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EC number: 950-123-7 | CAS number: 97849-65-5
- 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 (in vitro): Key study. Test method according to OECD 439, GLP study. The mean corrected percent viability of the treated tissues was 112.6 %, versus 1.3% in the positive control (5% SDS). Therefore, the test item is not irritant to the skin.
Eye irritation (in vitro): Key study. Test method according to OECD 438, GLP study. No prediction can be made for the test item in the ICE test.
Eye irritation (in vivo): Key study. Test method according to the OECD 405, GLP study. Based on the results of an acute eye irritation study performed on New Zealand White rabbits, the test substance is classified as eye damage (category 1).
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:
- 05 September 2019 - 17 September 2019
- 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:
- yes
- Remarks:
- Standard deviation between the 2 NSCkilled replicates treated with the test item was 35.4% instead of 18% at the maximum as defined in OECD test guideline. This deviation is considered as without impact on the conclusion of the study (see reasoning below)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Remarks:
- (SkinEthic RHE® model)
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: foreskin (number of donors not specified)
- Source strain:
- not specified
- Justification for test system used:
- The SkinEthic RHE® model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 19-RHE-156 (living epidermis); 17-RHE-124, 18-RHE-002, 18-RHE-142 and 19-RHE-096 (killed epidermis).
- Production date: N/A
- Shipping date: 17/09/2019
- Delivery date: 17/09/2019
- Expiration date: 23/09/2019
- Date of initiation of testing: 17/09/2019
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 37ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.4 (CV = 2.7%) specification OD > 0.7. Historical negative control mean OD range = 0.489-1.217 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.5)
- Barrier function: 5.8 h (Specification 4.0h < ET50< 10.0h)
- Morphology: 8 Cell layers (specification > 4.), highly differentiated epidermis consisting of organized basal, spinous and granular layers and a multilayered stratum corneum.
- Contamination: No.
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
- Fresh tissues / killed tissues: 2 killed tissues ( NSMTT control); 2 living tissues ((NSCliving control) and 2 additional killed tissues (NSCkilled control).
- N. of replicates : 2
- Method of calculation used: True viability % = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT + OD of killed tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 100
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- The test item is considered as non-irritant to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg (32mg/cm2)
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS - Duration of treatment / exposure:
- 42 minutes
- Duration of post-treatment incubation (if applicable):
- 41 hours and 15 minutes
- Number of replicates:
- 3
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean
- Value:
- 112.6
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- (distilled water)
- Positive controls validity:
- valid
- Remarks:
- 1.3% viability (5% SDS)
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: assessed by NSMTT controls (see table below).
- Colour interference with MTT: assessed by NSCliving controls and NSCkilled controls (see table below).
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, mean OD = 0.999 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.5).
- Acceptance criteria met for positive control: yes, mean viability = 1.3% (acceptability criteria should be < 40%).
- Acceptance criteria met for variability between replicate measurements: Standard deviation between the 2 NSCkilled control replicates treated with the test item was 35.4% instead of 18% at the maximum as defined in OECD test guideline. This deviation is considered as without impact on the conclusion of the study because regardless of the addition of the viability obtained by the NSCkilled control, the viability obtained with the test item remains higher than 50% and does not change the classification of the product. - Interpretation of results:
- other: Not classified (CLP Regulation EC no. 1272/2008)
- Conclusions:
- In the vitro human reconstructed epidermis test the mean corrected percent viability of the treated tissues was 112.6%. Therefore, the test item can be considered as not irritant to skin.
- Executive summary:
An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic™ RHE epidermis model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 41 hours and 15 minutes in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction. Moreover, 2 living and 2 killed Human skin model surfaces were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate non-specific living and killed colour controls. Under the test conditions, the mean corrected percent viability of the treated tissues was 112.6%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is considered as not irritant to the skin.
