Registration Dossier

Administrative data

Description of key information

Skin irritation

The dermal irritation potential of target chemical was assessedin various in-vivo experimental studies which were conducted for test chemical and its structurally similar read across substances.Based on the available key data and supporting studies,it can be concluded that the testchemical is unable to cause skin irritation and considered as not irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Not Classified”.

 

Eye Irritation:

Various studies were performed on rabbits to assess the ocular irritation potential of test chemical. The results obtained from these studies lead to a conclusion that Test chemical is not likely to cause irritation to the eye and can be considered as not irritating. Hence, comparing the above annotations with the criteria of CLP regulation, the Test chemical can be classified under the category “Not Classified”.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
data is from experimental report
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 402 (Acute Dermal Toxicity)
Principles of method if other than guideline:
To assess the toxicological profile of test chemical on application as a single semi-occlusive dermal application to rats
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Details on test animals
Sex: females, Females were nulliparous and non-pregnant
Source : Geniron Biolabs Pvt. Ltd., Bengaluru, India
Age at treatment: 10 to 12 weeks
Body weight range at treatment: Females: 235.18 to 238.33 g
Identification :By rat accession number. Identification of individual rats is by cage card and crystal violet colour body markings. The temporary body marking during acclimatization period was done with crystal violet. The rat accession numbers were allotted during the course of the study and was included in raw data and reported.
Housing: Animals were housed individually in standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottle. Steam sterilized corn cob was used and changed once a week along with the cage.
Diet:Hypro Rat & Mice pellet feed, manufactured at Sangli, Maharashtra, was provided to ad libitum to animals
Water: Purified water in polycarbonate bottles with stainless steel sipper tubes was provided ad libitum
Acclimatization:After physical examination for good health and suitability for experiment, the rats were acclimatized for six, eight, twelve and fourteen days before treatment for dose range finding and main study respectively under standard laboratory conditions. Animals were observed once daily during acclimatization period.

Environmental Conditions:

Temperature: 22 to 25°C
Relative humidity: 66 to 68%
Air changes: air conditioned with adequate fresh air supply (12.4 air changes/hour)
Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

In Life dates: Start: 26 June 2018 , End: 11 September 2018

Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
Dose range finding study:200 (0.20 mL/kg body weight), 1000 (1 mL/kg body weight) and 2000(1.99 mL/kg body weight)
Main Study: 2000 mg/kg
Duration of treatment / exposure:
24 hours
Observation period:
14 days
Number of animals:
5 females (3 females for dose range finding study followed by 2 females for main study)
Details on study design:
TEST SITE
- Area of exposure: clipped skin of the torso
- % coverage: 10% of the body surface of the animal (semi-occlusive).
- Type of wrap if used: The area of application was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it was secured in position by adhesive tape wound around the torso

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towels

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after removal of test item using the Draize criteria

SCORING SYSTEM:
- Method of calculation: Draize method
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
72 h
Score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
No skin irritation was observed.

TABLE 1.            Individual body weight, body weight changes and pre-terminal deaths

Group and

Dose

(mg/kg body weight)

Rat

No.

S

e

x

Body weight (g)

Pre-terminal deaths

Initial

(Day 1 - at treatment)

8th  

day

Weight change

(day 8 – Initial)

15th

day

Weight change

(day 15 – Initial)

G1 and

200

DRF

Rw283

F

235.18

242.68

7.50

248.19

13.01

0

G2 and

1000

DRF

Rw284

F

238.33

245.45

7.12

251.67

13.34

0

G3 and

2000

DRF

Rw285

F

238.16

244.73

6.57

249.14

10.98

0

G3 and

2000

Main study

Rw286

F

230.34

236.59

6.25

241.08

10.74

0

Rw287

F

234.62

242.93

8.31

247.63

13.01

0

 DRF: Dose Range Finding   F: Female

APPENDIX 1.      Individual test item application, clinical signs, skin reactionand necropsy findings

Dose range finding study

 

Group & Dose

(mg/kg

body weight)

Date and time of application

Rat

Number

S

e

x

Initial

Bwt

(g)

Quantity

(mL)

applied

Observations and skin reaction

Days

1

2

3

4

5

30

min

1 h #

2 h #

3 h #

4 h

#

5 h #

6 h

#

*

Er

@

Ed

@

*

Er

@

Ed

@

*

Er

@

Ed

@

G1 and

200

DRF

11 April 2018

and

10.55 AM

Rm8907

F

223.58

0.04

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose

(mg/kg

body weight)

