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EC number: 246-788-1 | CAS number: 25279-09-8
- 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: Key study: OECD TG 439. GLP study. Tissue viability after exposure and post-treatment incubation was 27.6%, i.e. less that 50%. Therefore, the test item is irritating to the skin (Category 2).
Key study: OECD TG 431. GLP study. The test item is not corrosive to the skin (viability = 92.7 and 105.3% after 3 and 60 min exposure).
Eye irritation: Key study: OECD TG 437. GLP study. The IVIS score for the corneas treated with test item was found to be 0.87 (No category according to OECD 437). Thus, the test item was determined to be not irritating to the eye.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 February 2018 - 07 May 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- This study addresses the human health endpoint skin irritation. It makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RhE) is also recommended by the OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies. The results of the study are believed to be of value in predicting the potential of inducing skin irritation by the test item in humans.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model
- Tissue batch number(s): N°18-RHE-029
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ºC
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 times in a constant soft stream of 1 mL DPBS from 5-8 cm distance from the insert.
- Observable damage in the tissue due to washing: No.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 180 minutes
- Wavelength: 570 nm
NUMBER OF REPLICATE TISSUES: 3
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The test is considered to be irritant to skin, if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
The test item is considered as non-irritant to skin, if the tissue viability after exposure and post-treatment incubation is more than (>) 50%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- 16 ± 2 mg of test item/0.5 cm2
- Duration of treatment / exposure:
- 42 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- 3
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 (mean of 3 replicates)
- Value:
- 27.6
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: Test item did not produce direct MTT reduction when compared to concurrent negative control (Maintenance meduim).
- Colour interference with MTT: Difference in absorbance due to color interference was not observed between negative control (isopropanol) and dihydromyrcenyl formate. The test item did not form color in isopropanol, therefore results shows no interference in OD due to test item.
DEMONSTRATION OF TECHNICAL PROFICIENCY: JRF Study Number: 618-1-06-9641
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The data met the acceptance criteria since the mean OD value of the 3 tissues was ≥ 1.2 at 570 ± 30 nm according to the historical database. The Standard Deviation value is considered as valid if it is ≤ 18%. The OD values (Corrected ODs) of negative controls in all tissues were between 1.221 to 1.273
- Acceptance criteria met for positive control:
The data met the acceptance criteria since the mean viability, expressed as % of the NC, was < 40 % and the Standard Deviation value is ≤ 18 %. Mean % viability of the positive control was 1.5%
- Acceptance criteria met for variability between replicate measurements:
Standard deviation of each intra-batch mean (3 Replicates/Tissue and 3 Tissue/Run) was < 18%.
- Range of historical values if different from the ones specified in the test guideline:
Optical Density at 570±30 nm
Exposure time: 42-minute exposure time
Negative Control (Dulbecco's phosphate-buffered saline):
Mean: 2.081
Standard deviation: 0.208
Minimum: 1.964
Maximum:2.776
Positive Control (Sodium dodecyl sulfate, 5% aqueous)
Mean: 0.028
Standard deviation: 0.007
Minimum: 0.021
Maximum: 0.053 - Interpretation of results:
- Category 2 (irritant) based on GHS criteria
- Conclusions:
- The substance was determined to be irritating to the skin.
- Executive summary:
An in-vitro Skin Irritation test with Reconstructed Human Epidermis (RHE) Tissues was performed according to the OECD Guideline 439 (GLP study). Tissues were exposed to the negative control (Dulbecco’s Phosphate Buffered Saline (DPBS)), positive control (sodium dodecyl sulfate, 5% aqueous (SDS)) and test item in triplicate for 42 minutes at room temperature. The mean cell viability in tissues treated with the test item was 27.6% after 42 minutes exposure. A significant reduction in percent cell viability was observed in treated tissues when compared with the concurrent negative control. The Optical density (OD) values for the negative control replicates were between 1.221 to 1.273, against the guideline requirement of ≥ 0.8 and ≤ 3.0 (≥ 1.2 as per SkinEthic SOP). The OD of the blank was between 0.041 to 0.045 which met the guideline requirement of OD < 0.1. The positive control showed a 1.5% cell viability, against the acceptance criteria of <40% for the SkinEthic RHE model, compared to concurrent negative control. Variation between tissue replicates (i.e. CV% value) was 4.54% for the test item group, 0.00% for positive control and 0.81% for negative control against the guideline requirement of ≤ 18%, which demonstrate the efficiency of the test system, SkinEthicTM RHE model. All criteria for a valid study were met. Based on these results, the test item was determined to be irritating to the skin (category 2).
