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EC number: 950-576-0 | CAS number: -
- 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 (OECD TG 439): not irritating
Skin corrosion (OECD TG 431): not corrosive
Eye irritation (OECD TG 437): not irritating
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30 April - 05 July 2019
- 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)
- Version / remarks:
- 2016
- Deviations:
- no
- GLP compliance:
- yes
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- other: epidermal keratinocytes
- Cell source:
- other: MatTek Corporation, Ashland MA, U.S.A.
- Source strain:
- other: not applicable
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): Lot no.: 30903 kit A and kit B
- Production date: 3 July 2019
- Date of initiation of testing: 30 April
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 62 - 95%, containing 5.0 ± 0.5% CO2 in air in the dark at 36.3 - 37.1°C.
- Temperature of post-treatment incubation: 37.0 ± 1.0ºC
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one step, washing done with PBS
- 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: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
NUMBER OF REPLICATE TISSUES:
- 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to SHR 1396 and two for a 1-hour exposure.
- For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point.
ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
DECISION CRITERIA
A test item is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
- In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount applied: 50 µL of the undiluted test item was added into the 6-well plates on top of the skin tissues
- Duration of treatment / exposure:
- 3 minutes and 60 minutes
- Number of replicates:
- 2
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 minutes treatment
- Value:
- 107
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- mean tissue viability: 7.4%
- Remarks on result:
- other: not corrosive
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 hour treatment
- Value:
- 101
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- mean tissue viability: 5.7%
- Remarks on result:
- other: not corrosive
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range
- Acceptance criteria met for positive control: The mean relative tissue viability following the 1-hour exposure to the positive control was 5.7%.
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 13%, indicating that the test system functioned properly. - Interpretation of results:
- other: not corrosive
- Remarks:
- According to Regulation (EC) No. 1272/2008 and its amendments.
- Conclusions:
- The results of an in vitro skin corrosion test showed that SHR 1396 was not corrosive to the skin.
- Executive summary:
An in vitro skin corrosion test was performed following OECD guidelines and GLP principles. The test item SHR 1396 was applied undiluted (50 µL) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 5.7% after the 1-hour exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues waswithin the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 13%,indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with SHR 1396 compared to the negative control tissues was 107% and 101%, respectively. Because the mean relative tissue viability for SHR 1396 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment SHR 1396 is considered to be not corrosive and does not need to be classified.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 June 2019 - 17 June 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)
- Version / remarks:
- 28 July 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- 20 July 2012
- Deviations:
- no
- GLP compliance:
- yes
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- other: epidermal keratinocytes
- Cell source:
- other: SkinEthic Laboratories, Lyon, France.
- Source strain:
- other: not applicable
- Justification for test system used:
- In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model TM (EPISKIN-SM TM, 0.38 cm^2)
- Tissue batch numbers: 19-EKIN-024
- Production date: 17 June 2019
- Date of initiation of testing: 11 June 2019
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 81 - 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.2°C).
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 2 (phosphate buffered saline)
- Observable damage in the tissue due to washing: no
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader.
- Wavelength: 570 nm
NUMBER OF REPLICATE TISSUES: 3 tissues per test item per experiment, together with positive and negative control.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 exposure time.
EVALUATION
The corrected OD (ODc) for each sample or control will be calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
SHR 1396 was checked for possible color interference and possible direct MTT reduction before the study was started. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount applied: 25 μL, undiluted
NEGATIVE CONTROL
- Amount applied: 25 μL
POSITIVE CONTROL
- Amount applied: 25 μL
- Concentration (in PBS): 5% - Duration of treatment / exposure:
- 15 ± 0.5 minutes at room temperature
- Duration of post-treatment incubation (if applicable):
- 42 hours at 37°C
- Number of replicates:
- 3 tissues per test item per experiment together with negative and positive controls
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Experiment 1
- Value:
- 100
- Negative controls validity:
- valid
- Remarks:
- 100 %
- Positive controls validity:
- valid
- Remarks:
- 8.9 %
- Remarks on result:
- no indication of irritation
- Remarks:
- The standard deviation value of the percentage viability of three tissues treated identically was < 9%, indicating that the test system functioned properly.