Rat

Number

S

e

x

Observation

Necropsy

findings

Days

6

7

8

9

10

11

12

13

14

15

G1 and

200

DRF

Rm8907

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female             N: Normal          h: hour    min: minutes                      NAD: No abnormality detected      Er: Erythema                      Ed: Edema  

Score 0: No Erythema / Edema       

    

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

 

 

APPENDIX 2 contd. Individual test item application, clinical signs, skin reaction and necropsy findings

 

Dose range finding study

Group & Dose

(mg/kg

body weight)

Date and time of application

Rat

Number

S

e

x

Initial

Bwt

(g)

Quantity

(mg)

applied

Observations and skin reaction

Days

1

2

3

4

30

 min

1 h

2 h

3 h 

4 h

5 h

6 h

*

Er

@

Ed

@

*

Er

@

Ed@

G1- DRF

 and

200

 

03 July 2018

and

11.36 AM

Rw283

F

235.18

47

N

N

N

N

N

N

N

N

N

0

0

N

0

0

Group & Dose

(mg/kg

body weight)

Animal

Number

S

e

x

Observation/ Days

Necropsy

findings

5

 

*

Er

@

Ed

@

6

7

8

9

10

11

12

13

14

15

G1- DRF

 and

200

 

Rw283

F

N

0

0

N

N

N

N

N

N

N

N

N

N

NAD

 

F: Female             N: Normal          h: hour    min: minutes                       NAD: No abnormality detected      Er: Erythema                       Ed: Edema  

Score 0: No Erythema / Edema       

    

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

APPENDIX 2 contd. Individual test item application, clinical signs, skin reaction and necropsy findings

 

Dose range finding study

 

 

Group & Dose

(mg/kg

body weight)

Date and time of application

Rat

Number

S

e

x

Initial

Bwt

(g)

Quantity

(mg)

applied

Observations and skin reaction

Days

1

2

3

4

30

 min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er

@

Ed

@

*

Er

@

Ed

@

G2- DRF

 and

1000

 

05 July 2018

and

11.56 AM

Rw284

F

238.33

238

N

N

N

N

N

N

N

N

N

0

0

N

0

0

Group & Dose

(mg/kg

body weight)

Animal

Number

S

e

x

Observation/ Days

Necropsy

findings

5

 

*

Er

@

Ed

@

6

7

8

9

10

11

12

13

14

15

G2- DRF

 and

1000

 

Rw284

F

N

0

0

N

N

N

N

N

N

N

N

N

N

NAD

F: Female             N: Normal          h: hour    min: minutes                       NAD: No abnormality detected      Er: Erythema                       Ed: Edema  

Score 0: No Erythema / Edema       

    

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

 

 

APPENDIX 2 contd. Individual test item application, clinical signs, skin reaction and necropsy findings

 

Main study

 

Group & Dose

(mg/kg

body weight)

Date and time of application

Rat

Number

S

e

x

Initial

Bwt

(g)

Quantity

(mg)

applied

Observations and skin reaction

Days

1

2

3

4

30

min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er @

Ed @

*

Er @

Ed @

G3- Main

and

2000

 

12 July 2018

and

11.41 AM to 11.42 AM

Rw286

F

230.34

461

N

N

N

N

N

N

N

N

N

0

0

N

0

0

Rw287

F

234.62

469

N

N

N

N

N

N

N

N

N

0

0

N

0

0

 

Group & Dose

(mg/kg

body weight)

Animal

Number

S

e

x

Observation/ Days

Necropsy

findings

5

 

*

Er

@

Ed

@

6

7

8

9

10

11

12

13

14

15

G3- Main

 and

2000

 

Rw286

F

N

0

0

N

N

N

N

N

N

N

N

N

N

NAD

Rw287

F

N

0

0

N

N

N

N

N

N

N

N

N

N

F: Female             N: Normal          h: hour    min: minutes                       NAD: No abnormality detected      Er: Erythema                       Ed: Edema  

Score 0: No Erythema / Edema          

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

 


Interpretation of results:
other: not irritating
Conclusions:
There was no skin reaction observed at test item applied area. The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that test chemical was Non-Irritating to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.
Executive summary:

The study designed and conducted to determine the dermal reaction profile of test chemical in Wistar rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures.

The test chemical was tested in 5 females (3 females for dose range finding study followed by 2 females for main study) Wistar rats at the doses of 200, 1000 and 2000 mg/kg body weight.

