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Registration Dossier
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EC number: 200-270-1 | CAS number: 56-37-1
Endpoint summary
Administrative data
Description of key information
Skin Irritation:
Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Also, the erythema and edema score of rats was calculated as 0. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.
Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and can be classified as “Not Classified” as per CLP criteria.
Eye Irritation:
Treatment of the eyes with fluorescein 24 hours after instillation of the test substance did not reveal any epithelial damage. Signs of systemic intoxication were not observed.
Based on these observations and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as an Eye Irritant.
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 study report.
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 402 (Acute Dermal Toxicity)
- Principles of method if other than guideline:
- The study was designed and conducted to determine the dermal Irritation/corrosion potential of the test chemical in Sprague Dawley rats.
- GLP compliance:
- yes
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): Benzyltriethylammonium chloride
- Molecular formula: C13H22NCl
- Molecular weight: 227.777 g/mol
- Smiles notation: [N+](CC)(CC)(Cc1ccccc1)CC.[ClH-]
- InChl: 1S/C13H22N.ClH/c1-4-14(5-2,6-3)12-13-10-8-7-9-11-13;/h7-11H,4-6,12H2,1-3H3;1H/q+1;/p-1
- Substance type: Organic
- Physical state: Solid
SOURCE OF TEST MATERIAL
- Test Item: Benzyl triethylammonium chloride (CAS No. 56-37-1)
- Source of test material: Sustainability Support Services (Europe) AB
- Batch No.of test material: ATEBA16081
- Manufacturing Date: FEB; 2016
- Expiration date of the lot/batch: JUNE; 2017
- Purity test date: No data available
- Consistency: Solid, crystalline powder
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: Ambient Temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item was grounded to fine powder prior to application. The particulates were moistened
with distilled water before application.
- Preliminary purification step (if any):No data
- Final dilution of a dissolved solid, stock liquid or gel: No data
- Final preparation of a solid: No data
FORM AS APPLIED IN THE TEST (if different from that of starting material): No data
OTHER SPECIFICS: Safety Precautions : Safety precautions included use of protective clothing, gloves, masks and eye protection (glasses). - Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: National Institute of Biosciences, Pune.
- Females (if applicable) nulliparous and non-pregnant: No data available
- Age at study initiation: Young adult male and female rats aged between 8 – 12 weeks were used.
- Weight at study initiation: The weight range of approximately 224.2 to 264.5 grams at initiation of dosing.
Body weights at the start :
Male
Mean : 258.06 g (= 100 %)
Minimum : 248.5 g (- 3.70 %)
Maximum : 264.5 g (+ 2.50 %)
Total No. of animals : 5
Female
Mean : 230.42 g (= 100 %)
Minimum : 224.2 g (- 2.70 %)
Maximum : 237.7 g (+ 3.16 %)
Total No. of animals : 5
- Identification: Each rat was individually identified by the cage number.
- Fasting period before study: No data
- Housing: The rats were individually housed in polycarbonate cages with paddy husk as bedding.
- Diet (e.g. ad libitum): Rodent feed supplied by the Nutrivet Life Sciences, Pune, was provided ad libitum from individual feeders.
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. All water was from a local source and passed through the reverse osmosis membrane before use.
- Acclimation period: 5 days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 22.5 degree centigrade.
- Humidity (%): 55.4% to 58.8%.
- Air changes (per hr): Ten to fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.
IN-LIFE DATES: 06-06-2017 to 21-06-2017 - Type of coverage:
- semiocclusive
- Preparation of test site:
- clipped
- Vehicle:
- water
- Remarks:
- (Distilled water)
- Controls:
- not specified
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- Concentration (if solution): No data available
VEHICLE
- Amount(s) applied (volume or weight with unit): No data available
- Concentration (if solution): No data available
- Lot/batch no. (if required): No data available
- Purity: No data
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): No data available
- Concentration (if solution): No data available
POSITIVE CONTROL
- Amount(s) applied (volume or weight): No data available
- Concentration (if solution): No data available - Duration of treatment / exposure:
- 24 hours
- Observation period:
- Dermal reaction was observed daily for study period of 14 days.
- Number of animals:
- 10 (5/sex).
- Details on study design:
- TEST SITE
- Area of exposure: Trunk (dorsal surface and sides from scapular to pelvic area)
- % coverage: Approximately 10% of the body surface area.
- Type of wrap if used: Porous gauze dressing and non-irritating tape.
REMOVAL OF TEST SUBSTANCE
- Washing (if done): Distilled water was used to remove residual test item.
OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : Dermal reaction was observed daily for study period of 14 days.
SCORING SYSTEM: Draize Method. - Irritation parameter:
- erythema score
- Basis:
- mean
- Time point:
- 14 d
- Score:
- 0
- Max. score:
- 4
- Reversibility:
- not specified
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- edema score
- Basis:
- mean
- Time point:
- 14 d
- Score:
- 0
- Max. score:
- 4
- Reversibility:
- not specified
- Remarks on result:
- no indication of irritation
- Irritant / corrosive response data:
- Overall result:
Sex : Male
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days.
Sex : Female
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any skin reaction during the study period of 14 days. - Other effects:
- Other effects:
Clinical Signs of Toxicity and Mortality
Sex : Male
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.
Sex : Female
Group I -
Animal treated at the dose level of 2000 mg/kg body weight did not result in any signs of toxicity during the study period of 14 days. All animals survived through the study period of 14 days.
Body Weight
Sex : Male
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 9.03% and 16.52% respectively.
Sex : Female
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 5.99% and 10.57% respectively.
Gross Pathological Findings
Gross pathological examination did not reveal any abnormalities in animals from 2000 mg/kg dose group. - Interpretation of results:
- other: Not irritating
- Conclusions:
- Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Also, the erythema and edema score of rats was calculated as 0. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.
Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and can be classified as “Not Classified” as per CLP criteria. - Executive summary:
The study was designed and conducted to determine the dermal Irritation/corrosion potential of the test chemical in Sprague Dawley rats. This study was performed as per OECD guideline No. 402. Ten rats (5 male and 5 female) were used for conducting dermal irritation/corrosion study.
