Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate

Endpoint summary

Description of key information

Skin Irritation

There were no clinical signs of toxicity and mortality. There were no skin reactions at the site of application at 24, 48 and 72 hours after test patch removal (as per Draize method). The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that the test chemical was "Not Irritating" to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.

Eye Irritation

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 1.2 passing the acceptance criteria. The mean % tissue viability of test substance Test chemical was determined to be 3.4%.

Hence, under the experimental test conditions it was concluded that test chemical was considered to be an eye irritant and being classified as “Irritating to eyes in Category 2” as per CLP Regulation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 05, 2017 to July 17, 2017

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 articles 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 (MatTek Corp., Ashland, MA).

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ 3-dimensional human tissues used in this study

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 Identification
All 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, Ashland, MA) 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 Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate neat (undiluted) on June 28, 2017 (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-tests
Pretesting for MTT auto-reduction and coloring was not performed for this study but was based on the results obtained from another study (CYP1690_R1b).

MTT Assay
Following 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 2 hours, 57 minute and 25 second 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 2 hours 04 minutes and 11 seconds with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer 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 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 articles are applied topically to tissues by pipette. Tissues were exposed to controls and the test articles for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.

a) Controls
30 µL of negative control DPBS, 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 Article
For Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate, the tissues were moistened with 25 μL of ultrapure water to improve contact of the tissue surface with the test article. Approximately 25 mg of Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl) benzoate was evenly applied to the apical surface of each tissue (n=3). All the tissues were placed into the ~37°C incubator with 5% CO2. The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.

3.Post-exposure treatment
After the 1 hour exposure, the tissues were rinsed 20 to 25 times with 1 mL of 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 either 25 hours, 38 minutes and 23 seconds or for 24 hours, 10 minutes and 09 seconds (as there were numerous tissues, they had to be broken down into 2 sets to complete dosing in a timely manner). 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 17 hours, 03 minutes and 34 seconds prior to performing the MTT assay, 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: 6/08/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Twice

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL MTT medium (1.0 mg/mL).
- Incubation time: After 2 hours, 57 minute and 25 second MTT incubation
- Spectrophotometer: Synergy H4 spectrophotometer
- Wavelength: 570 nm
- Filter: No data
- Filter bandwidth: No data
- Linear OD range of spectrophotometer: No data

NUMBER OF REPLICATE TISSUES: 3

CALCULATIONS and STATISTICAL METHODS
All data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows:

MTT Assay
Blanks:
·        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 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 (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 media
ODvt= 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 VivoPrediction
Mean tissue viability ≤50% Irritant (I), R38
Mean 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 (undiluted)

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
- 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:

Run 1

Value:

8.4

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, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 9.0 passing the acceptance criteria.

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The dermal irritation potential of test chemical 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 8.4 %. Thus, the test chemical was considered to be a skin irritant in Category 1 as per CLP Regulation.

Executive summary:

The dermal irritation potential of test chemical was determined according to the OECD 439 test guideline followed 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. The objective of this study was to assess the dermal irritation potential of test article. Tissues were exposed to test articls 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, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 9.0 passing the acceptance criteria.

 

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 8.4 %.

 

Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to human skin and being classified as ''Irritating to skin in Category 1” as per CLP Regulation.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

data is from experimental reports

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 402 (Acute Dermal Toxicity)

Principles of method if other than guideline:

To assess the toxicological profile of the test chemical on application as a single semi-occlusive dermal application to rats

