Registration Dossier

Administrative data

Description of key information

Skin Irritation:

The dermal irritation potential of target chemical was assessed in various in- vitro and in-vivo experimental studies which were conducted for test chemical.Based on the available key data and supporting study,it can be concluded that the test chemical 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:

In the confirmatory test, the test chemical produced slight redness of conjunctivae after two hour of application of test compound upto 24 hours. However, there was no other clinical sign recorded in both of the animals during the whole observation period for 21 days.Also the overall irritation score was calculated to be 2.5/110. According to the irritation parameter propised by Kay & Calandra, the test chemical can be consdiered to be practically not irritating to eyes, It can be classified under the category "Not Classified".

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
data is from experimental reports
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Principles of method if other than guideline:
To assess the dermal irritation parameter of the test chemical in accordance with OECD 404
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Sex: female
- Age at study initiation: 10 to 12 weeks
- Weight at study initiation: 1.80kg±200g
- Housing: Animals were housed individually in stainless steel cages provided with stainless steel mesh bottom and facilities for food and water bottle.
- Identification: By cage tag and corresponding colour body marking
- Diet (e.g. ad libitum): Pelleted feed supplied by Pranav agro Industries Ltd., Delhi
- Water (e.g. ad libitum): Community tap water passed through ‘Aqua Guard on line water filter’, was kept in glass bottles, ad libitum
- Acclimation period:The healthy rabbits selected for study was acclimatized to standard laboratory condition for one week in experimental room under Veterinary examination.
- Randomization: After acclimatization and Veterinary examination three females were randomly selected.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Air conditioned rooms with temperature between 22-250C
- Humidity (%): elative humidity 40-60%
- Air changes (per hr): Air conditioned rooms with 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): illumination cycle set to 12 hours artificial fluorescent light and 12 hours dark

Type of coverage:
open
Preparation of test site:
shaved
Vehicle:
water
Controls:
not specified
Amount / concentration applied:
0.5 gm of test compound
Duration of treatment / exposure:
4 hours
Observation period:
60 min., 24, 48 and 72 hours after application.
Number of animals:
3 female rabbits
Details on study design:
TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage: small area (approximately 6 cm2) of intact skin site
- Type of wrap if used: impervious occlusive wrap

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: After patch removal, the dressing and unabsorbed test product was removed and the site of application was cleaned with lukewarm water.

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : 60 min., 24, 48 and 72 hours after application.

SCORING SYSTEM:
- Method of calculation: The site of application was observed for skin reaction if any. The intact skin site of application of each animal was observed for signs of erythema and oedema and the responses were scored following Draize’s method at 60 min., 24, 48 and 72 hours after application.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Skin reaction
The test chemical applied at the dose level of 0.5 gm on shaven back skin (approximately 6 cm2) of rabbit did not produce any clinical signs of irritation to skin during period of observation. The duration of application of test compound was 24th hour.

Other effects:
Clinical Signs
The test chemical applied on the shaven back skin of rabbit in the amount of 0.5 gm did not produce pain and any clinical signs of toxicity throughout the examination period of 14 days.

INDIVIDUAL ANIMAL DERMAL IRRITATION SCORES

 

Rabbit No.

Sex

INTACT SKIN

3 Min.

4 Hours

24 Hours

48 Hours

72 Hours

14 days

Erythema

Oedema

Erythema

Oedema

Erythema

Oedema

Erythema

Oedema

Erythema

Oedema

Erythema

Oedema

01

F

0

0

0

0

0

0

0

0

0

0

0

0

02

F

-

-

0

0

0

0

0

0

0

0

0

0

03

F

-

-

0

0

0

0

0

0

0

0

0

0

Total

0

0

0

0

0

0

0

0

0

0

0

0

Mean

0

0

0

0

0

0

0

0

0

0

0

0

Grand Total

0.00

 

Dermal Irritation Index: 0.0/4 = 0.0

CLINICAL SIGNS

SEX

ANIMAL NO.