The animals were kept in their cages for at least 5 days prior to administration for acclimatization to the laboratory condition and after acclimatization period, animals were randomly selected. Approximately 24 hours before application, the hair of each rat was closely clipped from the trunk (dorsal surface and sides from scapular to pelvic area) with an electric clipper, so as to expose at least 10% of the body surface area. The test item was moistened with distilled water. The test item was applied onto the exposed skin of the animal, taking care to spread the test item evenly over the entire area of approximately 10% of the total body surface area or as much of the area as can reasonably be covered. The test item was held in contact with the skin using a porous gauze dressing and non irritating tape around the animal to cover the exposure site for first 24 hours exposure period. Elizabethan collar was placed on each animal for first 24 hours after application of the test item. These collars prevent ingestion of test item. Following 24 hours of exposure, the wrapping was removed and the test site wiped free of excess test item. Distilled water was used to remove residual test item.
Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Also, the erythema and edema score of rats was calculated as 0. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.
Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and can be classified as “Not Classified” as per CLP criteria.
- 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
- 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: 6/08/2017TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL 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 dataNUMBER OF REPLICATE TISSUES: 3CALCULATIONS and STATISTICAL METHODSAll data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT AssayBlanks:· The optical density (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. 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 mean (SEM) and the percent coefficient of variation (% CV) was calculated. Tested compound :· Calculate the blank corrected value ODTT= ODTTraw– ODblank. · The OD mean per tissue was calculated. · The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100. · The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues. · The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Data Correction Procedure for MTT Interfering Compounds (if applicable)True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).ODtvt= optical density of treated viable tissueODkt= optical density of killed tissuesODtkt= optical density of treated killed tissueODukt= 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 mediaODvt= 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 VitroResults In VivoPredictionMean tissue viability ≤50% Irritant (I), R38Mean tissue viability >50% Non-irritant (NI)- 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: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 30 µL sterile DPBS- Concentration (if solution): neatPOSITIVE 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:
- 1
- Value:
- 45.1
- Vehicle controls validity:
- not specified
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Other effects / acceptance of results:
- The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 1.767 to 2.218. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was SD>18 passing the acceptance criteria.
- Interpretation of results:
- Category 2 (irritant) based on GHS criteria
- 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 chemical was determined to be 45.1%. Thus, test chemical was considered to be irritating to the human skin.
- Executive summary:
The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. 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 mean of OD for test chemical was determined to be 1.280. The standard deviation of viabilities for test chemical were calculated to be 22.11.
The MTT data show the assay quality controls were met and passed the acceptance of criteria.
The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 45.1%. Thus, test chemical was considered to be irritating to the human skin.
Referenceopen allclose all
Summary of Evaluation of Dermal Reaction
Laboratory Test Item Code :TAS/122/023
Test System : Sprague Dawley Rat
Sex : Male
Group No. |
Dose mg/kg |
Dermal Reaction |
Total Number of Animals |
Animal Nos. |
Period of signs in days From - to |
Mortality |
I |
2000 |
No dermal reaction observed |
5 |
1 - 5 |
Day 0 - Day 14 |
0/5 |
Sex : Female
Group No. |
Dose mg/kg |
Dermal Reaction |
Total Number of Animals |
Animal Nos. |
Period of signs in days From - to |
Mortality |
I |
2000 |
No dermal reaction observed |
5 |
6 - 10 |
Day 0 - Day 14 |
0/5 |
Individual Animal - Evaluation of Dermal Reaction
Laboratory Test Item Code :TAS/122/023
Test System : Sprague Dawley Rat
Sex : Male
Group : I
Dose : 2000 mg/kg body weight
Animal |
Dermal |
D A Y S |
||||||||||||||
No. |
Reaction |
0 |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
14 |
1 |
Erythema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Oedema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
Erythema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Oedema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
3 |
Erythema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Oedema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
4 |
Erythema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Oedema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
5 |
Erythema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Oedema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Sex : Female
Group : I
Dose : 2000 mg/kg body weight
Animal |
Dermal |
D A Y S |
||||||||||||||
No. |
Reaction |
0 |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
14 |
6 |
Erythema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Oedema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
7 |
Erythema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Oedema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
8 |
Erythema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Oedema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
9 |
Erythema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Oedema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
10 |
Erythema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
Oedema |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Individual Animal - Body Weight and Percent Body Weight Gain (g)
Laboratory Test Item Code :TAS/122/023
Test System : Sprague Dawley Rat
Sex : Male
Group : I
Dose : 2000 mg/kg body weight
Animal No. |
Body weight Day 0 |
Body weight Day 7 |
% body weight gain day 0-7 |
Body weight Day 14 |
% body weight gain day 7- 14 |
% body weight gain day 0- 14 |
1 |
248.5 |
267.2 |
7.53 |
290.8 |
8.83 |
17.02 |
2 |
256.4 |
277.7 |
8.31 |
295.8 |
6.52 |
15.37 |
3 |
258.4 |
282.8 |
9.44 |
300.3 |
6.19 |
16.22 |
4 |
262.5 |
288.3 |
9.83 |
306.1 |
6.17 |
16.61 |
5 |
264.5 |
291.1 |
10.06 |
310.5 |
6.66 |
17.39 |
Sex : Female
Group : I
Dose : 2000 mg/kg body weight
Animal No. |
Body weight Day 0 |
Body weight Day 7 |
% body weight gain day 0-7 |
Body weight Day 14 |
% body weight gain Day 7- 14 |
% body weight gain day 0- 14 |
6 |
