Okra, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2017, February 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

-The objective of this study was to evaluate the in vitro sensitization potential of the test item applied onto 3D human reconstructed epidermis (EpiSkin™) using the SENS-IS assay, based on the quantitative analysis of specific gene biomarkers.
The expression profile of 61 genes divided in three sets were analysed: one set of 23 genes related to the irritation process and the two other sets of genes, named “SENS-IS” and “ARE”, with 21 and 17 biomarkers respectively, involved in skin sensitization [1, 2].
The expression level of these separate sets of genes was determined by qRT-PCR (quantitative real-time polymerase chain reaction) after a 15 min incubation period with the test item and a 6 hours post-incubation period at 37°C.
The fold expression of each gene were calculated as a ratio between the mRNA level of test item-treated epidermis versus control (vehicle treated) epidermis.


METHODS
Preliminary test: Solubility test
The test item was solubilized in phosphate buffered saline (PBS), olive oil and dimethylsulfoxide (DMSO) at a concentration of 50 and 10% (v/v) at room temperature and at 37°C (if not soluble at RT).
The solubility of the test item was assessed by visual inspection of each preparation.

Main test: SENS-IS assay
The test item (30 µL) was deposited on the epidermis surface and gently spread on the entire surface [3]. After 15 minutes of exposure, the epidermis was rinsed with PBS and then incubated at 37°C for 6 hours.
After incubation, reconstructed epidermis was removed from the inserts with forceps and placed in a cryotube for freezing in liquid nitrogen. The epidermis was then transferred in a tube containing 1 mL of Qiazol reagent and 2 steel beads and was homogenized using the TissueLyser II. After centrifugation, the supernatant was collected and stored at -20°C until RNA extraction.
After addition of bromochloropropane, total RNA were purified using the miRNeasy extraction Kit according to the manufacturer’s instructions (Qiagen, Courtaboeuf, France). RNA quality was assessed by measuring 260/280 absorbance ratio. The mRNA was then reversed as cDNA using SuperScript III Reverse Transcriptase kit and RNase inhibitor.
After reverse transcription, quantitative gene expression was measured by qRT-PCR using a SYBRGreen® buffer and specific primers defined for the SENS-IS test. The housekeeping genes (Glucuronidase ß, ß2 microglobuline, and Nono « non-POU domain containing octamer-binding ») were analyzed in parallel.

Data analysis and interpretation
Samples were analyzed by the LightCycler 480 software using the second derivative maximum method. This method allows the calculation of the crossing point (Cp) of each sample defined as the number of cycles from which the fluorescence signal enters the exponential phase of the reaction. This value is dependent of the amount of the mRNA present in the sample.
For each sample, the mRNA content for each gene of interest was normalized to the mean mRNA content of the 3 house-keeping genes. For each gene, the fold increase in mRNA level over vehicle controls was calculated as followed:
E = Expression level for the test item / Expression level for the vehicle controls*
* Mean of OO and PBS samples
The endpoint values are the number of positive genes (i.e., genes obtaining a E > 1.25) in each group.

Validation of the study:
alidation of the experiment
The following criteria must be met for an experiment to be considered valid:
- Negative sensitization control (DMSO): this control should induce the over-expression of 6 genes at most in the SENS-IS and ARE groups of genes.
- Positive irritation control and negative sensitization control (SLS at 5%): this control should induce the over expression of at least 16 genes in the IRRITATION group of genes and 6 genes at most in the SENS-IS and ARE groups of genes.
- Positive sensitization control (TNBS at 1M): this control should induce the over expression of at least 7 genes in the SENS-IS or ARE group of genes.

Validation of the sample
The following criteria must be met for sample result to be considered valid:
- The Cp value of HSP90AA1 gene must be ≤ 21.
- If more than 20 genes are overexpressed in the “IRRITATION” set of genes for a given concentration, the result is classified as false positive to take into account non-specific genes up regulation that could be due to cell stress.

Test item classification
A test item is classified as irritant if at least 16/23 genes of the “IRRITATION” group are significantly overexpressed.
A test item is classified as sensitizer if at least 7/17 genes of the “ARE” group, and/or 7/21 genes in the “SENS-IS” group are significantly overexpressed.
Moreover, the results obtained with the different concentrations allow the classification of the test item according to the lowest concentration that gives a positive result. Thus a test item is classified in:
- category 1A: strong to extreme skin sensitizer, when a positive result is obtained at concentrations of 0.1 and/or 1%,
- category 1B: weak to moderate sensitizer, when a positive result is obtained at concentrations of 10 and/or 50%.
A test item is classified as a non-sensitizer when no positive result is observed for all tested concentrations (0.1, 1, 10, 50 and 100%).

GLP compliance:

yes (incl. QA statement)

Type of study:

other: SENS-IS test

Test material

Test material information

Constituent 1

Reference substance name:

Okra, ext.

EC Number:

294-510-2

EC Name:

Okra, ext.

Cas Number:

91723-07-8

Molecular formula:

not applicable

IUPAC Name:

Hibiscus abelmoschus, ext.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0016468911
- Expiration date of the lot/batch: December 9, 2019


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

In vitro test system

Details on the study design:

The objective of this study was to evaluate the in vitro sensitization potential of the test item applied onto 3D human reconstructed epidermis (EpiSkin™) using the SENS-IS assay, based on the quantitative analysis of specific gene biomarkers.
The expression profile of 61 genes divided in three sets were analysed: one set of 23 genes related to the irritation process and the two other sets of genes, named “SENS-IS” and “ARE”, with 21 and 17 biomarkers respectively, involved in skin sensitization [1, 2].
The expression level of these separate sets of genes was determined by qRT-PCR (quantitative real-time polymerase chain reaction) after a 15 min incubation period with the test item and a 6 hours post-incubation period at 37°C.
The fold expression of each gene were calculated as a ratio between the mRNA level of test item-treated epidermis versus control (vehicle treated) epidermis.


