Registration Dossier

Administrative data

Description of key information

1. Skin corrosion according to OECD 431:

The mean percent relative viability in tissues treated with the test item was 93.43% after 3-minute exposure and 94.50% after 60-minute exposure. No significant reduction in the percent cell viability was observed after 3-minute and 60-minute exposure in treated tissues when compared with that of the concurrent negative control. The difference between viability of treated tissues was less than 3%, i.e., % CV.

All criteria for a valid study were met. From the results of this study, under the specified experimental conditions, 2-Amino-5-Chloro-N,3-Dimethylbenzamide was concluded to be non-corrosive in the in vitro skin corrosion test, using reconstructed human epidermis (RhE) tissues.

2. Skin irritation according to OECD 431:

 

The mean percent cell viability in tissues treated with the test item showed no significant reduction in the percent cell viability observed in 2-Amino-5-Chloro-N,3-Dimethylbenzamide treated tissues, when compared with that of the concurrent negative control. The mean percent relative viability in tissues treated with the test item was 98.0 % after 42-minute exposure. From the results of this study, under the specified experimental conditions, 2-Amino-5-Chloro-N,3-Dimethylbenzamide was concluded to be not an irritant in the in vitro skin irritation test, using reconstructed human epidermis (RhE) tissues.

3. Eye irritation accoridng to OECD 492:

The mean per cent cell viability in tissues treated with 2-amino-5-chloro-N,3-dimethylbenzamide was found to be 82.97% in concurrent negative control (100%) after 4 hours exposure which is above the established tissue viability cut-off value of 50%. All criteria for a valid study were met.

From the results of this study, under the specified experimental conditions, 2 -amino-5 -chloro-N,3 -dimethylbenzamide was concluded to be non-irritating in the in vitro eye irritation test, using reconstructed human cornea-like epithelium (RhCE).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 January to 20 May
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2019
Deviations:
no
Qualifier:
according to
Guideline:
other: EU method B40 bis, “In Vitro Skin Corrosion:Human Skin Model Test”
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test Item Name 2-Amino-5-Chloro-N,3-Dimethylbenzamide
ADAMA Substance Code: M198
Batch/Lot Number: 628-042-00
Analysed Concentration: 99.0%
(Refer Certificate of Analysis in APPENDIX 5) Physical State: Beige Solid
Date of Manufacture: 16 October 2019
Date of Expiry: 16 October 2021
Storage Condition (at JRF): As per the instruction received from the Sponsor on storage of the test item, the test item was stored:
Storage Temperature: Room temperature (15 to 30 °C)
Storage Condition: Cool and dry conditions
Storage Container: In original container as supplied by the Sponsor
Storage Location: Test Item Control Office, JRF
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
other: human epidermis (RHE/S/17)
Justification for test system used:
This study addresses the human health endpoint skin corrosion. It makes use of reconstructed human epidermis (RhE) (human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RHE) is recommended by the OECD, EC and other regulatory authorities. The SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is a recommended model for conducting in vitro skin corrosion studies. The results of the study are believed to be predictive for the potential of inducing skin corrosivity in humans.
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model
- Tissue batch number: Lot N° 20-RHE-011
- Production date: 27 January 2020

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at 37 ± 1 °C, in 5±1% CO2 in a 95% humidified atmosphere for 60 minute exposure
- Positive control tissues: 40 µL/0.5 cm2 of 8N KOH was applied for an exposure period of 60 minutes at 37 ± 1 °C in 5 ± 1% CO2 in a humidified incubator.
- Negative control tissues: 40 µL/0.5 cm2 of sterile distilled water was applied for 3 minutes at room temperature and 60 minutes at 37±1 °C in 5±1% CO2 in a 95% humidified atmosphere.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amounts applied: 20 mg ± 3 mg of test item/0.5 cm2.

