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EC number: 216-223-3 | CAS number: 1530-32-1
- 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
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- 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
The dermal irritation potential of target chemical was assessedin an in- vitro and in-vivo experimental studies which were conducted for test chemical. Based on the available studies,it can be concluded thatchemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.
Eye Irritation:
The ocular irritation potential of target chemical was assessedin various in- vitro and in-vivo experimental studies which were conducted for test chemical in rabbits.Based on the available key data and supporting studies,it can be concluded thatchemical is able to cause severe irreversible eye damage and hence considered as severely irritating.Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 1 (irreversible effects on the eye)”.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- data is from experimental reports
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
- Principles of method if other than guideline:
- According to OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
- GLP compliance:
- yes
- Specific details on test material used for the study:
- Identification : ETHYL TRI-PHENYL PHOSPHONIUM BROMIDE
Appearance : White to off-white crystalline powder
Batch number : ETPB1/B/14016
CAS No. : 1530-32-1
AI Content (purity) : 99.79%
Manufactured date : February, 2014
Expiry Date : January, 2015 (Retest date)
Storage conditions : Room temperature (20 - 30 °C)
Safety precautions: Aprons, masks, caps, gloves and goggles were used to ensure the health and safety of the personnel.
Disposal : No test item remained unused after dosing. Hence no disposal was done - Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or test system and environmental conditions:
- Details on test animals
Source: Procured from LIVEON BIOLABS PVT. LTD
Age : 4 to 4.5 Months (Approximately)
Sex : Female
Number of Animals: Three
Health Status : Healthy young adults and nulliparous and non-pregnant rabbits were used for the study.
Body weight of animals : Minimum: 2.312 kg and Maximum: 2.480 kg (Prior to Treatment)
Acclimatisation : Rabbits were acclimatised to the test conditions for a period of 6 days (Animal No.-1) and 8 days (Animal No.-2 and 3) prior to the application of the test item.
Identification : During acclimatization marking was done with non toxic marker pen in the inside of left ear of rabbits and after acclimatization, animals were marked with permanent number in the inner side of right ear of rabbits. Permanent marker and cage card was used for identification. The individual cage card was labelled with at least study no., study type, test system, sex, dose, animal number, experimental start and experiment completion date.
Diet : All animals were provided conventional laboratory rabbit diet (Nutrivet Life Sciences, Pune) ad libitum. Batch No.: 200004.
Water : Aqua guard filtered tap water was provided ad libitum.
Husbandry : The animals were housed individually in stainless steel cages.
Room Sanitation : The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
Cages and water bottle : All the cages and water bottles were changed minimum twice a week.
Environmental Conditions:
Temperature : Minimum: 20.50 °C, Maximum: 22.50 °C
Relative humidity : Minimum: 51.50 %, Maximum: 66.30 %
Light-dark-rhythm : 12:12
Air Changes: More than 12 changes per hour - Type of coverage:
- semiocclusive
- Preparation of test site:
- clipped
- Vehicle:
- water
- Controls:
- yes, concurrent vehicle
- Amount / concentration applied:
- 0.5 gm of test item (pulverized form) moistened with 0.5 ml of distilled water
- Duration of treatment / exposure:
- 4 hours
- Observation period:
- 72 hours
- Number of animals:
- 3
- Details on study design:
- TEST SITE
- Area of exposure: approximately 6 X 6 cm at contralateral sites
- Type of wrap if used: porous gauze dressing and non-irritating tape (Micropore 3”)
REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test item was removed by using cotton soaked in distilled water
- Time after start of exposure: 4hr
- Control site: 0.5 ml distilled water was applied at control site.
SCORING SYSTEM: Draize Method - Irritation parameter:
- erythema score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 0
- Reversibility:
- not specified
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- edema score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 0
- Reversibility:
- not specified
- Remarks on result:
- no indication of irritation
- Irritant / corrosive response data:
- No erythema and edema (skin irritation) were found at the end of 72 hour observation period after patch removal.The individual mean score at 24, 48 and 72 hours for animal nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively.
- Other effects:
- Clinical Observation
No systemic toxicity was observed at treated rabbits during the experimental period.
Mortality
No mortality was observed during the observation period - Interpretation of results:
- other: not irritating
- Conclusions:
- In Animals No.1 revealed no erythema and oedema at 1, 24, 48 and 72 hours observation. Hence the confirmatory test was conducted on additional two animals (no. 2 and 3) to confirm the non irritant nature of the test item. After 4 hours of exposure, animal no. 2 and 3 revealed no erythema and oedema at 1, 24, 48 and 72 hours observation. The individual mean score at 24, 48 and 72 hours for animal nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively. Hence, under the experimental test conditions, it was concluded the test chemical was Non-Irritating to the skin of Female New Zealand White Rabbits and "Not Classified as Skin Irritant” as per CLP Classification.
- Executive summary:
An Acute Dermal Irritation/corrosion Study of the test chemical in Rabbits, was performed as per OECD guideline No. 404. Three healthy young adult female New Zealand White rabbits were used for conducting acute dermal irritation/corrosion study. Body weights were recorded on day 0 (prior to application) and at termination. Rabbits with good intact skin were selected for the study. The hairs of all the rabbits were clipped at contra lateral sites, approximately 24 hours prior to treatment. A dose of 0.5 gm of test item moistened with 0.5 ml of distilled water was applied to the skin, over an area of approximately 6 x 6 cm clipped of hair on one side of rabbits. The other untreated side was kept as control area and 0.5 ml of distilled water was applied at this site. At the end of 4 hours, the gauze patch was removed and test item application site was wiped with cotton soaked in distilled water without altering the integrity of the epidermis. Initially, the test item was applied to the clipped area of skin of one rabbit. The test site was covered with gauze patch. After 4 hours of exposure, animal no. 1 revealed no erythema and oedema at 1, 24, 48 and 72 hours observation. Hence the confirmatory test was conducted on additional two animals (no. 2 and 3) to confirm the non-irritant nature of the test item. After 4 hours of exposure, animal no. 2 and 3 revealed no erythema and oedema at 1, 24, 48 and 72 hours observation. In Animals No.1 revealed no erythema and oedema at 1, 24, 48 and 72 hours observation. Hence the confirmatory test was conducted on additional two animals (no. 2 and 3) to confirm the non-irritant nature of the test item. After 4 hours of exposure, animal no. 2 and 3 revealed no erythema and oedema at 1, 24, 48 and 72 hours observation. The individual mean score at 24, 48 and 72 hours for animal nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively. Hence under the experimental test conditions, it was concluded the test chemical was Non-Irritating to the skin of Female New Zealand White Rabbits and "Not Classified as Skin Irritant” as per CLP Classification.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from experimental study report
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Principles of method if other than guideline:
- The purpose of this study was to assess potential for the test article to be dermal irritants. The dermal irritation potential of test article may be predicted by measurement of their cytotoxic effect, as reflected in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiDerm™ model.