Based on the individual body weight, the undiluted test item at the doses of 200 (0.20 mL/kg body weight), 1000 (1 mL/kg body weight) and 2000(1.99 mL/kg body weight) - based on the density of the test item1.004 g/cm3 was applied directly to the clipped skin of the animal to cover about 10% of the body surface of the animal (semi-occlusive). The area of application was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it was secured in position by adhesive tape wound around the torso. The test item contact period with the skin was for 24 hours. After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towels. All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after removal of test item using the Draize criteria. There were no clinical signs of toxicity and mortality. There was no skin reaction observed at test item applied area. Body weight was measured on days 1, 8 and 15 and all rats gained weight during experimental period. At the end of observation period, all surviving animals were euthanized and subjected to necropsy. There were no abnormalities detected at the necropsy.

There was no skin reaction observed at test item applied area. The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that the test chemical was Non-Irritating to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article may be predicted by measurement of its cytotoxic effect, as reflected in
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiOcular™ model
GLP compliance:
no
Specific details on test material used for the study:
- Name of test material: 12H-phthaloperin-12-one
- Common name: C.I. Solvent Orange 60
- Molecular formula: C18H10N2O
- Molecular weight: 270.29 g/mol
- Smiles notation: O=C1N2c3c4c(cccc4N=C2c2ccccc12)ccc3
- InChl: 1S/C18H10N2O/c21-18-13-8-2-1-7-12(13)17-19-14-9-3-5-11-6-4-10-15(16(11)14)20(17)18/h1-10H
- Substance type: Organic
- Physical state: Solid


RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature / Fridge storage
- Stability under test conditions: No data available
- Solubility and stability of the test substance in the solvent/vehicle: No data available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prior to the main test, the test articles are tested for their ability to reduce/interact with MTT and their ability to stain the tissues itself. All tests are performed according to the test protocol provided by MatTek In Vitro Life Science Lab. (Bratislava, Slovakia)
- Preliminary purification step (if any): No data available
- Final dilution of a dissolved solid, stock liquid or gel: No data available
- Final preparation of a solid: No data available

FORM AS APPLIED IN THE TEST:solid
Species:
human
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
- Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

- Test System Identification
All of the EpiOcular™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues is included in this report. Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.

- Justification of the test method and considerations regarding applicability
EpiOcularTM Eye Irritation (OCL) by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakien

The test articles and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakien.The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
growing body of evidence indicates that EpiOcular™ effectively provides a non-animal means to assess potential irritancy. The EpiOcular™ model closely mimics the human corneal mucosa and thus provides an important in vitro approach in the evaluation of ocular irritancy and toxicity. Each lot of tissues was Quality Assuurance by MatTek In Vitro Life Science Laboratories regarding to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg of the test chemical
- Concentration (if solution): neat (undiluted)

VEHICLE (no vehicle)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): neat
Duration of treatment / exposure:
Tissues were exposed for 6 hrs ± 15 min for solid test articles and controls, at approximately 37°C, 5% CO2 in a humidified incubator.
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Following the washing and the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time of 18 hours for solid test articles and controls
Number of animals or in vitro replicates:
2 tissues were used for test compound and control.
Details on study design:
- Details of the test procedure used
The tissues were exposed to the test article neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 30 minutes for liquid test articles and controls, at approximately 37°C, 5% CO2 in a humidified incubator. After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~12 minutes for liquid test articles and controls. Following the washing step and the, the tissues were incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~2 hours for liquid test articles and controls. Tissue viability was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

- MTT Auto reduction and colouring assessment
MTT Pre-test
The test article was assessed for the potential to interfere with the assay. Approximately 50 µL of liquid test article was added to 1 mL of MTT media (~1 mg/mL) and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 3 hours. 50 µL of ultrapure water was used as a negative control.
- Test Article Color Test
Approximately 50 µL of liquid test article was added to 1.0 mL of ultrapure water and 2.0 mL isopropanol and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 04 minutes and 35 seconds. Samples were then added to the wells of a clear 96-well plate and the plate was read on a Thermo Scientific Multiskan FC Microplate Photometer to 570 nm. Test articles that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions).