METHODS
Preliminary test: Solubility test
The test item was solubilized in phosphate buffered saline (PBS), olive oil and dimethylsulfoxide (DMSO) at a concentration of 50 and 10% (v/v) at room temperature and at 37°C (if not soluble at RT).
The solubility of the test item was assessed by visual inspection of each preparation.

Main test: SENS-IS assay
The test item (30 µL) was deposited on the epidermis surface and gently spread on the entire surface [3]. After 15 minutes of exposure, the epidermis was rinsed with PBS and then incubated at 37°C for 6 hours.
After incubation, reconstructed epidermis was removed from the inserts with forceps and placed in a cryotube for freezing in liquid nitrogen. The epidermis was then transferred in a tube containing 1 mL of Qiazol reagent and 2 steel beads and was homogenized using the TissueLyser II. After centrifugation, the supernatant was collected and stored at -20°C until RNA extraction.
After addition of bromochloropropane, total RNA were purified using the miRNeasy extraction Kit according to the manufacturer’s instructions (Qiagen, Courtaboeuf, France). RNA quality was assessed by measuring 260/280 absorbance ratio. The mRNA was then reversed as cDNA using SuperScript III Reverse Transcriptase kit and RNase inhibitor.
After reverse transcription, quantitative gene expression was measured by qRT-PCR using a SYBRGreen® buffer and specific primers defined for the SENS-IS test. The housekeeping genes (Glucuronidase ß, ß2 microglobuline, and Nono « non-POU domain containing octamer-binding ») were analyzed in parallel.

Data analysis and interpretation
Samples were analyzed by the LightCycler 480 software using the second derivative maximum method. This method allows the calculation of the crossing point (Cp) of each sample defined as the number of cycles from which the fluorescence signal enters the exponential phase of the reaction. This value is dependent of the amount of the mRNA present in the sample.
For each sample, the mRNA content for each gene of interest was normalized to the mean mRNA content of the 3 house-keeping genes. For each gene, the fold increase in mRNA level over vehicle controls was calculated as followed:
E = Expression level for the test item / Expression level for the vehicle controls*
* Mean of OO and PBS samples
The endpoint values are the number of positive genes (i.e., genes obtaining a E > 1.25) in each group.

Validation of the study:
alidation of the experiment
The following criteria must be met for an experiment to be considered valid:
- Negative sensitization control (DMSO): this control should induce the over-expression of 6 genes at most in the SENS-IS and ARE groups of genes.
- Positive irritation control and negative sensitization control (SLS at 5%): this control should induce the over expression of at least 16 genes in the IRRITATION group of genes and 6 genes at most in the SENS-IS and ARE groups of genes.
- Positive sensitization control (TNBS at 1M): this control should induce the over expression of at least 7 genes in the SENS-IS or ARE group of genes.

Validation of the sample
The following criteria must be met for sample result to be considered valid:
- The Cp value of HSP90AA1 gene must be ≤ 21.
- If more than 20 genes are overexpressed in the “IRRITATION” set of genes for a given concentration, the result is classified as false positive to take into account non-specific genes up regulation that could be due to cell stress.

Test item classification
A test item is classified as irritant if at least 16/23 genes of the “IRRITATION” group are significantly overexpressed.
A test item is classified as sensitizer if at least 7/17 genes of the “ARE” group, and/or 7/21 genes in the “SENS-IS” group are significantly overexpressed.
Moreover, the results obtained with the different concentrations allow the classification of the test item according to the lowest concentration that gives a positive result. Thus a test item is classified in:
- category 1A: strong to extreme skin sensitizer, when a positive result is obtained at concentrations of 0.1 and/or 1%,
- category 1B: weak to moderate sensitizer, when a positive result is obtained at concentrations of 10 and/or 50%.
A test item is classified as a non-sensitizer when no positive result is observed for all tested concentrations (0.1, 1, 10, 50 and 100%).

Results and discussion

Positive control results:

SLS at 5% was classified as irritant (number of overexpressed irritant genes > 15) and non-sensitizer, the number of overexpressed genes in both the SENS-IS and ARE groups being below 7.
TNBS at 1M was classified as sensitizer since more than 6 genes are overexpressed in at least one of the two groups of genes (SENS-IS or ARE).
DMSO at 100% was classified as a non-sensitizer, the number of overexpressed genes in both the SENS-IS and ARE groups being below 7.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: run 1

Parameter:

other: Gene expression

Remarks:

SENS-IS and ARE

Value:

6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: run 2

Parameter:

other: gene expression

Remarks:

SENS-IS and ARE

Value:

7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Executive summary:

The results obtained for the positive and negative controls were within acceptance criteria and validated the study. Regarding the sensitization biomarkers, the test item gave negative results (less than 7 genes in the “SENS-IS” and “ARE” gene groups) when tested at 10% (w/v) in olive oil. At 50% (w/v) in olive oil, ambiguous results were obtained (negative in the first experiment and positive in the second experiment). The test item induced less than 7 genes in the “SENS-IS” and “ARE” gene groups when tested at 100% (not solubilized).

Under the experimental conditions of this SENS-IS assay performed with a partially solubilized or not solubilized test item, the test item is not classified in category 1A. In the absence of additional experiments due to a contamination of the test itemt, a classification of the test item as a non-sensitizeror as a sensitizer in category 1B can’t be determined.

For precautianal reasons, the test item will be classified with regard to skin sensitization.