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction/Colour interference with MTT: Because no color changes were observed it was concluded that SHR 1396 did not interact with the MTT endpoint.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 8.9%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was < 9%, indicating that the test system functioned properly. - Interpretation of results:
- other: Not skin irritating.
- Remarks:
- According to Regulation (EC) No. 1272/2008 and its amendments.
- Conclusions:
- The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
Since the mean relative tissue viability for SHR 1396 was above 50%, SHR 1396 is considered to be non-irritant. - Executive summary:
An in vitro skin irritation test was performed according to OECD 439 test guideline and GLP principles. SHR 1396 was a clear colorless liquid. The test item was applied undiluted (25 μL) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation was expressed as the remaining cell viability after exposure to the test item. The positive control (SDS) had a mean cell viability of 8.9% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control (PBS) tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 9%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 100%. Since the mean relative tissue viability for SHR 1396 was above 50% after 15 ± 0.5 minutes treatment SHR 1396 is considered to be non-irritant. In conclusion, SHR 1396 is non-irritant in the in vitro skin irritation test.
Referenceopen allclose all
Table 1: Mean Absorption values
|
3-minute application |
1-hour application |
||||||||
A (OD570) |
B (OD570) |
Mean (OD570) |
SD |
A (OD570) |
B (OD570) |
Mean (OD570) |
SD |
|||
Negative control |
1.647 |
1.657 |
1.652 |
± |
0.007 |
1.788 |
1.559 |
1.673 |
± |
0.162 |
SHR 1396 |
1.800 |
1.727 |
1.763 |
± |
0.051 |
1.715 |
1.677 |
1.696 |
± |
0.027 |
Positive control |
0.113 |
0.131 |
0.122 |
± |
0.013 |
0.086 |
0.103 |
0.095 |
± |
0.012 |
SD = Standard deviation
Duplicate exposures are indicated by A and B.
In this table the values are corrected for background absorption (0.045). Isopropanol was used to measure the background absorption.
Table 2: Mean Tissue Viability
|
3-minute application viability (percentage of control) |
1-hour application viability (percentage of control) |
Negative control |
100 |
100 |
SHR 1396 |
107 |
101 |
Positive control |
7.4 |
5.7 |
Table 3: Coefficient of Variation between Tissue Replicates
|
3 minute |
1 hour |
Negative control |
0.6 |
13 |
SHR 1396 |
4.0 |
2.2 |
Positive control |
14 |
16 |
CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]
Table 4: Individual OD measurements
|
3-minute application (OD570) A B |
1-hour application (OD570) A B |
||
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3 |
|
|
||
1.6880 |
1.6929 |
1.8350 |
1.6080 |
|
1.6925 |
1.7053 |
1.8375 |
1.5989 |
|
1.6959 |
1.7082 |
1.8254 |
1.6037 |
|
Test item OD570measurement 1 OD570measurement 2 OD570measurement 3 |
|
|
||
1.8530 |
1.7640 |
1.7682 |
1.7250 |
|
1.8358 |
1.7813 |
1.7528 |
1.7271 |
|
1.8444 |
1.7710 |
1.7578 |
1.7123 |
|
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3 |
|
|
||
0.1580 |
0.1774 |
0.1313 |
0.1477 |
|
0.1581 |
0.1761 |
0.1316 |
0.1482 |
|
0.1580 |
0.1754 |
0.1308 |
0.1469 |
OD = Optical density
Duplicate exposures are indicated by A and B.
Table 5: Historical control data
Negative control |
Positive control |
|||
3-minute treatment (OD570) |
1-hour treatment (OD570) |
3-minute treatment (OD570) |
1-hour treatment (OD570) |
|
Range |
1.258 – 2.615 |
1.371 – 2.371 |
0.092 – 0.56 |
0.046 – 0.339 |
Mean |
1.73 |
1.78 |
0.19 |
0.14 |
SD |
0.24 |
0.21 |
0.09 |
0.05 |
n |
116 |
119 |
114 |
112 |
SD = Standard deviation
n = Number of observations
The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2015 to November 2018.
