Registration Dossier

Administrative data

Description of key information

Skin irritation

The dermal irritation potential of test chemical was assessedin various experimental studies which were conducted in rabbits, guinea pigs and humans.Based on the available key data and supporting studies,it can be concluded that the testchemical is able to cause skin irritation and considered as slightly irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category ““Category 2”.

 

Eye irritation

The ocular irritation potential of target chemical was assessedin various in vivo and in-vitro experimental studies which were conducted for test chemical and its structurally similar read across chemical.Based on the available key data and supporting studies,it can be concluded thatchemical is able to cause eye irritation and considered as slightly irritating. Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category “Category 2”.

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 report
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Principles of method if other than guideline:
To evaluate the dermal irritation potential of the test chemical in New Zealand white rabbits
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Source : Institute for Industrial Research & Toxicology
Age : 10 to 12 weeks
Sex : Female
Body weight range : 1.80kg±200g
Identification : By cage tag and corresponding colour body marking
Environmental conditions : Air conditioned rooms with 10-15 air changes per hour, temperature between 22-25 deg C, relative humidity 40-60% and illumination cycle set to 12 hours artificial fluorescent light and 12 hours dark.
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., B7/6 Ramesh Nagar, Delhi
Water : Community tap water passed through ‘Aqua Guard on line water filter’, was kept in glass bottles, ad libitum
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
unchanged (no vehicle)
Controls:
not specified
Amount / concentration applied:
0.5 gm of test compound
Duration of treatment / exposure:
no data
Observation period:
upto 14 days after application
Number of animals:
Three
Details on study design:
Details on study design
TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage:approx 6 square cm
- Type of wrap if used: impervious dressing
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: after patch removal, the site of application was cleaned with luke warm
water
OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : 1, 24,48 and 72 hours
SCORING SYSTEM: The intact skin site of application was observed for erythema and edema and scored according to Draize method
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
The test compound when applied at the dose level of 0.5 gm on shaven back skin of rabbit produced slight redness after 4 hours of patch removal. However, no other skin reaction was observed throughout the study period.
Other effects:
The test compound tert-butyl-4-methoxyphenol applied on the shaven back skin of rabbit did not produce any clinical signs of toxicity throughout the examination period of 14 days.

TABLE – 1

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

1

0

1

0

0

0

0

0

0

0

02

F

-

-

1

0

0

0

0

0

0

0

0

0

03

F

-

-

1

0

1

0

0

0

0

0

0

0

Total

0

0

3

0

2

0

0

0

0

0

0

0

Mean

0

0

1

0

0.67

0

0

0

0

0

0

0

Grand Total

1.67

Dermal Irritation Index: 1.67/4 = 0.42

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The dermal irritation index of the test chemical in New Zealand white rabbits was calculated as 0.42 and test compound can be considered as slightly irritating to the skin of the New Zealand white rabbits under test condition.
Executive summary:

A dermal irritation study was conducted on 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 2.00 kg 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. Slight redness was observed at the site of application of test compound after four hour patch removal. This condition was recovered after 24 hours. 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. After four hours the patch was removed and the skin reactions were graded according to Draize’s method. The test compound produced slightly redness after four hour patch removal upto 24 hours. There were no other clinical signs and symptoms recorded during the entire observation period.

 The dermal irritation index of the test chemical in New Zealand white rabbits was calculated as 0.42 and test compound can be classified under "Slight irritant ".

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:
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 assay, in the MatTek EpiDerm™ model.
GLP compliance:
no
Specific details on test material used for the study:
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 or 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 by MatTek provided test protocol.
- 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:
SolidOTHER SPECIFICS: No data available
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: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 30 µL sterile DPBS- Concentration (if solution): neatPOSITIVE 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:
Run 1
Value:
72
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Not irritating
Other effects / acceptance of results:
The MTT data show the assay quality controls were met.

Code N°

Tissue

Raw data

 

Blank corrected data

mean

% of viability

 

n

Aliq. 1

Aliq. 2

Aliq. 1

Aliq. 2

of aliquotes

 

NC

1

2.0377

2.1005

2.003

2.066

2.034

93.0

 

2

1.944

1.9615

1.909

1.927

1.918

87.7

 

3

2.6474

2.646

2.613

2.611

2.612

119.4

PC

1

0.0883

0.088

0.053

0.053

0.053

2.4

 

2

0.0796

0.0789

0.045

0.044

0.044

2.0

 

3

0.0809

0.0804

0.046

0.046

0.046

2.1

C4

1

1.7777

1.7334

1.743

1.699

1.721

78.6

 

2

1.3925

1.4253

1.358

1.390

1.374

62.8

 

3

1.6539

1.6728

1.619

1.638

1.629

74.4

Interpretation of results:
other: not Iirritating
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 substance was determined to be 72 %. 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 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 substance was determined to be 72%.