224.2 |
239.8 |
6.96 |
249.7 |
4.13 |
11.37 |
7 |
228.4 |
242.4 |
6.13 |
253.7 |
4.66 |
11.08 |
8 |
229.4 |
243.0 |
5.93 |
250.6 |
3.13 |
9.24 |
9 |
232.4 |
245.3 |
5.55 |
257.6 |
5.01 |
10.84 |
10 |
237.7 |
250.5 |
5.38 |
262.2 |
4.67 |
10.31 |
Individual Animal -Clinical Signs of Toxicity and Mortality
Laboratory Test Item Code :TAS/122/023
Test System : Sprague Dawley Rat
Sex : Male
Group No. |
Dose mg/kg |
Observed Signs |
Total Number of Animals |
Animal Nos. |
Period of signs in days From - to |
Mortality |
I |
2000 |
No clinical signs observed |
5 |
1 |
Day 0 - Day 14 |
0 |
2 |
Day 0 - Day 14 |
0 |
||||
3 |
Day 0 - Day 14 |
0 |
||||
4 |
Day 0 - Day 14 |
0 |
||||
5 |
Day 0 - Day 14 |
0 |
Sex : Female
Group No. |
Dose mg/kg |
Observed Signs |
Total Number of Animals |
Animal Nos. |
Period of signs in days From - to |
Mortality |
I |
2000 |
No clinical signs observed |
5 |
6 |
Day 0 - Day 14 |
0 |
7 |
Day 0 - Day 14 |
0 |
||||
8 |
Day 0 - Day 14 |
0 |
||||
9 |
Day 0 - Day 14 |
0 |
||||
10 |
Day 0 - Day 14 |
0 |
| Code N° | Tissue | Raw data | Blank corrected data | mean | % of viability | ||
| n | Aliq. 1 | Aliq. 2 | Aliq. 1 | Aliq. 2 | of aliquotes | ||
| NC | 1 | 1.7913 | 1.8155 | 1.756 | 1.781 | 1.768 | 92.2 |
| 2 | 2.232 | 2.2748 | 2.197 | 2.240 | 2.218 | 115.7 | |
| 3 | 1.7837 | 1.8199 | 1.749 | 1.785 | 1.767 | 92.1 | |
| PC | 1 | 0.0802 | 0.0773 | 0.045 | 0.042 | 0.044 | 2.3 |
| 2 | 0.0758 | 0.0767 | 0.041 | 0.042 | 0.041 | 2.2 | |
| 3 | 0.0722 | 0.0695 | 0.037 | 0.035 | 0.036 | 1.9 | |
| C1 | 1 | 0.9143 | 0.9043 | 0.879 | 0.869 | 0.874 | 45.6 |
| 2 | 1.0095 | 0.9638 | 0.975 | 0.929 | 0.952 | 49.6 | |
| 3 | 0.802 | 0.806 | 0.767 | 0.771 | 0.769 | 40.1 | |
| mean | SD | mean of | SD | CV % | |
| of OD | of OD | viabilities [%] | of viabilities | [%] | |
| NC | 1.918 | 0.260 | 100.0 | 13.57 | 13.57 |
| PC | 0.040 | 0.004 | 2.1 | 0.21 | 10.02 |
| C1 | 0.865 | 0.092 | 45.1 | 4.78 | 10.60 |
| Classification | ||
| NC | NI | qualified |
| PC | I | qualified |
| C1 | I | qualified |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01-04-1986 - 08-04-1986
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Experimental test result performed using standard test guidelines
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 405 (Acute Eye Irritation / Corrosion)
- Principles of method if other than guideline:
- The purpose of the study was to evaluate the ability of the test chemical to produce ocular irritation/corrosion in rabbits following a single application.
- GLP compliance:
- not specified
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): Benzyltriethylammonium chloride
- Molecular formula: C13H22NCl
- Molecular weight: 227.777 g/mol
- Smiles notation: [N+](CC)(CC)(Cc1ccccc1)CC.[ClH-]
- InChl: 1S/C13H22N.ClH/c1-4-14(5-2,6-3)12-13-10-8-7-9-11-13;/h7-11H,4-6,12H2,1-3H3;1H/q+1;/p-1
- Substance type: Organic
- Physical state: Solid
- Chemical name: Benzyltriethyl ammonium chloride
- Trade name/code: BETEC-DRY
- Batch No. HH-85-102A
- CAS No.: 56-37-1
- Purity: 100.1-100.2% based on Cl-
- Solubility: Water, ethanol
- Melting point:185°C dec.
- Appearance: Off-white crystalline powder
- Storage: At ambient temperature in the dark
- Comments: Material is hygroscopic, avoid undue exposure to moist air - Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or tissues and environmental conditions:
- TEST ANIMALS
- Source: The Broekman Institute, Someren, The Netherlands
- Age at study initiation: 8 to 10 weeks old
- Weight at study initiation: Three young adult female rabbits weighed as 2851, 2907 and 2873 grams
- Housing: individually housed in metal cages with perforated floors (RUCO, Valkenswaard)
- Diet (e.g. ad libitum): fed standard laboratory animal diet (100 g per day), obtained from Hope Farms, Woerden (LK-01, pellet diameter 4 mm)
- Water (e.g. ad libitum): free access to tap-water
- Acclimation period: 16 weeks of acclimation
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22°C
- Humidity (%): 55 - 75 per cent
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 69 mg (equivalent to 0.1 ml)
- Concentration (if solution): not applicable
VEHICLE
- Amount(s) applied (volume or weight with unit): not applicable
- Concentration (if solution):not applicable
- Lot/batch no. (if required): not applicable
- Purity: not applicable - Duration of treatment / exposure:
- The test substance is applied in a single dose to one of the eyes of the experimental animals
- Observation period (in vivo):
- the eyes were examined approximately 1, 24, 4 and 72 hours, and 7 days after instillation of the test substance.
- Duration of post- treatment incubation (in vitro):
- Not applicable
- Number of animals or in vitro replicates:
- Not applicable
- Details on study design:
- REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not specifed
- Time after start of exposure: Not specifed
TOOL USED TO ASSESS SCORE: fluorescein - Irritation parameter:
- cornea opacity score
- Basis:
- mean
- Time point:
- other: 1/24/48/72 hrs and 7 days
- Score:
- 0
- Max. score:
- 4
- Reversibility:
- not specified
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- iris score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 0
- Max. score:
- 2
- Reversibility:
- not specified
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- conjunctivae score
- Remarks:
- Redness
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 1.3
- Max. score:
- 3
- Reversibility:
- fully reversible within: Seven days after dosing the conjunctival effects had disappeared.
- Remarks on result:
- other: in one of the eyes of three female rabbits, slight to moderate redness (persisting only for 72 hours) was observed
- Irritation parameter:
- chemosis score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 0.6
- Max. score:
- 4
- Reversibility:
- fully reversible within: Seven days after dosing the conjunctival effects had disappeared.
- Remarks on result:
- other: In one of the eyes of three female rabbits, acute severe swelling of the conjunctivae was observed which diminished after 24 hrs;
- Irritant / corrosive response data:
- Table 1 shows the scores for irritation of the cornea, iris and conjunctivae approximately 1, 24, 48 and 72 hours, and 7 days after instillation of the test substance. Immediately after dosing the (animals were observed to squeeze their eyelids together, and lacrimation was excessive. One hour later slight conjunctival redness was observed in all animals, as well as moderate to severe swelling of the eyelids. At this time lacrimation was still increased, but this effect disappeared during the next twenty-four hours. The swelling of the conjunctivae diminished during this period, whereas the erythema became more pronounced. Forty-eight hours after dosing the blood vessels in the nictating membrane were injected, and in one animal (no. 251) this membrane was still slightly swollen. These effects were still present 72 hours after dosing, but disappeared completely during the next four days. Adverse effects on the cornea and iris were not observed in any of the rabbits during the entire observation period. Treatment of the eyes with fluorescein 24 hours after instillation of the test substance did not reveal any epithelial damage. Signs of systemic intoxication were not observed.