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:
- 25 Apr 2019 - 25 Apr 2019
- 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)
- Version / remarks:
- 2017
- Deviations:
- no
- GLP compliance:
- yes
- Species:
- cattle
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- 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 applied: 750 µL, undiluted
CONTROLS
Positive control: Ethanol
Amount applied: 750 µL
Negative control: Physiological saline
Amount applied: 750 µL - Duration of treatment / exposure:
- 10 ± 1 minutes at 32 ± 1°C.
- Duration of post- treatment incubation (in vitro):
- 120 ± 10 minutes at 32 ± 1°C.
- Details on study design:
- SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
QUALITY CHECK OF THE ISOLATED CORNEAS : Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.
NUMBER OF REPLICATES : 3 corneas per treatment group.
NEGATIVE CONTROL USED : Physiological saline
POSITIVE CONTROL USED : Ethanol
APPLICATION DOSE AND EXPOSURE TIME
TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
POST-INCUBATION PERIOD: yes/no. If YES please specify duration
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
- POST-EXPOSURE INCUBATION:
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected
opacity values of the treated corneas for each treatment group.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein /mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were
incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C. After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader).
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
DECISION CRITERIA: The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in table 1 (see other information on materials and methods) - Irritation parameter:
- in vitro irritation score
- Remarks:
- mean
- Run / experiment:
- Experiment 1
- Value:
- 0.5
- Negative controls validity:
- valid
- Remarks:
- -0.1
- Positive controls validity:
- valid
- Remarks:
- 48
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- cornea opacity score
- Remarks:
- mean
- Run / experiment:
- Experiment 1
- Value:
- 0.5
- Negative controls validity:
- valid
- Remarks:
- 0.5
- Positive controls validity:
- valid
- Remarks:
- 22
- Remarks on result:
- no indication of irritation
- Irritation parameter:
- other: mean permeability
- Run / experiment:
- Experiment 1
- Value:
- 0.002
- Negative controls validity:
- valid
- Remarks:
- -0.001
- Positive controls validity:
- valid
- Remarks:
- 1.748
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
DEMONSTRATION OF TECHNICAL PROFICIENCY: The mean in vitro irritancy score of the positive control (Ethanol) was 48 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes - Interpretation of results:
- other: Not eye irritating/corrosive
- Remarks:
- According to Regulation (EC) No. 1272/2008 and its amendments.
- Conclusions:
- In conclusion, since SHR 1396 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
- Executive summary:
A Bovine Corneal Opacity and Permeability (BCOP) test was performed following OECD guidelines and GLP principles. The test item was applied as it is (750 µL) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The meanin vitro irritancy score of the positive control (Ethanol) was 48 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. SHR 1396 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.5 after 10 minutes of treatment. In conclusion, since SHR 1396 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Reference
Table 1: Summary of Opacity, Permeability and In Vitro Scores
Treatment |
Mean Opacity1 |
Mean Permeability1 |
MeanIn vitroIrritation Score1, 2 |
Negative control |
-0.1 |
-0.001 |
-0.1 |
Positive control (Ethanol) |
22 |
1.748 |
48 |
SHR 1396 |
0.5 |
0.002 |
0.5 |
1 Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test item.
2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490value).
Table 2: Opacity Score
Treatment |
Opacity before treatment |
Opacity after treatment |
Final Opacity1 |
Negative control corrected Final Opacity2 |
Mean Final Opacity |
|
|
||||||
Negative control |
2.1 |
2.4 |
0.3 |
|
-0.1 |
|
3.1 |
2.5 |
-0.6 |
||||
2.9 |
3.1 |
0.2 |
||||
|
||||||
Positive control |
3.5 |
27.2 |
23.8 |
24 |
22 |
|
3.5 |
24.4 |
21.0 |
21 |
|||
4.0 |
25.4 |
21.4 |
21 |
|||
|
||||||
SHR 1396 |
3.2 |
2.9 |
-0.3 |
-0.3 |
0.5 |
|
3.0 |
4.1 |
1.1 |
1.1 |
|||
3.1 |
3.8 |
0.7 |
0.7 |
|||
Calculations are made without rounding off.