Reference
Table 1. Table of results
Well ID |
OD |
Mean OD / |
Mean OD / |
Viability |
Mean viability |
SD |
Conclusion |
|
Negative |
SPL 1 |
0.964 |
0.955 |
0.999 |
95.6 |
100.0 |
4.5 |
|
0.945 |
||||||||
0.956 |
||||||||
SPL 2 |
1.044 |
1.045 |
104.6 |
|||||
1.046 |
||||||||
1.045 |
||||||||
SPL 3 |
0.981 |
0.997 |
99.8 |
|||||
1.017 |
||||||||
0.992 |
||||||||
Positive |
SPL 4 |
0.012 |
0.013 |
0.013 |
1.3 |
1.3 |
0.1 |
Irritant |
0.012 |
||||||||
0.013 |
||||||||
SPL 5 |
0.013 |
0.013 |
1.3 |
|||||
0.013 |
||||||||
0.013 |
||||||||
SPL 6 |
0.011 |
0.012 |
1.2 |
|||||
0.011 |
||||||||
0.012 |
||||||||
Test item |
SPL 13 |
1.080 |
1.087 |
0.995 |
108.8 |
99.6 |
9.4 |
|
1.080 |
||||||||
1.100 |
||||||||
SPL 14 |
0.985 |
1.000 |
100.1 |
|||||
1.017 |
||||||||
0.998 |
||||||||
SPL 15 |
0.888 |
0.899 |
90.0 |
|||||
0.919 |
||||||||
0.889 |
||||||||
Test item |
SPL 18 |
0.054 |
0.055 |
0.069 |
5.5 |
6.9 |
1.9 |
|
0.057 |
||||||||
0.052 |
||||||||
SPL 19 |
0.083 |
0.082 |
8.2 |
|||||
0.083 |
||||||||
0.078 |
||||||||
Test item |
SPL 20 |
0.021 |
0.020 |
0.270 |
2.0 |
27.0 |
35.4 |
|
0.020 |
||||||||
0.019 |
||||||||
SPL 21 |
0.514 |
0.520 |
52.1 |
|||||
0.513 |
||||||||
0.531 |
||||||||
Test item |
SPL 16 |
0.060 |
0.062 |
0.072 |
6.2 |
7.2 |
1.4 |
|
0.062 |
||||||||
0.062 |
||||||||
SPL 17 |
0.082 |
0.082 |
8.2 |
|||||
0.082 |
||||||||
0.080 |
||||||||
Test item |
|
112.6 |
|
Non irritant |
# mean of 3 values (triplicate of the same extract).
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:
- 16 September 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 438 (Isolated Chicken Eye 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
- Qualifier:
- according to guideline
- Guideline:
- EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- chicken
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption.
- Number of animals: Not specified.
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old. 1.5 - 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The heads have been collected on 16 September 2019 at 8: 26 am.The eyes were enucleated at Phycher on 16 September 2019 at 10:05 am.
- indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no - 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):30 mg of test item - Duration of treatment / exposure:
- 10 seconds
- Duration of post- treatment incubation (in vitro):
- No post-treatment incubation is performed.
- Number of animals or in vitro replicates:
- 3
- Details on study design:
- SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled at 31.9°C.
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure using sodium fluorescein. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope.
Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that were not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes were rejected. (see table appendix No.4 on "Any other information on results incl. tables")
Once all eyes had been examined and approved, the eyes were incubated between 45 and 64 minutes to equilibrate them to the test system prior to dosing.
EQUILIBRATION AND BASELINE RECORDINGS:
Eyes were incubated between 45 and 64 minutes to equilibrate them to the test system prior to dosing (TG indicates approximately 45 to 60 min. This deviation is considered as without impact on the conclusion of the study)
Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.
NUMBER OF REPLICATES:3
NEGATIVE CONTROL USED
30 μL physiological saline - Dutscher Batch No. 3013524 (one eye)
SOLVENT CONTROL USED: not applicable.
POSITIVE CONTROL USED
Sodium hydroxide – Fisher Scientific, Batch No. 1550248 - 30 mg (three eyes)
APPLICATION DOSE AND EXPOSURE TIME
30 mg of the test item was applied for 10 seconds.
OBSERVATION PERIOD:Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.
REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: NO
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points (see table 4)
- Damage to epithelium based on fluorescein retention: Fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item (see table 5)
- Swelling: optical pachymeter on a slit-lamp microscope ((HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I). The slit-width was set at 9 1/2 equalling 0.095 mm. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item (see table 3).