- Interpretation of results:
- other: Not irritating
- Conclusions:
- Immediately after dosing the (animals were observed to squeeze their eyelids together, and lacrimation was excessive. One hour later slight conjunctival redness was observed in all animals, as well as moderate to severe swelling of the eyelids. These effects were still present 72 hours after dosing, but disappeared completely during the next four days. Adverse effects on the cornea and iris were not observed in any of the rabbits during the entire observation period. Treatment of the eyes with fluorescein 24 hours after instillation of the test substance did not reveal any epithelial damage. Signs of systemic intoxication were not observed.
Based on these observations and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as an Eye Irritant. - Executive summary:
The purpose of the study was to evaluate the ability of the test chemical to produce ocular irritation/corrosion in rabbits following a single application. The study was performed according to OECD 405 Guidelines and evaluated according to EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).3 young adult female New Zealand White rabbits were used for the study.
Before dosing the test substance was ground to a fine powder using a mortar and pestle. The bulk density of the test substance was determined by NOTOX and amounted to 0.698 g/ml. On the day of dose administration, three portions of 69±1.5 mg of the powder were dispensed in glass containers with screw caps. One portion of the dispensed amount (equivalent to 0.1 ml) was instilled into the conjunctival sac of the right eye of the rabbit using a spatula. The lids were then gently held together for two seconds and then released immediately after instillation of the test substance, the animals were observed and abnormalities were recorded. The eyes were examined approximately 1, 24, 48, 72 hours and 7 days after instillation of the test chemical. Immediately after treatment, the animals were transferred to metal cages. The left eye remained untreated and served as control. Immediately after instillation of the test substance, the animals were observed and abnormalities were recorded. The eyes were examined approximately 1, 24, 48, 72 hours and 7 days after instillation of the test chemical. Approximately 24 hours after instillation of the test substance (immediately after scoring the corneal opacity and the alterations of the iris and conjunctivae), a solution of 2% sodium fluorescien in water (pH adjusted to 7.0) was applied to both eyes of the test animals to examine quantitatively the potential for corneal injury. The brightly green staining area indicating epithelial damage was estimated as a percentage of total corneal area. Any observed local effects other than those indicated above were recorded. The ocular lesions were scored according to OECD 405 Guidelines and the results were evaluated according to the EEC criteria for classification and labeling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).
Immediately after dosing the (animals were observed to squeeze their eyelids together, and lacrimation was excessive. One hour later slight conjunctival redness was observed in all animals, as well as moderate to severe swelling of the eyelids. At this time lacrimation was still increased, but this effect disappeared during the next twenty-four hours. Forty-eight hours after dosing the blood vessels in the nictating membrane were injected, and in one animal (no. 251) this membrane was still slightly swollen. These effects were still present 72 hours after dosing, but disappeared completely during the next four days. Adverse effects on the cornea and iris were not observed in any of the rabbits during the entire observation period. Treatment of the eyes with fluorescein 24 hours after instillation of the test substance did not reveal any epithelial damage. Signs of systemic intoxication were not observed.
Based on these observations and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as an Eye Irritant.
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2006-06-22 to 2006-06-22
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Justification for type of information:
- data is from experimental reports
- Qualifier:
- according to guideline
- Guideline:
- other: INVITTOX Protocol no. 98 "Bovine Corneal Opacity and Permeability Assay", dated February 1994.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Bovine Corneal Opacity and Permeability Assay (BCO-P) SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.
- Deviations:
- no
- Principles of method if other than guideline:
- The eye irritation potential of the test chemical was assessed in an In- vitro study conducted equivalent to OECD 437 (BCOP) Guidelines
- GLP compliance:
- not specified
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0000008753
- Expiration date of the lot/batch: 2008-03-01
- Purity: 100%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (range of 20 +-5°C), light proteced
- Solubility and stability of the test substance in the solvent/vehicle:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test item was tested at a concentration of 20% in saline. Strong stirring with a magnetic stirrer resulted in a solution. Until administration, the solution was stirred with a magnetic stirrer. - Species:
- other: Freshly isolated bovine cornea
- Strain:
- other: not applicable
- Details on test animals or tissues and environmental conditions:
- TEST ANIMALS
- Source: Freshly isolated bovine eyes were collected from the abattoir: Abattoir Basel, Schlachthofstrasse 55, CH-4055 Basel, Switzerland. After excess tissue was removed from the excised eyes, they were stored at room temperature in Hank's balanced salt solution containing penicillin/streptomycin and then transported for further preparation. The eyes were used immediately after delivery in the laboratory and within four hours after slaughtering.
ENVIRONMENTAL CONDITIONS
- Temperature (deg C): Throughout the whole experiment the eyes were maintained in a water bath at 32 °C +- 2 °C. - Vehicle:
- physiological saline
- Controls:
- yes, concurrent vehicle
- yes, concurrent positive control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% solution in physiological saline
VEHICLE
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9%
POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): no data
- Lot/batch no. (if required): 054063/2
- Purity: >99.5% (Assay) - Duration of treatment / exposure:
- 240 minutes
- Duration of post- treatment incubation (in vitro):
- after the final opacity measurement, corneas were incubated for about 90 minutes and optical density at 490 nm was determined
- Number of animals or in vitro replicates:
- 3 corneas per test group
- Details on study design:
- SELECTION AND PREPARATION OF CORNEAS
- All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. Each cornea was dissected from the eye using scalpel and rounded scissors. A rim of about 2 -3 mm of tissue (sclerea) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in complete minimum essential medium (cMEM) and were checked finally with a view box for defects listed before.
- Since the bovine eyes were delivered in the afternoon, corneas were stored in a preservation medium overnight in a refrigerator at about 4 °C. The preservation medium was composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine. Shortly before use, Dextran was added.
- each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring, but stretching had to be avoided. After the anterior part of the holder was positioned on the top of the cornea and fixed in place with screws, both compartments of the holder were filled with cMEM. Care was taken to assure no air bubbles were present within the compartments.
- For equilibrium, the corneas in the holder were incubated for about one hour at 32 deg. C +- 2 deg. C in a water bath.
- At the end of the incubation period the medium was removed from both compartments and replaced by fresh cMEM, and the basal opacity was determined (t0min). For measurement, the posterior compartment was plugged while the anterior compartment remained unplugged.
