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EC number: 201-642-6 | CAS number: 85-91-6
- 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 overall irritation score of the substance was determined to be 0 and no erythema and edema (skin irritation) were found at the end of 14 days observation period after patch removal.
Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.
Eye Irritation:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline . The mean % tissue viability of test chemical was determined to be 57.5%. Thus, the test chemical was considered to be irritating to MatTek EpiOcular Tisssue Model OCL-200
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:
- To determine the dermal reaction profile 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): Methyl N-methyl anthranilate
- Molecular formula : C9H11NO2
- Molecular weight : 165.191 g/mole
- Substance type: Organic
- Physical State: Liquid
SOURCE OF TEST MATERIAL
- Test Item: Methyl-N-methyl anthranilite (CAS No. 85-91-6)
- Batch No.of test material: 0009
- Manufacturing Date: May; 2016
- Expiration date of the lot/batch: May; 2021
- Purity test date: No data available
- Consistency: Liquid
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: Test articles is tested as provided (neat).
- 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: Liquid
OTHER SPECIFICS: No data available - 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 217.8 to 251.8 grams at initiation of dosing.
Body weights at the start :
Male
Mean : 246.46 g (= 100 %)
Minimum : 238.5 g (- 3.23 %)
Maximum : 251.8 g (+ 2.17 %)
Total No. of animals : 5
Female
Mean : 221.36 g (= 100 %)
Minimum : 217.8 g (- 1.61 %)
Maximum : 224.3 g (+ 1.33 %)
Total No. of animals : 5
- Identification: Each rat was individually identified by the cage number.
- Fasting period before study: No data available
- 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 22.3 degree centigrade.
- Humidity (%): 54.8% 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: 18-07-2017 to 02-08-2017 - Type of coverage:
- semiocclusive
- Preparation of test site:
- clipped
- Vehicle:
- unchanged (no vehicle)
- 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:
- 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.
OTHER OBSERVATIONS
Type and Frequency of Tests, Analyses and Measurements
Viability:Twice daily.
Clinical Observations and General Appearance:
Animals were observed for clinical signs, mortality, until sacrifice.
Onset, duration and severity of any sign were recorded. The clinical signs and mortality observations were conducted at 10, 30, 60 minutes, 2, 4 and 6 hours on the day of dosing and once daily thereafter for 14 day. Daily observation was done as far as possible at the same time.
The observations were included general clinical signs, observations of eyes, mucous membranes, respiratory, circulatory system and behavior pattern.
Body weights:
Individual animal body weights were recorded pre-test (prior to administration of the test item), day 7 and at termination on day 14.
Gross Pathology:
Necropsy was performed on animals surviving at the end of the study. Macroscopic examination of all the orifices, cavities and tissues were made and the findings were recorded. All animals surviving the study period were sacrificed by the carbon dioxide asphyxiation technique (day 15).
Histopathology:
No gross abnormalities were observed in animals sacrificed terminally hence, no histopathology was performed. - 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 8.14% and 16.70% respectively.
Sex : Female
Group I (2000 mg/kg) - Percent body weight gain after 7 days and 14 days was found to be 4.74% and 9.61% 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:
- The overall irritation score of the substance was determined to be 0 and no erythema and edema (skin irritation) were found at the end of 14 days observation period after patch removal.
Hence, it was concluded that the test chemical was Non-Irritating to the skin of Sprague Dawley rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification. - Executive summary:
A study was designed and conducted to determine the dermal reaction profile of the test chemical in Sprague Dawley rats. The study was performed as per OECD Guidelines 402 and complying
to the GLP procedures. 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 applied directly 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.
The test chemical was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. 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. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days. 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 classified as “Category-Not Classified” as per CLP Classification.