Time (Min.)

Time (Hours)

Time (Day)

3

1

4

24

48

72

14

 

FEMALE

 

01

N

N

N

N

N

N

N

02

N

N

N

N

N

N

N

03

N

N

N

N

N

N

N

 

N: No Clinical Signs

C: Clinical Signs Observed

Interpretation of results:
other: not irritating
Conclusions:
The test chemical applied at the dose level of 0.5 gm on shaven back skin (approximately 6 cm2) of rabbit did not produce any clinical signs of irritation to skin during period of observation. The overall irritation score was calculated to be 0.00. Hence, the test chemicalk can be considered to be not irritating to skin. It can be classified under the category "Not Classified".
Executive summary:

A dermal irritation study was conducted on female New Zealand white rabbits in accordance with OECD 404 to assess the irritation parameter of the test chemical.

In the initial test one healthy rabbit of body weight 1.80kg±200gm was selected for the study after acclimatization. The test compound in the amount of 0.5 gm was applied at the different sites on the shaven back skin of animal. The hairs of back sides were removed (approximately 6 cm2) one day earlier before the treatment.

The test substance in the amount of 0.5 gm was applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which was held in place with non irritating tape. The first patch was applied on the shaven back skin of rabbit and removed after three minutes. No serious reaction was observed at the site of application. The second patch was applied on the different shaven back side and removed after one hour. There were no signs of skin reaction observed at this site of application. Finally, a third patch was applied at a different site and was removed after four hour. No skin reaction was observed after four hours patch removal.Finally, the animal was observed for 14 days, for any irritation and corrosion.

Because no corrosive effect observed in the initial test, a confirmatory test was done in order to confirm the irritant or negative response of the test substance by using two additional animals. In the confirmatory test the test compound in the amount of 0.5 gm was applied on the shaven back skin of two animals, each with one patch, for an exposure period of four hours.

The test chemical applied at the dose level of 0.5 gm on shaven back skin (approximately 6 cm2) of rabbit did not produce any clinical signs of irritation to skin during period of observation. The overall irritation score was calculated to be 0.00. Hence, the test chemical can be considered to be not irritating to skin. It can be classified under the category "Not Classified".

Endpoint:
skin 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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be dermal irritants. The dermal irritation potential of test article may be predicted by measurement of their cytotoxic effect, as reflected in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiDerm™ model.
GLP compliance:
no
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: as provided by MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Source strain:
other: Not applicable
Details on animal used as source of test system:
- 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 epidermis. 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 IdentificationAll of the EpiDerm™ 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 are included in this report. Tissue plates were appropriately labeled with study information.
Justification for test system used:
The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, In Vitro Life Science Laboratories, Bratislava, Slovakia) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
The tissues were exposed to the test article neat (undiluted) on April 25, 2018 (Run 1 of 1). EpiDerm™ 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 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.MTT and Color Pre-testsPretesting has actually been conducted for all chemicals, although the first intitial 8 test chemicals a pretesting was not conducted (for skin).MTT AssayFollowing the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 3 hours 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 time was approximately 3 hours with gentle shaking. 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 is 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 EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight (18 ± 3 hrs) at ~37°C, 5% CO2 in a humidified incubator.