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

- Source: Geniron Biolabs Pvt. Ltd., Bengaluru
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 to 12 Weeks
- Weight at study initiation: Females: 217.20 to 222.36 g
- Identification:By rat accession number. Identification of individual rats is by cage card and crystal violet colour body markings. The temporary body marking during acclimatization period was done with crystal violet. The rat accession numbers were allotted during the course of the study and was included in raw data and reported.
- Housing: Animals were housed individually in standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill. Additionally, polycarbonate rat huts were placed inside the cage as enrichment objects and were changed along with the cage once a week. Bedding: Steam sterilized corn cob was used and changed once a week along with the cage.
- Diet (e.g. ad libitum): Rat & Mice pellet feed, ad libitum
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier, ad libitum
- Acclimation period: The animals were acclimatized six days for G1-DRF, eleven days for G2-DRF, thirteen days G3-DRF and fifteen days for G3-Main before treatment. Animals were observed once daily during acclimatization period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 24°C
- Humidity (%): 56 to 67 %
- Air changes (per hr): air conditioned with adequate fresh air supply (12.9 air changes/hour).
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 22 June 2018 To: 31 August 2018

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: Milli-Q water

Controls:

not specified

Amount / concentration applied:

DRF = 200, 100, 2000 mg/kgbw
Main = 2000 mg/kg

Duration of treatment / exposure:

24 hourd

Observation period:

treatment site was observed 24, 48 and 72 hours after removal of test chemical using the Draize criteria

Number of animals:

G1 – DRF - 1
G2 – DRF - 1
G3 – DRF and G3-Main – 3

Details on study design:

TEST SITE
- Area of exposure: clipped skin of the torso
- % coverage: 10% of the body surface of the animal (semi-occlusive).
- Type of wrap if used: The area of application was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it was secured in position by adhesive tape wound around the torso
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towels
OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : All the rats were observed for clinical signs of toxicity and mortality
for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after removal of test item using the Draize criteria
SCORING SYSTEM:
- Method of calculation: Draize method

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

There were no skin reactions at the site of application at 24, 48 and 72 hours after test patch removal (as per Draize method).

Other effects:

There were no clinical signs and pre-terminal deaths (mortality) observed during the study

Dose Range Finding Study -DRF

Group & Dose (mg/kg body weight)

Date and time of application

Rat Number

Sex

Initial Bwt (g)

Quantity (mg) applied

Observation and skin reaction

Days

1

2

3

4

5

30 min

1h

2h

3h

4h

5h

6h

*

Er@

Ed@

*

Er@

Ed@

*

Er@

Ed@

G1 and 200

28 JUNE 2018 10:23 to 10: 24 AM

Rw223

F

238.66

221.66

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

Group & Dose (mg/kg body weight)

Animal Number

Sex

Observation

Necropsy findings

Days

6

7

8

9

10

11

12

13

14

15

G1 and 200

Rw223

F

N

N

N

N

N

N

N

N

N

N

NAD

Group & Dose (mg/kg body weight)

Date and time of application

Rat Number

Sex

Initial Bwt (g)

Quantity (mg) applied

Observation and skin reaction

Days

1

2

3

4

5

30 min

1h

2h

3h

4h

5h

6h

*

Er@

Ed@

*

Er@

Ed@

*

Er@

Ed@

G2 and 1000

03 July 2018 and 11.24 AM

Rw224

F

217.20

217

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

Group & Dose (mg/kg body weight)

Animal Number

Sex

Observation

Necropsy findings

Days

6

7

8

9

10

11

12

13

14

15

G2 and 1000

Rw224

F

N

N

N

N

N

N

N

N

N

N

NAD

Group & Dose (mg/kg body weight)

Date and time of application

Rat Number

Sex

Initial Bwt (g)

Quantity (mg) applied

Observation and skin reaction

Days

1

2

3

4

5

30 min

1h

2h

3h

4h

5h

6h

*

Er@

Ed@

*

Er@

Ed@

*

Er@

Ed@

G3 and 2000

05 July 2018 and 11.30 AM

Rw225

F

221.40

442

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

Group & Dose (mg/kg body weight)

Animal Number

Sex

Observation

Necropsy findings

Days

6

7

8

9

10

11

12

13

14

15

G3 and 2000

Rw225

F

N

N

N

N

N

N

N

N

N

N

NAD

Main study

Group & Dose (mg/kg body weight)

Date and time of application

Rat Number

Sex

Initial Bwt (g)

