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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 January to 16 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to
Guideline:
other: EC No. 440/2008, L 112, method B60: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method
Version / remarks:
14 February 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
The test system used for the in vitro KeratinoSensTM assay was KeratinoSensTM (HaCaT) cell line, an immortalised adherent human keratinocyte cell line. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The KeratinoSensTM test method has been validated by EURL ECVAM and considered scientifically valid to support the discrimination of skin sensitisers and non-sensitisers for the purpose of hazard identification

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
CAS Number: 890707-28-5
Batch/Lot Number: 628-042-00
Active Ingredient Content: 99.0%
Physical State: Beige Solid
Molecular Weight: 198.65
Date of Manufacture: 16 October 2019
Date of Expiry: 16 October 2021
Storage Condition:
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

In vitro test system

Details on study design:
DETAILS OF TEST SYSTEM
- The genetically modified HaCaT cell line (KeratinoSensTM) obtained from Givaudan Schweiz AG
- The KeratinoSensTM cell line contains a stable insertion of the luciferase gene under the transcriptional control of a constitutive SV40 promoter fused with an antioxidant response element (ARE) of the human AKR1C2 gene
- Source: Mutagenicity section, Jai Research Foundation, India
- Cell line Lot No.: JRF/HaCaT/2016/01

VEHICLE
- Dimethyl sulfoxide (DMSO)
- . 2-amino-5-chloro-N,3-dimethylbenzamide was found to be soluble in dimethyl sulfoxide, at the concentration of 200 mM. Hence, dimethyl sulfoxide was selected as the vehicle for this study.

NEGATIVE (SOLVENT) CONTROL: yes
DMSO was used as the concurrent negative control for each set of experiments.
Name: DMSO
CAS Number: 67-68-5
Manufactured by: Qualigens
Lot N°: 3491020519
Retest Date: May 2024
Appearance (Colour): Colourless Liquid
Assay: 99.95%
Storage: Room temperature

POSITIVE CONTROL USED: yes
Trans-cinnamaldehyde was used as a positive control for which stock concentrations from 0.4 to 6.4 mM were prepared in DMSO, which were further diluted to achieve final concentration of positive control ranging from 4 to 64 µM.
CAS Number: 14371-10-9
Manufactured by: Sigma Aldrich
Batch N°:STBF4119V
Date of Receipt: 06 October 2015
Date of Expiry: 05 October 2020
Appearance (Colour): Light yellow liquid
Purity: 99.4%
Storage: 2–8 °C
Density: 1.05 g/mL

NUMBER OF REPLICATES, APPLICATION DOSE AND EXPOSURE TIME
- KeratinosensTM (HaCaT) cells were exposed to 2-Amino-5-Chloro-N,3-Dimethylbenzamide at concentrations: 0.98 µM to 2000 µM
- KeratinosensTM (HaCaT) cells were exposed to Positive control (Trans-cinnamaldehyde) at concentrations: 4 to 64 µM.
- KeratinosensTM (HaCaT) cells were exposed to Negative control (DMSO)
- Cells were incubated for 48 ± 2 hours in 5 ± 1% CO2 at 37 ± 1 °C. After incubation, cells were analysed for luciferase activity.
- Cell viability of the concurrently treated cells was also evaluated using the MTT test with a separate set of plates.
- For each test item and positive control, one experiment consisting of at least two independent repetitions, each containing three replicates of each concentration.
- Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells.