2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test article are applied topically to tissues by pipette(liquid) Tissues were exposed to controls and the test article for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.a) Controls30 µL of negative control DPBS and 30 μL of the positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.b) Test Articles 25 mg of the test article was applied topically to the tissue 3. Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 15 times with sterile DPBS. After the 15th rinse from washing bottle, each insert wasw completely submerge 3 times in 150 ml DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for 24 ± 2 hours. After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 18 ± 3 hours, for a total of an approximately 42 hour post-exposure incubation.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The EpiDerm™ 3 dimensional human tissue model- Tissue Lot number(s): 26459- Date of initiation of testing: 6/08/2017TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: The test substance was rinsed from the tissues with sterile DPBS by filling and emptying the tissue insert 15 times to remove any residual test material. This was followed by completely submerge the insert 3 times in 150 ml DPBS.MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 300 µL MTT medium (1.0 mg/mL).- Incubation time: After 3 hours- Spectrophotometer: Synergy H4 spectrophotometer - Wavelength: 570 nm- Filter: No data- Filter bandwidth: No data- Linear OD range of spectrophotometer: No dataNUMBER OF REPLICATE TISSUES: 3CALCULATIONS and STATISTICAL METHODSAll data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT AssayBlanks:·        The optical density (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. 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. Tested compound :·        Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·        The OD mean per tissue was calculated. ·        The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Data Correction Procedure for MTT Interfering Compounds (if applicable)True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).ODtvt= optical density of treated viable tissueODkt= optical density of killed tissuesODtkt= optical density of treated killed tissueODukt= optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored Compounds (if applicable)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 mediaODvt= optical density of viable tissues incubated in media alone - Evaluation of data The results of the assay was evaluated and compared to negative control. Table: Criteria for in vitro Interpretation: In VitroResults In VivoPredictionMean tissue viability ≤50% Irritant (I), R38Mean tissue viability >50% Non-irritant (NI)- Assay quality controls- Negative Controls (NC)The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.  - Positive Controls (PC)5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.   - Standard Deviation (SD)The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit):25 mg
- Concentration (if solution): neat

VEHICLE (Not used)
- 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): 30 µL sterile DPBS
- Concentration (if solution): neat

POSITIVE CONTROL- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% solution of sodium dodecyl sulfate
Duration of treatment / exposure:
The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
For a total of an approximately 42 hour post-exposure incubation.
Number of replicates:
3 tissues were used for test compound and control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
103.5
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Mean O.D. :2.160 ; Non-Irritatnt
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.

Code N°

Tissue No.

Raw data

Blank corrected data

mean of OD

% of viability

Aliq. 1

Aliq. 2

Aliq. 1

Aliq. 2

NC

 

1

2.2457

2.1514

2.211

2.116

2.164

103.7

2

2.1558

2.2157

2.121

2.181

2.151

103.0

3

2.004

1.9602

1.969

1.925

1.947

93.3

PC

1

0.0828

0.0819

0.048

0.047

0.047

2.3

2

0.0792

0.0786

0.044

0.044

0.044

2.1

3

0.0875

0.0858

0.053

0.051

0.052

2.5

Test chemical

1

1.8047

1.8249

1.770

1.790

1.780

85.3

2

2.2468

2.3414

2.212

2.306

2.259

108.2

3

2.5173

2.4352

2.482

2.400

2.441

117.0

Interpretation of results:
other: not irritating
Conclusions:
The dermal irritation potential of test article was determined according to the OECD 439 test guideline followed for this study.The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 103.5%. Thus, test chemical was considered to be not irritating to the human skin.
Executive summary:

The dermal irritation potential of test article was determined according to the OECD 439 test guideline for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article (25mg) and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. 

 