- Macroscopic morphological damage to the surface: The aim of this evaluation was to determine whether any “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea were visible.These findings can vary in severity and may occur simultaneously.
SCORING SYSTEM:
- Mean corneal swelling: It was expressed as a percentage and was calculated from corneal thickness measurements according to the following formula:
(corneal thickness measurement at time t - corneal thickness at time=0 / corneal thickness at ime=0 )*100
- Mean maximum opacity score:
0 - No opacity,
0.5 -Very faint opacity
1- Scattered or diffuse areas; details of the iris clearly visible
2- Easily discernible translucent area; details of the ris are slightly obscured,
3-Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4-Complete corneal opacity; iris invisible
- Mean fluorescein retention score at 30 minutes post-treatment :
0-No fluorescein retention,
0.5-Very minor single cell staining,
1-Single cell staining scattered throughout the treated area of the cornea,
2-Focal or confluent dense single cell staining,
3-Confluent large areas of the cornea retaining fluorescein
DECISION CRITERIA: Decision criteria was used as indicated in the TG. - Irritation parameter:
- cornea opacity score
- Run / experiment:
- Highest mean
- Value:
- 0.8
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- ICE Class II
- Irritation parameter:
- fluorescein retention score
- Run / experiment:
- Mean
- Value:
- 2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- ICE class III
- Irritation parameter:
- percent corneal swelling
- Run / experiment:
- Highest mean
- Value:
- 14
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- ICE class II
- Irritation parameter:
- morphological effects
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: No effects
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: NO, no morphological effects were noted, whatever the examination time.
DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES, the combination of the three endpoints for the negative control, physiological saline, was 3xI classified as “No Category”
- Acceptance criteria met for positive control: YES, the combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV,classified as “Corrosive/Severe Irritant ” - Interpretation of results:
- other: No prediction can be made (CLP Regulation EC no. 1272/2008)
- Conclusions:
- Under experimental conditions, no prediction can be made for the test item in the ICE test.
- Executive summary:
An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hydroxide (positive control) or 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Under experimental conditions, no prediction can be made for the test item in the ICE test since the combinations of the 3 endpoints were 1 x III, 2 x II.
- Endpoint:
- eye irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 04 November 2019 - 08 November 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- According to OECD 405 with GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 405 (Acute Eye Irritation / Corrosion)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or tissues and environmental conditions:
- TEST ANIMALS
- Source: Granja San Bernardo (Tulebras, Navarra – Spain).
- Age at study initiation:15 weeks old
- Weight at study initiation: 3.17 kg.
- Housing: The animal was kept in an individual box installed in conventional air conditioned animal husbanding.
- Diet (e.g. ad libitum): ad libitum. ENVIGO – 2030C.
- Water (e.g. ad libitum): ad libitum. Drinking water (tap-water from public distribution system).
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 ºC
- Humidity (%): 30 to 70%. A relative humidity higher than the maximum recommended value was registered on 04 November 2019 (81%). As no effect was noted on the health of the animals, this deviation is considered as without impact on the conclusion of the study.
- Air changes (per hr): ≥ 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (07.00 to 19.00) / 12 hours dark. - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent no treatment
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg.
- Concentration (if solution): N/A - Duration of treatment / exposure:
- Residual test item was noted at the reading time of 1 hour after treatment, thus the eye was rinsed with physiological saline.
- Observation period (in vivo):
- Ocular examinations were performed on both eyes (treated and control) 1 hour, 24, 48 and 72 hours following treatment.
- Number of animals or in vitro replicates:
- 1 animal.
- Details on study design:
- REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with physiological saline because residual test item remained in the eye affter one hour.
