Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin corrosion: Key study. OECD 431, GLP study. The test item is not skin corrosive since the mean percent viabilities of the treated tissues were 99.3% (for 3 min exposure) and 80.7% (for 60 min exposure), i.e. >50% and >15% respectively.

Skin irritation: Key study: OECD 439, GLP study. The test item is irritating to the skin (category 2) since the tissue viability after exposure and post-treatment incubation was 9.3%, i.e. less that 50%.

Eye Irritation: Key study: OECD 437, GLP study. The test item was determined to be classified for Category 1 since the mean IVIS value of tested substance was found to be 161.84, i.e. higher than 55.

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:
December 12, 2017 - December 16, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
SkinEthicTM RHE
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Justification for test system used:
The human skin model, epidermis has been validated for corrosion testing and it can be use for skin corrosion test accordiing the OECD guideline 431.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model
- Tissue batch number(s): 17-RHE-127
- Delivery date: 12/12/2017
- Date of initiation of testing: 12/12/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1 °C for 60 minute exposure and room temperature for 3 minute exposure.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times in constant soft stream of 1 mL DPBS
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 uL of 1.00 mg/mL
- Incubation time: 180 min.
- Spectrophotometer: absorbance microplate reader
- Wavelength: 570 nm
- Filter: no

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Specification O.D. >0.7, Results: O.D.=1.1
- Barrier function: Using TRITON X-100 1%. Specification: 4.0 h<=ET50>=10.0 h. Results:4.5 h
- Morphology: Specification: number of cell layers >=4. Results: 6 cell layers (absence of significant histological abnormalities)
- Contamination: No

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
- Freeze killed tissues (for positive control)-Non-specific MTT reduction calculation (NSMTT)
- Procedure used to prepare the killed tissues (if applicable): Killed tissues were prepared by incubating the viable tissues at -20 ± 5°C for 48 hours.
- N. of replicates: 2
- Method of calculation used: The true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the positive minus the percent non-specific MTT reduction obtained with the killed tissues exposed to the same, calculated relative to the negative control run concurrently (%NSMTT). NSMTT was < 0% relative to the negative control, hence true MTT metabolic conversion of treated tissue was not determined.

- Live tissues (negative control and test item): Non-specific Color (NSC)
- N. of replicates : 2
- Method of calculation used: The true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solutions minus the percent non specific color obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT.For positive control test item, %NSC was between 0 to 0.1 % relative to the negative control, hence TOD and relative viability calculation was not determined.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
If the viability after 3 minutes exposure is strictly less than 50% or greater or equal to 50% and the viability after 1 hour exposure is strictly less than 15 %, test item has to be classified as CORROSIVE.

If the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15 %, test item has to be classified as NON-CORROSIVE.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg ± 3 mg of test item/0.5 cm2

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL/0.5 cm2 of sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 40 µL/0.5 cm2 of 8N KOH
Duration of treatment / exposure:
2 exposure periods: 30 and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours (incubation in MTT solution)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
ca. 99.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure
Value:
ca. 80.7
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: For adapted positive control treated tissues, NSMTT was < 0% relative to the negative control, therefore true MTT metabolic conversion of treated tissue is not determined.
- Colour interference with MTT: For adapted controls to correct colour interference due to test item, treated tissues were exposed for period of 3 minute at room temperature and 60 minutes at 37±1 °C and 5±1% CO2 in a 95% humidified incubator. %NSC was between 0 to 0.1% relative to the negative control, hence TOD and relative viability calculation were not determined.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes. It was performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Criteria: 0.8>=OD<=3.0. Results:2.271-2.404. Criteria met.
- Acceptance criteria met for positive control: Cell viability should be less than 15% according the guideline. The results: Cell viability =5.31%. Criteria met.
- Acceptance criteria met for variability between replicate measurements: The viability should not exceed 30% between tissue replicates. Viability was less than 1.46%.Criteria met.