NEGATIVE CONTROL
- Amounts applied: 40 µL/0.5 cm2 of sterile distilled water

POSITIVE CONTROL
- Amounts applied: 40 µL/0.5 cm2 of 8N KOH
Duration of treatment / exposure:
3 minutes at room temperature and 60 minutes at 37 ± 1 °C
Number of replicates:
three replicates//time point
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 Minutes Exposure
Value:
97.67
Negative controls validity:
valid
Remarks:
Viability: 100%
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 Minutes Exposure
Value:
94.5
Negative controls validity:
valid
Remarks:
Viability: 100%
Positive controls validity:
valid
Remarks:
Viability: 0.34%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No visible damage
- Direct-MTT reduction: No interaction
- Colour interference with MTT: No interaction

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: OD values (corrected ODs) of the negative controls in all tissues were between 1.537 and 1.918 i.e., within the test guideline optical density requirement of ≥ 0.8 and ≤ 3.0 (the acceptance criteria for SkinEthicTM RHE model). Results of the negative control met the OECD 431 guideline acceptance range for the prediction model SkinEthicTM RHE.
- Acceptance criteria met for positive control: The mean percent viability of the positive control was 0.34%, which met OECD 431 acceptance criteria, i.e., <15% viability.
- the observed results were within the acceptance/historical range of SkinEthic Laboratories/OECD TG acceptance criteria.

Pre-Tests

 

Colour Interference Test:

No significant difference in the absorbance was observed between the negative control (distilled water) and the test item. Therefore results of the colour interference test shows that interference was not observed due to test item. The results of the colour interference test are provided in the below-mentioned table:

Treatment

Optical Density (nm)

Interaction

Negative Control (distilled water)

0.041

No

0.041

2-Amino-5-Chloro-N,3-Dimethylbenzamide

0.046

No

0.044

Direct MTT Reduction Test:

The test item did not produce any direct MTT reduction (in the absence of tissues) when compared with that of the concurrent negative control (distilled water). Results of the direct MTT reduction test are summarised below:

Treatment

Interaction

Negative Control (distilled water)

No

2-Amino-5-Chloro-N,3-Dimethylbenzamide

No

Main Study:

The mean percent viability of tissues treated with 2-Amino-5-Chloro-N,3-Dimethylbenzamide, negative control and positive control are summarised below:

Treatment

Viability

3 Minutes Exposure

60 Minutes Exposure

Negative control (Sterile distilled water)

100%

100%

2-Amino-5-Chloro-N,3-Dimethylbenzamide

93.43%

94.50%

Positive control(8N KOH)

-

0.34%

Interpretation of results:
GHS criteria not met
Conclusions:
From results of this study, it is concluded that 2-Amino-5-Chloro-N,3-Dimethylbenzamide is predicted to be non-corrosive to the skin as indicated in OECD 431, under the specified conditions of this study using reconstructed human epidermis (RhE) tissues. The substance is not classified as skin corrosive according to EC regulation 1272/2008 (CLP).
Executive summary:

This study was performed to evaluate the corrosive potential of 2-Amino-5-Chloro-N,3-Dimethylbenzamide, using reconstructed human epidermis (RhE) tissue, in accordance with OECD 431.

Tissues were exposed to 2-Amino-5-Chloro-N,3-Dimethylbenzamide (test item) and sterile distilled water (negative control) for 3 minutes at room temperature and 60 minutes at 37 ± 1 °C in 5 ± 1% CO2using three replicates/time point. Positive control tissues were exposed for 60 minutes at 37 ± 1 °C in 5 ± 1% CO2.

The mean percent relative viability in tissues treated with the test item was 93.43% after 3-minute exposure and 94.50% after 60-minute exposure. No significant reduction in the percent cell viability was observed after 3-minute and 60-minute exposure in treated tissues when compared with that of the concurrent negative control. The difference between viability of treated tissues was less than 3%, i.e., % CV.

Treatment

Viability

3 Minutes Exposure

60 Minutes Exposure

Negative control (Sterile distilled water)

100%

100%

2-Amino-5-Chloro-N,3-Dimethylbenzamide

93.43%

94.50%

Positive control (8N KOH)

-

0.34%

All Optical Density (OD) values (corrected OD) for negative control replicates were between 1.537 and 1.918, against a guideline requirement of ≥ 0.8 and ≤ 3.0 (the acceptance criteria for SkinEthicTM RHE model). The positive control showed 0.34 % cell viability, against a guideline requirement of <15%, when compared with that of the concurrent negative control, demonstrating the efficiency of the SkinEthicTM RHE model.

All criteria for a valid study were met. From the results of this study, under the specified experimental conditions, 2-Amino-5-Chloro-N,3-Dimethylbenzamide was concluded to be non-corrosive in the in vitro skin corrosion test, using reconstructed human epidermis (RhE) tissues.