- MTT Assay:
After the recovery period, the MTT assay was performed on run 1 tissues by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 3 hours of MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator.The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction for liquid exposed tissues was overnight incubation. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissues

- Evaluation of Test Article in the cell Models
1. Cell System:
Upon receipt, the MatTek EpiOcular™ tissue cultures were placed in 1.0 mL of fresh Maintenance medium (in a 6-well plate) for 60 minutes. After the 60 minutes incubation, the Maintenance medium was exchanged with fresh medium and the tissues were incubated overnight (16-24 hrs) at approximately 37°C, approximately 5% CO2 in a humidified incubator.
2. Control and Test Article Exposures:
20 µL of calcium and magnesium free DPBS was added to each tissue and the tissues placed back into the incubator for 30 minutes. The controls and the test article will be applied topically to tissues by pipette. Three tissues will be used per test compound and control.
a)Controls: 50 µL of negative control sterile ultrapure water and positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
b)Test Article: 50 µL of liquid test article were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
3. Post exposure treatment:
After the exposure, the tissues were rinsed 20 times with sterile of DPBS to remove test material. The apical surface was gently blotted with a cotton swab and cultures were immediately transferred to a 12-well plate containing 5 mL of media per well. Tissues exposed to liquid test articles (and the respective control) were incubated, submerged in the media for ~12 minutes at room temperature.For liquid test articles, tissues, Tissuses were then transferred to 6-well plates containing 1.0 mL fresh Maintenance medium per well and incubated for a post-exposure recovery period for 2 hours at approximately 37 degC, 5% CO2 in a humidified incubator.
- Doses of test chemical and control substances used
Test Article:
50 µL of liquid test article were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
Controls: 50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Tissues were exposed for approximately 30 minutes for liquid test articles and controls, at approximately 37°C, 5% CO2 in a humidified incubator. Following the washing step and the, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~2 hours for liquid test articles and controls.
- Justification for the use of a different negative control than ultrapure H2O (Not applicable
- Justification for the use of a different positive control than neat methyl acetate (Not applicable)
- Number of tissue replicates used per test chemical and controls: 2 tissues were used for test compound and control.
- Description of the method used to quantify MTT formazan
The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction for liquid exposed tissues was overnight incubation with a 20 minute 24 second shake the following morning. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm. The blue formazan salt was extracted by placing the tissue insterts in 1 mL isopropanol in a 6-well plate. The extraction for solid exposed tissues was 3 hrs incubation. After an addition of 1 ml isopropanol and mixing, the optical density of the extracted formazan (200μL/well of a 96-well plate) was determined using a Thermo Scientific Multiskan FC Microplate Photometer at 570 nm.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
· The OD mean from all replicates for each plate (ODblank).
Negative Controls (NC):
· The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
· The OD mean per NC tissue was calculated.
· The mean OD for all tissues corresponds to 100% viability.
· The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.
Positive Control (PC):
· Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
· The OD mean per PC tissue was calculated.
· The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
· The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
· The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.
Tested Articles:
· Calculate the blank corrected value ODTT= ODTTraw– ODblank.
· The OD mean per tissue is calculated.
· The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
· The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
· The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.
Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)

Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.
Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.
- Evaluation of data
The results of the assay was evaluated and compared to negative control.
Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category
- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density (OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
- Standard Deviation (SD)Each test of ocular irritancy potential is predicted from the mean viability determined on 3 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the replicates is <18% for three replicate tissues.
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
83.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.
Interpretation of results:
other: not irritating
Conclusions:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance was determined to be 83.2%. Thus, test substance was considered to be not irritating to the human eyes.
Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours recovery after the post-soak.The viability of each tissue was determined by MTT assay.

The MTT data show the assay quality controls were met, passing the acceptance criteria.

The mean % tissue viability of test substance was determined to be 83.2%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be not irritating to the human eyes and can thus be classified as “Not Classified’’ as per CLP Regulation

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation:

Various studieshas been investigated for the test chemical to observe the potential for dermal irritation to a greater or lesser extent. The studies are based on in vivo experiments conducted for target chemicaland its structurally similar read across substances which have been summarized as below;

 

The study designed and conducted to determine the dermal reaction profile of test chemical in Wistar rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures. The test chemical was tested in 5 females (3 females for dose range finding study followed by 2 females for main study) Wistar rats at the doses of 200, 1000 and 2000 mg/kg body weight. Based on the individual body weight, the undiluted test item at the doses of 200 (0.20 mL/kg body weight), 1000 (1 mL/kg body weight) and 2000(1.99 mL/kg body weight) - based on the density of the test item1.004 g/cm3 was applied directly to the clipped skin of the animal to cover about 10% of the body surface of the animal (semi-occlusive). The area of application was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it was secured in position by adhesive tape wound around the torso. The test item contact period with the skin was for 24 hours. After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towels. All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after removal of test item using the Draize criteria. There were no clinical signs of toxicity and mortality. There was no skin reaction observed at test item applied area. Body weight was measured on days 1, 8 and 15 and all rats gained weight during experimental period. At the end of observation period, all surviving animals were euthanized and subjected to necropsy. There were no abnormalities detected at the necropsy.