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 July 2018 - 14 July 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- This study addresses the human health endpoint skin corrosion. It makes use of reconstructed human epidermis (RHE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RhE) is also recommended by the OECD and other regulatory authorities. SkinEthic™ RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies. The results of the study are believed to be of value in predicting the potential of inducing skin corrosivity by the test item in humans.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE model
- Tissue batch number(s): N°18-RHE-077
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ± 1 ºC
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 20 times in a constant soft stream of 1 mL DPBS from 5-8 cm distance from the insert.
- Observable damage in the tissue due to washing: No.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 180 minutes
- Wavelength: 570 nm
NUMBER OF REPLICATE TISSUES: 3
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than (<) 50%, or if the viability after 3 minutes exposure is greater than or equal to (≥) 50 % and the viability after 1 hour exposure is less than (<) 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to (≥) 50% and the viability after 1 hour exposure is greater than or equal to (≥) 15%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- 40 μL of test item/0.5 cm2
- Duration of treatment / exposure:
- 3 minutes / 1 hour
- Number of replicates:
- 3
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 minutes exposure (mean of 3 replicates)
- Value:
- 92.7
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 100%
- Positive controls validity:
- not applicable
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 60 minutes exposure (mean of 3 replicates)
- Value:
- 105.3
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 100%
- Positive controls validity:
- valid
- Remarks:
- 0.24%
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: Test item did not produce direct MTT reduction when compared to concurrent negative control (Maintenance meduim).
- Colour interference with MTT: Difference in absorbance due to color interference was not observed between negative control (isopropanol) and dihydromyrcenyl formate. The test item did not form color in isopropanol, therefore results shows no interference in OD due to test item.
DEMONSTRATION OF TECHNICAL PROFICIENCY: JRF Study Number: 616-1-06-9542
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The data met the acceptance criteria since the mean OD value of the 3 tissues was ≥ 0.8 and ≤ 3.0 for each exposure time.
- Acceptance criteria met for positive control: Yes. The data met the acceptance criteria since the mean viability of the PC, expressed as % of the NC, was < 15 %.
- Acceptance criteria met for variability between replicate measurements: Standard deviation of each intra-batch mean (3 Replicates/Tissue and 3 Tissue/Run) was < 30%.
- Range of historical values if different from the ones specified in the test guideline (OD at 560 nm):
Negative Control (Distilled water), 3-min exposure: mean = 2.375, SD = 0.264, min = 1.610, max = 2.594
Negative Control (Distilled water), 3-min exposure: mean = 2.470, SD = 0.224, min = 1.806, max = 2.693
Positive Control (8N KOH), 60-min exposure: mean = 0.061, SD = 0.072, min = 0.004, max = 0.284 - Interpretation of results:
- Category 2 (irritant) based on GHS criteria
- Conclusions:
- The substance was determined to be non-corrosive to the skin.
- Executive summary:
An in-vitro Skin Corrosion test with Reconstructed Human Epidermis (RHE) Tissues was performed according to the OECD Guideline 431 (GLP study). SkinEthic™ RHE tissues were exposed to the test item for 3 minutes at room temperature and 60 minutes at 37ºC in an incubator with 5% CO2. Negative (distilled water) and positive (8N KOH, 60 min) controls were run in parallel. Three replicates units per group and time point were used. Exposure of the test item was terminated by rinsing with 20 x 1 mL of DPBS. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals with isopropanol overnight and measuring the concentration of formazan by determining the OD at 570 nm. All criteria for a valid study were met. Under test conditions, the mean corrected percent viability of the treated tissues was 92.7 and 105.3 % after 3 and 60 minutes exposure, respectively, versus 0.24% in the positive control. From results of this study, it is concluded that the test item is non-corrosive to the skin.