Quantity (mg) applied

Observation and skin reaction

Days

1

2

3

4

5

30 min

1h

2h

3h

4h

5h

6h

*

Er@

Ed@

*

Er@

Ed@

*

Er@

Ed@

G3 and 2000

07 July 2018 and 11:32 AM and 11:33 AM

Rw226

F

219.14

438

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

Rw227

F

222.36

444

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

Group & Dose (mg/kg body weight)

Animal Number

Sex

Observation

Necropsy findings

Days

6

7

8

9

10

11

12

13

14

15

G3 and 2000

Rw226

F

N

N

N

N

N

N

N

N

N

N

NAD

Rw227

F

N

N

N

N

N

N

N

N

N

N

NAD

N: Normal

h: hour

min: minutes

NAD: No abnormality detected

Er: Erythema

Ed: Edema Score

0: No Erythema / Edema

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

Interpretation of results:

other: not irritating

Conclusions:

There were no skin reactions at the site of application at 24, 48 and 72 hours after test patch removal (as per Draize method). The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that the test chemical was "Not Irritating" to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.

Executive summary:

A study designed and conducted to determine the dermal reaction profile of the test chemical in Wistar rats. The study was performed as per OECD Guidelines 402 and complying to the GLP procedures.Wistar rats were tested with 200 mg/kg, 1000 mg/kg and 2000 mg/kg with 1 female for the dose range finding study, followed by additional 2 females for main study at the dose of 2000 mg/kg body weight. Based on the individual body weight, the test item at the dose of 200 mg/kg, 1000 mg/kg and 2000 mg/kg body weight was weighed on an aluminium foil and made into a paste by adding sufficient volume of the Milli-Q water and completely transferred on to the cotton gauze (size: Females: 8 x 5 cm of 6 ply) and applied directly (semi-occlusive) to the clipped skin of the rat to cover about 10% of body surface of the rat. It was secured in position by adhesive tape wound around the torso. The

test item contact period with the skin was for 24 hours. After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towels.

All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after removal of test item using the Draize criteria.

There were no clinical signs of toxicity and mortality. There were no skin reactions at the site of application at 24, 48 and 72 hours after test patch removal (as per Draize method). The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that the test chemical was "Not Irritating" to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 05, 2017 to July 12, 2017

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 Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate (CAS No.- 17372-87-1) may be predicted by measurement of its cytotoxic effect, as reflected in the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiOcular™ model (MatTek Corp., Ashland, MA).

GLP compliance:

yes

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
Human Corneal Epithelia (HCE) by MatTek, Inc.:
The test article and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek Corporation (Ashland, MA). This model consists of normal human keratinocytes cultured on a permeable synthetic membrane at the air-liquid interface in a chemically defined medium. The cells form a stratified, squamous corneal epithelium resembling the corneal mucosa of the human eye. This model has been used with several common tests of cytotoxicity including MTT and interleukin 1-alpha (IL-1α). A growing body of evidence indicates that EpiOcular™ effectively provides a non-animal means to assess potential irritancy. The EpiOcular™ model closely mimics the human corneal mucosa and thus provides an important in vitro approach in the evaluation of ocular irritancy and toxicity. Each lot of tissues was Quality 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
- 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 hours for test article 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 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 test article and controls.

Number of animals or in vitro replicates:

3 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 Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate 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 hours for test article 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 test article and controls. 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 article 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
~50 mg of test article Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate 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 2 hours, 55 minutes and 02 seconds. 50 µL of ultrapure water was used as a negative control.

- Test Article Color Test
Approximately 50 mg of test article Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate 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 BioTek Synergy H4 (or equivalent) plate reader set to 570 nm. Test articles that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions).

- MTT Assay
After the recovery period, the MTT assay was performed on run 1 tissues by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 2 hours 55 minutes and 00 seconds (solids) of MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the tissues were rinsed twice with DPBS. The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was 2 hours 00 minutes with gentle shaking for solid exposed tissues. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer 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 59 minutes and 00 seconds. The tissues were not incubated overnight.