The MTT data show the assay quality controls were met and passed the acceptance of criteria. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 103.5%. Thus, test chemical was considered to be not irritating to the human skin.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
data is from experimental reports
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Principles of method if other than guideline:
To assess the ocular irritation parameter of the test chemical in accordance with OECD 405
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
-Source : In-bred
-Age : 10 to 12 weeks
-Body weight range : -Source : In-bred
-Age : 10 to 12 weeks
-Body weight range : 1.80kg ±200gg
-Identification : By cage tag and corresponding colour body marking.
-Acclimatization : The healthy rabbits selected for study was acclimatized to standard laboratory condition for one week in experimental room under Veterinary examination.
-Randomization : After acclimatization and Veterinary examination three females were randomly selected.
-Accommodation : Animals were housed individually in stainless steel cages provided with stainless steel mesh bottom and facilities for food and water bottle.
-Diet : Pelleted feed supplied by Pranav agro Industries Ltd., Delhi
-Water : Community tap water passed through ‘Aqua Guard on line water filter’, was kept in glass bottles, ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22-25 deg.C
- Humidity (%): 40-60%
- Air changes (per hr): 10-15 air changes per hour
- Light and Dark Cycle: illumination cycle set to 12 hours artificial fluorescent light and 12 hours dark.
Vehicle:
water
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 gm moistened with distilled water
Duration of treatment / exposure:
the eyelids were gently pulled together for about one second to prevent loss of material
Observation period (in vivo):
1, 24, 48 and 72 hours after test substance application
Duration of post- treatment incubation (in vitro):
no data available
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): not washed
SCORING SYSTEM:Scale of weighted scores for grading the severity of ocular lesions developed by Draize et al

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein: hand-slit lamp
Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. After recording the observations at 24 hours, the eyes were further examined with the aid of fluorescein.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24/48/72 h
Score:
2.5
Max. score:
110
Reversibility:
not specified
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
In the initial test, the test chemical produced slight redness and mild discharge after 2 hours of application of test compound. This condition was recovered after 24 hours. Furthermore, no other clinical signs were recorded after application of test compound such as cage side activity, pain etc
In the confirmatory test, the test chemical produced slight redness of conjunctivae after two hour of application of test compound upto 24 hours. However, there was no other clinical sign recorded in both of the animals during the whole observation period for 21 days.
Other effects:
The test compound when applied in conjunctival sac of rabbits did not show any observable clinical signs such as cage side activity and pain or stress etc. throughout the observation period of 21 days

TABLE- 1     GRADING OF OCULAR LESIONS

 

S.NO/

SEX

 

OBSERVATION

Score

Total

Total Score

1/F

 

1 hour

24hours

48 hours

72 hours

Cornea

A.       Opacity-Degree of Density

0

0

0

0

0

0×0×5=0

B.       Area of Cornea Involved

0

0

0

0

0

Iris

A.       Values

0

0

0

0

0

0

Conjunctivae

A.       Redness

1

0

0

0

0

1+0+1×5=10

B.       Chemosis

0

0

0

0

0

C.       Discharge 

0

1

0

0

0

2/F

Cornea

A.       Opacity-Degree of Density

0

0

0

0

0

0×0×5=0

B.       Area of Cornea Involved

0

0

0

0

0

Iris

A.       Values

0

0

0

0

0

0

Conjunctivae

A.       Redness

1

0

0

0

0

1+0+1×5=10

B.       Chemosis

0

0

0

0

0

C.       Discharge 

0

1

0

0

0

3/F

Cornea

A.       Opacity-Degree of Density

0

0

0

0

0

0×0×5=0

B.       Area of Cornea Involved

0

0

0

0

0

Iris

A.       Values

0

0

0

0

0

0

Conjunctivae

A.       Redness

1

0

0

0

0

1+0+1×5=10

B.       Chemosis

0

0

0

0

0

C.       Discharge 

0

1

0

0

0

Grand total

30

Mean

10

Eye Irritation Scoring index

2.5

Interpretation of results:
other: not irritating
Conclusions:
In the initial test, the test chemical produced slight redness and mild discharge after 2 hours of application of test compound. This condition was recovered after 24 hours. Furthermore, no other clinical signs were recorded after application of test compound such as cage side activity, pain etc
In the confirmatory test, the test chemical produced slight redness of conjunctivae after two hour of application of test compound upto 24 hours. However, there was no other clinical sign recorded in both of the animals during the whole observation period for 21 days.
Also the overall irritation score was calculated to be 2.5/110. According to the irritation parameter propised by Kay & Calandra, the test chemical can be consdiered to be practically not irritating to eyes,
It can be classified under the category "Not Classified".
Executive summary:

The acute eye irritation study of the test chemical was conducted in New Zealand White Rabbit as per the OECD- 405 Guideline. 3 female young adult New Zealand White rabbits were used for the study.