1 Final Opacity = Opacity after treatment – Opacity before treatment.
2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control. 3
3 In case the mean final opacity of the negative control is below zero, no correction will be made.
Table 3: Permeability Score Individual Values (Corrected)
Treatment |
Dilution factor |
Negative control corrected OD49011 |
Negative control corrected OD49021 |
Negative control corrected OD49031 |
Negative control corrected OD490 Average |
Negative control corrected final OD490 |
Average OD |
|
|||||||
Positive control |
6 |
0.405 |
0.397 |
0.396 |
0.399 |
2.397 |
1.748 |
6 |
0.347 |
0.350 |
0.352 |
0.350 |
2.099 |
||
1 |
0.752 |
0.747 |
0.748 |
0.749 |
0.749 |
||
|
|||||||
SHR 1396 |
1 |
0.002 |
-0.001 |
-0.002 |
0.000 |
0.000 |
0.002 |
1 |
0.002 |
0.003 |
0.002 |
0.002 |
0.002 |
||
1 |
0.003 |
0.003 |
0.006 |
0.004 |
0.004 |
Calculations are made without rounding off.
1 OD490values corrected for the mean final negative control permeability (-0.001).
Table 4: In Vitro Irritancy Score
Treatment |
Final Opacity2 |
Final OD4902 |
In vitroIrritancy Score1 |
|
|||
Negative control |
0.3 |
-0.001 |
0.2 |
-0.6 |
-0.002 |
-0.7 |
|
0.2 |
0.000 |
0.2 |
|
|
|||
Positive control |
24 |
2.397 |
60 |
21 |
2.099 |
52 |
|
21 |
0.749 |
33 |
|
|
|||
SHR 1396 |
-0.3 |
0.000 |
-0.3 |
1.1 |
0.002 |
1.1 |
|
0.7 |
0.004 |
0.8 |
1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490value).
2 Positive control and test item are corrected for the negative control.
Table
5: Historical Control Data for the BCOP Studies
Negative control |
Positive control |
|||
Opacity |
Permeability |
In vitroIrritancy Score |
In vitroIrritancy Score |
|
Range |
-2.0 – 3.0 |
-0.034 – 0.100 |
-2.2 – 3.0 |
26.0 – 89.6 |
Mean |
0.49 |
0.00 |
0.54 |
52.96 |
SD |
1.24 |
0.01 |
1.28 |
13.46 |
n |
118 |
118 |
118 |
114 |
SD = Standard deviation
n = Number of observations
The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Mar 2016 to Mar 2019.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin irritation:
An in vitro skin irritation test was performed according to OECD 439 test guideline and GLP principles. SHR 1396 was a clear colorless liquid. The test item was applied undiluted (25 μL) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation was expressed as the remaining cell viability after exposure to the test item. The positive control (SDS) had a mean cell viability of 8.9% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control (PBS) tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 9%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 100%. Since the mean relative tissue viability for SHR 1396 was above 50% after 15 ± 0.5 minutes treatment SHR 1396 is considered to be non-irritant. In conclusion, SHR 1396 is non-irritant in the in vitro skin irritation test.
Skin corrosion:
An in vitro skin corrosion test was performed following OECD guidelines and GLP principles. The test item SHR 1396 was applied undiluted (50 µL) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 5.7% after the 1-hour exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 13%,indicating that the test system functioned properly. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with SHR 1396 compared to the negative control tissues was 107% and 101%, respectively. Because the mean relative tissue viability for SHR 1396 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment SHR 1396 is considered to be not corrosive and does not need to be classified.
Eye irritation:
A Bovine Corneal Opacity and Permeability (BCOP) test was performed following OECD guidelines and GLP principles. The test item was applied as it is (750 µL) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 48 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. SHR 1396 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.5 after 10 minutes of treatment. In conclusion, since SHR 1396 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Justification for classification or non-classification
Based on the available study results, SHR 1396 does not have to be classified and has no obligatory labelling requirement for skin irritation/corrosion and eye irritation according to Regulation (EC) No 1272/2008 and its amendments.
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