- GLP compliance:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia; Lot No; E1131
- Expiration date of the lot/batch: NA
- Purity test date: NA
RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature or Fridge storage
- Stability under test conditions: No data available
- Solubility and stability of the test substance in the solvent/vehicle: No data available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data available
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prior to the main test, the test articles are tested for their ability to reduce/interact with MTT and their ability to stain the tissues itself. All tests are performed according to the by MatTek provided test protocol.
- Preliminary purification step (if any): No data available- Final dilution of a dissolved solid, stock liquid or gel: No data available
- Final preparation of a solid: No data available
FORM AS APPLIED IN THE TEST: Solid
OTHER SPECIFICS: No data available - Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: as provided by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Source strain:
- other: Not applicable
- Details on animal used as source of test system:
- - Description of the cell system used:The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.Test System IdentificationAll of the EpiDerm™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues are included in this report. Tissue plates were appropriately labeled with study information.
- Justification for test system used:
- The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, In Vitro Life Science Laboratories, Bratislava, Slovakia) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- The tissues were exposed to the test article neat (undiluted) on April 25, 2018 (Run 1 of 1). EpiDerm™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.MTT and Color Pre-testsPretesting has actually been conducted for all chemicals, although the first intitial 8 test chemicals a pretesting was not conducted (for skin).MTT AssayFollowing the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 3 hours MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was approximately 3 hours with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean negative control tissues.Evaluation of Test Article in the Cell Models:1. Cell system: Upon receipt, the MatTek EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight (18 ± 3 hrs) at ~37°C, 5% CO2 in a humidified incubator.2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test article are applied topically to tissues by pipette(liquid) Tissues were exposed to controls and the test article for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.a) Controls30 µL of negative control DPBS and 30 μL of the positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.b) Test Articles 25 mg of the test article was applied topically to the tissue 3. Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 15 times with sterile DPBS. After the 15th rinse from washing bottle, each insert wasw completely submerge 3 times in 150 ml DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for 24 ± 2 hours. After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 18 ± 3 hours, for a total of an approximately 42 hour post-exposure incubation.
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The EpiDerm™ 3 dimensional human tissue model
- Tissue Lot number(s): 26459
- Date of initiation of testing: 6/08/2017
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The test substance was rinsed from the tissues with sterile DPBS by filling and emptying the tissue insert 15 times to remove any residual test material. This was followed by completely submerge the insert 3 times in 150 ml DPBS.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL MTT medium (1.0 mg/mL).
- Incubation time: After 3 hours
- Spectrophotometer: Synergy H4 spectrophotometer
- Wavelength: 570 nm
- Filter: No data
- Filter bandwidth: No data
- Linear OD range of spectrophotometer: No data
NUMBER OF REPLICATE TISSUES: 3
CALCULATIONS and STATISTICAL METHODS
All data were background subtracted before analysis. MTT data are represented as % viable compared to negative control. Data was generated as follows:
Blanks:
• The optical density (OC) mean from all replicates for each plate (ODblank)
Negative Controls (NC):
• The blank corrected value was calculated: ODNC = ODNCraw – ODblank
• The OD mean per NC tissue was calculated.
• The mean OD for all tissues corresponds to 100% viability
• The mean, standard deviation (SD) and the percent coefficient of variation (% CV)
Positive Controls (PC):
• Calculation of the blank corrected value: ODPC = ODPCraw – ODblank
• The OD mean per PC tissue was calculated.
• The viability per tissue was calculated: %PC = [ODPC / mean ODNC] x 100
• The mean viability for all tissues was calculated: Mean PC = S %PC / number of
tissues
• The standard deviation (SD) and the percent coefficient of variation (% CV)
Tested Compound (TC):
• Calculation of the blank corrected value: ODTC = ODTCraw – ODblank
• The OD mean per treated tissue was calculated.
• The viability per tissue was calculated: %TC = [ODTC / mean ODNC] x 100
• The mean viability for all tissues was calculated: Mean TC = S %TC / number of
tissues
• The standard deviation (SD) and the percent coefficient of variation (% CV)
Data Correction Procedure for MTT Interfering Compounds (if applicable)
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt
where ODkt = (mean ODtkt – mean ODukt)
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissue
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)
Data Correction Procedure for Colored Compounds (if applicable)
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt – ODvt
ODtvt= optical density of treated viable tissue incubated in MTT media
ODvt= optical density of viable tissues incubated in media alone
- Evaluation of data The results of the assay was evaluated and compared to negative control.
Table: Criteria for in vitro Interpretation:
In vitro Result In vivo Classification
EU GHS
Mean tissue viability ≤ 50% Irritant (I), Category 2
Risk Phrase (R38)
Mean tissue viability > 50% Non-Irritant (NI) No category
Assay quality controls
- Negative Controls (NC)The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.
- Positive Controls (PC)5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control. - Standard Deviation (SD)The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit):25 mg
- Concentration (if solution): neat
VEHICLE (Not used)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none- Lot/batch no. (if required): none
- Purity: none
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL sterile DPBS
- Concentration (if solution): neat
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% solution of sodium dodecyl sulfate - Duration of treatment / exposure:
- The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.
- Duration of post-treatment incubation (if applicable):
- For a total of an approximately 42 hour post-exposure incubation.
- Number of replicates:
- 3 tissues were used for test compound and control.
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Run 1
- Value:
- 54.6
- Vehicle controls validity:
- not specified
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Remarks:
- Mean O.D.1.250 ; SD= 4.83 ;%CV= 8.85; Non-Irritant
- Other effects / acceptance of results:
- The MTT data show the assay quality controls were met.
- Interpretation of results:
- other: not irritating
- Conclusions:
- The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study. The Mean % tissue viability compared to negative control (n=3) of the test substance.was determined to be 54.6 %. Thus, substance was considered to be not irritating to the human skin.