Pre-Tests

Color Interference Test

Treatment

Optical Density (nm)

Interaction

Negative Control (Isopropanol)

0.043

No

0.049

4-Trifluoromethylsalicylic Acid (ATFMS)

0.257

Yes

0.261

Direct MTT Reduction Test

Treatment

Interaction

Negative Control (Maintenance medium)

No

4-Trifluoromethylsalicylic Acid (ATFMS)

No

Mean percent viability of the 4-trifluoromethylsalicylic acid (ATFMS)

Treatment

Viability

3 Minutes Exposure

60 Minutes Exposure

Negative control

(Sterile distilled water)

100%

100%

4-Trifluoromethylsalicylic Acid (ATFMS)

99.3%

80.7%

Positive control

(8N KOH)

-

5.31%

Individual results (positive and negative control)

Treatment

Exposure

Time

Tissue Replicate

O.D.

Corrected O.D.

Mean of Corrected O.D.

Mean O.D. of Replicate tissues

% Viability/

Tissue

Mean % Viability

S.D. of % Viability

% C.V. of % Viability

Corrosivity Class

Negative Control

(Sterile Distilled water)

3 Minutes

1

2.352

2.309

2.294

2.328

100

100

NA

NA

NA

2.331

2.288

2.328

2.285

2

2.411

2.368

2.353

2.434

2.391

2.343

2.3

3

2.329

2.286

2.336

2.433

2.39

2.374

2.331

60 Minutes

1

2.408

2.365

2.306

2.324

100

100

NA

NA

2.324

2.281

2.314

2.271

2

2.335

2.292

2.299

2.332

2.289

2.360

2.317

3

2.447

2.404

2.368

2.342

2.299

2.444

2.401

Positive Control

(8N KOH)

60 Minutes

1

0.172

0.129

0.125

0.123

5.38

5.31

0.07

1.32

Corrosive

0.165

0.122

0.166

0.123

2

0.172

0.129

0.123

5.29

0.167

0.124

0.160

0.117

3

0.150

0.107

0.122

5.25

0.179

0.136

0.167

0.124

Individual results (test item)

Treatment

Exposure

Time

Tissue Replicate

O.D.

Corrected O.D.

Mean of Corrected O.D.

Mean O.D. of Replicate tissues

% Viability/

Tissue

Mean % Viability

S.D. of % Viability

% C.V. of % Viability

Corrosivity Class

4-Trifluoromethylsalicylic acid (ATFMS)

3 Minutes

1

2.453

2.411

2.343

2.312

100.6

99.3

1.15

1.16

Non-corrosive

2.356

2.314

2.347

2.305

2

2.332

2.29

2.290

98.4

2.345

2.303

2.318

2.276

3

2.324

2.282

2.303

98.9

2.351

2.309

2.36

2.318

60 Minutes

1

1.923

1.881

1.908

1.877

82.1

80.7

1.18

1.46

1.98

1.938

1.948

1.906

2

1.899

1.857

1.862

80.1

1.895

1.853

1.918

1.876

3

1.882

1.84

1.86

80

1.901

1.859

1.922

1.88

Adapted Control forNon-Specific MTT Reduction (NSMTT)

Treatment

Exposure Time

Tissue Replicate

O.D.

Corrected O.D.

Mean of Corrected O.D.

Mean OD of Replicate tissues

% NSMTT

Mean % NSMTT

TODTT

% Relative Viability

Negative Control

(Untreated killed tissues)

60 minutes

1

0.17

0.128

0.127

0.129

NA

NA

NA

NA

0.17

0.128

0.168

0.126

2

0.175

0.133

0.13

0.168

0.126

0.172

0.13

Positive Control

(Positive control treated killed tissues)

60 minutes

1

0.046

0.004

0.005

0.005

-5.3

-5.4

NA

NA

0.047

0.005

0.047

0.005

2

0.046

0.004

0.004

-5.4

0.045

0.003

0.046

0.004

Adapted Control for Non-Specific Color (NSC)

Treatment

Exposure Time

Tissue Replicate

O.D.

Corrected O.D.

Mean of Corrected O.D.