Referenceopen allclose all
Data summary of percent viability:
Treatment |
Tissue Replicate |
O.D. at 570 nm |
Blank Corrected O.D. |
Mean of Corrected O.D. |
Mean O.D. of Three Tissues |
% Viability/ Tissue |
Mean % Viability |
S.D. of % Viability |
C.V. of % Viability |
Corrosivity Class |
Negative Control (Dulbecco’s Phosphate Buffered Saline (DPBS)) |
1 |
1.274 |
1.231 |
1.238 |
1.238 |
100 |
100 |
0.01 |
0.81 |
NA |
1.282 |
1.239 |
|||||||||
1.287 |
1.244 |
|||||||||
2 |
1.280 |
1.237 |
1.231 |
|||||||
1.264 |
1.221 |
|||||||||
1.277 |
1.234 |
|||||||||
3 |
1.270 |
1.227 |
1.244 |
|||||||
1.276 |
1.233 |
|||||||||
1.316 |
1.273 |
|||||||||
Dihydromyrcenyl Formate |
1 |
0.361 |
0.319 |
0.336 |
0.342 |
27.1 |
27.6 |
0.61 |
2.21 |
Category 2 |
0.384 |
0.342 |
|||||||||
0.388 |
0.346 |
|||||||||
2 |
0.381 |
0.339 |
0.34 |
27.5 |
||||||
0.384 |
0.342 |
|||||||||
0.382 |
0.340 |
|||||||||
3 |
0.395 |
0.353 |
0.35 |
28.3 |
||||||
0.388 |
0.346 |
|||||||||
0.393 |
0.351 |
|||||||||
Positive control (Sodium dodecyl sulphate (5% aq.)) |
1 |
0.061 |
0.018 |
0.018 |
0.018 |
1.5 |
1.5 |
0 |
0.00 |
Category 2 |
0.061 |
0.018 |
|||||||||
0.060 |
0.017 |
|||||||||
2 |
0.062 |
0.019 |
0.019 |
1.5 |
||||||
0.062 |
0.019 |
|||||||||
0.061 |
0.018 |
|||||||||
3 |
0.061 |
0.018 |
0.018 |
1.5 |
||||||
0.061 |
0.018 |
|||||||||
0.061 |
0.018 |
Keys: O.D. = Optical Density, S.D. = Standard Deviation, C.V. = Coefficient of Variation, NA = Not Applicable
Note: For Negative control, SD and CV of % viability was calculated using corrected OD at 570 nm and for the test item and positive control SD and CV of % viability was calculated using % viability/tissue."
Data summary of percent viability:
Treatment |
Exposure Time |
Tissue Replicate |
O.D. |
Corrected O.D. |
Mean of Corrected O.D. |
Mean O.D. of Replicate tissues |
% Viability/ Tissue |
Mean % Viability |
S.D. of % Viability |
% C.V. of % Viability |
Corrosivity Class |
Test item |
3 Minutes |
1 |
1.009 |
0.966 |
0.970 |
0.969 |
92.80 |
92.70 |
0.115 |
0.12 |
Non-corrosive |
1.015 |
0.972 |
||||||||||
1.016 |
0.973 |
||||||||||
2 |
1.008 |
0.965 |
0.968 |
92.60 |
|||||||
1.013 |
0.970 |
||||||||||
1.013 |
0.970 |
||||||||||
3 |
1.017 |
0.974 |
0.970 |
92.80 |
|||||||
1.012 |
0.969 |
||||||||||
1.009 |
0.966 |
||||||||||
60 Minutes |
1 |
1.076 |
1.033 |
1.030 |
1.028 |
105.50 |
105.30 |
0.208 |
0.20 |
||
1.070 |
1.027 |
||||||||||
1.072 |
1.029 |
||||||||||
2 |
1.070 |
1.027 |
1.029 |
105.40 |
|||||||
1.076 |
1.033 |
||||||||||
1.069 |
1.026 |
||||||||||
3 |
1.072 |
1.029 |
1.026 |
105.10 |
|||||||
1.063 |
1.020 |
||||||||||
1.073 |
1.030 |
Treatment |
Exposure Time |
Tissue Replicate |
O.D. |
Corrected O.D. |
Mean of Corrected O.D. |
Mean O.D. of Replicate tissues |
% Viability/ Tissue |
Mean % Viability |
S.D. of % Viability |
% C.V. of % Viability |
Corrosivity Class |