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 31 minutes 00 seconds. The controls and the test article will be applied topically to tissues by pipette. Three tissues will be used per test compound and control.

a)Controls:
50 µL of negative control sterile ultrapure water, 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 6 hour exposure time.

b)Test Article:
Approximately 50 mg of the test article Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate were added to the tissues as a fine powder. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time.

3. Post exposure treatment
After the exposure, the tissues were rinsed 20 to 25 times with ~1 mL 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 solid test article (and the respective control) were incubated, submerged in the media for ~25 minutes at room temperature. 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 17 hours 48 minutes 01 seconds at approximately 37 degC, 5% CO2 in a humidified incubator.

- Doses of test chemical and control substances used
a)Test Article:
Approximately 50 mg of the Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate were added to the tissues as a fine powder. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time.

b)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 6 hour exposure time.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Tissues were exposed for approximately ~6 hours for test article and controls, at approximately 37°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 test article 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: 3 tissues were used for test compound and control.

- Description of the method used to quantify MTT formazan
The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was 2 hours 00 minutes with gentle shaking for solid exposed tissues. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
·  The OD mean from all replicates for each plate (ODblank).

Negative Controls (NC):
·  The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
·  The OD mean per NC tissue was calculated.
·  The mean OD for all tissues corresponds to 100% viability.
·  The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.

ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.

Positive Control (PC):
·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
·        The OD mean per PC tissue was calculated.
·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.

Tested Articles:
·  Calculate the blank corrected value ODTT= ODTTraw– ODblank.
·  The OD mean per tissue is calculated.
·  The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
·  The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
·  The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.

Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)

Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.

Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.

- Evaluation of data
The results of the assay was evaluated and compared to negative control.

Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category

- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density (OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
 
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
 
- Standard Deviation (SD)
Each test of ocular irritancy potential is predicted from the mean viability determined on 3 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the replicates is <18% for three replicate tissues.

 

Irritation parameter:

other: mean % tissue viability

Run / experiment:

Run 1

Value:

3.4

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, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 1.2 passing the acceptance criteria.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The ocular irritation potential of test chemical was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance Disodium 2-(2,4,5,7- tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate was determined to be 3.4%. Thus, the test chemical was considered to be an eye irritant.

Executive summary:

The ocular irritation potential of test chemical was determined according to the OECD 492 test guideline followed 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. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 hour 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, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 1.2 passing the acceptance criteria.

 

The mean % tissue viability of test chemical was determined to be 3.4%.

 

Hence, under the experimental test conditions it was concluded that test chemical was considered to be an eye irritant and being classified as “Irritating to eyes in Category 2” 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 summarized to determine the degree of irritation caused by the test chemical in living organisms. These results include in vivo as well as in vitro experimental data for the test chemicals.

The dermal irritation potential of test chemical was determined according to the OECD 439 test guideline followed 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. The objective of this study was to assess the dermal irritation potential of test article. Tissues were exposed to test articls 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, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 9.0 passing the acceptance criteria.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 8.4 %.

 Hence, under the experimental test conditions it was concluded that test chemical was considered to be irritating to human skin and being classified as ''Irritating to skin in Category 1” as per CLP Regulation.

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

Wistar rats were tested with 200 mg/kg, 1000 mg/kg and 2000 mg/kg with 1 female for the dose range finding study, followed by additional 2 females for main study at the dose of 2000 mg/kg body weight. Based on the individual body weight, the test item at the dose of 200 mg/kg, 1000 mg/kg and 2000 mg/kg body weight was weighed on an aluminium foil and made into a paste by adding sufficient volume of the Milli-Q water and completely transferred on to the cotton gauze (size: Females: 8 x 5 cm of 6 ply) and applied directly (semi-occlusive) to the clipped skin of the rat to cover about 10% of body surface of the rat. It was secured in position by adhesive tape wound around the torso. The test item contact period with the skin was for 24 hours. After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towels.