- Time after start of exposure: 1 h
SCORING SYSTEM:
CHEMOSIS (A):
0-No swelling
1-Slight swelling, including the nictitating membrane
2-Swelling with eversion of the eyelid
3-Swelling with eyelid half-closed
4-Swelling with eyelid more than half-closed
DISCHARGE (B):
0-No discharge
1-Slight discharge (normal slight secretions in the inner corner not to be taken into account)
2-Discharge with moistening of the eyelids and neighbouring hairs
3-Discharge with moistening of the eyelids and large areas around the eye
REDNESS (C):
0-Blood vessels normal
1-Vessels significantly more prominent than normal
Vessels individually distinguishable with difficulty
2-Generalised red coloration
3-Generalised deep red coloration
IRIS (D):
0-Normal
1-Iris significantly more wrinkled than normal, congestion, swelling of the iris which continues to react to light, even slowly
2-No reaction to light, haemorrhage, significant damage (any or all of these characteristics)
CORNEA: DEGREE OF OPACITY (E):
0- No modification visible either directly or after instillation of fluorescein (no loss of glint or polish)
1- Translucent areas (diffuse or disseminated), iris details clearly visible
2- Easily identifiable translucent area, iris details slightly obscured
3- Opalescent area, no iris details visible, pupil outline scarcely distinguishable
4- Total corneal opacity, completely obscuring the iris and pupil
CORNEA: EXTENT OF OPACITY (F):
1- Opaque area present but covering one quarter or less
2- Between one quarter and half
3- Between half and three quarters
4- Between three quarters and the entire surface
TOOL USED TO ASSESS SCORE: not specified - Irritation parameter:
- cornea opacity score
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 3.7
- Max. score:
- 4
- Reversibility:
- not reversible
- Irritation parameter:
- iris score
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 0.7
- Max. score:
- 2
- Irritation parameter:
- conjunctivae score
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Reversibility:
- not reversible
- Remarks on result:
- not determinable
- Remarks:
- (black coloration preventing the redness quotation)
- Irritation parameter:
- chemosis score
- Basis:
- animal #1
- Time point:
- 24/48/72 h
- Score:
- 3
- Max. score:
- 3
- Reversibility:
- not reversible
- Irritant / corrosive response data:
- The ocular reactions observed during the study were important to severe:
- at the conjunctivae level: a black coloration of palpebral and bulbar conjunctivae preventing quotation of redness. A moderate chemosis noted 24 hours after the test item instillation and remaining on the last day of the test (Day 4) with same intensity (grade 3).
- at the iris level: no reaction to light was noted on Day 1.
- at the corneal level: a moderate opacity noted 24 hours after the test item instillation and remaining on the last day of the test (Day 4) with severe intensity (grade 4).
Considering the severity of the reactions, the animal was euthanized on day 4 in accordance with the principles of animal welfare as the study was stopped. - Interpretation of results:
- other: classified as serious eye damage (Cat 1) (CLP Regulation EC no. 1272/2008)
- Conclusions:
- Based on the results of an acute eye irritation study performed on New Zealand White rabbits, the test substance has to be classified in category 1 (serious eye damage) as irreversible effects were found on the eye.
- Executive summary:
The eye irritation potential of the test substance was determined in accordance with the OECD guideline 405 with GLP. One adult New Zealand White rabbit was given a single ocular application of 0.1 g test substance in one eye while the contralateral eye remained untreated and served as the control. Animal was observed at 1, 24, 48, 72 h after the test item was applied. The corneal opacity, iris and the conjunctivae (redness and chemosis) scores were recorded. The individual eye irritation scores at 24, 48 and 72 h post-application observations were 3, 4 and 4 for corneal opacity, 2, 0 and 0 for iris effects, 3, 3 and 3 for conjunctival chemosis. Conjunctival redness could not be measured due to a black coloration of palpebral and bulbar conjunctivae. Considering the severity of the reactions, the animal was euthanized on day 4 in accordance with the principles of animal welfare as the study was stopped. The moderate chemosis and opacity noted 24 hours after the test item instillation remained on the last day of the test (Day 4) with intensity of grade 3 and 4 respectively. Based on these results, the test substance has to be classified in category 1 (serious eye damage) according to CLP Regulation.