The substance is not classified as skin corrosive according to EC regulation 1272/2008 (CLP).

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 January 2020 to 12 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
June 2019
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from multiple donors
Source strain:
other: human: SkinEthicTM RHE
Justification for test system used:
This study addresses the human health endpoint skin irritation. It makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RhE) is also recommended by the OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies. The results of the study are believed to be of value in predicting the potential of inducing skin irritation by the test item in humans.
Vehicle:
water
Details on test system:
The test system used for the in vitro skin irritation test was reconstructed human epidermis (SkinEthicTM RHE) as recommended by the OECD 439 guideline. The SkinEthicTM RHE model consists of normal human keratinocytes cultured for 17-days on an insert of 0.5 cm2 polycarbonate filter at the air-liquid interface in a chemically defined growth medium. The cells form a multilayered, highly differentiated and stratified epidermis model of the human epidermis that consists of main basal, supra basal, spinous and granular layers and a functional stratum corneum:
Reconstructed human epidermis tissues, SkinEthicTM RHE model
- procured from SkinEthic Laboratories, Episkin 4, Rue Alexander Fleming, 69366 Lyon Cedex 07, France;
- Lot N°20-RHE-011

Negative control
tissues were treated with sterile Dulbecco’s Phosphate Buffered Saline (DPBS,
Lot N°: RNBH6544)

Positive control
tissues were treated with sterile sodium dodecyl sulfate (5% aq.):
CAS Number: 151-21-3
Purity: 100%
Lot N°: 0000000192
Date of receip : May 06, 2016
Date of Expiry: May 05, 2021
Appearance: White powder
Storage: Room temperature
1g sodium dodecyl sulfate was dissolved in 20 mL sterile distilled water (5% aq.) for the preparation
of 5% sodium dodecyl sulfate (5% aq.).

Solvents and Chemicals
MTT: Sigma (MKCB8895)
Isopropanol: Qualigens (3591580819)
Dulbecco's phosphatebuffered saline-10X: Sigma (RNBH6544)
SkinEthic Maintenance Medium : SkinEthic Laboratories (20 SMM 003)
SkinEthic Growth Medium : SkinEthic Laboratories (20 SGM 008)

EXPERIMENTAL PROCEDURES

Pre-Tests
Prior to the main experiment, preliminary tests such as colour interference test, and direct MTT reduction tests were performed to select the appropriate adapted controls.

Colour Interference Test
To identify colour interference, spectral analysis of the test item in distilled water was tested to evaluate if the test item requires adapted controls. To check colour interference, 16 µL of the distilled water as negative control and 16 mg of 2-Amino-5-Chloro-N,3-Dimethylbenzamide was added into 0.3 mL of distilled water in a transparent glass test tube and incubated at 37 ± 1 °C in 5% CO2 in a 95% humidified atmosphere for 60 minutes. Absorbance (OD) was measured at 570 nm.

Direct MTT Reduction Test
To assess non-specific reduction of MTT by the test item, direct MTT reduction test was performed. For negative control 16 µL of maintenance medium and 16 mg of 2-Amino-5-Chloro-N,3-Dimethylbenzamide was added into 0.3 mL of MTT solution (1 mg/mL) in 24 well plate and incubated at room temperature in dark for 180 minutes. Then visual examination was performed to check the MTT reduction.

Main Study

Pre-incubation
Upon receipt, the RhE tissues were cleaned to remove basal agarose using Kim wipes and then transferred to 6-well plates containing 1 mL fresh growth medium. Tissues were pre-incubated overnight, at 37±1 °C in 5±1% CO2 in a 95% humidified incubator.

Treatment
For treatment of negative and positive control tissues, 16 µL/0.5 cm2 sterile Dulbecco’s phosphate buffered saline (DPBS) and 16 µL/0.5 cm2 of 5% sodium dodecyl sulphate (5% aq.) were applied, respectively. Nylon mesh was placed on the apical surface of each tissue to assist the uniform spreading of liquid materials (i.e. for negative and positive controls). For test item treatment, the epidermal surface of the tissue was wetted using 10 µL of sterile distilled water (using a positive displacement pipette) followed by application of 16 mg (± 2 mg) of test item/0.5 cm2 and gently spread across the surface using a blunt spatula. All treated tissues were incubated at room temperature for 42 minute exposure.