Results and discussion

Positive control results:
Luciferase activity induction obtained with the positive control, trans-cinnamaldehyde, was found to be >1.5 (the threshold value) at concentrations of 32 µM, and 64 µM in both repetitions. The induction for positive control at concentrations of 32 µM and 64 µM was found to be 1.77, and 2.85 µM in experiment 2 and 1.57 and 2.19 µM in experiment 4, respectively. The EC1.5 for positive control was 20.11 in experiment 2 and 28.15 in experiment 4. The average gene induction for positive control at 64 µM was found to be 2.85 and 2.19 for experiment 2 and 4, respectively (which was within the guideline acceptable limits of 2 and 8). A clear dose response, with increasing gene induction at increasing dose, was observed for trans-cinnamaldehyde in experiment 2 and experiment 4.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Luciferase gene induction
Remarks:
Imax: maximal fold gene-induction (Imax) value observed at any concentration of the test item and positive control.
Run / experiment:
Imax
Value:
1.14
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
KeratinoSensTM Prediction: Non-Sensitiser
Key result
Parameter:
other: Luciferase gene induction
Remarks:
EC1.5: value representing the concentration for which a gene induction above the 1.5-fold threshold.
Run / experiment:
EC1.5
Value:
> 2 000
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
KeratinoSensTM Prediction: Non-Sensitiser
Key result
Parameter:
other: Cellular viability
Remarks:
IC50: concentration value for 50% reduction of cellular viability.
Run / experiment:
IC50
Value:
> 2 000
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
KeratinoSensTM Prediction: Non-Sensitiser
Key result
Parameter:
other: Cellular viability
Remarks:
IC30: concentration value for 30% reduction of cellular viability.
Run / experiment:
IC30
Value:
1 571.2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
KeratinoSensTM Prediction: Non-Sensitiser
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Negative Control

The coefficient of variation observed for the negative control, during experiment 2 and 4 for 2-Amino-5-Chloro-N,3-Dimethylbenzamide was 11.11% and 12.01%, respectively, which was below 20%. Since variation amongst the replicates was less than 20%, results of this run were considered as valid.

 

Luciferase Activity

The maximal average fold induction of luciferase activity (Imax) following treatment with 2-Amino-5-Chloro-N,3-Dimethylbenzamide was 1.14, which is less than the 1.5-fold increase required for a positive result. The EC1.5 mean values, representing the concentration where induction of luciferase activity is above the 1.5 fold threshold (i.e 50% enhanced luciferase activity), for 2-Amino-5-Chloro-N,3-Dimethylbenzamide were >2000 µM and there was no dose dependent increase in luciferase induction

 

In Vitro Skin Sensitisation: Keratinocyte-Based ARE-Nrf2 Luciferase Reporter Gene Test of 2-Amino-5-Chloro-N,3-Dimethylbenzamide Using KeratinoSensTM (HaCat) Cell line.

 

Mean Summary of Luciferase Activity Induction and Cellular Viability Values:

Imax(Maximal average fold induction of luciferase activity):

Test Item

Rep2

Rep4

Average

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

1.02

1.27

1.14

trans-Cinnamaldehyde

2.85

2.19

2.52

EC1.5(Concentration for which induction of luciferase activity is above the 1.5-fold threshold:

Test Item

Rep2

Rep4

GeoMean

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

>2000µM

>2000µM

>2000µM

trans-Cinnamaldehyde

20.11

28.15

23.79

IC50(Concentration value for 50% reduction of cellular viability):

Test Item

Rep2

Rep4

GeoMean

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

1831.78µM

>2000µM

>2000µM

IC30(Concentration value for 30% reduction of cellular viability):

Test Item

Rep2

Rep4

GeoMean

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

1403.00µM

1759.55µM

1571.20µM

Rep = Repetition/Experiment – each comprising 3 plates.

Values shown for each repetition are the mean values of 3 replicates.

IC50and IC30cannot be determined for positive control.

In Vitro Skin Sensitisation: Keratinocyte-Based ARE-Nrf2 Luciferase Reporter Gene Test of 2-Amino-5-Chloro-N,3-Dimethylbenzamide Using KeratinoSensTM (HaCat) Cell line

 

Summary of Mean Luciferase Activity Induction Values (Mean of Two Repetitions /Experiments):

2-Amino-5-Chloro-N,3-Dimethylbenzamide(µM)

Fold Luciferase Activity Induction

Mean

SD

0.98

1.14

0.18

1.95

1.10

0.22

3.91

1.07

0.29

7.81

1.00

0.25

15.63

1.04

0.24

31.25

1.08

0.16

62.5

1.04

0.18

125

1.00

0.11

250

0.85

0.17

500

0.82

0.08

1000

0.80

0.04

2000

0.51

0.24

Cytotoxicity Assessment

Toxicity was observed, as cellular viability was below 70%, at a concentration of 2000 µM. Cellular viability was 53.78% at the tested concentrations of 2000 µM.