Referenceopen allclose all
Appendix No. 4: Selected eyes for the performance of the ICE test
Chamber |
Flurescein retention |
Corneal opacity |
Morphological effects |
Corneal thickness (e) |
1 |
0.5 |
0 |
N.t.R |
0.55 |
2 |
0.5 |
0 |
N.t.R |
0.52 |
3 |
0.5 |
0 |
N.t.R |
0.50 |
4 |
0.5 |
0 |
N.t.R |
0.52 |
5 |
0.5 |
0 |
N.t.R |
0.56 |
6 |
0.5 |
0 |
N.t.R |
0.50 |
7 |
0.5 |
0 |
N.t.R |
0.52 |
8 |
0.5 |
0 |
N.t.R |
0.51 |
9 |
0.5 |
0 |
N.t.R |
0.50 |
10 |
0.5 |
0 |
N.t.R |
0.50 |
11 |
0.5 |
0 |
N.t.R |
0.52 |
12 |
0.5 |
0 |
N.t.R |
0.53 |
13 |
0.5 |
0 |
N.t.R |
0.52 |
14 |
0.5 |
0 |
N.t.R |
0.52 |
15 |
0.5 |
0 |
N.t.R |
0.48 |
16 |
0.5 |
0 |
N.t.R |
0.51 |
N.t.R: Nothing to report
Table 9: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT
Test item
Endpoint measured |
Eye No. |
0 |
30 |
75 |
120 |
180 |
240 |
Corneal opacity |
10 |
0 |
1 |
1 |
1 |
1 |
1 |
11 |
0 |
1 |
1 |
1 |
1 |
1 |
|
12 |
0 |
0.5 |
0.5 |
0.5 |
0.5 |
0.5 |
|
Mean |
|
0.0 |
0.8 |
0.8 |
0.8 |
0.8 |
0.8 |
ICE class |
|
|
|
|
II |
|
|
Fluorescein retention |
10 |
0.5 |
2 |
|
|
|
|
11 |
0.5 |
2 |
|
|
|
|
|
12 |
0.5 |
2 |
|
|
|
|
|
Mean |
|
0.5 |
2 |
|
|
|
|
ICE class |
|
|
III |
|
|
|
|
Corneal thickness |
10 |
0.54 |
0.54 |
0.56 |
0.56 |
0.60 |
0.60 |
11 |
0.52 |
0.53 |
0.53 |
0.54 |
0.60 |
0.60 |
|
12 |
0.53 |
0.56 |
0.56 |
0.56 |
0.56 |
0.56 |
|
Corneal swelling (%) |
10 |
- |
8 |
12 |
12 |
20 |
20 |
11 |
- |
2 |
2 |
4 |
15 |
15 |
|
12 |
- |
6 |
6 |
6 |
6 |
6 |
|
Mean |
|
|
5 |
7 |
7 |
14 |
14 |
ICE class |
|
|
|
|
II |
|
|
Combination of the 3 Endpoints |
2 x II, 1 x III |
||||||
CLASSIFICATION |
No prediction can be made |
Note: No morphological effects were noted, whatever the examination time.
Table 8: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT
Positive control
Endpoint measured |
Eye No. |
0 |
30 |
75 |
120 |
180 |
240 |
Corneal opacity |
1 |
0 |
4 |
4 |
4 |
4 |
4 |
2 |
0 |
4 |
4 |
4 |
4 |
4 |
|
3 |
0 |
4 |
4 |
4 |
4 |
4 |
|
Mean |
|
0.0 |
4.0 |
4.0 |
4.0 |
4.0 |
4.0 |
ICE class |
|
|
|
|
IV |
|
|
Fluorescein retention |
1 |
0.5 |
3 |
|
|
|
|
2 |
0.5 |
3 |
|
|
|
|
|
3 |
0.5 |
3 |
|
|
|
|
|
Mean |
|
0.5 |
3.0 |
|
|
|
|
ICE class |
|
|
IV |
|
|
|
|
Corneal thickness |
1 |
0.55 |
- |
- |
- |
- |
- |
2 |
0.52 |
- |
- |
- |
- |
- |
|
3 |
0.50 |
- |
- |
- |
- |
- |
|
Corneal swelling (%) |
1 |
(-) |
(-) |
(-) |
(-) |
(-) |
(-) |
2 |
(-) |
(-) |
(-) |
(-) |
(-) |
(-) |
|
3 |
(-) |
(-) |
(-) |
(-) |
(-) |
(-) |
|
Mean |
|
- |
- |
- |
- |
- |
- |
ICE class |
|
|
|
|
IV |
|
|
Combination of the 3 Endpoints |
3 x IV |
||||||
CLASSIFICATION |
Category 1 : Corrosive/Severe irritant |
Note:
(-): Severe loosening of the corneal epithelium noted from 30 minutes post-dose in eyes No. 1, No. 2 and No. 3
Table 7: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT
Negative control
Endpoint measured |
Eye No. |
0 |
30 |
75 |
120 |
180 |
240 |
Corneal opacity |
16 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
ICE class |
|
|
|
|
I |
|
|
Fluorescein retention |
16 |
0.5 |
0.5 |
- |
- |
- |
- |
ICE class |
|
|
I |
|
|
|
|
Corneal thickness |
16 |
0.51 |
0.53 |
0.53 |
0.53 |
0.53 |
0.53 |
Corneal swelling (%) |
16 |
- |
4 |
4 |
4 |
4 |
4 |
ICE class |
|
|
|
|
I |
|
|
Combination of the 3 Endpoints |
3 x I |
||||||
CLASSIFICATION |
No Category |
Note: No morphological effects were noted, whatever the examination time.