Reference
Individual Animal - Evaluation of Dermal Reaction
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 |
Table No. I
Summary of Clinical Signs of Toxicity and Mortality
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 - 5 |
Day 0 - Day 14 |
0/5 |
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 - 10 |
Day 0 - Day 14 |
0/5 |
Table No. II
Summary of Evaluation of Dermal Reaction
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 |
Table No.III
Mean Body Weight and Percent Body Weight Gain (g)
Test System : Sprague Dawley Rat
Sex : Male
Group No. |
Dose (mg/kg body weight) |
|
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 |
I |
2000 |
Mean |
246.46 |
266.50 |
8.14 |
287.54 |
7.91 |
16.70 |
± SD |
5.29 |
4.67 |
1.08 |
3.15 |
1.48 |
1.82 |
Sex : Female
Group No. |
Dose (mg/kg body weight) |
|
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 |
I |
2000 |
Mean |
221.36 |
231.84 |
4.74 |
242.62 |
4.65 |
9.61 |
± SD |
2.63 |
2.75 |
0.95 |
3.32 |
0.60 |
1.42 |
Table No.IV
Summary of Gross Pathological Findings
Test System : Sprague Dawley Rat
Sex : Male
Group No. |
Dose mg/kg |
Animal Numbers |
Animal Fate |
Gross Pathological Findings |
I |
2000 |
1 - 5 |
TS |
No abnormality detected |
Sex : Female
Group No. |
Dose mg/kg |
Animal Numbers |
Animal Fate |
Gross Pathological Findings |
I |
2000 |
6 - 10 |
TS |
No abnormality detected |
TS = Terminal Sacrifice
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- December 06, 2017 to February 16, 2018
- 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 is to provide classification of chemicals concerning their eye irritation potential using an alternative to the Draize Rabbit Eye Test, according to the OECD Test Guideline No. 492, “Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”. The EpiOcular™ EIT is intended to differentiate those materials that are UN GHS No Category (i.e., do not meet the requirements for UN GHS classification) from those that would require labeling as either UN GHS Category 1 or 2. This assay is not intended to differentiate between UN GHS Category 1 / Hazard
Code 318 and UN GHS Category 2 / Hazard Codes 319 and 320. - GLP compliance:
- yes
- Specific details on test material used for the study:
- - Name of test material (as cited in study report): Methyl N-methyl anthranilate
- Molecular formula : C9H11NO2
- Molecular weight : 165.191 g/mole
- Substance type: Organic
- Physical State: Liquid
SOURCE OF TEST MATERIAL
- Test Item: Methyl-N-methyl anthranilite (CAS No. 85-91-6)
- Batch No.of test material: 0009
- Manufacturing Date: May; 2016
- Expiration date of the lot/batch: May; 2021
- Purity test date: No data available
- Consistency: Liquid
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: Test articles is tested as provided (neat).
- 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: Liquid
OTHER SPECIFICS: No data available - Species:
- other: MatTek EpiOcular Tisssue Model OCL-200
- Strain:
- other: Not applicable
- Details on test animals or tissues and environmental conditions:
- -Test System: MatTek EpiOcular™ Tissue Model OCL-200
Storage:EpiOcular™ tissues and assay medium will be refrigerated at approximately 2-8°C upon arrival and until use.
Supplier:MatTek Corporation, Ashland, MA
- Justification of the test method and considerations regarding applicability
The EpiOcular™ Tissue Model closely parallels human ocular tissue, thus providing a useful in vitro means to assess ocular irritancy and toxicology - 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 μl
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 will be topically exposed to the test article and control articles for 30 ± 2 minutes.
- Observation period (in vivo):
- Not applicable
- Duration of post- treatment incubation (in vitro):
- Following the post soak,Tissues will be incubated in 1 ml fresh assay medium in a humidified 37±1°C, 5±1% CO2 incubator.
- Number of animals or in vitro replicates:
- 2 tissues were used for test compound and control.
- Details on study design:
- -Plate Reader Linearity Check:
The linearity of the plate reader or spectrophotometer used for optical density (OD) determination will be verified prior to its use the same week the EIT assay is being
performed.
A dilution series of trypan blue or thiazolyl blue tetrazolium bromide (MTT) formazan will be prepared and 200 μl aliquots will be pipetted into a 96-well plate.
The optical density of the plate wells will be measured at a wavelength of 570 nm (OD570), with no reference wavelength.
A regression line and an R-squared value will be generated using Microsoft Excel®. Verification will be considered acceptable if the R-squared value is >0.999.
Assessment of Direct MTTReduction:No assessment of the direct MTT (methyl thiazole tetrazolium) reduction potential of each test article will be made.