All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after removal of test item using the Draize criteria.

There were no clinical signs of toxicity and mortality. There were no skin reactions at the site of application at 24, 48 and 72 hours after test patch removal (as per Draize method). The mean erythema and edema scores were 0.0 at all observation times. Hence, it was concluded that the test chemical was "Not Irritating" to the skin of Wistar rats under the experimental conditions tested and classified as “Category-Not Classified” as per CLP Classification.

The above Guideline study is further supported by a study performed according to OECD 404 Guidelines to evaluate the dermal irritation potential of the test chemical in rabbits. 3 animals (1 male, 2 females) New Zealand White rabbits were used for the study.

 500 mg/0.5g of the test compound wasapplied onto the left flank of each of three young adult rabbits. The duration of the treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours, as well as 7, 10 and 14 days after removal of the dressing.

The test item did not elicit any skin reactions at the application site of any animal (all scores = 0). The individual mean score for erythema/eschar and oedema for each of the three animals was 0. A light to marked red staining was apparent in all animals for the majority of the observation period and was still present in one animal 14 days after removal of the dressing, the end of the observation period for all animals. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals.

The test chemical was not irritating to the skin of New Zealand White Albino rabbits.

The above results are supported by a study performed which uses a single rabbit ear to indicate the Comedogenicity and irritancy of the test chemical. The test chemical was mixed in propylene glycol at a 9 to 1 dilution for testing unless otherwise indicated (10% concentration). A colony of New Zealand albino rabbits that have genetically good ears and free from mites were used. Three rabbits, weighing two to three kilograms, were used for each assay. Animals were housed singly in suspended cages and fed Purina Rabbit Chow and water ad libitum. Animals were maintained on a 12-hour light and 12-hour dark cycle. A dose of 1 ml of the test material was applied and spread once daily to the entire inner surface of once for five days per week for two weeks. The opposite untreated ear of each animal served as an untreated control.

The irritancy produced by repeated application of the chemical on the surface epidermis in the rabbit ear is evaluated on a scale of 0 to 5. The grades are summarized as follows:

 0 = No irritation; 1 = few scales, no Erythema; 2 = diffuse scaling, no Erythema; 3 = Generalized scaling with Erythema; 4 = Scaling, Erythema and Edema; 5 = Epidermal necrosis and slough.

The test chemical falls under Grade 0 (no irritation observed).

Hence it can be concluded that the test chemical was not irritating to rabbit ears.

Eventhough the results of the in vitro Guideline study gave indication of irritation/corrosive property of the test chemical, but the in vivo results claim otherwise. Also, when tested at the maximum applicable dose of 2000mg/kg in rats, no signs of erythema or edema were observed till 14 days of observation. Keeping all these factors in to consideration, the test chemical can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Eye Irritation

Various studies have been summarized to determine the extent of ocular damage caused by the test chemical in living organisms. The results include experimental in vivo and invitro studies for the target chemical.

The ocular irritation potential of test chemical was determined according to the OECD 492 test guideline followed 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. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 hour 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, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 1.2 passing the acceptance criteria. The mean % tissue viability of test substance Test chemical was determined to be 3.4%.

Hence, under the experimental test conditions it was concluded that test chemical was considered to be an eye irritant and being classified as “Irritating to eyes in Category 2” as per CLP Regulation.

This result is supported by another in vitro study conducted according to OECD 492 EpiOcular™ (MatTek ,MA) assay. EpiOcular™ (MatTek, USA) tissues were transferred to a six-well plate containing 1.0 mL of pre-warmed assay medium. Tissues were then incubated overnight at 37℃in a humidified atmosphere of 5% CO2. After incubation, tissues were pre-treated with 20 mL of Dulbecco’s Phosphate Buffered Saline (D-PBS) and incubated for 30 min, prior to exposure to the test chemical. The experimental steps after pre- treatment of solid materials, was as follows:

1) Treatment, 50 mg of solid material for 360 min;

 2) Rinse, for rinsing the tissues, three clean beakers, each containing 100 mL of Dulbecco’s Phosphate Buffered Saline, were prepared; two tissues that were treated with the same chemical were washed at a time; and each beaker was rinsed three times;

 3) Post-soak, after the final rinse, tissues were immersed in 5 mL of assay medium in a 12-well plate for 25 min for solid materials at room temperature;

4) Post-incubation, tissues were then transferred to a six-well plate containing 1 mL of warm assay medium (37℃) and incubated for 18 hrsfor solid materials.