 

Interpretation

For 2-Amino-5-Chloro-N,3-Dimethylbenzamide, four experiments were conducted and out of these, two valid experiments (experiment 2 and 4) were considered for the final evaluation. The reason for conducting the repeat experiments was due to the variation between each replicates of test item and failure of positive control to meet the acceptance criteria in experiment 1 and experiment 3, respectively (results of experiments 1 and 3 are on file but not included in this report).

The mean results are summarised below:

Test Item Name

Luciferase gene induction

Cellular viability

KeratinoSensTM

Prediction

Imax

EC1.5

IC50

IC30

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

1.14

>2000µM

>2000µM

1571.20µM

Non-Sensitiser

Positive Control

(Trans-Cinnamaldehyde)

2.52

23.79

-

 

-

Sensitiser

Results of this KeratinoSensTM assay indicate that 2-Amino-5-Chloro-N,3-Dimethylbenzamide met all the evaluation criteria to predict that it is a non-sensitiser in this test. The negative and positive controls met the acceptance criteria and were correctly identified as the non-sensitiser and sensitiser, respectively. This showed the suitability of the test system and procedures used.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
From the results of this KeratinoSensTM assay, under the specified experimental conditions, 2-Amino-5-Chloro-N,3-Dimethylbenzamide is predicted to be a non-sensitiser to skin according to EC regulation 1272/2008 (CLP).
Executive summary:

This study was conducted to evaluate the skin sensitisation potential of 2-Amino-5-Chloro-N,3-Dimethylbenzamide, by assessing keratinocyte activation, using a on Keratinocyte-Based ARE-Nrf2 Luciferase Reporter Gene method, as recommended by the OECD Test guideline 442D.

2-Amino-5-Chloro-N,3-Dimethylbenzamide was found to be soluble at 200 mM in dimethyl sulfoxide (DMSO). Therefore DMSO was selected as a vehicle. The test item was tested in two independent experiments. KeratinosensTM (HaCaT) cells were exposed to 2-Amino-5-Chloro-N,3-Dimethylbenzamide at concentrations of 0.98 µM to 2000 µM, to the positive control (trans-cinnamaldehyde) at concentrations of 4 to 64 µM or to the negative control (DMSO). Cells were incubated for 48 ± 2 hours in 5 ± 1% CO2at 37 ± 1 °C. After incubation, cells were analysed for luciferase activity. Cell viability of the concurrently treated cells was also evaluated using the MTT test with a separate set of plates. The Imax, and EC1.5value was calculated based on luciferase activity, i.e., luminescence measured (reading of three plates) while IC50and IC30were calculated based on results of cytotoxicity (OD values, reading of one plate).

Test Item Name

Luciferase gene induction

Cellular viability

KeratinoSensTM

Prediction

Imax

EC1.5

IC50

IC30

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

1.14

>2000µM

>2000µM

1571.20 µM

Non-Sensitiser

Positive Control

(Trans-Cinnamaldehyde)

2.52

23.79

-

 

-

Sensitiser

2-Amino-5-Chloro-N,3-Dimethylbenzamide met all the evaluation criteria to be concluded as a non-sensitiser in the KeratinoSens assay. Negative and positive controls met the acceptance criteria for the controls and were correctly identified as non-sensitiser and sensitiser, respectively. All criteria for a valid study were met, as described in the study plan.

From the results of this KeratinoSens assay, under the specified experimental conditions, 2-Amino-5-Chloro-N,3-Dimethylbenzamide is predicted to be a non-sensitiser to skin.