Negative Control (Sterile Distilled water) |
3 Minutes |
1 |
1.081 |
1.038 |
1.043 |
1.045 |
100 |
100 |
0.002 |
0.19 |
NA |
1.066 |
1.023 |
||||||||||
1.112 |
1.069 |
||||||||||
2 |
1.085 |
1.042 |
1.045 |
||||||||
1.093 |
1.050 |
||||||||||
1.085 |
1.042 |
||||||||||
3 |
1.086 |
1.043 |
1.046 |
||||||||
1.089 |
1.046 |
||||||||||
1.092 |
1.049 |
||||||||||
60 Minutes |
1 |
1.010 |
0.967 |
0.973 |
0.976 |
100 |
100 |
0.004 |
0.41 |
||
1.026 |
0.983 |
||||||||||
1.011 |
0.968 |
||||||||||
2 |
1.017 |
0.974 |
0.981 |
||||||||
1.034 |
0.991 |
||||||||||
1.022 |
0.979 |
||||||||||
3 |
1.035 |
0.992 |
0.975 |
||||||||
1.008 |
0.965 |
||||||||||
1.012 |
0.969 |
||||||||||
Positive Control (8N KOH) |
60 Minutes |
1 |
0.045 |
0.002 |
0.002 |
0.002 |
0.20 |
0.24 |
0.064 |
26.67 |
Corrosive |
0.045 |
0.002 |
||||||||||
0.045 |
0.002 |
||||||||||
2 |
0.047 |
0.004 |
0.003 |
0.31 |
|||||||
0.046 |
0.003 |
||||||||||
0.046 |
0.003 |
||||||||||
3 |
0.045 |
0.002 |
0.002 |
0.20 |
|||||||
0.045 |
0.002 |
||||||||||
0.045 |
0.002 |
Key: O.D. = Optical Density, S.D. = Standard Deviation, C.V. = Coefficient of Variation, NA = Not Applicable
Note: For Negative control, SD and CV of % viability was calculated using corrected OD at 570 nm and for the test item and positive control SD and CV of % viability was calculated using % viability/tissue."
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (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:
- 02 January 2018 - 20 January 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- other: Cow
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra
- Characteristics of donor animals (e.g. age, sex, weight): Between 1 to 5 years
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported (in a sealed plastic container) under cold condition in Hanks’ Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL]
- Time interval prior to initiating testing: The eyes were used within 24 hours from slaughter
- indication of any existing defects or lesions in ocular tissue samples: Corneas from eyes free of visible defects were used. - 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): 750 µL
- Duration of treatment / exposure:
- 10 minutes
- Duration of post- treatment incubation (in vitro):
- 2 h
- Number of animals or in vitro replicates:
- 3 replicates
- Details on study design:
- SELECTION AND PREPARATION OF CORNEAS
Eyes were examined prior to use. Corneas from eyes free of visible defects were used. Corneas that have opacity lesser than seven opacity units or equivalent for the opacitometer were used in the study.
QUALITY CHECK OF THE ISOLATED CORNEAS
NUMBER OF REPLICATES : 3
NEGATIVE CONTROL USED : Yes (nomral saline)
POSITIVE CONTROL USED : Yes (dimethylformamide)
APPLICATION DOSE AND EXPOSURE TIME : 750 µL of test item; corneas were exposed for approx. 10 min.