Tables of results. Assessment of acute eye irritation. Individual and mean scores of conjunctivae, iris and cornea
TEST ITEM: Violet LCC6D
Instillation: 0.1 g of the test item
Instillation dates (D0): 04 November 2019
Animal Nº: A7755
Table 1
Observation |
CONJUNCTIVAE |
IRIS |
CORNEA |
Individual |
||||||
A |
B |
C |
(A+B+C)x2 |
D |
Dx5 |
E |
F |
ExFx5 |
||
|
|
|
|
X= |
|
Y= |
|
|
Z= |
X+Y+Z= |
1 Hour (D0) |
2 |
3 |
* |
Not calculable |
* |
Not calc. |
* |
* |
Not calc. |
Not calculable |
24 Hours (D1) |
3 |
1 |
* |
Not calculable |
2 |
10 |
3 |
4 |
60 |
Not calculable |
48 Hours (D2) |
3 |
1 |
* |
Not calculable |
0 |
0 |
4 |
3 |
60 |
Not calculable |
72 Hours (D3) |
3 |
1 |
* |
Not calculable |
0 |
0 |
4 |
3 |
60 |
Not calculable |
Day 4 (D4) |
3 |
1 |
* |
Not calculable |
0 |
0 |
4 |
3 |
60 |
Not calculable |
Notes:
(*) black coloration of palpebral and bulbar conjunctivae preventing quotation of redness, corneal opacity reading and the iris observation.
Considering the severe reactions observed, the animal was euthanized on day 4.
Table 2
Animal n° |
Time after |
CONJUNCTIVAE |
IRIS |
CORNEA |
|
CHEMOSIS (A) |
REDNESS ( C) |
LESION (D) |
OPACITY ( E) |
||
A7755 |
24 hours |
3 |
* |
2 |
3 |
48 hours |
3 |
* |
0 |
4 |
|
72 hours |
3 |
* |
0 |
4 |
|
Start: 3.17 |
TOTAL |
9 |
Not calc. |
2 |
11 |
End: 3.16 |
Mean |
3.0 |
Not calc. |
0.7 |
3.7 |
CLASSIFICATION |
Taking into account the severity of the reactions the test item has to be classified in Category 1 "Irreversible effects on the eyes" with the hazard statement H318 "Causes serious eye damage" |
Notes:
(*) black coloration of palpebral and bulbar conjunctivae preventing quotation of redness.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irreversible damage)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Serious eye damage/eye irritation: In vitro studies were first performed in order to decide on the classification of the substance. Results from in vitro test were inconclusive thus an acute eye irritation/corrosion test in accordance with OECD Guideline 405 was started. Irreversible effects were found on the eye treated with the test substance and it was concluded that the test substance should be classified in category 1 (serious eye damage) according to CLP Regulation.
Justification for classification or non-classification
Skin irritation/corrosion. Based on the available information, the substance is not classified for skin irritation/corrosion in accordance with CLP, Regulation (EC) No. 1272/2008.
Eye irritation/serious eye damage: Based on the available data, the substance is classified as serious eye damage (Cat 1) according to CLP Regulation no. 1272/2008.
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