One healthy rabbits of body weight 1.96kg was selected for study after acclimatization. Both eyes of rabbits were examined for any abnormal discharge such as eye irritation, ocular defects or pre-existing corneal injury from eye 24 hours prior to application of test compound.

The test compound was applied in the conjunctival sac of rabbit after gently pulling the lower lid away from the eyeball at the dose rate of0.1gm.The lids were then gently held for about one second in order to prevent loss of the material. The other eye which remain untreated, served as a control. The acute irritation to eye conjunctiva, cornea and iris was evaluated at 1, 24, 48 and 72 hoursafter the treatment. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animal was observed normally for 21 days. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weight before and during the study was observed.

The test chemical produced slight redness and mild discharge after 2 hours of application of test compound. This condition was recovered after 24 hours. Furthermore, no other clinical signs were recorded after application of test compound such as cage side activity, pain etc

A confirmatory test was performed using two additional animals according to the same procedure. The acute irritation to eye conjunctiva, cornea and iris was evaluated at 1, 24, 48 and 72 hours after the treatment. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animals were observed normally for 21 days. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weight before and during the study was observed.

In the confirmatory test, the test chemical produced slight redness of conjunctivae after two hour of application of test compound upto 24 hours. However, there was no other clinical sign recorded in both of the animals during the whole observation period for 21 days.

Also the overall irritation score was calculated to be 2.5/110. According to the irritation parameter propised by Kay & Calandra, the test chemical can be consdiered to be practically not irritating to eyes,

It can be classified under the category "Not Classified".

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 (MTT) assay, in the MatTek EpiOcular™ model
GLP compliance:
no
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 Lab. (Bratislava, Slovakia).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. Each lot of tissues was Quality Assured by MatTek, Inc. according 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 solid 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 approximately 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 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 chemicals 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 6 hrs ± 15 min for solid 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 ~25 minutes for solid test articles and controls.Following the washing and 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 chemicals 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:
Inserts are removed from the 24-well plate after 3 hrs of incubation and the bottom of the insert is blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 1 ml isopropanol in each well so that no isopropanol is flowing into the insert. At the end of the non-submerged extraction inserts and tissues are discarded without piercing and 1 ml of isopropanol is added into each well. The extract solution is mixed and the optical density of the extracted formazan (200 μL/well of a 96-well plate) was determined using 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.2 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: When a solid was tested, 50 mg of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
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:
When a solid was tested, 6 hours of the solid were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hrs ± 15 min.
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 6 hours for solid test articles and controls, at approximately37°C, 5% CO2 in a humidified incubator.
Following 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 totaling 18 hours for solid 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 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 meanthe 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 2 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:
28.4
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
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 28.2%. Thus, the test chemical was considered to be irritating to the human eyes.
Executive summary:

The ocular irritation potential of test chemical 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 28.4%. Hence, under the experimental test conditions it was concluded that test substance was considered to be irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” 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 studies have 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-vitro and in-vivo experiments conducted for target chemical which have been summarized as below:

 

A dermal irritation study was conducted on female New Zealand white rabbits in accordance with OECD 404 to assess the irritation parameter of the test chemical. In the initial test one healthy rabbit of body weight 1.80kg±200gm was selected for the study after acclimatization. The test compound in the amount of 0.5 gm was applied at the different sites on the shaven back skin of animal. The hairs of back sides were removed (approximately 6 cm2) one day earlier before the treatment. The test substance in the amount of 0.5 gm was applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which was held in place with non irritating tape. The first patch was applied on the shaven back skin of rabbit and removed after three minutes. No serious reaction was observed at the site of application. The second patch was applied on the different shaven back side and removed after one hour. There were no signs of skin reaction observed at this site of application. Finally, a third patch was applied at a different site and was removed after four hour. No skin reaction was observed after four hours patch removal. Finally, the animal was observed for 14 days, for any irritation and corrosion. Because no corrosive effect observed in the initial test, a confirmatory test was done in order to confirm the irritant or negative response of the test substance by using two additional animals. In the confirmatory test the test compound in the amount of 0.5 gm was applied on the shaven back skin of two animals, each with one patch, for an exposure period of four hours. The test chemical applied at the dose level of 0.5 gm on shaven back skin (approximately 6 cm2) of rabbit did not produce any clinical signs of irritation to skin during period of observation. The overall irritation score was calculated to be 0.00. Hence, the test chemical can be considered to be not irritating to skin.