TREATMENT METHOD: Open chamber
A volume of 750 μL was introduced into the anterior chamber through the dosing holes on the top surface of the chamber. The anterior compartment was then plugged. Post application of the test item the holders were turned to a horizontal position and slightly rotated to ensure uniform covering of the test item over the cornea.
POST-INCUBATION PERIOD: yes (2 h)
Once the medium was free of test item, the corneas were given a final rinse with EMEM (without phenol red). Media in the anterior and the posterior chamber was removed and fresh EMEM (without phenol red) was filled. The compartments were plugged and post treatment opacity of each cornea was recorded. Once the medium was free of test item, the corneas were given a final rinse with EMEM (without phenol red). Anterior chamber was then refilled with fresh EMEM without phenol red. After rinsing, the corneas were incubated for an additional period of approximately 2 hours ± 10 minutes at 32 ± 1ºC. At the end of post-exposure incubation period the media in the anterior and the posterior chamber were removed and fresh EMEM (without phenol red) was filled. The compartments were plugged and post treatment opacity of each cornea recorded.
After opacity measurement, the medium was removed from the anterior chamber and was filled with 1 mL of 4 mg/mL fluorescein solution whilst the posterior chamber was filled with fresh EMEM (without phenol red). The holders were then incubated in a horizontal position for approximately 90 ± 5 min at 32 ± 1 ºC. After the incubation the medium in the posterior chamber was transferred into labelled tubes. An aliquot of the medium from the posterior chamber was transferred to a 96-well plate. The plate reader was set to read at OD490. A reading of EMEM (without phenol red) was also recorded which served as blank.
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Once, until no visual evidence of test item.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: passage of sodium fluorescein dye (OD490)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA:
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1 - Irritation parameter:
- in vitro irritation score
- Run / experiment:
- 1
- Value:
- 0.34
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- 2
- Value:
- 1.31
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- 3
- Value:
- 0.96
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- The mean IVIS score for the corneas treated with test item was found to be 0.87.
OTHER EFFECTS:
- Visible damage on test system: No.
DEMONSTRATION OF TECHNICAL PROFICIENCY: JRF Study nº 530-01-01-10123 (2015)
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes (mean IVIS = -0.49)
- Acceptance criteria met for positive control: Yes (mean IVIS = 160.97)
- Range of historical values if different from the ones specified in the test guideline:
Dimethylformide (positive control): Mean ± SD = 120.37 ± 30.51 (min 58.94, max 165.18) - Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item was determined to be not irritating to the eye.
- Executive summary:
An in-vitro eye irritation bovine corneal opacity and permeability test was performed according to OECD Guideline 437 (GLP study). Three sets, each consisting of three corneas were tested. The first set served as control and was treated with 750 μL normal saline. The second set served as positive control and was treated with 750 μL dimethylformamide with the third set treated with 750 μL test item. Post application the corneas were incubated for approximately 10 minutes after which the test item was washed-off the cornea and the corneas kept in incubation for approximately 2 h at 32 ± 1 ºC. At the end of the incubation period opacity readings were taken. Post opacity permeability reading was measured by applying 1 mL of fluorescein sodium solution (4 mg/mL) on to the anterior surface of the cornea and was incubated for approximately 90 min at 32 ºC. At the end of the incubation period the Optical Density (OD) was measured at 490 nm for the fluid collected from the posterior chamber. The mean In-Vitro Irritancy Score (IVIS) of normal saline (control) and dimethylformamide (positive control) treated corneas were found to be -0.49 and 160.97 which confirmed the reliability of the test procedure. The IVIS score for the corneas treated with test item was found to be 0.87 (No category according to OECD 437). Thus, the test item was determined to be not irritating to the eye.
Reference
Corneal Opacity
The mean final opacity values for dihydromyrcenyl formate treated eyes (0.68) did not show any observable increase in comparison to the control group (-0.78). An observable marked increase in final mean opacity was observed in the corneas treated with N,N-dimethylformamide (124.96).
Corneal Permeability
The mean final corneal permeability values for dihydromyrcenyl formate treated eyes (0.012) did not show any observable increase in comparison to the control group (0.019). An observable marked increase in mean final corneal permeability was observed in the corneas treated with N,N- dimethylformamide (2.400).