Rinsing and Drying
After exposure, tissues were rinsed and then dried with cotton buds. The test item was removed by rinsing 25 times in a constant soft stream of 1 mL DPBS from 5–8 cm distance from the insert to remove the entire test item from the epidermal surface. Mesh (applied on negative and positive control tissues) was removed during washing all tissues. The bottom of the tissue inserts were dried on a sterile absorbent paper (Kim wipes) for 1–2 seconds. The surface of the stratum corneum was gently swept up using both ends of a cotton tip (5–6 turns per end). After washing, inserts were transferred to holding plates containing 300 µL maintenance medium.

Post-Incubation
After washing and drying, tissues were incubated in 6-well plate containing 2 mL growth medium at 37±1°C in 5±1% CO2 in a humidified incubator for 42 hours.

MTT Test
Post incubation, the MTT test was performed. Tissues were placed in MTT (1.0 mg/mL) solution and incubated for 180 minutes at 37 ± 1°C in 5 ± 1% CO2 in a 95% humidified incubator. At the end of MTT test, tissues were observed for MTT reduction.

Formazan Extraction
At the end of the MTT incubation period, the blue formazan salt was extracted by submerging tissues in 1.5 mL isopropanol in a 24-well plate. Tissues were incubated (without shaking) overnight in refrigerator, protected from light. After extraction, any residual tissues were autoclaved and sent to En-Cler biomedical waste Pvt. Ltd for disposal.

Optical Density Measurements
The optical density (OD) of the extracted formazan (200 μL/well of a 96 well plate) was determined in triplicate per tissue using a SynergyHT microplate reader (BioTek Instuments, USA) at 570 nm.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 mg (± 2 mg) of test item/0.5 cm2

NEGATIVE CONTROL
- Concentration: 16 μL/0.5 cm2 sterile Dulbecco’s phosphate
buffered saline (DPBS)

POSITIVE CONTROL
- Concentration: 16 μL/0.5 cm2 of 5% sodium dodecyl sulphate (5% aq.)
Duration of treatment / exposure:
42 minute exposure
Duration of post-treatment incubation (if applicable):
In a humidified incubator for 42 hours.
Number of replicates:
three replicates test item/positive control/negative control
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean Percent Viability/ 42 minutes exposure
Value:
98
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No interaction
- Colour interference with MTT: No interaction

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The OD values (corrected ODs) of negative controls in all tissues were between 2.316 and 2.388. Therefore, the results of the negative control met the acceptance range of the OECD test guideline 439 for the prediction model SkinEthicTM RHE. Test guideline requirement of optical density is ≥ 0.8 and ≤ 3.0
- Acceptance criteria met for positive control: The mean % viability of the positive control was 0.9%, which met the acceptance criteria of the OECD test guideline 439, i.e. <40% viability.

Pre-Tests

 

Colour Interference Test:

Treatment

Optical Density (nm)

Interaction

Negative Control (distilled water)

0.041

No

0.041

2-Amino-5-Chloro-N,3-Dimethylbenzamide

0.054

No

0.053

Direct MTT Reduction Test:

Treatment

Interaction

Negative Control (Maintenance Medium)

No

2-Amino-5-Chloro-N,3-Dimethylbenzamide

No

Main Study:

Treatment

Mean Percent Viability

42 Minutes Exposure

Negative control

(Dulbecco’s Phosphate Buffered Saline (DPBS))

100

2-Amino-5-Chloro-N,3-Dimethylbenzamide

98.0

Positive control

(Sodium dodecyl sulfate (5% aq.))

0.9

Data Summary of Percent Viability:

Treatment

Tissue Replicate

OD at 570 nm

Blank Corrected OD

Mean of Corrected OD

Mean OD of Three Tissues

% Viability/Tissue

Mean % Viability

SD. of % Viability

CV of % Viability

Irritant Class

Negative Control

(Dulbecco’s Phosphate Buffered Saline (DPBS))

1

2.431

2.388

2.364

2.343

100

100

0.019

0.81

NA

2.399

2.356

2.392

2.349

2

2.369

2.326

2.338

2.391

2.348

2.382

2.339

3

2.367

2.324

2.326

2.359

2.316

2.380

2.337

2-Amino-5-Chloro-N,3-Dimethylbenzamide

1

2.359

2.316

2.308

2.295

98.5

98.0

0.473

0.48

No Category

2.356

2.313

2.339

2.296

2

2.348

2.305

2.291

97.8

2.335

2.292

2.318

2.275

3

2.337

2.294

2.286

97.6

2.324

2.281

2.326

2.283

Positive control

(Sodium dodecyl sulfate (5% aq.))

