Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
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 animals / tissue source

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

Test system

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)

Results and discussion

In vitro

Results
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.

Any other information on results incl. tables

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%.

Applicant's summary and conclusion

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.