-Assessment of Coloring or Staining Materials:
No assessment of each test article’s ability to absorb light at the wavelength (570 nm) used for MTT determination will be made.
- Pre-Incubation:
EpiOcular™ tissues will be placed in six-well plates containing warmed assay media and will be equilibrated in a humidified 37±1°C, 5 ±1% CO2 incubator for at least one hour. The media will then be changed and the tissues incubated overnight (16-24 hours).
Any tissues not being incubated the same day will be allowed to re-equilibrate at 37±1°C, 5±1% CO2 and will be stored at approximately 2-8°C..
-Pre-Treatment:After the overnight incubation, the tissues will be moistened with 20 μl of phosphatebuffered saline (PBS) and incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.
-Dosing:50 μl of a liquid test article will be applied topically to duplicate tissues and incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.
After dosing and incubation, the tissues will be thoroughly rinsed with PBS and soaked in 5 ml of room-temperature assay medium in a 12-well plate for the appropriate amount of time.
Tissues will be soaked for 12±2 minutes.
Incubation in MTT:The tissues will be incubated with 300 μl of 1 mg/ml MTT in DMEM for 3 hours ± 10 minutes at 37±1°C, 5±1% CO2
-MTT Extraction:
Following the three-hour MTT incubation period, each tissue will be removed individually and gently rinsed with PBS to remove any residual MTT solution.
The extraction plate will be covered and sealed to reduce evaporation of extractant.
For solid, colored, or staining test articles, 2.0 ml of extractant solution will be used in a six-well plate, allowing extraction to occur through the bottom of the insert.
Extraction Conditions:The extraction will be allowed to proceed overnight at room temperature in the dark.
Alternatively, the extraction can proceed for at least two hours, with shaking, at room temperature.
-Decant Extractant:
Tissues immersed in extractant solution in a 24-well plate: After the extraction period is complete, the liquid within each tissue insert will be decanted back into the well
from which it was taken, i.e., the solution will be mixed with the extractant in the well.
The tissue inserts will then be discarded.
-Transferring to 96-Well Plate:Two 200 μl aliquots from each well of the extraction plate(s) will be pipetted into a 96- well microtiter plate.
-Measuring Optical Density:The optical density of the extracted samples will be determined at a single wavelength of 570 nm and using eight 200 μl aliquots of the Extractant as blanks.
Calculating Percent Viability:
The percent viability of the test tissues will be determined using the following formula:
% Viability = 100 x (ODsample / ODNegative Control)
Quality Controls:
Negative Controls: The assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5.
Positive Controls: The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability.
Tissue Variability: The difference in viability between identically treated tissues must be less than 20%. This applies to tissues treated with the same test article as well as living and killed controls.
Ocular Irritation Potential:
An irritant is predicted if the mean relative tissue viability of two individual tissues exposed to the test substance is less than or equal to 60% of the mean viability of the
negative control-treated tissue viability.
In Vitro Result In Vivo Prediction (GHS3)
Mean tissue viability ≤ 60% Category 1 / Hazard Code 318, or
Category 2 / Hazard Codes 319 and 320
Mean tissue viability > 60% No Category (Non-Irritating)
Borderline Results:
If the test article-treated tissue viability is 60±5%, a second EIT should be performed. If the results of the second test disagree with the first, then a third test should be performed. The conclusion will be based on the agreement of two of the three tests.
Duration:The duration of the EpiOcularTM Eye Irritation Test is approximately five days. - Irritation parameter:
- other: mean % tissue viability
- Run / experiment:
- Run 1
- Value:
- 57.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Other effects / acceptance of results:
- Negative Controls: The assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5.
Positive Controls: The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability.
Tissue Variability: The difference in viability between identically treated tissues must be less than 20%. - Interpretation of results:
- Category 2 (irritating to eyes) based on GHS criteria
- Conclusions:
- The ocular irritation potential of test article was determined according to the OECD 492 test guideline . The mean % tissue viability of test chemical was determined to be 57.5%. Thus, the test chemical was considered to be irritating to MatTek EpiOcular Tisssue Model OCL-200
- Executive summary:
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 mean OD570 of the negative control tissues was 1.665 and 1.589, which met the acceptance criteria of greater than 0.8 and less than 2.5. The mean relative viability of the positive control tissues was 45.2 and 36.5, which met the acceptance criterion of less than 50%. The differences in viability between identically treated tissues were 0.03 to 5.60, which met the acceptance criterion of less than 50%. All controls passed the acceptance criteria for a valid study.