 

There was no skin reaction observed at test item applied area. The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that the test chemical was Non-Irritating to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.

 

The skin irritation study was conducted on a single rabbit ear to indicate the Comedogenicity and irritancy of the test chemical. The test chemical was mixed in propylene glycol at a 9 to 1 dilution for testing unless otherwise indicated (10% concentration). A colony of New Zealand albino rabbits that have genetically good ears and free from mites were used. Three rabbits, weighing two to three kilograms, were used for each assay. Animals were housed singly in suspended cages and fed Purina Rabbit Chow and water ad libitum. Animals were maintained on a 12-hour light and 12-hour dark cycle. A dose of 1 ml of the test material was applied and spread once daily to the entire inner surface of once for five days per week for two weeks. The opposite untreated ear of each animal served as an untreated control. The irritancy produced by repeated application of the chemical on the surface epidermis in the rabbit ear is evaluated on a scale of 0 to 5. The test chemical falls under Grade 0 (no irritation observed). Hence it can be concluded that the test chemical was not irritating to rabbit ears.

 

The above study was supported by the results of a skin irritation test performed on rabbits to determine the irritation potential of the test chemical. 0.1 mL of 25% or 50% test chemical in physiological saline solution was applied to the skin of rabbits observed for effects(duration of exposure and observation period not mentioned). No known skin reactions were observed. Hence, the test chemical was considered to be not irritating to rabbits’ skin.

 

These results are further supported by another skin irritation study performed to assess the irritation potential of the test chemical. The test chemical was applied to rabbit skin and observed for signs of irritation (dose, duration not mentioned).The test chemical did not cause any irritation to rabbit skin. Hence, the test chemical can be considered not irritating to rabbit skin.

 

All these studies lead to a conclusion that Test chemical is indeed not irritating to skin. Hence, comparing the above annotations with the criteria of CLP regulation, Test chemical can be classified under the category “Not Classified”.

 

Eye Irritation:

Various studies were performed on rabbits to assess the ocular irritation potential of test chemical which have been summarized as follows:

 

The ocular irritation potential of test article was determined according to the OECD 492 test guideline for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours recovery after the post-soak. The viability of each tissue was determined by MTT assay. The MTT data show the assay quality controls were met, passing the acceptance criteria. The mean % tissue viability of test substance was determined to be 83.2%. Hence, under the experimental test conditions it was concluded that test chemical was considered to be not irritating to the human eyes and can thus be classified as “Not Classified’’ as per CLP Regulation.

 

The results from in-vitro experiment was supported by the case study which reports of an accidental exposure to the test chemical. A man accidently splashed 7% alkaline solution of reduced form of the test chemical into his eyes. His conjunctiva appeared blue several hours later; cornea was somewhat turbid but not stained. In course of 10 days cornea gradually got cleared & some fine blue dots were seen in stroma. The test chemical appeared to be rather inert & nontoxic in human tissues. Hence, the test chemical can be considered not irritating to human eyes.

 

These results were supported by an eye irritation test carried out to evaluate the irritation parameter of the other test chemical. 100 mg or 100 mg of 25 or 50% the test chemical suspension in water was instilled into rabbit eyes and the effects were observed (duration not specified). No changes were observed in rabbits. Hence, the test chemical was considered to be not irritating to rabbit eyes.

 

  The above results were further supported by an ocular irritation study conducted to evaluate the eye irritation potential of the test chemical. The test chemical was used in 0.25 ml of a filtrate from a digest in aqueous saline prepared at 40°C.The rabbits were observed for signs of irritation. After application slight redness was observed which was reversible. No other ocular irritation reaction were observed. Hence, the test chemical was considered to be not irritating to eye.

 

All these studies lead to a conclusion that Test chemical is indeed not irritating to eye. Hence, comparing the above annotations with the criteria of CLP regulation, Test chemical can be classified under the category “Not Classified”.

Justification for classification or non-classification

The skin and eye irritation potential of test chemical was observed in various studies. The results obtained from these studies indicate that the chemical is not likely to cause skin and eye irritation. Hence the test chemical can be classified under the category “Not Classified” for skin and eye as per CLP.