- Executive summary:
The dermal irritation potential of test article was examined according to the OECD 439. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met and passed the acceptance of criteria. The mean of OD for test chemical was determined to be 1.250. The standard deviation of viabilities for test chemical were calculated to be 4.83. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 54.6%. Hence, under the current experimental test conditions the test chemical was considered to be non-irritating to human skin.
Referenceopen allclose all
Skin Reaction
In Treated area Dose:0.5 gm of test item (moistened with 0.5 ml distilled water) Sex:Female
Animal No. |
Test |
Treated area* |
Erythema score |
Oedema score |
||||||
1h |
24h |
48h |
72h |
1h |
24h |
48h |
72h |
|||
1 |
Initial |
Right |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
Confirmatory |
Left |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
3 |
Right |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
In Control area Dose:0.5 ml of distilled water Sex:Female
Animal No. |
Test |
Treated area* |
Erythema score |
Oedema score |
||||||
1h |
24h |
48h |
72h |
1h |
24h |
48h |
72h |
|||
1 |
Initial |
Left |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
Confirmatory |
Right |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
3 |
Left |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Key: h = Hour.
Erythema Oedema
0 =No erythema 0 =No oedema
Table 1 Continued…
Mean Individual Animal Score at 24, 48 and 72 hours
Animal Number Observations |
1 |
2 |
3 |
Erythema |
0.00 |
0.00 |
0.00 |
Oedema |
0.00 |
0.00 |
0.00 |
Individual Animal BodyWeight
Sex:Female
Animal No. |
Body Weight (kg) |
|
Prior to Dosing |
At termination |
|
1 |
2.480 |
2.566 |
2 |
2.354 |
2.418 |
3 |
2.312 |
2.370 |
Individual AnimalClinicalSigns
Sex:Female
Animal No. |
Days (Post dosing Observation) |
|||
0 |
1 |
2 |
3 |
|
1 |
1 |
1 |
1 |
1 |
2 |
1 |
1 |
1 |
1 |
3 |
1 |
1 |
1 |
1 |
Key: 1 = Normal.
Observation Table:
Code N° |
Tissue No. |
Raw data |
Blank corrected data |
mean of OD |
% of viability |
||
Aliq. 1 |
Aliq. 2 |
Aliq. 1 |
Aliq. 2 |
||||
NC
|
1 |
2.5235 |
2.5252 |
2.488 |
2.490 |
2.489 |
108.6 |
2 |
2.2737 |
2.2435 |
2.239 |
2.208 |
2.224 |
97.0 |
|
3 |
2.1558 |
2.2403 |
2.121 |
2.205 |
2.163 |
94.4 |
|
PC |
1 |
0.0806 |
0.0793 |
0.046 |
0.044 |
0.045 |
2.0 |
2 |
0.0901 |
0.0847 |
0.055 |
0.050 |
0.052 |
2.3 |
|
3 |
0.0947 |
0.0918 |
0.060 |
0.057 |
0.058 |
2.5 |
|
Test chemical |
1 |
1.4276 |
1.3965 |
1.393 |
1.361 |
1.377 |
60.1 |
2 |
1.2302 |
1.2437 |
1.195 |
1.209 |
1.202 |
52.4 |
|
3 |
1.2011 |
1.2137 |
1.166 |
1.179 |
1.172 |
51.1 |
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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from experimental study report.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
- Principles of method if other than guideline:
- The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected in the (MTT) assay, in the MatTek EpiOcular™ model
- GLP compliance:
- no
- Species:
- human
- Strain:
- other: Not applicable
- Details on test animals or tissues and environmental conditions:
- - Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.
- Test System Identification
All of the EpiOcular™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues is included in this report. Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
- Justification of the test method and considerations regarding applicability
EpiOcularTM Eye Irritation (OCL) by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakien
The test articles and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek,In Vitro Life Science Lab. (Bratislava, Slovakia).The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD). - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg of solid test chemical
- Concentration (if solution): neat (undiluted)
VEHICLE (no vehicle)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat - Duration of treatment / exposure:
- Tissues were exposed for approximately 6 hrs ± 15 min for solid test articles, and controls, at approximately 37°C, 5% CO2 in a humidified incubator.
- Observation period (in vivo):
- Not applicable
- Duration of post- treatment incubation (in vitro):
- Following the washing and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals and controls
- Number of animals or in vitro replicates:
- 2 tissues were used for test compound and control.
- Details on study design:
- - Details of the test procedure used
The tissues were exposed to the test article neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA)
for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 6 hrs ± 15 min for solid test articles and controls at approximately 37°C, 5% CO2 in a humidified incubator.
After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~25 minutes for solid test articles and controls.Following the washing and post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test chemicals and controls.Tissue viability was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
- MTT Auto reduction and colouring assessment
MTT Pre-test
The test article was assessed for the potential to interfere with the assay. Approximately 50 µL of liquid test article was added to 1 mL of MTT media (~1 mg/mL) and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 3 hours. 50 µL of ultrapure water was used as a negative control.
- Test Article Color Test
Approximately 50 µL of liquid test article was added to 1.0 mL of ultrapure water and 2.0 mL isopropanol and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 04 minutes and 35 seconds. Samples were then added to the wells of a clear 96-well plate and the plate was read on a Thermo Scientific Multiskan FC Microplate Photometer to 570 nm. Test articles that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions).
- MTT Assay:
Inserts are removed from the 24-well plate after 3 hrs of incubation and the bottom of the insert is blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 1 ml isopropanol in each well so that no isopropanol is flowing into the insert. At the end of the non-submerged extraction inserts and tissues are discarded without piercing and 1 ml of isopropanol is added into each well. The extract solution is mixed and the optical density of the extracted formazan (200 μL/well of a 96-well plate) was determined using Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissues.
- Evaluation of Test Article in the cell Models
1. Cell System:
Upon receipt, the MatTek EpiOcular™ tissue cultures were placed in 1.0 mL of fresh Maintenance medium (in a 6-well plate) for 60 minutes. After the 60 minutes incubation, the Maintenance medium was exchanged with fresh medium and the tissues were incubated overnight (16-24 hrs) at approximately 37°C, approximately 5% CO2 in a humidified incubator.
