Irritation Scores - Mean Values of all Animals (Treated Eye)

Eye reaction

Hours

1

24

48

72

Corneal Opacity

0

0

0

0

Iris

0

0

0

0

Redness

0

0

0

0

Chemosis

0

0

0

0


TABLE 4.   Detailsof Administration of Analgesic and Anesthetic

Rabbit No.

Sex

Date

Administration of Systemic Analgesic (SC)

Administration of Topical Anesthetic agent

Test Item instillation

Time

(AM)

Volume

(mL)

 

Time

 

Volume

Time

(AM)

Volume

(mg)

RBa4451

M

29 August 2018

 

9:15

0.07

10:10 AM

2 drops

 

10:15

100

RBa4452

M

30 August 2018

 

10:20

0.07

11.15 AM

2 drops

 

11:20

100

RBa4453

M

30 August 2018

 

10:25

0.07

11.20 AM

2 drops

 

11:25

100

SC: Subcutaneous injection   AM: antemeridian      

 

Details of post application

 

Rabbit No.

Sex

Date

Administration of Systemic Analgesic (SC)

Buprenorphine

Administration of Systemic Analgesic (SC)

Meloxicam

Time

 

Volume

(mL)

 

Time

 

Volume

(mL)

RBa4451

M

29 August 2018

 

6:15 PM

0.07

6:15 PM

0.23

RBa4452

M

30 August 2018

 

7:20 PM

0.07

7:20 PM

0.23

RBa4453

M

30 August 2018

 

7:25 PM

0.07

7:25 PM

0.23

SC: Subcutaneous injection  PM: post meridian

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the present study results the test Licocare RBW 300 FL TP is “Non-Irritant” on the eye of New Zealand white rabbits.
Executive summary:

The acute eye irritation / corrosion study in New Zealand White rabbits was conducted to evaluate the eye irritation / corrosion potential of Licocare RBW 300 FL TP when a single dose of the test item was instilled in to the conjunctival sac of the rabbit’s eye.On test day one (treatment day), a quantity of 100 mg of the finely ground test item was instilled into the conjunctival sac of the left eye of the animal after gently pulling the lower lid away from the eyeball. The lids were then held together gently for about one second to prevent loss of test item. The right eye remained untreated and served as the reference control. All the rabbits were treated in a similar manner.The eyes of each rabbit were examined at 1, 24, 48 and 72 hours post-instillation and scored.

Mean Eye reactions observed were as follows:

Eye reaction

Hours

1

24

48

72

Corneal Opacity

0

0

0

0

Iris

0

0

0

0

Redness

0

0

0

0

Chemosis

0

0

0

0

There were no clinical signs of toxicity and pre-terminal deaths and no abnormality was detected at necropsy in any of the animals.

Based on the above, the test itemLicocare RBW 300 FL TPis classified as follows:

The test itemis not subject of classification according toRegulation (EC) No 1272/2008 of the European Parliament and of the council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006,as there were no eye reactions in any of the rabbits.

The test itemis not subject of classification according toOECD Harmonised Integrated Classification System OECD, 2001, as there were no eye reactions in any of the rabbits. 

The test itemis not subject ofclassification Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Seventh Revised Edition, United Nations (2017). ST/SG/AC.10/30/Rev.7, as there were no eye reactions in any of the rabbits.

Based on the present study results the test item Licocare RBW 300 FL TP is“Non-Irritant”on the eyeof New Zealand white rabbits.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item, Licocare RBW 300 FL TP was tested for its possible skin irritation potential using a three dimensionalReconstructed Human Epidermis model,EpiSkin, through topical application for 15 minutes. Under the experimental conditions described in the report, the mean relative tissue viability for the test item, Licocare RBW 300 FL TP was 145.54 % and hence, in according with UN GHS it is predicted to be non-irritant to skin.

The occular irritancy of the test item “Licocare RBW 300 FL TP (Solid)” was carried out using Bovine Corneal Opacity and Permeability assay. The Licocare RBW 300 FL TP was applied directly (neat) to the corneas by means of open chamber method. Based on the experimental conditions described in the report and theIn VitroIrritancy Score (IVIS), it is concluded that the test item Licocare RBW 300 FL TP (Solid) is occular non-irritant, classified under ‘No Category’ as per UN GHS.

The acute eye irritation / corrosion study in New Zealand White rabbits was conducted to evaluate the eye irritation / corrosion potential of Licocare RBW 300 FL TP when a single dose of the test item was instilled in to the conjunctival sac of the rabbit’s eye (study was performed according to OECD 405).There were no eye ractoions, clinical signs of toxicity and pre-terminal deaths and no abnormality was detected at necropsy in any of the animals.

Justification for classification or non-classification

Under the experimental in vitro and in vivo conditions described in the reports and Licocare RBW 300 FL TP is predicted to be non-irritant to the skin and eye.