Registration Dossier

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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

Referenceopen allclose all

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.

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.


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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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

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.


 


 

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. 

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.