Hence, under the current experimental test conditions it was concluded that test substance was considered to be irritating to human skin and can thus be classified as ''Non irritating” as per CLP Regulation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed journal.
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
An eye irritation study was condcuted to determine the adverse effects caused by the chemical six albino New Zealand rabbits.
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Remarks:
albino
Vehicle:
not specified
Controls:
yes
Amount / concentration applied:
0.1ml
Duration of treatment / exposure:
48 hours
Observation period (in vivo):
24 and 48 hours
Details on study design:
Not specified
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 24
Score:
2
Max. score:
110
Reversibility:
not specified
Remarks on result:
other: minimal or mild eye irritation in rabbits.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 48
Score:
0
Max. score:
110
Reversibility:
not specified
Irritant / corrosive response data:
Minimal irritation was observed in treated rabbits.
Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
The test chemical was considered to be mildly irritating to the skin of treated albino rabbits.
Executive summary:

The eye-irritating ability of a face powder containing 0.2 percent test chemical was studied in six albino New Zealand rabbits. The test material was instilled into one eye of each animal in a single 0.1 ml dose; the untreated eye of each rabbit served as control.

Average eye irritation scores 24 and 48 hours following exposure to the face powder were 2 and 0, respectively (the maximum possible score per observation was 110).

The author concluded the test chemical as minimal irritating to the eye of treated albino rabbits.

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:
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:
86.1
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Remarks:
Mean O.D.:1.837; Non-irritant
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

2.2532

2.2372

2.219

2.203

2.211

103.6

2

2.098

2.0888

2.064

2.054

2.059

96.4

PC

1

0.5744

0.5927

0.540

0.558

0.549

25.7

2

0.7763

0.7786

0.742

0.744

0.743

34.8

Test chemical

1

1.8724

1.9032

1.838

1.869

1.853

86.8

2

1.8439

1.8665

1.809

1.832

1.821

85.3

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 86.1%. Thus, the test chemical 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 (50mg) 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 86.1%.

Hence, under the experimental test conditions it was concluded that test substance 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:
adverse effect observed (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 vivo and in-vitro experiments in rabbits, guinea pigs and humans for target chemical which have been summarized as follows;

 

A dermal irritation study was conducted on 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 2.00 kg 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. Slight redness was observed at the site of application of test compound after four hour patch removal. This condition was recovered after 24 hours. 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. After four hours the patch was removed and the skin reactions were graded according to Draize’s method. The test compound produced slightly redness after four hour patch removal upto 24 hours. There were no other clinical signs and symptoms recorded during the entire observation period.The dermal irritation index of the test chemical in New Zealand white rabbits was calculated as 0.42 and test compound can be classified under "Slight irritant ".

 

Further, an in-vitro stud has been reported for test chemical to determine the dermal irritation potential of test article was determined 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 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 substance was determined to be 72%. Hence, under the current experimental test conditions it was concluded that test substance was considered to be irritating to human skin.

 

A skin irritation study of suntan preparation containing 0.1 percent test chemical and an eye shadow and a face powder containing 0.2 percent of the test chemical were evaluated for their skin-irritating ability. In this study, the test formulation (0.1 ml) was applied under an occlusive filter disc to the clipped skin of the back of each of 9 albino rabbits. The discs were removed after 24 hours of skin contact, and the test sites were graded for irritation on a scale of 0 (no effect) to 4 (severe irritation). The grading of skin reactions was repeated 48 to 72 hours following the initial reading. The primary irritation indices were 0.46 (suntan preparation) and 0.1 1 (face powder), indicating that the formulation were minimal or slight skin irritant. Thus it can be concluded that the test substance is minimal irritating to skin of albino rabbits.

 

Another dermal irritation study was conducted in which an eye makeup preparation containing 0.1 percent of test chemical was evaluated in skin irritation study by means of the procedures specified in 16 CFR 1500.41.The test material (0.5 g) was applied under an occlusive patch to the intact and abraded skin of each of 6 albino rabbits. After a 24-hour exposure period, the patch was removed, and the test sites were graded for irritation. Skin reactions were evaluated again 48 hours following the first reading. The primary irritation index was 2.75, indicating that the product was a moderate skin irritant. Hence the test chemical can be considered as moderately irritating to the skin of treated rabbits.

 

The above results were supported by two guinea pig immersion tests conducted to evaluate dermal-irritating ability of two different bubble bath formulations each containing 0.1 percent test chemical. Six albino guinea pigs in each study were clipped free of abdominal hair and immersed up to their axillae in a 0.5 percent aqueous solution of the product. The 12 animals (6 animals/product) BHA concentrations for 4 hours a day for 3 consecutive days. Forty-eight hours following the last exposure, the skin reactions on the abdomen were graded on a scale of 10 (normal skin) to 1 (moribund due to skin injuries). The average irritation indices for the two formulations were 7.9 and 5.0, indicating mild (moderate scaling, no loss of skin elasticity) and moderate (cracking and fissuring, considerable loss in skin elasticity), skin irritation, respectively. No evidence of systemic toxicity was observed in either test. Thus on the basis of calculated irritation indices, the test chemical was considered to be mildly irritating to the skin of treated guinea pigs.