1

0.066

0.023

0.022

0.022

0.9

0.9

0.000

0.00

Category 2

0.065

0.022

0.065

0.022

2

0.066

0.023

0.022

0.9

0.065

0.022

0.064

0.021

3

0.066

0.023

0.022

0.9

0.064

0.021

0.065

0.022

Key: OD = Optical Density, SD = Standard Deviation, CV = Coefficient of Variation, NA = Not Applicable

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

Based on the results of this study, 2-Amino-5-Chloro-N,3-Dimethylbenzamide is classified under “No Category (Non Irritant)”, as per the “United Nations Globally Harmonized System of Classification and Labelling of Chemicals”.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of this study, 2-Amino-5-Chloro-N,3-Dimethylbenzamide is predicted not to be a skin irritant. The substance is not classified as skin irritating according to EC regulation 1272/2008 (CLP).
Executive summary:

This study was performed to evaluate the skin irritation potential of 2-Amino-5-Chloro-N,3-Dimethylbenzamide, using reconstructed human epidermis (RHE) tissue, in accordance with OECD 439.

Tissues were exposed to the negative control (Dulbecco’s Phosphate Buffered Saline (DPBS)), positive

control (sodium dodecyl sulfate, 5% aqueous (SDS)) and 2-Amino-5-Chloro-N,3-Dimethylbenzamide in triplicate for 42 minutes, at room temperature.

The mean percent cell viability in tissues treated with the test item is shown below. There was no significant reduction in the percent cell viability observed in 2-Amino-5-Chloro-N,3-Dimethylbenzamide treated tissues, when compared with that of the concurrent negative control.

Treatment

Mean Percent Viability

42 Minutes Exposure

Negative control

(Dulbecco’s Phosphate Buffered Saline (DPBS))

100

2-Amino-5-Chloro-N,3-Dimethylbenzamide

98.0

Positive control

(Sodium dodecyl sulfate (5% aq.))

0.9

The negative and positive controls met the acceptance range for the OECD guideline and the efficiency of the

test system was demonstrated. All criteria for a valid study were met as described in the OECD 439

guideline.

Based on the results of this study, 2-Amino-5-Chloro-N,3-Dimethylbenzamide is predicted not to be a skin

irritant according to EC regulation 1272/2008 (CLP).

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:
29 January 2020 to 13 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Version / remarks:
2019
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Test Item Name 2-Amino-5-Chloro-N,3-Dimethylbenzamide
ADAMA Substance Code: M198
Batch/Lot Number: 628-042-00
Analysed Concentration: 99.0%
(Refer Certificate of Analysis in APPENDIX 5) Physical State: Beige Solid
Date of Manufacture: 16 October 2019
Date of Expiry: 16 October 2021
Storage Condition (at JRF): As per the instruction received from the Sponsor on storage of the test item, the test item was stored:
Storage Temperature: Room temperature (15 to 30 °C)
Storage Condition: Cool and dry conditions
Storage Container: In original container as supplied by the Sponsor
Storage Location: Test Item Control Office, JRF
Species:
human
Strain:
other: Reconstructed human cornea epithelium (RhCE)
Details on test animals or tissues and environmental conditions:
Test System: Reconstructed human cornea epithelium (RhCE)
Model: SkinEthicTM HCE
Source: EPISKIN– 4 Rue Alexander Fleming, 69366 Lyon Cedex 07 – France
Lot No.: 20-HCE-012
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 30 μL DPBS + 30 mg 2-Amino-5-Chloro-N,3-Dimethylbenzamide

NEGATIVE CONTROL:
Name: Dubelcco’s Phosphate Buffer Saline (DPBS)
- Amounts applied: 30 μL DPBS
Batch Nº: 1918956
Expiry Date: September 30, 2020
Manufactured by: Gibco