2. Control and Test Article Exposures:
20 µL of calcium and magnesium free DPBS was added to each tissue and the tissues placed back into the incubator for 30 minutes. The controls and the test article will be applied topically to tissues by pipette.2 tissues will be used per test compound and control.
a)Controls: 50 µL of negative control sterile ultrapure water and positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
b)Test Article: When a solid was tested, 50 mg of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
3. Post exposure treatment:
After the exposure, the tissues were rinsed 20 times with sterile of DPBS to remove test material. The apical surface was gently blotted with a cotton swab and cultures were immediately transferred to a 12-well plate containing 5 mL of media per well. Tissues exposed to liquid test articles (and the respective control) were incubated, submerged in the media for ~12 minutes at room temperature.For liquid test articles, tissues, Tissuses were then transferred to 6-well plates containing 1.0 mL fresh Maintenance medium per well and incubated for a post-exposure recovery period for 2 hours at approximately 37 degC, 5% CO2 in a humidified incubator.
- Doses of test chemical and control substances used
Test Article:
When a solid was tested, 6 hours of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
Controls: 50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Tissues were exposed for approximately 6 hours for solid test articles and controls, at approximately37°C, 5% CO2 in a humidified incubator.
Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling 18 hours for solid test articles and controls.
- Justification for the use of a different negative control than ultrapure H2O (Not applicable)
- Justification for the use of a different positive control than neat methyl acetate (Not applicable)
- Number of tissue replicates used per test chemical and controls: 2 tissues were used for test compound and control.
- Description of the method used to quantify MTT formazan
The blue formazan salt was extracted by placing the tissue insterts in 1 mL isopropanol in a 6-well plate. The extraction for solid exposed tissues was 3 hrs incubation. After an addition of 1 ml isopropanol and mixing, the optical density of the extracted formazan (200μL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for
the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
· The OD mean from all replicates for each plate (ODblank).
Negative Controls (NC):
· The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
· The OD mean per NC tissue was calculated.
· The mean OD for all tissues corresponds to 100% viability.
· The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.
Positive Control (PC):
· Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
· The OD mean per PC tissue was calculated.
· The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
· The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
· The standard deviation (SD), standard error of the meanthe mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.
Tested Articles:
· Calculate the blank corrected value ODTT= ODTTraw– ODblank.
· The OD mean per tissue is calculated.
· The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
· The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
· The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.
Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt =
(mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)
Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in
media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.
Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.
- Evaluation of data
The results of the assay was evaluated and compared to negative control.
Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category
- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density(OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
- Standard Deviation (SD)
Each test of ocular irritancy potential is predicted from the mean viability determined on 2 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the
replicates is <18% for three replicate tissues. - Irritation parameter:
- other: mean % tissue viability
- Run / experiment:
- Run 1
- Value:
- 2.5
- Vehicle controls validity:
- not specified
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Remarks:
- Mean O.D.=0.049; Irritant
- Other effects / acceptance of results:
- The MTT data show the assay quality controls were met.
- Interpretation of results:
- Category 1 (irreversible effects on the eye) based on GHS criteria
- Conclusions:
- The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance was determined to be 2.5%. Thus, the test chemical was considered to be irritating to the human eyes.
- Executive summary:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours recovery after the post-soak. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met, passing the acceptance criteria. The mean % tissue viability of test substance was determined to be 2.5 %. Hence, under the experimental test conditions it was concluded that test substance was considered to be severely irritating to the human eyes.
- Endpoint:
- eye irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from experimental study report.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 405 (Acute Eye Irritation / Corrosion)
- Principles of method if other than guideline:
- The objective of the study was to assess the irritant and/or corrosive effects of the given test chemical on eye when exposed by the ocular route in rabbits.
- GLP compliance:
- yes
- Specific details on test material used for the study:
- AI Content (purity) : 99.7%
Batch number : ETPB1/B/14016
Identification : ETHYL TRI-PHENYL PHOSPHONIUM BROMIDE
Appearance : White to off-white crystalline powder
Manufactured date : February, 2014
Expiry Date : January, 2015 (Retest date)
Storage conditions : Room temperature (20 - 30 °C)
Safety precautions : Aprons, masks, caps, gloves and goggles were used to ensure the health and safety of the personnel.
Disposal : No test item remained unused after dosing.No disposal was done. - Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or tissues and environmental conditions:
- TEST ANIMALS
- Sex: Male
- Source: Procured from LIVEON BIOLABS PVT. LTD.
- Age at study initiation: 2.5 to 4.5 Months (Approximately), healthy young adult rabbits were used for the study.
- Weight at study initiation: Minimum: 2.018 kg and Maximum: 2.566 kg (Prior to Treatment)
- Housing: The animals were housed individually in stainless steel cages.
- Identification: During acclimatization marking was done with non toxic marker pen in the inside of left ear of rabbits and after acclimatization, animals were marked with permanent number in the inner side of right ear of rabbits. Permanent marker and cage card was used for identification. The individual cage card was labelled with at least study no., study type, test system, sex, dose, experiment start date and experiment completion date.
- Room Sanitation: The experimental room floor and work tops were swept and mopped with disinfectant solution every day.
- Cages and water bottle: All the cages and water bottles were changed minimum twice a week.
- Diet (e.g. ad libitum): All animals were provided conventional laboratory rabbit diet (Nutrivet Life Sciences, Pune) ad libitum.
- Water (e.g. ad libitum): Aqua guard filtered tap water was provided ad libitum.
- Acclimation period: Rabbits were acclimatised to the test conditions for a period of 7 days (Animal No.-1) and 10 days (Animal No. 2 and 3) prior to the application of the test item.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Minimum: 19.60°C Maximum: 22.20°C
- Humidity (%):Minimum - 56.50% Maximum - 69.20%
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12:12 - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent no treatment
- Amount / concentration applied:
- Quantity of 100 mg test item (in pulverized form) was placed in the conjunctival sac of eye in three rabbits, after gently pulling the lower lid away from the eyeball.
- Duration of treatment / exposure:
- 24 hours
- Observation period (in vivo):
- 1, 24, 48, 72 hours and on day 7, day 14 and on day 21 after instillation of test item.
- Number of animals or in vitro replicates:
- Three male rabbits
- Details on study design:
- REMOVAL OF TEST SUBSTANCE
- Washing (if done): the treated eye of rabbit was washed with normal saline.
