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EC number: - | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
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- Endpoint summary
- Stability
- Biodegradation
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- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
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
- 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
- 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.
Referenceopen allclose all
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
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- 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
- 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.
Referenceopen allclose all
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
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.
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