Mean OD of Replicate tissues

% NSC

Mean % NSC

TODCT

% Relative Viability

Negative Control

 

3 minutes

1

0.043

0.001

0.001

0.001

NA

NA

NA

NA

0.044

0.002

0.042

0

2

0.043

0.001

0.000

0.041

-0.001

0.042

0

Negative Control

 

60 minutes

1

0.042

0

0.000

0.001

NA

NA

NA

NA

0.043

0.001

0.041

-0.001

2

0.043

0.001

0.001

0.042

0

0.043

0.001

4-Trifluoromethylsalicylic acid (ATFMS)

3 minutes

1

0.046

0.004

0.003

0.003

0.1

0.1

NA

NA

0.045

0.003

0.045

0.003

2

0.045

0.003

0.002

0.0

0.044

0.002

0.044

0.002

4-Trifluoromethylsalicylic acid (ATFMS)

60 minutes

1

0.045

0.003

0.002

0.002

0.0

0.0

NA

NA

0.043

0.001

0.043

0.001

2

0.043

0.001

0.001

0.0

0.044

0.002

0.043

0.001

Interpretation of results:
GHS criteria not met
Conclusions:
Under experimental conditions, the test item was concluded to be non-corrosive in IN VITRO skin corrosion test using reconstructed human epidermins (RHE) tissues.
Executive summary:

An in vitro skin corrosion test for the test item was performed in a reconstructed human epidermis SkinEthic RHE model, according to OECD TG 431 (GLP study). The tissues were exposed to test item and sterile distilled water (negative control) for 3 minutes at room temperature and 60 minutes at 37±1 °C and 5±1% CO2. Since potassium hydroxide is direct MTT reducer, adapted control with two freeze killed tissues were used and treated with 8N KOH for exposure of 60 minutes. To evaluate the non-specific OD due to the residual test item staining, adapted control with two live tissues were used for test item and negative control for exposure of 3 minutes and 60 minutes. Percent relative viability in the tissues treated with the test item was 99.3% at 3 minute exposure period and 80.7% at 60 minute exposure period. Significant reduction in percent cell viability was not observed at the either 3 minute or 60 minute exposure period in the treated tissues when compared with the concurrent negative control. Differences between the viability of treated tissues was ≤ 1.46% i.e. %CV. OD values for negative control replicates were between 2.271 -2.404 and positive control showed 5.31 % cell viability. All criteria were valid. From the results of this study, the test item is concluded as non-corrosive using reconstructed human epidermins (RHE) tissues.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 February 2018 - 04 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This study addresses the human health endpoint skin irritation. It makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RhE) is also recommended by the OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies. The results of the study are believed to be of value in predicting the potential of inducing skin irritation by the test item in humans.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model
- Tissue batch number(s): N°18-RHE-029

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 25 times in a constant soft stream of 1 mL DPBS from 5-8 cm distance from the insert.
- Observable damage in the tissue due to washing: No.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 180 minutes
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues:Yes.
- N. of replicates : 2
- Method of calculation used: As test item detected as able to stain the tissues, was evaluated the non-specific OD due to the residual test item staining (unrelated to any mitochondrial activity) and subtract it before calculations of the “true” viability %.


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
The test is considered to be irritant to skin, if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
The test item is considered as non-irritant to skin, if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
16 ± 2 mg of test item/0.5 cm2
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 (mean of 3 replicates)
Value:
9.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: Test item did not produce direct MTT reduction when compared to concurrent negative control (Maintenance meduim).
- Colour interference with MTT: Difference in absorbance due to color interference was observed between negative control (isopropanol) and test item. Therefore results of the color interference test shows slight interference in optical density due to the test item.

DEMONSTRATION OF TECHNICAL PROFICIENCY: JRF Study Number: 618-1-06-9641

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The data met the acceptance criteria since the mean OD value of the 3 tissues was ≥ 1.2 at 570 ± 30 nm according to the historical database. The Standard Deviation value is considered as valid if it is ≤ 18%. The OD values (Corrected ODs) of negative controls in all tissues were between 1.221 to 1.273

- Acceptance criteria met for positive control:
The data met the acceptance criteria since the mean viability, expressed as % of the NC, was < 40 % and the Standard Deviation value is ≤ 18 %. Mean % viability of the positive control was 1.5%

- Acceptance criteria met for variability between replicate measurements:
Standard deviation of each intra-batch mean (3 Replicates/Tissue and 3 Tissue/Run) was < 18%.