- Time after start of exposure: 24 hours
SCORING SYSTEM:Draize Method
TOOL USED TO ASSESS SCORE: Ophthalmoscope and fluorescein strips. - Irritation parameter:
- cornea opacity score
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 1
- Reversibility:
- not fully reversible within: 21 days
- Remarks on result:
- positive indication of irritation
- Irritation parameter:
- cornea opacity score
- Basis:
- animal: #2, #3
- Time point:
- 24/48/72 h
- Score:
- 1.33
- Reversibility:
- not fully reversible within: 21 days
- Remarks on result:
- positive indication of irritation
- Irritation parameter:
- iris score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 0
- Reversibility:
- not fully reversible within: 21 days
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- conjunctivae score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 2
- Reversibility:
- not fully reversible within: 21 days
- Remarks on result:
- positive indication of irritation
- Irritation parameter:
- chemosis score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 1.67
- Reversibility:
- not fully reversible within: 21 days
- Remarks on result:
- positive indication of irritation
- Irritant / corrosive response data:
- The following were observed in treated rabbits.
In the initial test, 0.1 gm of test item was applied into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 0.1 gm of test item was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. All animals were observed till day 21 post test item installation.
Untreated eye of all the three rabbits was normal throughout the experimental period.
The following grading scores were observed in treated eye of tested rabbits.
Observation at 1 hour after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- Zero in all 3 animals; Iris: Normal in all 3 animals. Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in all 3 animals; Chemosis: some swelling above normal (includes nictitating membranes) was seen in all animal.
Observation at 24 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity- One quarter (or less) but not zero; Iris: Normal in all 3 animals; Conjunctivae - Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was seen in all the animals.
At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 40 %, 50 % and 40 % damage in animal no. 1, 2 and 3, resectively.
Observation at 48 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity- One quarter (or less) but not zero; Iris: Normal in all 3 animals; Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in animal number 1 whereas diffuse, crimson color; individual vessels not easily discernible in animal number 2 and 3; Chemosis: Obvious swelling with partial eversion of lids was seen in animal numbers. 2 and 3 whereas some swelling above normal (includes nictitating membranes) was seen in animal no.1.
Observation at 72 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity- One quarter (or less) but not zero; Iris: Normal in all 3 animals; Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in animal numbers 1 and 3 whereas diffuse, crimson color; individual vessels not easily discernible in animal number 2; Chemosis: some swelling above normal (includes nictitating membranes) was noticed in all the animals.
Observation at day 7 after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in all 3 animals; Area of Opacity- One quarter (or less) but not zero; Iris: Normal in all 3 animals; Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in animal number 3 whereas diffuse, crimson color; individual vessels not easily discernible in animal numbers 1 and 2; Chemosis: some swelling above normal (includes nictitating membranes) was noticed in animal numbers 2 and 3 whereas obvious swelling with partial eversion of lids was seen in animal number 1.
Observation at day 14 after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible in animal numbers 1 and 2 whereas animal number 3 recovered to normal; Area of Opacity- One quarter (or less) but not zero in animal numbers 1 and 2 whereas animal number 3 recovered to normal; Iris: Normal in all 3 animals; Conjunctivae - Some blood vessels definitely hyperaemic (injected) was seen in animal number 3 whereas diffuse, crimson color; individual vessels not easily discernible in animal numbers 1 and 2; Chemosis: some swelling above normal (includes nictitating membranes) was noticed in animal numbers 1 and 2 whereas animal number 3 recovered to normal.
Observation at day 21 after instillation of test item revealed: Cornea- No ulceration or opacity in all 3 animals; Area of Opacity- No opacity was seen in all 3 animals; Iris: Normal in all 3 animals. Conjunctivae: recovered to normal in all the animals; Chemosis: No swelling was observed in all the animals.
The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.00, 0.00, 2.00, 1.67; 1.33, 0.00, 2.00, 1.67 and 1.33, 0.00, 2.00, 1.67, respectively. - Other effects:
- Clinical Observation
No systemic toxicity was observed in treated rabbits during the experimental period.
Mortality
No mortality was observed during the observation period.
Body weight
Increase in body weights of all the animals weighed at termination as compared to day 0 body weights. - Interpretation of results:
- Category 1 (irreversible effects on the eye) based on GHS criteria
- Conclusions:
- The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.00, 0.00, 2.00, 1.67; 1.33, 0.00, 2.00, 1.67 and 1.33, 0.00, 2.00, 1.67, respectively. Under the experimental conditions tested, all the three animals were fully irreversible within an observation period of 21 days. Hence, the given test chemical is being classified as an eye irritant in 'Category 1' (irreversible effects on the eye) as per the CLP regulation.
- Executive summary:
An Acute Eye Irritation/Corrosion Study of the given test chemical in rabbits was conducted as per OECD test guideline no. 405. 3 young adult male New Zealand White rabbits were used for the study. Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 0.1 g (100 mg) of test item was instilled in the other (treated) eye of each rabbit. The eye was observed at 1, 24, 48, 72 hour, day 7, day 14 and day 21 for all the three animals post, test item instillation. Ophthalmoscope was used for scoring of eye lesions. In the initial test, 0.1 g (100 mg) of test item was instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions; hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 0.1 g (100 mg)of test item was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. All the animals were observed till day 21 post, test item instillation. Untreated eye of all the three rabbits were normal throughout the experimental period. The following grading scores were observed in treated eye of tested rabbits. Observation at 1 hour after instillation of test item revealed: Cornea-No ulceration or opacity was observed in all the animals; Area of Opacity-Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Some blood vessels definitely hyperaemic (injected) was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all the animals. Observation at 24 hours after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal luster); details of iris clearly visible in all the animals; Area of Opacity-One quarter (or less) but not zero in all the animals ;Iris: Normal in all the animals; Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all the animals. At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 40%, 50% and 40% damage in animal no. 1, 2 and 3 respectively. Observation at 48 hours after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal luster); details of iris clearly visible in all the animals; Area of Opacity-One quarter (or less) but not zero in all the animals; Iris: Normal in all the animals; Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Obvious swelling with partial eversion of lids was observed in all the animals. Observation at 72 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal luster); details of iris clearly visible was observed in animal no. 1 and easily discernible translucent area; details of iris slightly obscured in animal no. 2 and 3;Area of Opacity-One quarter (or less) but not zero was observed in animal no. 1 whereas greater than one quarter, but less than half was observed in animal no. 2 and 3;Iris:Normal in all the animals; Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Obvious swelling with partial eversion of lids was observed in all the animals. Observation on day 7 after instillation of test item revealed: Cornea-Easily discernible translucent area; details of iris slightly obscured was observed in animal no. 1 whereas nacrous area; no details of iris visible; size of pupil barely discernible was observed in animal no. 2 and 3;Area of Opacity-Greater than one quarter, but less than half was observed in animal no. 1 whereas greater than half, but less than three quarters was observed in animal no. 2 and 3;Iris:Normal in animal no. 1 whereas details of iris was not visible in animal no. 2 and 3;Conjunctivae-Diffuse, crimson color; individual vessels not easily discernible was observed in animal no. 1 and 3 whereas diffuse beefy red was observed in animal no. 2;Chemosis:Obvious swelling with partial eversion of lids was observed in animal no. 1 and 3 whereas swelling, with lids about half closed was observed in animal no. 2. Observation on day 14 after instillation of test item revealed: Cornea-Nacrous area; no details of iris visible; size of pupil barely discernible was observed in all the animals; Area of Opacity-Greater than half, but less than three quarters was observed in all the animals; Iris: Details of iris were not visible in all the animals; Conjunctivae -Diffuse beefy red was observed in animal no. 3 whereas diffuse, crimson color; individual vessels not easily discernible was observed in animal no. 2 and 3;Chemosis:Swelling, with lids about half closed was observed in animal no. 1 whereas obvious swelling with partial eversion of lids was observed in animal no. 2 and 3. Observation on day 21 after instillation of test item revealed: Cornea-Nacrous area; no details of iris visible; size of pupil barely discernible was observed in all the animals; Area of Opacity-Greater than half, but less than three quarters was observed in all the animals; iris: Details of iris were not visible in all the animals; Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Obvious swelling with partial eversion of lids was observed in all the animals. The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.00, 0.00, 2.00, 1.67; 1.33, 0.00, 2.00, 1.67, and 1.33, 0.00, 2.00, 1.67, respectively. Under the experimental conditions tested, all the three animals were fully irreversible within an observation period of 21 days. Hence, the given test chemical is likely to be classified as an eye irritant in 'Category 1' (irreversible effects on the eye) as per the CLP regulation.