The mean % tissue viability of test chemical was determined to be 57.5%.
Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the MatTek EpiOcular Tissue Model OCL-200 and being classified as ‘’Irritating to eyes in Category 2”
Reference
|
Tissue Viability |
Irritancy Classification |
GHS Category
|
|||
Mean |
SD |
|||||
85-91-6 |
57.5 |
3.36 |
Irritant |
Category 1 or 2 |
Test and control article identity |
Tissue no. |
Raw data |
Blank corrected data |
Mean of aliquots |
% viability |
OD |
Viabilities (%) |
||||
MEAN |
SD |
Mean |
SD |
||||||||
Aliq 1 |
Aliq 2
|
Aliq 1 |
Aliq 2 |
|
|
|
|
||||
85-91-6 |
1
|
1.039 |
1.024 |
0.993 |
0.978 |
0.985 |
59.2 |
0.957 |
0.056 |
57.5 |
3.36 |
2 |
0.983 |
0.968 |
0.937 |
0.922 |
0.929 |
55.8 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin Irritation
In different studies,the test chemicalhas been investigated for its potential to cause dermal irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits, rats, humans, mice along with in vitro experiments for the test chemical. The results are summarized as follows:
A study was designed and conducted to determine the dermal reaction profile of the test chemical in Sprague Dawley rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures. 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 applied directly 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.
The test chemical was applied to shorn skin of 5 male and 5 female animals at 2000 mg/kg body weight. 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. Administration of the test item did not result in any signs of toxicity and mortality during the study period of 14 days. 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 classified as “Category-Not Classified” as per CLP Classification.
This is supported by the in vitro study performed according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” to assess 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. The mean tissue viabilities for the Positive control, Methyl acetate were 6.5%, 10.7% respectively in the first and second run, whereas the tissue viabilities of the negative control, Tissue culture water remained at 100% in the both the runs.
The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 28.7%.
Hence, under the experimental test conditions it was concluded the test chemical was considered to be irritating to the human skin and being classified as “Category 2'' as per CLP Regulation.
The above in vitro study was lent support by another dermal irritation study performed in humans to determine its irritation potential.
10% in petrolatum of test sample was administrated on two different panels of human subject for 48 hours closed patch test. The test site was observed for signs of irritation till 48 hours. No known cutaneous reaction was observed after 48 hours. Therefore, the test chemical can be considered as non irritant on human skin.
These results are supported by a Skin irritation phototoxicity study conducted on hairless mouse to evaluate the toxic nature of the test compound. Open application of 100% test chemical (20 μl/5cm2) was not irritating at non-irradiated locations of the skin. The test chemical was not irritating to the skin of hairless mouse.
The above results are further supported by a skin irritation study performed on rabbits to assess the dermal irritation potential of the test chemical. The neat test sample was applied on intact or abraded rabbit skin for 24 hours occlusion period. Since there was no evidence of any skin reaction, the test chemical can be considered as non irritant on rabbit skin.
These results are also supported by a Human phototoxicity study carried out to evaluate the irritation potential of the test chemical.
A single 24 hour occluded application of duplicate patches was made to naïve sites. One of the duplicate sites was exposed to UVB and UVA radiation for evaluation of phototoxic potential, and one was used to evaluate primary irritation. The concentrations used were 0.1-0.5% for study 1 and 1% for study 2 in vehicle 25%:75% DEP:EToH.
No effects were observed between test material and either vehicle or blank controls. The test chemical was not irritating to human skin.
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 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 2.156.The standard deviation of viabilities for test chemical were calculated to be 1.21.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 96.0%.Hence, under the current experimental test conditions it was concluded that test chemical was considered to be not irritating to human skin.
Even though the results from the in-vitro experimental study claims that the test chemical was irritating to skin, but the in-vivo studies give a stronger evidence of its lack of irritation potential. Therefore, the test chemical can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.