POSITIVE CONTROL:
Reference Substance Name: Methyl acetate
- Amounts applied: 10 μL DPBS + 30 μL methyl acetate (undiluted)
CAS No: 79-20-9
Analysed Purity: 99.5%
Batch No: STBG5814V
Supplied by: Sigma
Manufactured by: Sigma
Date of Receipt: May 08, 2017
Date of Expiry : May 07, 2022
Physical Appearance: Colourless liquid
Storage Condition (at JRF): Room temperature
Duration of treatment / exposure:
Tissues were exposed to test item, negative and positive control for 4 h
Duration of post- treatment incubation (in vitro):
For post incubation, the MTT test was performed. Tissues were placed in MTT (1.0 mg/mL) solution and incubated for 180 minutes at 37 ± 1°C in 5 ± 1% CO2 and saturated with humidity
Number of animals or in vitro replicates:
Two replicates in each group (test item, positive/negative control)
Details on study design:
NEGATIVE CONTROL USED: yes
- Name: Dubelcco’s Phosphate Buffer Saline
- Batch Nº: 1918956

POSITIVE CONTROL USED: yes
- Reference Substance Name: Methyl acetate
- Analysed Purity: 99.5%
- Batch No: STBG5814V

NUMBER OF REPLICATES , APPLICATION DOSE AND EXPOSURE TIME
- Test material: 30 mg 2-Amino-5-Chloro-N,3-Dimethylbenzamide+30 μL DPBS
- Positive control: 10 μL DPBS + 30 μL methyl acetate (undiluted)
- Negative control: 30 μL DPBS
- two replicates in each group (test item, positive/negative control)
Irritation parameter:
other: optical density
Run / experiment:
Mean Percent Viability
Value:
82.97
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Validity of the Test
- Negative control OD values were found to be within the range of > 1.0 to ≤ 2.5
- Mean tissue viability values for positive control was found < 20%
- Variation within the replicates was acceptable (< 20%)
Therefore, the experiment is considered valid.

Pre-Tests

Direct MTT Reduction Test

The test item did not produce direct MTT reduction when compared with that of the concurrent negative control (distilled water). Results of direct MTT reduction test are summarised in the table mentioned below:

Treatment

Interaction

Negative Control (distilled water)

No

2-Amino-5-Chloro-N,3-Dimethylbenzamide

No

Colour Interference Test

Any difference in the absorbance, due to colour interference, was not observed visibly for the test item. Results of direct MTT reduction test are summarised in the table mentioned below:

Treatment

Interaction

2-Amino-5-Chloro-N,3-Dimethylbenzamide

No

Negative Control

The individual optical density, corrected optical density, mean of corrected optical density, viability, and mean percent viability, are summarised in the following table.

The OD values (corrected mean ODs) of the negative control was 1.678 and 1.751. Therefore, results of the negative control met the acceptance range of the OECD test guideline 492 for the prediction model SkinEthicTM HCE. Test guideline requirement of optical density is > 1.0 and ≤ 2.5 (the acceptance criteria for SkinEthicTM HCE model, as per the OECD TG 492). Variation between the two negative controls tissues was < 20%, which met the acceptance range of the OECD test guideline 492 for the prediction model SkinEthicTM HCE. As per the certificate of analysis for the batch of tissues used in the study (Batch# 20-HCE-012), average optical density observed was 2.0 in QC testing at SkinEthic Laboratories. Therefore, the observed results were within the acceptable range of the SkinEthic Laboratories/OECD TG acceptance criteria.

Positive Control

The individual optical density, corrected optical density, mean of corrected optical density, viability, and mean percent viability, are summarised in the following table.

The mean % viability of the positive control was 18.51%, which met the acceptance criteria of the OECD test guideline 492, i.e., <20% viability.

 

Main Study

The individual optical density, corrected optical density, mean of corrected optical density, viability and mean per cent viability for test item are summarised in the following table.