- Range of historical values if different from the ones specified in the test guideline:
Optical Density at 570±30 nm
Exposure time: 42-minute exposure time
Negative Control (Dulbecco's phosphate-buffered saline):
Mean: 2.081
Standard deviation: 0.208
Minimum: 1.964
Maximum:2.776
Positive Control (Sodium dodecyl sulfate, 5% aqueous)
Mean: 0.028
Standard deviation: 0.007
Minimum: 0.021
Maximum: 0.053

Data summary of percent viability:

Treatment

Tissue Replicate

O.D. at 570 nm

Blank Corrected O.D.

Mean of Corrected O.D.

Mean O.D. of Three Tissues

% Viability/

Tissue

Mean % Viability

S.D. of % Viability

C.V. of % Viability

Corrosivity Class

Negative Control

(Dulbecco’s Phosphate Buffered Saline (DPBS))

1

1.274

1.231

1.238

1.238

100

100

0.01

0.81

NA

1.282

1.239

1.287

1.244

2

1.280

1.237

1.231

1.264

1.221

1.277

1.234

3

1.270

1.227

1.244

1.276

1.233

1.316

1.273

4-Trifluoromethylsalicylic Acid (ATFMS)

1

0.157

0.115

0.114

0.12

9.2

9.7

0.44

4.54

Category 2

0.159

0.117

0.152

0.110

2

0.164

0.122

0.123

9.9

0.165

0.123

0.165

0.123

3

0.164

0.122

0.124

10.0

0.168

0.126

0.165

0.123

Positive control

(Sodium dodecyl sulphate (5% aq.))

1

0.061

0.018

0.018

0.018

1.5

1.5

0

0.00

Category 2

0.061

0.018

0.060

0.017

2

0.062

0.019

0.019

1.5

0.062

0.019

0.061

0.018

3

0.061

0.018

0.018

1.5

0.061

0.018

0.061

0.018

Keys: O.D. = Optical Density, S.D. = Standard Deviation, C.V. = Coefficient of Variation, NA = Not Applicable

Note: For Negative control, SD and CV of % viability was calculated using corrected OD at 570 nm and for the test item and positive control SD and CV of % viability was calculated using % viability/tissue."

Treatment

Exposure Time

Tissue Replicate

O.D.

Corrected O.D.

Mean of Corrected O.D.

Mean OD of Replicate tissues

% NSC

Mean % NSC

TODCT

% Relative Viability

Negative Control

(Dulbecco’s Phosphate Buffered Saline (DPBS))

42 minutes

1

0.044

0.001

0.001

0.001

-

-

NA

NA

0.043

0.000

0.044

0.001

2

0.045

0.002

0.001

0.045

0.002

0.043

0.000

4-Trifluoromethylsalicylic Acid (ATFMS)

42 minutes

1

0.042

0.000

0.000

0

-0.1

-0.1

NA

NA

0.043

0.001

0.042

0.000

2

0.042

0.000

0.000

-0.1

0.042

0.000

0.043

0.001

Key: O.D. = Optical Density, S.D. = Standard Deviation, C.V. = Coefficient of Variation, NA = Not Applicable

Note: NSC was -0.1% relative to the negative control hence considered as “0”, hence TOD and relative viability calculation was not determined;

 

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The substance was determined to be irritating to the skin.
Executive summary:

An in-vitro Skin Irritation test with Reconstructed Human Epidermis (RHE) Tissues was performed according to the OECD Guideline 439 (GLP study). Tissues were exposed to the negative control (Dulbecco’s Phosphate Buffered Saline (DPBS)), positive control (sodium dodecyl sulfate, 5% aqueous (SDS)) and test item in triplicate for 42 minutes at room temperature. The mean cell viability in tissues treated with the test item was 9.7% after 42 minutes exposure. A significant reduction in percent cell viability was observed in treated tissues when compared with the concurrent negative control. The Optical density (OD) values for the negative control replicates were between 1.221 to 1.273, against the guideline requirement of ≥ 0.8 and ≤ 3.0 (≥ 1.2 as per SkinEthic SOP). The OD of the blank was between 0.041 to 0.045 which met the guideline requirement of OD < 0.1. The positive control showed a 1.5% cell viability, against the acceptance criteria of <40% for the SkinEthic RHE model, compared to concurrent negative control. Variation between tissue replicates (i.e. CV% value) was 4.54% for the test item group, 0.00% for positive control and 0.81% for negative control against the guideline requirement of ≤ 18%, which demonstrate the efficiency of the test system, SkinEthicTM RHE model. All criteria for a valid study were met. Based on these results, the test item was determined to be irritating to the skin (category 2).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
November 17, 2017 - November 18, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra.
- Characteristics of donor animals (e.g. age, sex, weight): Age between 1 to 5 years (age of animals was determined based on the teeth count and horn ring count).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported under cold condition in Hank's Balanced Salt Solution containing antibiotics [e.g., penicillin at 100 IU/mL and streptomycin at 100 µg/mL].
- Time interval prior to initiating testing: The eyes were used within 24 h from the slaughtering.
- indication of any existing defects or lesions in ocular tissue samples: No. Eyes were examined prior to use. Corneas from eyes free of visible defects were used. Corneas that have opacity lesser than seven opacity units or equivalent for the opacitometer were used in the study.
- Indication of any antibiotics used: Antibiotic used during transport in buffered Hanks medium [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].
Vehicle:
other: Corn oil
Remarks:
(Sigma Aldrich; Batch MKBZ9899V)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL applied on each cornea.
- Concentration (if solution): 20% (w/v) in corn oil.

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL.
Duration of treatment / exposure:
4 hours (± 5 minutes) at 32 ± 1 ºC.
Duration of post- treatment incubation (in vitro):
90 min (± 5 min) at 32 ± 1 ºC.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS : Corneas, free from defects were dissected to a 2 to 3 mm rim and were transferred to container containing Hank's Balanced Salt Solution containing antibiotics (e.g., penicillin at 100 IU/mL and streptomycin at 100 µg/mL).

QUALITY CHECK OF THE ISOLATED CORNEAS : Selected corneas were mounted on the corneal holders with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then placed on the top of the cornea and fixed in place with screws. Both chambers will then be filled to excess with prewarmed phenol red free Eagle's Minimum Essential Medium (EMEM) (posterior chamber first to return the cornea to its natural concave position), ensuring that no bubbles were present within the holders. The device was then equilibrated at 32 ± 1 ºC for at least one hour to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, the medium was removed from both the chambers and fresh pre-warmed phenol red free EMEM is added to both chambers and baseline opacity readings were taken for each cornea. Corneas that have opacity lesser than seven opacity units or equivalent for the opacitometer were used in the study.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : Yes, normal saline.

SOLVENT CONTROL USED (if applicable) : Yes, corn oil.

POSITIVE CONTROL USED : Yes, Imidazole 20% (w/v) concentration in normal saline

APPLICATION DOSE AND EXPOSURE TIME : 750 µL of 20% (w/v) in corn oil for 4 hours exposure.

TREATMENT METHOD: [closed chamber]

POST-INCUBATION PERIOD: yes, holders are incubated in a horizontal position for 90 ± 5 min at 32 ± 1ºC.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the corneal epithelium was washed until no visual evidence of test item was observed using EMEM (containing phenol red). Once the medium was free of test item, the corneas were given a final rinse with EMEM (without phenol red). Media in the anterior and the posterior chamber was removed and fresh EMEM (without phenol red) was filled.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: An opacitometer BASF-OP3.0 was used. Opacity value calculation for selecting the cornea for the experiment:
Mean Opacity value =
Step 1. Baseline opacity reading (with medium without cornea)/post incubation opacity reading (with medium and cornea)
Step 2. [Step 1 value of each cornea – b]/a
Where b = 0.9894 and a = 0.0251 (a and b values are empirical derived for the instrument)