Referenceopen allclose all
Code N° |
Tissue No. |
Raw data |
Blank corrected data |
mean of OD |
% of viability |
||
Aliq. 1 |
Aliq. 2 |
Aliq. 1 |
Aliq. 2 |
||||
NC
|
1 |
2.0043 |
2.0745 |
1.970 |
2.040 |
2.005 |
99.4 |
2 |
2.0651 |
2.0633 |
2.030 |
2.029 |
2.029 |
100.6 |
|
PC |
1 |
0.5856 |
0.6029 |
0.551 |
0.568 |
0.559 |
27.7 |
2 |
0.6292 |
0.6432 |
0.594 |
0.608 |
0.601 |
29.8 |
|
Test chemical |
1 |
0.0835 |
0.0846 |
0.049 |
0.050 |
0.049 |
2.4 |
2 |
0.0846 |
0.0841 |
0.050 |
0.049 |
0.050 |
2.5 |
Table 1 : Individual Animal Eye Irritation Scores
Treated Dose:0.1 g (100 mg) of test item (in pulverized form) Sex:Male
Animal Number |
1 |
|||||||
Application Side |
Right |
|||||||
Eye Reactions |
* |
Hour(s) |
Day |
|||||
1 |
24 |
48 |
72 |
7 |
14 |
21 |
||
Cornea |
0 |
0 |
1 |
1 |
1 |
2 |
3 |
3 |
Area of Opacity |
0 |
0 |
1 |
1 |
1 |
2 |
3 |
3 |
Iris |
0 |
0 |
0 |
0 |
0 |
0 |
0# |
0# |
Conjunctiva |
0 |
1 |
2 |
2 |
2 |
2 |
3 |
2 |
Chemosis |
0 |
1 |
1 |
2 |
2 |
2 |
3 |
2 |
Corneal Damage (%) |
40 |
Animal Number |
2 |
|||||||
Application Side |
Right |
|||||||
Eye Reactions |
* |
Hour(s) |
Day |
|||||
1 |
24 |
48 |
72 |
7 |
14 |
21 |
||
Cornea |
0 |
0 |
1 |
1 |
2 |
3 |
3 |
3 |
Area of Opacity |
0 |
0 |
1 |
1 |
2 |
3 |
3 |
3 |
Iris |
0 |
0 |
0 |
0 |
0 |
0# |
0# |
0# |
Conjunctiva |
0 |
1 |
2 |
2 |
2 |
3 |
2 |
2 |
Chemosis |
0 |
1 |
1 |
2 |
2 |
3 |
2 |
2 |
Corneal Damage (%) |
50 |
Animal Numbers |
3 |
|||||||
Application Side |
Right |
|||||||
Eye Reactions |
* |
Hour(s) |
Day |
|||||
1 |
24 |
48 |
72 |
7 |
14 |
21 |
||
Cornea |
0 |
0 |
1 |
1 |
2 |
3 |
3 |
3 |
Area of Opacity |
0 |
0 |
1 |
1 |
2 |
3 |
3 |
3 |
Iris |
0 |
0 |
0 |
0 |
0 |
0# |
0# |
0# |
Conjunctiva |
0 |
1 |
2 |
2 |
2 |
2 |
2 |
2 |
Chemosis |
0 |
1 |
1 |
2 |
2 |
2 |
2 |
2 |
Key:*= Pre-treatment eye examination.
# = Details of iris was not visible Corneal Damage (%) 40 Key:*= Pre-treatment eye examination. # = Details of iris was not visible
Table 1 Continued…
Untreated Dose: Control Sex:Male
Animal Number |
1 |
|||||||
Application Side |
Left |
|||||||
Eye Reactions |
* |
Hour(s) |
Day |
|||||
1 |
24 |
48 |
72 |
7 |
14 |
21 |
||
Cornea |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Area of Opacity |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Iris |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Conjunctiva |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Chemosis |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Corneal Damage (%) |
0 |
Animal Number |
2 |
|||||||
Application Side |
Left |
|||||||
Eye Reactions |
* |
Hour(s) |
Day |
|||||
1 |
24 |
48 |
72 |
7 |
14 |
21 |
||
Cornea |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Area of Opacity |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Iris |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Conjunctiva |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Chemosis |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Corneal Damage (%) |
0 |
Animal Number |
3 |
|||||||
Application Side |
Left |
|||||||
Eye Reactions |
* |
Hour(s) |
Day |
|||||
1 |
24 |
48 |
72 |
7 |
14 |
21 |
||
Cornea |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Area of Opacity |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Iris |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Conjunctiva |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Chemosis |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Corneal Damage (%) |
0 |
Key:*= Pre-treatment eye examination.