TREATMENT METHOD
- Fresh cMEM was filled into the posterior compartment, while the anterior compartment has received 0.75 mL-aliquots of the test item or negative or positive control respectively and were evenly distributed on the surface of the corneas. The holder was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item and was incubated in a horizontal position in a water-bath at 32°C +/- 2°C.
REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the test item was rinsed off from the application side by changing cMEM several times until precipitates of the test substance could be observed no longer, fresh cMEM was replaced in both compartments and the opacity was measured (t240min).
- Time after start of exposure: 240 minutes
METHODS FOR MEASURED ENDPOINTS
Corneal opacity:
- An opacitometer was used to determine the changes in light transmission passing through the corneas. The opacitometer was calibrated with a standardized opaque polyester sheet as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated corneas.
- After recording the basal opacity of all corneas, the mean value of all corneas was calculated. No cornea deviated from this by more than +/- 3 units and no cornea was discarded.
- The change of opacity value of each treated cornea or positive or negative control corneas was calculated by subtracting the initial basal opacity from the post treatment opacity reading, for each individual cornea (t240min-t0min). The average change in opacity of the negative control corneas was calculated and this value subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity. The mean corrected opacity value of each treatment group was then calculated from the individual corrected opacity values of the treated corneas for each treatment condition.
Corneal permeability
- To demonstrate possible treatment-induced transepithelial permeability of the cornea, the permeability test with fluorescein sodium dye was performed in a second step. Corneal permeability is quantified by measuring the amount of fluorescein sodium dye diffusing into the medium in the posterior chamber. After the final opacity measurement was performed, the medium was removed from the anterior compartment and replaced by 1 mL of a fluorescein solution, 0.5% dissolved in Dulbecco's phosphate-buffered saline. Corneas were incubated again in a horizontal position for about 90 minutes in a water-bath at 32°C +/- 2°C. Medium from the posterior compartment was removed with a 5 mL-syringe, well mixed and transferred to a cuvette of 10 mm path length and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
- The dye solution is valid for use if a dilution of the stock solution containing 10 ug/mL shows an optical density (OD490) of 1.610 to 1.910.
- The corrected OD490 value of each treated cornea or positive control corneas was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea. The mean corrected permeability values of each treatment group were calculated from the individual corrected permeability values of the treated corneas for each treatment condition.
SCORING SYSTEM:
In Vitro Score Calculation:
The following formula was used to determine the In Vitro Score:
In-Vitro Score = opacity value + (15 X OD490 value)
The In-Vitro Score was calculated for each individual treatment and positive control cornea. The mean In Vitro Score value of each treated group was calculated from the individual In-Vitro Score Values:
Negative control:
In-Vitro Score = opacity value + (15 X OD490 value)
Positive control and test substance cornea:
In-Vitro Score = corrected opacity value + (15 X corrected OD490 value)
Depending on the score obtained, the test substance was classified into one of the following categories:
In-Vitro Score: (Proposed In-Vitro Irritation Scale)
0-3 (non eye irritant)
3.1-25 (mild eye irritant)
25.1-55 (moderate eye irritant)
55.1 -80 (severe eye irritant)
> 80.1 (very severe eye irritant) - Irritation parameter:
- in vitro irritation score
- Run / experiment:
- 1
- Value:
- 1
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: range: 0.2 - 2.1
- Irritation parameter:
- cornea opacity score
- Run / experiment:
- 1
- Value:
- 1.3
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: range: 0.3 - 2.3
- Irritation parameter:
- other: permeability
- Run / experiment:
- 1
- Value:
- -0.024
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: range: -0.048 - -0.012
- Other effects / acceptance of results:
- IVIS (range)
- negative control: 2.2 (0.6-3.3)
- positive control: 82.5 (71.3-97.0)
cornea opacity score (range)
- negative control: 0.7 (0-1)
- positive control: 52.3 (39.3-62.3)
permeability (range)
- negative control: 0.102 (0.038-0.156)
- positive control: 2.012 (1.594-2.310)
According to the results obtained in the experiment, the positive control has an IVIS > 55. The acceptability criteria was met. - Interpretation of results:
- other: not irritating
- Conclusions:
- The In-Vitro Irritation Score for the test chemical was 1.0. The In-vitro Irritation score for positive control was 82.5 and negative control was 2.2, which fulfill the validity criteria of the assay.
Based on the scale for interpretation of irritation potential according to In-Vitro Irritation score, the test chemical can be considered to be not irritating to Bovine Corneas. - Executive summary:
The eye irritation potential of the test chemical was assessed in an In- vitro study conducted equivalent to OECD 437 (BCOP) Guidelines. Freshly isolated bovine eyes were collected from the abattoir. After excess tissue was removed from the excised eyes, they were stored at room temperature in Hank's balanced salt solution containing penicillin/streptomycin and then transported for further preparation. The eyes were used immediately after delivery in the laboratory and within four hours after slaughtering. Throughout the whole experiment the eyes were maintained in a water bath at 32 °C +- 2 °C. Since the bovine eyes were delivered in the afternoon, corneas were stored in a preservation medium overnight in a refrigerator at about 4 °C. The preservation medium was composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine. Shortly before use, Dextran was added. Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring, but stretching had to be avoided. After the anterior part of the holder was positioned on the top of the cornea and fixed in place with screws, both compartments of the holder were filled with cMEM. Care was taken to assure no air bubbles were present within the compartments.For equilibrium, the corneas in the holder were incubated for about one hour at 32 deg. C +- 2 deg. C in a water bath. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. Each cornea was dissected from the eye using scalpel and rounded scissors. A rim of about 2 -3 mm of tissue (sclerea) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in complete minimum essential medium (cMEM) and were checked finally with a view box for defects. At the end of the incubation period the medium was removed from both compartments and replaced by fresh cMEM, and the basal opacity was determined (t0min). For measurement, the posterior compartment was plugged while the anterior compartment remained unplugged. Fresh cMEM was filled into the posterior compartment, while the anterior compartment has received 0.75 mL-aliquots of the test item or negative(natrium chloratum) or positive control(imidazole) respectively and were evenly distributed on the surface of the corneas. The holder was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item and was incubated in a horizontal position in a water-bath at 32°C +/- 2°C. After the test item was rinsed off from the application side by changing cMEM several times until precipitates of the test substance could be observed no longer, fresh cMEM was replaced in both compartments and the opacity was measured (t240min). 3 corneas per test group were used for the assay.