The above in-vivo result was supported by an in-vitro test conducted for test chemical according to the OECD 439 test guideline. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. Tissues were exposed to the test article (25mg) and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay.  The MTT data show the assay quality controls were met and passed the acceptance of criteria. The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 103.5%. Thus, test chemical was considered to be not irritating to the human skin.

 

Based on the above summarized studies for target chemical,it can be concluded that the test chemical 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 have been performed to extent of ocular irritation caused by the test chemical in living organisms. These include in vivo as well as in vitro experimental studies performed for the test chemical. The results are summarized below:

The acute eye irritation study of the test chemical was conducted in New Zealand White Rabbit as per the OECD- 405 Guideline. 3 female young adult New Zealand White rabbits were used for the study. One healthy rabbits of body weight 1.96kg was selected for study after acclimatization. Both eyes of rabbits were examined for any abnormal discharge such as eye irritation, ocular defects or pre-existing corneal injury from eye 24 hours prior to application of test compound.The test compound was applied in the conjunctival sac of rabbit after gently pulling the lower lid away from the eyeball at the dose rate of0.1gm.The lids were then gently held for about one second in order to prevent loss of the material. The other eye which remain untreated, served as a control. The acute irritation to eye conjunctiva, cornea and iris was evaluated at 1, 24, 48 and 72 hoursafter the treatment. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animal was observed normally for 21 days. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weight before and during the study was observed. The test chemical produced slight redness and mild discharge after 2 hours of application of test compound. This condition was recovered after 24 hours. Furthermore, no other clinical signs were recorded after application of test compound such as cage side activity, pain etc A confirmatory test was performed using two additional animals according to the same procedure. The acute irritation to eye conjunctiva, cornea and iris was evaluated at 1, 24, 48 and 72 hours after the treatment. The grades of ocular reaction (conjunctiva, cornea and iris) were recorded at each observation. To determine the reversibility of the effect the animals were observed normally for 21 days. Any other lesions in the eye viz pannus, staining were observed and scored accordingly. Examination of reactions was facilitated by use of biomicroscope and hand slit lamp. Individual animal weight before and during the study was observed.

In the confirmatory test, the test chemical produced slight redness of conjunctivae after two hour of application of test compound upto 24 hours. However, there was no other clinical sign recorded in both of the animals during the whole observation period for 21 days. Also the overall irritation score was calculated to be 2.5/110. According to the irritation parameter propised by Kay & Calandra, the test chemical can be consdiered to be practically not irritating to eyes, It can be classified under the category "Not Classified".

The ocular irritation potential of test chemical was also 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 28.4%. Hence, under the experimental test conditions it was concluded that test substance was considered to be irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation.

There was an indication of positive effects in vitro but when tested in vivo it was confirmed that the test chemical was indeed negative. Since in vivo studies are always of a higher scientific value than in vitro so the in vivo studies override in vitro studies.

Taking these factors into considerations, the test chemical can be considered to be not irritating to eyes.

Justification for classification or non-classification

Available results for the test chemical indicate a very strong possibility that the test chemical lacks the potential to cause irritation/corrosion to skin and eyes.

Hence, the test chemical can be considered to be not irritating to eyes and skin. It can be classified under the category "Not Classified" under CLP Regulation.