 

The overall results were supported by the skin irritation study performed to evaluate the skin irritating ability of three cosmetic products containing 0.2% of test chemical. During the study, the test material was applied under an occlusive patch in a single 0.1 ml dose of the volar surface of the forearm and/or inner aspect of the upperarm of 19 to 20 subjects. Ages of those tested ranged from 18 to 65. After 24 hours, the patches were removed and the test sites graded for skin irritation on a scale of 0 (no irritation) to 4 (severe irritation). For the three products each containing 0.2 percent test chemical, the primary irritation indices were 0.03 (20 subjects), 0.05 (20 subjects), and 0.15 (20 subjects), respectively, indicating in each case minimal skin irritation. Thus the test chemical can considered as mildly irritating to the skin of treated humans.

 

Even though the in-vitro study suggests that the test chemical might not be able to cause dermal effects, but it was strongly contradicted by the other in-vivo studies available for test chemicalthat strongly indicates that the test has potential to cause skin reactions when applied dermally.Hence based on the above summarized studies and based on majority of results obtained,it can be concluded that the testchemical is able to cause skin irritation and thus considered as irritating.Comparing the above annotations with the criteria of CLP regulation, it can be classified under the category“Category 2 (irritant)”.

Eye irritation

In different studies, the test chemical has been investigated for potential for ocular irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits for target chemical and its structurally similar read across substance that have been summarized as below;

 

 The eye-irritating ability of a face powder containing 0.2 percent of test chemical was studied in six albino New Zealand rabbits. The test material was instilled into one eye of each animal in a single 0.1 ml dose; the untreated eye of each rabbit served as control. Average eye irritation scores 24 and 48 hours following exposure to the face powder were 2 and 0, respectively (the maximum possible score per observation was 110). The author concluded the test chemical as minimal irritating to the eye of treated albino rabbits.

 

Another ocular irritation study was conducted on six albino New Zealand rabbits to test the eye irritating ability of an eye shadow containing 0.2 percent of test chemical. The test material (0.1 ml) was instilled three times a day for an unspecified number of days into the conjunctival sac of one eye of each animal by means of a +second spray held 6 inches from the face. It was not reported whether or not the treated eyes were given a water rinse. Average eye irritation scores on Days 1, 2, 3, 4, and 7 were 2, 1, 2, 1, and 0, respectively (maximum score per reading, 110). The product was considered a “mild” eye irritant by the investigators. Thus the test chemical was concluded to be moderately irritating to rabbit eyes.

 

Further, an in-vitro eye irritation study was conducted to determine 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 (50mg) 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 86.1%. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human eyes.

 

In next eye irritation test, an eye makeup preparation containing 0.1 percent of test chemical was assessed to determine its irritation potential according to the methods outlined in 16 CFR 1500.42. One-tenth ml of the test material was placed into one eye of each of six albino rabbits in a single application; the untreated eyes served as controls. Treated eyes did not receive a water rinse. Eyes were graded for ocular reaction 24, 48, and 72 hours following instillation of the test preparation. All treated eyes were negative for conjunctival redness, conjunctival chemosis, keratitis, and iritis. Hence the test chemical was considered to be not irritating to the eyes of treated albino rabbits.

 

The positive results were further supported by an eye irritation test of similar read across chemical performed in six New Zealand White rabbits. The chemical was applied as finely ground powder at a dose equivalent to 55 mg (0.1 ml) for 24 hours and reactions were scored at 24 , 48 and 72 hours according to Federal Hazardous Substances Act (FHSA). Mild irritating effects were observed with the irritation score of 18.8 (out of maximum score of 110). All scored zero after 168 hours. Since the chemical caused slight discomfort and ocular lesions during the 72 hours observation period, the test chemical was considered to be mildly irritating to the eyes of six New Zealand White rabbits.

 

Even though few references indicate that the test chemical may not be able to cause ocular irritation, which are contradicted by other in-vivo studies that strongly suggests that the test has potential to cause ocular reactions when instilled into the eyes. The dose and duration of contact may affect the occurrence or severity of skin reactions. Thus on the basis of majority of result obtained, it is concluded that the test chemical is able to cause eye irritation and thus can be considered as irritating. Hence, comparing the above annotations with the criteria of CLP regulation, Test chemical can be classified under the category “Category 2 (irritant)”.

 

 

Justification for classification or non-classification

The skin and eye irritation potential of test chemical were observed in various studies. The results obtained from these studies indicates that the chemical is able to cause slight skin and eye irritation. Hence the test chemical can be classified under the category “Category 2” for skin and eye as per CLP.