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Toxicological information

Skin irritation / corrosion

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Administrative data

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

Data source

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

Materials and methods

Test guidelineopen allclose all
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)

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

In vitro test system

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

Results and discussion

In vitro

Resultsopen allclose all
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.

Any other information on results incl. tables

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%

Applicant's summary and conclusion

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).