In vitro Irritancy Score (IVIS)
The mean In-Vitro Irritancy Score (IVIS) of normal saline (control) and N,N-dimethylformamide (positive control) treated corneas were found to be -0.49 and 160.97 which confirmed the reliability of the test procedure.
The mean IVIS score for the corneas treated with dihydromyrcenyl formate was found to be 0.87.
Dihydromyrcenyl Formate (0.75 mL) |
|||||||||||
Cornea holder N° |
Io (LUX) |
I (Initial) (LUX) |
Initial Opacity Value |
I (Post Treatment) (LUX) |
Post Treatment Opacity Value |
Corr. Opacity Value |
Final Opacity Value |
OD490 Value |
Corr. OD490Value |
Final OD490 Value |
IVIS |
20 |
1083 |
1019 |
2.92 |
1029 |
2.51 |
-0.41 |
0.37 |
0.065 |
0.017 |
-0.002 |
0.34 |
21 |
1103 |
1011 |
4.05 |
1011 |
4.05 |
0.00 |
0.78 |
0.102 |
0.054 |
0.035 |
1.31 |
22 |
1112 |
1061 |
2.34 |
1058 |
2.46 |
0.12 |
0.90 |
0.071 |
0.023 |
0.004 |
0.96 |
Mean |
0.68 |
- |
0.031 |
0.012 |
0.87 |
||||||
SD |
0.28 |
- |
0.020 |
0.020 |
0.49 |
Normal Saline (0.75 mL) |
|||||||||
Cornea Holder N° |
Io (LUX) |
I (Initial) (LUX) |
Initial Opacity Value |
I (Post Treatment) (LUX) |
Post Treatment Opacity Value |
Corr. Opacity Value |
OD490 Value |
Corr. OD490 Value |
IVIS |
13 |
1112 |
1054 |
2.61 |
1053 |
2.65 |
0.04 |
0.060 |
0.012 |
0.22 |
14 |
1067 |
961 |
4.82 |
1021 |
2.22 |
-2.60 |
0.091 |
0.043 |
-1.96 |
15 |
1052 |
990 |
2.92 |
985 |
3.13 |
0.21 |
0.051 |
0.003 |
0.26 |
Mean |
-0.78 |
- |
0.019 |
-0.49 |
|||||
SD |
1.58 |
- |
0.021 |
1.27 |
N,N-Dimethylformamide (0.75 mL) |
|||||||||||
Cornea holder N° |
Io (LUX) |
I (Initial) (LUX) |
Initial Opacity Value |
I (Post Treatment) (LUX) |
Post Treatment Opacity Value |
Corr. Opacity Value |
Final Opacity Value |
OD490 Value |
Corr. OD490 Value |
Final OD490 Value |
IVIS |
17 |
1088 |
1016 |
3.25 |
233 |
146.62 |
143.37 |
144.15 |
1.908 |
1.860 |
1.841 |
171.77 |
18 |
1094 |
969 |
5.56 |
287 |
112.45 |
106.89 |
107.67 |
3.385 |
3.337 |
3.318 |
157.44 |
19 |
1103 |
1024 |
3.50 |
266 |
125.79 |
122.29 |
123.07 |
2.108 |
2.060 |
2.041 |
153.69 |
Mean |
124.96 |
- |
2.419 |
2.400 |
160.97 |
||||||
SD |
18.31 |
- |
0.801 |
0.801 |
9.54 |
Keys: IVIS = In Vitro Irritation Score, Io =Baseline Reading (With medium but without cornea), I = LUX Reading with Medium and Cornea, OD490= Optical Density at 490 Wave Length, - = Not Applicable, Corr. = Corrected. Blank OD490value = 0.048.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Skin irritation: Based on available data, the substance is classified as Skin Irritant, Category 2 (H315) according to the CLP Regulation (EC) No. 1272/2008.
Eye irritation: Based on available data, the substance is not classified for Eye Damage according to the CLP Regulation (EC) No. 1272/2008.
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