Table 1 Continued Eye Irritation Scores - Mean Values after 24, 48, 72 Hours (Treated eye)
Animal No. Eye Reaction |
1 |
2 |
3 |
Corneal Opacity |
1.00 |
1.33 |
1.33 |
Iris |
0.00 |
0.00 |
0.00 |
Conjunctiva |
2.00 |
2.00 |
2.00 |
Chemosis |
1.67 |
1.67 |
1.67 |
Table 2 : Individual AnimalClinicalSigns
Sex:Male
Animal No. |
Days (Post application observation) |
|||||||||||||||||||||
0 |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
14 |
15 |
16 |
17 |
18 |
19 |
20 |
21 |
|
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
2 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
3 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
Key:1 = Normal
Table 3: Individual Animal Body Weight
Sex :Male
Animal No. |
Animal Body Weight (kg) |
|
Prior to application |
At termination |
|
1 |
2.566 |
2.742 |
2 |
2.228 |
2.670 |
3 |
2.018 |
2.498 |
Key:kg = Kilogram
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irreversible damage)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin Irritation:
Study 1:
An Acute Dermal Irritation/corrosion Study of the test chemical in Rabbits, was performed as per OECD guideline No. 404. Three healthy young adult female New Zealand White rabbits were used for conducting acute dermal irritation/corrosion study. Body weights were recorded on day 0 (prior to application) and at termination. Rabbits with good intact skin were selected for the study. The hairs of all the rabbits were clipped at contra lateral sites, approximately 24 hours prior to treatment. A dose of 0.5 gm of test item moistened with 0.5 ml of distilled water was applied to the skin, over an area of approximately 6 x 6 cm clipped of hair on one side of rabbits. The other untreated side was kept as control area and 0.5 ml of distilled water was applied at this site. At the end of 4 hours, the gauze patch was removed and test item application site was wiped with cotton soaked in distilled water without altering the integrity of the epidermis. Initially, the test item was applied to the clipped area of skin of one rabbit. The test site was covered with gauze patch. After 4 hours of exposure, animal no. 1 revealed no erythema and oedema at 1, 24, 48 and 72 hours observation. Hence the confirmatory test was conducted on additional two animals (no. 2 and 3) to confirm the non-irritant nature of the test item. After 4 hours of exposure, animal no. 2 and 3 revealed no erythema and oedema at 1, 24, 48 and 72 hours observation. In Animals No.1 revealed no erythema and oedema at 1, 24, 48 and 72 hours observation. Hence the confirmatory test was conducted on additional two animals (no. 2 and 3) to confirm the non-irritant nature of the test item. After 4 hours of exposure, animal no. 2 and 3 revealed no erythema and oedema at 1, 24, 48 and 72 hours observation. The individual mean score at 24, 48 and 72 hours for animal nos. 1, 2 and 3 were 0.00, 0.00, 0.00 and 0.00, 0.00, 0.00, for erythema and oedema formation, respectively. Hence under the experimental test conditions, it was concluded the test chemical was Non-Irritating to the skin of Female New Zealand White Rabbits and "Not Classified as Skin Irritant” as per CLP Classification.
Study 2:
The dermal irritation potential of test article was examined according to the OECD 439. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met and passed the acceptance of criteria. The mean of OD for test chemical was determined to be 1.250. The standard deviation of viabilities for test chemical were calculated to be 4.83. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 54.6%. Hence, under the current experimental test conditions the test chemical was considered to be non-irritating to human skin.
Study 3:
The Acute Dermal Irritation study of the test chemical was conducted on New Zealand white rabbits. The study was conducted according to OECD test guideline 404. In the initial test one healthy rabbit of body weight 2.18 kg selected for study after acclimatization. The test compound in the amount of 0.5 gm was applied uniformly at the different sites on the shaven back skin of animal. The hairs of back sides were removed (approximately 6 cm’) one day earlier before the treatment. The test chemical in the amount of 0.5 gm was applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which was held in place with non-irritating tape. The first patch was applied on the shaven back skin of rabbit and removed after three minutes. No serious reaction was observed at the site of application. The second patch was applied on the different shaven back side and removed after one hour. There were no signs of skin reaction observed at this site of application. Finally, a third patch was applied at a different site and was removed after four hour. Redness was observed at the site of application of the test compound after 4 hours of patch removal. Finally, the animal was observed for 14 days, for any irritation and corrosion. Because of there was no corrosive effect was observed in the initial test, a confirmatory test was done in order to confirm the irritant or negative response of the test substance by using two additional animals. In the confirmatory test the test chemical in the amount of 0.5 gm was applied on the shaven back skin (approximately 6 cm’) of two animals, each with one patch, for an exposure period of four hours. After four hours the patch was removed and the skin reactions were graded according to Draize’s method. The test compound when applied dermally on New Zealand White rabbit in the amount of 0.5 gm produced erythema up to 24 hours. Furthermore, no clinical signs were observed during the entire observation period. The dermal irritation index of the test compound for New Zealand white rabbits was calculated as 2.25 and test compound can be classified under moderately irritant under test condition.
Although the last dermal irritation study shows positive result, but the observed effects were mild and not enough to classify the chemical as skin irritant. Also, based on recorded scores, the observed effects were completely reversible in 14 days (erythema and edema score 0.0). Thus, considering the results of the other studies and based on majority of results obtained,it can be concluded that the testchemical is unlikely to cause skin irritation and hence considered as not irritating.Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.