An opacitometer was used to determine the changes in light transmission passing through the corneas. The opacitometer was calibrated with a standardized opaque polyester sheet as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated corneas.After recording the basal opacity of all corneas, the mean value of all corneas was calculated. No cornea deviated from this by more than +/- 3 units and no cornea was discarded. The change of opacity value of each treated cornea or positive or negative control corneas was calculated by subtracting the initial basal opacity from the post treatment opacity reading, for each individual cornea (t240min-t0min). The average change in opacity of the negative control corneas was calculated and this value subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity. The mean corrected opacity value of each treatment group was then calculated from the individual corrected opacity values of the treated corneas for each treatment condition. The corrected OD490 value of each treated cornea or positive control corneas was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea. The mean corrected permeability values of each treatment group were calculated from the individual corrected permeability values of the treated corneas for each treatment condition.
The following formula was used to determine the In Vitro Score:
In-Vitro Score = opacity value + (15 X OD490 value)
The In-Vitro Score was calculated for each individual treatment and positive control cornea. The mean In Vitro Score value of each treated group was calculated from the individual In-Vitro Score Values:
Negative control:
In-Vitro Score = opacity value + (15 X OD490 value)
The In-Vitro Irritation Score for the test chemical was 1.0. The In-vitro Irritation score for positive control was 82.5 and negative control was 2.2, which fulfill the validity criteria of the assay.
Based on the scale for interpretation of irritation potential according to In-Vitro Irritation score, the test chemical can be considered to be not irritating to Bovine Corneas.
Referenceopen allclose all
Table1:
EYE IRRITATION SCORES FOR BETEC-DRY IN THE RABBIT
Rabbit no. and sex |
Body weight (grams) |
Observation time after instillation (hours) |
Cornea opacity |
Iris lesion |
Conjunctivae |
|
redness |
swelling (chemosis) |
|||||
249; Female |
2851 |
1 |
0 |
0 |
1 |
3 |
24 |
0 |
0 |
2 |
1 |
||
48 |
0 |
0 |
1 |
0 |
||
72 |
0 |
0 |
1 |
0 |
||
Day 7 |
0 |
0 |
0 |
0 |
||
Subtotala)/ Mean valuec) |
0/0 |
0/0 |
4/1.3 |
1/0.3 |
||
Rabbit no. and sex |
Body weight (grams) |
Observation time after instillation (hours) |
Cornea opacity |
Iris lesion |
Conjunctivae |
|
redness |
swelling (chemosis) |
|||||
250; Female |
2907 |
1 |
0 |
0 |
1 |
2 |
24 |
0 |
0 |
1 |
1 |
||
48 |
0 |
0 |
1 |
0 |
||
72 |
0 |
0 |
1 |
0 |
||
Day 7 |
0 |
0 |
0 |
0 |
||
Subtotala)/ Mean valuec) |
0/0 |
0/0 |
3/1.0 |
1/0.3 |
||
Rabbit no. and sex |
Body weight (grams) |
Observation time after instillation (hours) |
Cornea opacity |
Iris lesion |
Conjunctivae |
|
redness |
swelling (chemosis) |
|||||
251; Female |
2873 |
1 |
0 |
0 |
1 |
3 |
24 |
0 |
0 |
2 |
1 |
||
48 |
0 |
0 |
2 |
1 |
||
72 |
0 |
0 |
1 |
1 |
||
Day 7 |
0 |
0 |
0 |
0 |
||
Subtotala)/ Mean valuec) |
0/0 |
0/0 |
5/1.7 |
3/1.0 |
||
Totalb)/ Mean valuec) |
0/0 |
0/0 |
12/1.3 |
5/0.6 |
||
a) Subtotal = sum of 24-, 48-
and 72-hours scores for each animal individually
b) Total = sum of 24-, 48- and 72-hours scores calculated over all animals.
c) Mean value = mean score of 24-, 48- and 72-hours reading time.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin Irritation:
Various studies have been investigated for assessing the dermal irritation potential of the test chemical to a greater or lesser extent. The studies are based on in vivo and in vitro experiments in rabbits along with in vitro data for test chemical.
The study was designed and conducted to determine the dermal Irritation/corrosion potential of the test chemical in Sprague Dawley rats. This study was performed as per OECD guideline No. 402. Ten rats (5 male and 5 female) were used for conducting dermal irritation/corrosion study.
The animals were kept in their cages for at least 5 days prior to administration for acclimatization to the laboratory condition and after acclimatization period, animals were randomly selected. Approximately 24 hours before application, the hair of each rat was closely clipped from the trunk (dorsal surface and sides from scapular to pelvic area) with an electric clipper, so as to expose at least 10% of the body surface area. The test item was moistened with distilled water. The test item was applied onto the exposed skin of the animal, taking care to spread the test item evenly over the entire area of approximately 10% of the total body surface area or as much of the area as can reasonably be covered. The test item was held in contact with the skin using a porous gauze dressing and non irritating tape around the animal to cover the exposure site for first 24 hours exposure period. Elizabethan collar was placed on each animal for first 24 hours after application of the test item. These collars prevent ingestion of test item. Following 24 hours of exposure, the wrapping was removed and the test site wiped free of excess test item. Distilled water was used to remove residual test item.
Administration of the test item at 2000 mg/kg did not result in any skin reaction at the site of application during the study period of 14 days. Also, the erythema and edema score of rats was calculated as 0. Animals exhibited normal body weight gain through the study period of 14 days. Gross pathological examination did not reveal any abnormalities attributable to the treatment.
Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and can be classified as “Not Classified” as per CLP criteria.
The dermal irritation potential of test article was also determined according to the OECD 439 test guideline for this study. 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 mean of OD for test chemical was determined to be 1.280. The standard deviation of viabilities for test chemical were calculated to be 22.11.
The MTT data show the assay quality controls were met and passed the acceptance of criteria.
The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 45.1%. Thus, test chemical was considered to be irritating to the human skin.
This is supported by the results of an in-vivo GLP study conducted according to OECD Guideline 404 and Commission Directive 2004/73/EC, B.4 for assessing the dermal irritation potential of the test chemical.3 New Zealand White rabbits (2 females, 1 male) were used for this study
The pH of the test item was measured before the study initiation date. A formulation of the 1% (w/w) solution was prepared. The pH was found to be 5. 0.5 g (per animal) of the test substance was weighed as delivered and then moistened with approximately 0.5 mL of purified water before application. The test item was placed on a surgical gauze patch (2.5 cm x 2.5 cm) and applied to the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with tape. The animals were exposed to the chemical for 4 hours. After 4 hours, skin was flushed with lukewarm tap water to clean the application site. The skin reactions were read at approximately 1, 24, 48 and 72 hours after exposure (removal of the dressing, gauze patch and test item).Body weights, viability, clinical signs were also observed from acclimation period till termination of the study. The skin reaction was assessed according to the numerical scoring system listed in the Commission Directive 2004/73/EC, April 29, 2004. The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. An animal is positive when the mean score is 2 or greater. The test is positive for irritation when at least 2 animals are positive for the same endpoint (erythema/eschar or oedema). No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
The test chemical did not elicit any skin reactions at the application site of any animal at any of the observation times (all scores 0). The individual mean score for erythema/eschar and oedema for each of the three animals was therefore 0.
Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 2001), the test chemical was considered to be "not irritating" to rabbit skin.
To further support the results obtained in in-vivo studies, an in-vitro dermal irritation study was performed to assess the irritation potential of the test chemical. The study was conducted according to OECD Guideline 430, In Vitro Skin Corrosion: Transcutaneous Electrical Resistance (TER), without deviations.
Wistar rats were used for the preparation of the skin discs. The dorsal and flank hair was carefully removed. The animals were then washed by careful wiping, whilst submerging the area in antibiotic solution containing streptomycin and penicillin to inhibit bacterial growth. Animals were washed with antibiotics again three days after the first wash, and were used within 3 days. Animals were anaesthetised with CO2 and subsequently decapitated. The dorsal skin of each animal was removed and stripped of excess fat by carefully peeling it away from the skin. The skin was placed over the end of a PTFE (polytetrafluoroethylene) tube, ensuring that the epidermal surface was in contact with the tube. A rubber ‘O’ ring was press-fitted over the end of the tube to hold the skin in place and excess tissue was trimmed away. The tube was supported by a spring clip inside a receptor chamber containing magnesium sulphate solution (154 mM). 153.9 mg, 112.1 mg and 129.9 mg (enough material of the ground test item ) was applied to cover the complete surface of the skin discs. 3 skin discs were used for each test group. The exposure period was 24 hours. Deionised water and hydrochloric acid were used as concurrent negative and positive controls respectively. After 24 hours, Test item and controls were subsequently removed by washing with a jet of tap water at up to 30 °C until no further material could be removed. - The TER was measured by using a low-voltage, alternating current impedance meter. Prior to measuring the electrical resistance, the surface tension of the skin was reduced by adding a sufficient volume of 70% ethanol to cover the epidermis. After a few seconds, the ethanol was removed by inverting the tube, and the tissue was then hydrated by the addition of 3 mL magnesium sulphate solution (154 mM). The electrodes were placed on either side of the skin disc to take the conductivity measurement in µS/skin disc. The resistance values were calculated from conductivity by the following equation: resistance (ohm) = 1/conductivity (S). - The inner (thick) electrode clip was rested on the top of the PTFE tube during resistance measurement, to ensure that a consistent length of electrode was submerged in the magnesium sulphate solution. The outer (thin) electrode was positioned inside the receptor chamber so that it rested on the bottom of the chamber. The distance between the bottom of the spring clip and the bottom of the PTFE tube was maintained as a constant, since this distance affects the resistance value obtained. - The mean TER results are accepted on condition that concurrent positive and negative control values fall within the acceptable ranges for the method. The control substances and their associated resistance ranges for the methodology and apparatus described are: positive control: 10 M hydrochloric acid (36%) : 0.5 - 1 k Ω* negative control: deionised water: 8 - 25 k Ω* The decision criteria for classification of substances based on their TER values is :
- The test substance is considered to be corrosive to skin if the mean TER value is less than or equal to 5 kΩ.
- The test substance is considered to be non-corrosive to skin if the mean TER value obtained for the test substance is greater than 5 kΩ.
- If the mean disc dye content is greater than or equal to the mean disc dye content of the 36 % HCl positive control obtained concurrently, then the test item is a true positive and is therefore corrosive.
- If the mean disc dye content is well below the mean disc dye content of the 36 % HCl positive control obtained concurrently, then the test item is a false positive and is, therefore, non-corrosive.
The mean TER value for the test material was obtained to be 21.08 kΩ.
According to the decision criteria, Test chemical did not corrode rat skin under the experimental conditions used. Both positive and negative control reached typical impedance values as stated in the guideline and in a range of the historical controls. Therefore, the test chemical can be considered to be non-corrosive in the TER test.
The above invitro and invivo results are also supported by an in vitro study performed according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” to predict the irritation potential of the test chemical. 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. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to 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 shows that the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control.
The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 88.8.%.
Hence, under the experimental test conditions it was concluded that the test chemical was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.
The results of the in vivo studies and in vitro studies are in agreement with each other, eventhough one study suggests of a potential to cause irritation in vitro. As the remaining Guideline studies indicating a very strong possibility of the test chemical being not irritating to skin.,hence, the test chemical can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified.”
Eye Irritation:
Various studies have been investigated for assessing the ocular irritation potential of the test chemical to a greater or lesser extent. The studies are based on in vivo experiments in rabbits for the various test chemicals along with in vitro data.
The results are summarized as follows:
The purpose of the study was to evaluate the ability of the test chemical to produce ocular irritation/corrosion in rabbits following a single application. The study was performed according to OECD 405 Guidelines and evaluated according to EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).3 young adult female New Zealand White rabbits were used for the study.
Before dosing the test substance was ground to a fine powder using a mortar and pestle. The bulk density of the test substance was determined by NOTOX and amounted to 0.698 g/ml. On the day of dose administration, three portions of 69±1.5 mg of the powder were dispensed in glass containers with screw caps. One portion of the dispensed amount (equivalent to 0.1 ml) was instilled into the conjunctival sac of the right eye of the rabbit using a spatula. The lids were then gently held together for two seconds and then released immediately after instillation of the test substance, the animals were observed and abnormalities were recorded. The eyes were examined approximately 1, 24, 48, 72 hours and 7 days after instillation of the test chemical. Immediately after treatment, the animals were transferred to metal cages. The left eye remained untreated and served as control. Immediately after instillation of the test substance, the animals were observed and abnormalities were recorded. The eyes were examined approximately 1, 24, 48, 72 hours and 7 days after instillation of the test chemical. Approximately 24 hours after instillation of the test substance (immediately after scoring the corneal opacity and the alterations of the iris and conjunctivae), a solution of 2% sodium fluorescien in water (pH adjusted to 7.0) was applied to both eyes of the test animals to examine quantitatively the potential for corneal injury. The brightly green staining area indicating epithelial damage was estimated as a percentage of total corneal area. Any observed local effects other than those indicated above were recorded. The ocular lesions were scored according to OECD 405 Guidelines and the results were evaluated according to the EEC criteria for classification and labeling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC).