The mean per cent viability of the 2-amino-5-chloro-N,3-dimethylbenzamide treated tissues, and the control tissues, is tabulated below:

Treatment

Mean Percent Viability

Negative control (DPBS)

100.00

Positive control (methyl acetate)

18.51

2-Amino-5-Chloro-N,3-Dimethylbenzamide

82.97

The mean per cent cell viability in tissues treated with 2-amino-5-chloro-N,3-dimethylbenzamide was found to be 82.97% in concurrent negative control (100%) which is above the established tissue viability cut-off value of 50%.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the mean viability score of 82.97%, determined under the specified experimental conditions using SkinEthicTM HCE RhCE, the classification for 2-amino-5-chloro-N,3-dimethylbenzamide: In accordance with the provisions of regulation 1272/2008, Annex I, 3.3, it is proposed classification is not required.
Executive summary:

Study type: Reconstructed Human Cornea-like Epithelium (RhCE) Test, OECD 492.

Study title: In Vitro Eye Irritation Test of 2-Amino-5-Chloro-N,3-Dimethylbenzamide Using Reconstructed Human Cornea-Like Epithelium.

 

This study was performed to evaluate the ocular irritation potential of 2-amino-5-chloro-N,3-dimethylbenzamide, as measured by the potential to induce cytotoxicity in a reconstructed human cornea-like epithelium (RhCE) tissue construct which closely mimics the properties of the human corneal epithelium. The RhCE test can identify test items not requiring classification, according to EC regualtion 1272/2008 (CLP).

 

RhCE tissues (two tissues per set) were treated with 30 μL of either Dulbecco’s Phosphate Buffered Saline (DPBS) (Set 1 - control), 30 μL of undiluted methyl acetate (Set 2 - positive control), and 30 μL of Dulbecco’s Phosphate Buffered Saline (DPBS) along with 30 mg 2-amino-5-chloro-N,3-dimethylbenzamide (Set 3 – test group). Post treatment the tissues were incubated for 4 h ± 6 minutes. At the end of the exposure period, residue was removed, and the tissue were kept for 30 minutes in maintenance medium at room temperature followed by an incubation period of 18 h ± 30 minutes.

Tissue viability was measured, following the treatment and a post-treatment incubation period, by enzymatic conversion of the vital dye MTT into a blue MTT formazan salt, measured after extraction from tissues. The optical density (OD) of the extracted formazan was determined using a microplate reader at 570 nm. The viability of the RhCE tissue was determined in comparison to tissues treated with the negative control substance (% viability) and was then used to predict the ocular hazard potential of the test chemical.

The mean per cent cell viability in tissues is shown below. The mean per cent cell viability in tissues treated with 2-amino-5-chloro-N,3-dimethylbenzamide was found to be 82.97% in concurrent negative control (100%) which is above the established tissue viability cut-off value of 50%.

Treatment

Mean Percent Viability

Negative control (Dulbecco’s Phosphate Buffered Saline)

100.00

Positive control (methyl acetate)

18.51

2-Amino-5-Chloro-N,3-Dimethylbenzamide

82.97

The negative and positive controls met the acceptance criteria, as described in the study plan, and confirmed the reliability of the test procedure.

Based on the mean viability score of 82.97%, determined under the specified experimental conditions using SkinEthicTM HCE RhCE, the classification for 2-amino-5-chloro-N,3-dimethylbenzamide, it is proposed classification is not required.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

1. From results of the study “Skin corrosion – In vitro; OECD 431 and EC Method B40: Skin Corrosion test with Reconstructed Human Epidermis (RHE) Tissues”, 2-Amino-5-Chloro-N,3-Dimethylbenzamide is predicted to be non-corrosive to the skin under the specified conditions of this study.

2. From results of the study “Skin irritation – In Vitro; OECD 439 (June 2019), EC method B46 (July 2012): Skin Irritation test with Reconstructed Human Epidermis (RHE) Tissues”, 2-Amino-5-Chloro-N,3-Dimethylbenzamide is predicted not to be a skin irritant under the specified conditions of this study.

Based on these two studies the substance is not classified as skin corrosive nor as skin irritating according to EC regulation 1272/2008 (CLP).

3. From results of the study “ Reconstructed Human Cornea-like Epithelium (RhCE) Test, OECD 492: In Vitro Eye Irritation Test of 2-Amino-5 -Chloro-N,3-Dimethylbenzamide Using Reconstructed Human Cornea-Like Epithelium”, 2 -amino-5 -chloro-N,3 -dimethylbenzamide is predicted to be non-irritating to eyes under the specified conditions of this study.

Based on this study the substance is not classified as eye irritating according to EC regulation 1272/2008 (CLP).