Then, through MTT assay, the viability of tissues was measured to determine the eye irritation potential of test materials. Because colorants can interfere with MTT assay, additional color interference tests were conducted for EpiOcularTM according to TG 492. First, the light absorbance of colorants in water and isopropanol were examined for interference at 570 nm, which is in the same range with MTT for formazan, revealing significant color interferences. To consider the interference, an additional control, a non-specific color in living tissue (NSCliving) control, which underwent the entire testing procedure, although it was incubated with the medium instead of MTT solution during the MTT incubation step.

True tissue viability was calculated as [%Viability test] - [%NSCliving], and results showed similar tissue viabilities with those obtained without NSCliving. The treated tissues had viability greater than 60%, the test substance was classified as non-irritating, and if less than 60%, the test substance was classified as irritating.

Eosine YS decreased cell viability below 60%, which indicated ocular irritancy. The mean %tissue viability was <10%. Hence, the test chemical was considered to be an ocular irritant and it can be classified under the category “Category 2”.

The above in vitro Guideline studies were further supported by other in vitro study using MCTT HCE™ assay. MCTT HCE was prepared from primary human limbal cells. After receiving the tissues,900 mL of fresh maintenance medium was replenished and incubated at 37°C, 5% CO2 for 24 h. The experimental steps after pre- treatment of solid materials, was as follows:

Test material solutions at various concentrations were prepared by serial dilution in PBS and were treated at 50 mg for solids (100% coal-tar dyes) per well, after which MCTT HCE was incubated for 60 minutes. After the exposure, the models were washed four times with PBS and further incubated with 900 ml of a new maintenance medium for 16 hrs (post-incubation period) at 37°C, 5% CO2.For the measurement of tissue test chemical viability, tissues were incubated with 300 μL of WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3 -benzene disulfonate) and optical density was measured at 450 nm. For the measurement of tissue viability, tissues were incubated with 300 μL of WST-1 (4-[3-(4-iodophenyl)-2-(4 -nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and optical density was measured at 450 nm. Negative control, PBS, and coal-tar dyes showing cell viability over 45%, were determined as non-irritant materials. Positive control (2% of SLS) and coal-tar dyes that reduced cell viability below 45% were determined to be irritant materials. For histological evaluation, the MCTT HCETM was fixed in 10% neutral buffered formalin to process it for embedding in paraffin. Five-mm vertical sections were cut and stained with hematoxylin and eosin for examination by Olympus BX43 microscope.

 The test chemical decreased cell viability below 45%, which indicated ocular irritancy. The mean %tissue viability was <10%.

Hence, the test chemical was considered to be an ocular irritant and it can be classified under the category “Category 2”.

The results of the in vitro studies indicate a very strong possibility of the test chemical causing irritation to eyes. Hence, the test chemical can be considered to be irritating to eyes and can be classified under the category “Category 2”.

Justification for classification or non-classification

Eventhough the results of the in vitro Guideline study gave indication of irritation/corrosive property of the test chemical, but the in vivo results claim otherwise. Also, when tested at the maximum applicable dose of 2000mg/kg in rats, no signs of erythema or edema were observed till 14 days of observation. Keeping all these factors in to consideration, the test chemical can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

The results of the in vitro studies indicate a very strong possibility of the test chemical causing irritation to eyes. Hence, the test chemical can be considered to be irritating to eyes and can be classified under the category “Category 2”.