Immediately after dosing the (animals were observed to squeeze their eyelids together, and lacrimation was excessive. One hour later slight conjunctival redness was observed in all animals, as well as moderate to severe swelling of the eyelids. At this time lacrimation was still increased, but this effect disappeared during the next twenty-four hours. Forty-eight hours after dosing the blood vessels in the nictating membrane were injected, and in one animal (no. 251) this membrane was still slightly swollen. These effects were still present 72 hours after dosing, but disappeared completely during the next four days. Adverse effects on the cornea and iris were not observed in any of the rabbits during the entire observation period. Treatment of the eyes with fluorescein 24 hours after instillation of the test substance did not reveal any epithelial damage. Signs of systemic intoxication were not observed.
Based on these observations and applying the EEC criteria for classification and labelling of dangerous substances (Annex VI of the EEC Council directive 67/548/EEC as amended by Directive 83/467/EEC), the test chemical need not be labelled as an Eye Irritant.
This is supported by the results of an In- vitro study conducted equivalent to OECD 437 (BCOP) Guidelines. Freshly isolated bovine eyes were collected from the abattoir. After excess tissue was removed from the excised eyes, they were stored at room temperature in Hank's balanced salt solution containing penicillin/streptomycin and then transported for further preparation. The eyes were used immediately after delivery in the laboratory and within four hours after slaughtering. Throughout the whole experiment the eyes were maintained in a water bath at 32 °C +- 2 °C. Since the bovine eyes were delivered in the afternoon, corneas were stored in a preservation medium overnight in a refrigerator at about 4 °C. The preservation medium was composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine. Shortly before use, Dextran was added. Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring, but stretching had to be avoided. After the anterior part of the holder was positioned on the top of the cornea and fixed in place with screws, both compartments of the holder were filled with cMEM. Care was taken to assure no air bubbles were present within the compartments. For equilibrium, the corneas in the holder were incubated for about one hour at 32 deg. C +- 2 deg. C in a water bath. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. Each cornea was dissected from the eye using scalpel and rounded scissors. A rim of about 2 -3 mm of tissue (sclerea) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in complete minimum essential medium (cMEM) and were checked finally with a view box for defects. At the end of the incubation period the medium was removed from both compartments and replaced by fresh cMEM, and the basal opacity was determined (t0min). For measurement, the posterior compartment was plugged while the anterior compartment remained unplugged. Fresh cMEM was filled into the posterior compartment, while the anterior compartment has received 0.75 mL-aliquots of the test item or negative(natrium chloratum) or positive control(imidazole) respectively and were evenly distributed on the surface of the corneas. The holder was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item and was incubated in a horizontal position in a water-bath at 32°C +/- 2°C. After the test item was rinsed off from the application side by changing cMEM several times until precipitates of the test substance could be observed no longer, fresh cMEM was replaced in both compartments and the opacity was measured (t240min). 3 corneas per test group were used for the assay.
An opacitometer was used to determine the changes in light transmission passing through the corneas. The opacitometer was calibrated with a standardized opaque polyester sheet as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated corneas. After recording the basal opacity of all corneas, the mean value of all corneas was calculated. No cornea deviated from this by more than +/- 3 units and no cornea was discarded. The change of opacity value of each treated cornea or positive or negative control corneas was calculated by subtracting the initial basal opacity from the post treatment opacity reading, for each individual cornea (t240min-t0min). The average change in opacity of the negative control corneas was calculated and this value subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity. The mean corrected opacity value of each treatment group was then calculated from the individual corrected opacity values of the treated corneas for each treatment condition. The corrected OD490 value of each treated cornea or positive control corneas was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea. The mean corrected permeability values of each treatment group were calculated from the individual corrected permeability values of the treated corneas for each treatment condition.
The following formula was used to determine the In Vitro Score:
In-Vitro Score = opacity value + (15 X OD490 value)
The In-Vitro Score was calculated for each individual treatment and positive control cornea. The mean In Vitro Score value of each treated group was calculated from the individual In-Vitro Score Values:
Negative control:
In-Vitro Score = opacity value + (15 X OD490 value)
The In-Vitro Irritation Score for Test chemicalwas 1.0. The In-vitro Irritation score for positive control was 82.5 and negative control was 2.2, which fulfill the validity criteria of the assay.
Based on the scale for interpretation of irritation potential according to In-Vitro Irritation score, the test chemical can be considered to be not irritating to Bovine Corneas.
The above results are further supported by an eye irritation study conducted in rabbits to determine the irritation potential of the test chemical.
Exactly 0.1 ml of undiluted test material was instilled into the conjunctival sac of the right eye of one rabbit. Before a second rabbit could be similarly exposed, the first rabbit convulsed and died. The dose was reduced to 0.05ml and administrated to the second rabbit. This rabbit died 160 second later. The dose was reduced to 0.03 ml and administrated to each of 5 rabbits. All of the animals exhibited severe pharmacotoxic symptoms. Three of the animals died 2-3 minutes after dose administration. Transient conjunctival irritation (discharge) was noted shortly after instillation of the test material. The discharge subsided within 10-15minutes. No corneal lesions were noted. Eye irritation score was 0 in survived animals. Therefore, the test chemical was considered to be non-irritant to rabbit eye.
The results of the in vivo studies and in vitro study are in agreement with each other, thereby indicating a very strong possibility of the test chemical being not irritating to eyes.
The ocular irritation potential of test article was also determined according to the OECD 492 test guideline. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 hour 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, as the OD of the negative control tissues was between 0.8 to 2.5 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be below 50% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 0.45% passing the acceptance criteria.
The mean % tissue viability of the test chemical was determined to be 3.9%. Hence, under the experimental test conditions it was concluded that test substance was considered to be irritating to the MatTek EpiOcular Tissue Model OCL-200 and being classified as ‘’Irritating to eyes in Category 2”
Even though one in -vitro Guideline study for the test chemical claims that there is a possibility of test chemical to cause eye irritation, but it is strongly negated by the results of the invivo studies for the test chemicals and another in-vitro Guideline study.
Hence,the test chemical can be considered to be not irritating to eyes. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified.”
Justification for classification or non-classification
Based on the available data for the test chemical, it is not likely to cause any irritation to skin and eyes.
Hence, the test chemical can be classified under the category “Not Classified” as per CLP regulation.
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