Eye Irritation:
Various studies have been summarized to determine the extent of ocular damage caused by the test chemical in living organisms. The studies are based on in vivo experiments in rabbits along with in vitro data for the various test chemicals. The results have been summarized as follows:
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 mean OD570 of the negative control tissues was 1.665 and 1.589, which met the acceptance criteria of greater than 0.8 and less than 2.5. The mean relative viability of the positive control tissues was 45.2 and 36.5, which met the acceptance criterion of less than 50%. The differences in viability between identically treated tissues were 0.03 to 5.60, which met the acceptance criterion of less than 50%. All controls passed the acceptance criteria for a valid study.
The mean % tissue viability of test chemical was determined to be 57.5%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to the MatTek EpiOcular Tissue Model OCL-200 and being classified as ‘’Irritating to eyes in Category 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 liquid test articles and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 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, passing the acceptance criteria. The mean % tissue viability of test substance Dimethyl anthranilate (CAS No.85-91-6) was determined to be 98.2%. Hence, under the experimental test conditions it was concluded that test substance Dimethyl anthranilate (CAS No.85-91-6) was considered to be not irritating to the human eyes and can thus be classified as “Not classified’’ as per CLP Regulation
The in vitro result is supported by an eye irritation test conducted to assess the irritation potency of the test chemical.
Normal albino rabbit eyes were selected on the basis of absence of grossly visible staining by a 5-percent aqueous solution of fluorescein sodium, flushed with distilled water 20 seconds after application. A 2 hour interval was given so that the eye could return back to normal condition. Then, 0.005 ml of the undiluted test material was applied to the center of the cornea while the lids were retracted. About one minute later, the lids were released. This procedure is necessary to prevent the removal of a portion of the dose by the very efficient wiping system of the lids before intimate contact has been made with the eye. 18 to 24 hours later, the eyes were examined in strong diffuse daylight, then stained with fluorescein, and the injury was scored.
The test chemical was grouped under Grade 3 injury (0.1 ml undiluted gives injury of up to 5 points (0.5 ml gives over 5)) and Grade 9 injury (Excess of 1% solution gives injury up to 5 points (5% gives over 5)) when rabbit eyes were observed for injuries. The test chemical was known to have caused loss of vision or very slowly healing corneal burns. Based on these grades, it can be inferred that the test chemical was irritating to rabbit eyes.
These results are supported by another rabbit eye irritation test conducted in 5 healthy albino rabbits to assess the irritation potency of the test chemical. A 0.005 ml aliquot of neat test chemical was applied to the center of the cornea while the lids were retracted. One minute later the lids were released. The eyes were examined 18–24 h later in strong diffuse daylight and then stained with fluorescein. The test chemical was assigned grade 3 – necrosis since 13–37% of the cornea was visible after fluorescein staining.
Therefore, the test chemical can be considered to be irritating to rabbit eyes
The above results are further supported by an Eye irritation study performed according EPA OPP Series 81-4 to assess the irritation potential of the test chemical. The test procedure deviated from EPA OPP Series 81-4.
Eyes were examined with 2% sodium fluorescein 24 hours prior to application of test chemical.0.1 ml of Undiluted sample as a single instillation. Observations of the effects were made at 1, 24, 48 and 72 hours and days 4, 7, 10, 24.Irritancy scores were made after Kay, et al (1962).
The eye irritation scores after 72 hours were as follows-Cornea score -5/6, iris score – 4/6, conjunctivae redness score – 5/6, conjunctivae chemosis score -4/6, conjunctivae discharge – 2/6. Corneal Effects got cleared in 8 -21 days. Based on the test conditions, the test chemical was considered irritating to eyes. The eye irritation study was placed in TOX Category II according to EPA OPP regulations.
The in vivo and in vitro data are in mutual agreement with each other indicating a strong possibility of the test chemical being irritating to eye. Hence, the test chemical can be considered to be irritating to eye. Comparing the above annotations with the criteria of the CLP regulation the test chemical can be classified under the category 2
Justification for classification or non-classification
Even though the results from the in-vitro experimental study claims that the test chemical was irritating to skin, but the in-vivo studies give a stronger evidence of its lack of irritation potential. Therefore, the test chemical can be considered to be not irritating to skin and irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified” for skin irritation and category 2 for eye irritation.
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