Eye Irritation:
Study 1:
An Acute Eye Irritation/Corrosion Study of the given test chemical in rabbits was conducted as per OECD test guideline no. 405. 3 young adult male New Zealand White rabbits were used for the study. Rabbits free from injury of eye were selected for the study. The eyes of all the rabbits were examined 24 hours prior to treatment. One eye of each rabbit served as control and other as treated. Control eye was left untreated whereas; 0.1 g (100 mg) of test item was instilled in the other (treated) eye of each rabbit. The eye was observed at 1, 24, 48, 72 hour, day 7, day 14 and day 21 for all the three animals post, test item instillation. Ophthalmoscope was used for scoring of eye lesions. In the initial test, 0.1 g (100 mg) of test item was instilled into the conjunctival sac of the right eye of animal no.1 whereas the left eye of the rabbit served as the control. As animal no. 1 showed no severe ocular lesions; hence a confirmatory test was conducted on additional two rabbits (animal no. 2 and 3); 0.1 g (100 mg)of test item was instilled into the conjunctival sac of right eye of both the rabbits and left eye served as the control. All the animals were observed till day 21 post, test item instillation. Untreated eye of all the three rabbits were normal throughout the experimental period. The following grading scores were observed in treated eye of tested rabbits. Observation at 1 hour after instillation of test item revealed: Cornea-No ulceration or opacity was observed in all the animals; Area of Opacity-Zero in all the animals; Iris: Normal in all the animals; Conjunctivae - Some blood vessels definitely hyperaemic (injected) was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all the animals. Observation at 24 hours after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal luster); details of iris clearly visible in all the animals; Area of Opacity-One quarter (or less) but not zero in all the animals ;Iris: Normal in all the animals; Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Some swelling above normal (includes nictitating membranes) was observed in all the animals. At 24 hours observation the rabbits were examined for corneal epithelium cell damage using sodium fluorescein strips and noticed 40%, 50% and 40% damage in animal no. 1, 2 and 3 respectively. Observation at 48 hours after instillation of test item revealed: Cornea-Scattered or diffuse areas of opacity (other than slight dulling of normal luster); details of iris clearly visible in all the animals; Area of Opacity-One quarter (or less) but not zero in all the animals; Iris: Normal in all the animals; Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Obvious swelling with partial eversion of lids was observed in all the animals. Observation at 72 hours after instillation of test item revealed: Cornea- Scattered or diffuse areas of opacity (other than slight dulling of normal luster); details of iris clearly visible was observed in animal no. 1 and easily discernible translucent area; details of iris slightly obscured in animal no. 2 and 3;Area of Opacity-One quarter (or less) but not zero was observed in animal no. 1 whereas greater than one quarter, but less than half was observed in animal no. 2 and 3;Iris:Normal in all the animals; Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Obvious swelling with partial eversion of lids was observed in all the animals. Observation on day 7 after instillation of test item revealed: Cornea-Easily discernible translucent area; details of iris slightly obscured was observed in animal no. 1 whereas nacrous area; no details of iris visible; size of pupil barely discernible was observed in animal no. 2 and 3;Area of Opacity-Greater than one quarter, but less than half was observed in animal no. 1 whereas greater than half, but less than three quarters was observed in animal no. 2 and 3;Iris:Normal in animal no. 1 whereas details of iris was not visible in animal no. 2 and 3;Conjunctivae-Diffuse, crimson color; individual vessels not easily discernible was observed in animal no. 1 and 3 whereas diffuse beefy red was observed in animal no. 2;Chemosis:Obvious swelling with partial eversion of lids was observed in animal no. 1 and 3 whereas swelling, with lids about half closed was observed in animal no. 2. Observation on day 14 after instillation of test item revealed: Cornea-Nacrous area; no details of iris visible; size of pupil barely discernible was observed in all the animals; Area of Opacity-Greater than half, but less than three quarters was observed in all the animals; Iris: Details of iris were not visible in all the animals; Conjunctivae -Diffuse beefy red was observed in animal no. 3 whereas diffuse, crimson color; individual vessels not easily discernible was observed in animal no. 2 and 3;Chemosis:Swelling, with lids about half closed was observed in animal no. 1 whereas obvious swelling with partial eversion of lids was observed in animal no. 2 and 3. Observation on day 21 after instillation of test item revealed: Cornea-Nacrous area; no details of iris visible; size of pupil barely discernible was observed in all the animals; Area of Opacity-Greater than half, but less than three quarters was observed in all the animals; iris: Details of iris were not visible in all the animals; Conjunctivae -Diffuse, crimson color; individual vessels not easily discernible was observed in all the animals; Chemosis: Obvious swelling with partial eversion of lids was observed in all the animals. The individual mean score for animal nos. 1, 2 and 3 at 24, 48, 72 hours for corneal opacity, iris, conjunctiva and chemosis were found 1.00, 0.00, 2.00, 1.67; 1.33, 0.00, 2.00, 1.67, and 1.33, 0.00, 2.00, 1.67, respectively. Under the experimental conditions tested, all the three animals were fully irreversible within an observation period of 21 days. Hence, the given test chemical is likely to be classified as an eye irritant in 'Category 1' (irreversible effects on the eye) as per the CLP regulation.
Study 2:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours recovery after the post-soak. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met, passing the acceptance criteria. The mean % tissue viability of test substance was determined to be 2.5 %. Hence, under the experimental test conditions it was concluded that test substance was considered to be severely irritating to the human eyes.
Study 3:
An ocular irritation study was conducted on New Zealand white rabbits in accordance with OECD 405 to assess the irritation parameter of the test chemical 3 female New Zealand White rabbits were used for the study. In the initial test, 0.1 gm of the test chemical was instilled in the conjunctival sac of rabbits after gently pulling the lower lid away from the eyeball. The other eye which remained untreated served as a control. The ocular lesions were evaluated at 1, 24, 48 and 72 hours after the treatment. The grades of ocular reactions (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animals were observed normally for 21 days, any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weights before and during the study was observed. 0.1 gm of the test chemical produced scattered or diffuse areas of opacity in the cornea, obvious swelling with partial eversion of lids and conjunctival redness in rabbit’s up to 72 hours after application of the test chemical. Furthermore, no other clinical signs were recorded after application of the test chemical. The result from the initial test was confirmed in additional two animals of same sex at same dose level. In the confirmatory test, 0.1 gm of the test chemical produced scattered or diffuse areas of opacity in the cornea, obvious swelling with partial eversion of lids(+2) and conjunctival redness(+3) in rabbits upto 72 hours after application of the test chemical. There were no other major clinical signs observed throughout the observation period of 21 days. The eye irritation index was calculated to be 29.6. Based on the above findings and scores, the test chemical can be considered to be moderately irritating to eyes.
Based on the above summarized studies for target chemical, it can be concluded that the test chemical is able to cause severe irreversible eye damage and hence considered as severely irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 1 (irreversible effects on the eye)”.
Justification for classification or non-classification
The skin and eye irritation potential of test chemical was observed in various studies. The results obtained from these studies indicate that the chemical is not likely to cause skin irritation but able to cause severe eye damage. Hence the test chemical can be classified under the category for “Not Classified” skin and “Category 1 (irreversible effects on the eye)” for eye as per CLP.
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