Mean Opacity value =
Step 1. [Baseline opacity reading (with medium without cornea)/post incubation opacity reading (with medium and cornea)] – post treatment opacity reading (Calculate for each cornea)
Step 2. Calculate the mean of control group
Step 3. {[Step 1 value of each cornea (except control) – Step 2 value] – b}/a
Step 4. Calculate Mean of Step 3 value
Where b = 0.9894 and a = 0.0251 (a and b values are empirical derived for the instrument)

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader BioTek Instruments Inc. USA (OD490) .
Mean Permeability value =
Step 1. OD490 value of all the individual cornea – Blank OD490 value
Step 2. Calculate the mean of control group
Step 3. Step 1 value of each cornea (except control) – Step 2 value
Step 4. Calculate Mean of Step 3 value
Any dilution made to bring OD490 into linear range was multiplied by the dilution factor.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
In vitro irritancy score (IVIS) was calculated for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The in vitro irritancy score was calculated for each individual treatment.

DECISION CRITERIA: as indicated in the TG.
IVIS UN GHS
If the test item induces an IVIS ≤ 3 No category
If the test item induces an 3 < IVIS ≤ 55 No prediction can be made
If the test item induces an IVIS > 55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
159.78
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
151.54
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
174.19
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
161.84
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
153.69
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
2
Value:
145.48
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
3
Value:
161.72
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
153.63
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Yes, a proficiency study (study no. 530-1-01-10123) was performed from November 06, 2014 to February 06, 2015, which demonstrates the laboratory's proficiency to conduct Bovine corneal Opacity and Permeability test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes, the mean In-Vitro Irritancy Score (IVIS) of imidazole (20% w/v) (positive control) treated corneas were found to be 130.83 which is within the range of two standard deviation of the mean of the historical control data, confirming the reliability of the test procedure.

TABLE 1: In Vitro Irritation Score

 

Group : Normal Saline, 0.75 mL

Cornea Holder N°

Io

 (LUX)

I

(Initial)

(LUX)

Initial Opacity Value

I

(Post Treatment)

(LUX)

Post Treatment Opacity

Value

Corr. Opacity

Value

OD490

Value

Corr. OD490

Value

IVIS

2

1095

1032

2.85

999

4.25

1.40

0.125

0.084

2.66

7

1090

1012

3.49

977

5.03

1.54

0.114

0.073

2.64

8

1092

1035

2.62

987

4.66

2.04

0.123

0.082

3.27

Mean

1.66

-

0.080

2.86

SD

0.34

-

0.006

0.36

 

Group : Imidazole at 20% (w/v) in normal saline, 0.75 mL

Cornea Holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post

Treatment)

(LUX)

Post Treatment Opacity

Corr. Opacity

Final Opacity

OD490

Corr. OD490

Final OD490

IVIS

Score

3

1117

1066

2.33

277

121.24

118.91

117.25

1.564

1.523

1.443

138.90

9

1109

1034

3.31

289

113.46

110.15

108.49

2.870

2.829

2.749

149.73

15

1066

1002

2.97

309

98.03

95.06

93.40

0.819

0.778

0.698

103.87

Mean

106.38

-

1.710

1.630

130.83

SD

12.06

-

1.038

1.038

23.97

Group : Corn Oil, 0.75 mL

Cornea Holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post

Treatment)

(LUX)

Post Treatment Opacity

Value

Corr.

Opacity

Value

OD490

Value

Corr. OD490

Value

IVIS

4

1070

1024

2.21

989

3.69

1.48

0.14

0.099

2.97

11

1064

997

3.10

969

4.33

1.23

0.142

0.101

2.75

17

1066

1011

2.59

972

4.28

1.69

0.126

0.085

2.97

Mean

1.47

-

0.095

2.90

SD

0.23

-

0.009

0.13

Group : 4 - Trifluoromethylsalicylic acid (ATFMS) (suspension) at 20% (w/v) in Corn Oil, 0.75 mL

Cornea Holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post

Treatment)

(LUX)

Post Treatment Opacity

Value

Corr. Opacity

Value

Final Opacity

Value

OD490

Value

Corr. OD490

Value

Final OD490

Value

IVIS

5

1147

1071

3.25

231

158.41

155.16

153.69

0.542

0.501

0.406

159.78

6

1106

1045

2.75

233

149.70

146.95

145.48

0.540

0.499

0.404

151.54

12

1080

1008

3.27

209

166.46

163.19

161.72

0.967

0.926

0.831

174.19

Mean

153.63

-

0.642

0.547

161.84

SD

8.12

-

0.246

0.246

11.46

Keys: IVIS = In Vitro Irritation Score, Io =Baseline Reading (With medium but without cornea), I = LUX 

Reading with Medium and Cornea, OD490= Optical Density at 490 Wave Length, - = Not Applicable, Corr. = Corrected. Blank OD490value = 0.041.

 

InitialOpacity Value = [((Io⁄I)-b)/a)], Where I = Initial LUX, Io = Baseline Reading

Note: a (0.0251) and b (0.9894) are constant.

Example:Initial Opacity Value = [((1095/1032)-0.9894)/0.0251] =2.85

Post TreatmentOpacity Value = [((Io⁄I)-b)/a)], Where, I = Post Treatment LUX, Io = Baseline Reading.

Note: a (0.0251) and b (0.9894) are constant.

Example:Initial Opacity Value = [((1095/999)-0.9894)/0.0251] =4.25

Corr. Opacity Value = Post Treatment Opacity Value - Initial Opacity Value

Example:Corr. Opacity Value = 4.25 – 2.85 =1.40

Final Opacity Value = Corr. Opacity Value – Mean Corr. Opacity Value of Control (Group I)

Example:Final Opacity Value= 118.91 – 1.66 =117.25

Corr. OD490Value = OD490Value – Blank OD490Value, Where Blank Value for this trial was 0.044

Example:Corr. OD490Value = 0.125 -0.041 =0.084

Final OD490Value = Corr. OD490Value –Mean Corr.OD490Valueof Control (Group I)

Example:Corr. OD490Value 1.523 -0.080 =1.443

IVIS (Control) = Corr. Opacity Value + (15 x Corrected OD490)

Example:IVIS = 1.40 + [15 x 0.084] =2.66

IVIS (Treatment) = Final Opacity Value + (15 x Final OD490)

Example:IVIS = 117.25 + (15 x 1.443) =138.90

 

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the tested condition, the mean IVIS value of the test item was found to be 161.84. According to decision criteria for classification, test item has to be classified as eye irritant, category 1.
Executive summary:

An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in accordance with OECD TG 437 (GLP study) in order to determine the potential severe eye damaging effects of the test item. The corneas were obtained from freshly slaughtered cattles. An experiment was performed using three corneas for each treated series using the closed chamber method during 4 h ± 5 minutes. One set of corneas were treated with 750 μL of the prepared test item 20% (w/v) concentration in corn oil. One set of corneas served as positive control (750 μL of 20% (w/v) concentration in normal saline) and two set of corneas served as negative control and was treated with 750 μL of normal saline and corn oil, respectively. At the end of exposure period, the test item, positive and negative controls were removed from the anterior chamber and the corneal epithelium was washed until no visual evidence of test item was observed using EMEM (containing phenol red). Post opacity reading permeability was measured by applying1 mL of fluorescein sodium solution (5 mg/mL) on to the anterior surface of the cornea and was incubated for approximately 90 min at 32 ºC. At the end of the incubation period OD was measured at 490 nm for the fluid collected from the posterior chamber. The mean in-vitro irritancy score (IVIS) of normal saline, corn oil and 750 µL 20% (w/v) imidazole in normal saline (positive control) treated corneas were found to be 2.86, 2.90 and 130.83, respectively. The IVIS score for the corneas treated with 750 μL of test item at 20% (w/v) concentration in corn oil were found to be 161.8. Based on the results of this study, an indication of the classification for test item is Category 1.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin irritation/corrosion: Based on available data, the substance is classified for Skin Irritation Category 2 (H315) according to CLP Regulation (EC) No. 1272/2008.

Eye damage: Based on available data, the substance is classified for Eye Damage Category 1 (H318) according to CLP Regulation (EC) No. 1272/2008.