Esterification product of sunflower-oil fatty acids, with 1,4:3,6-dianhydro-D-glucitol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: SENS-IS assay

Version / remarks:

This study was conducted in compliance with the Study Plan n°DC20922/SI01 and standard operating procedures, with the deviation described below:
- The SDS quality control performed by the provider of the test system used in the two experiments was outside of our acceptance criteria. Nevertheless, the result for the histology scoring was satisfactory and these batches fulfilled our internal criteria for irritation (with SLS), sensitization (with TNBS) and the absence of sensitization (with DMSO). Therefore these batches were accepted.
This deviation was without impact on the validity of the study results.

Deviations:

yes

Remarks:

This deviation was without impact on the validity of the study results.

Principles of method if other than guideline:

- Principle of test:
The SENS-IS method for the detection of sensitisers is based on the 3D EpiSkin™ tissue model as a test system and on the analysis of the expression of a large panel of 61 genes relevant during the skin sensitisation process. The modulation of these biomarkers is measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).
The expression profile of 61 genes divided in three sets will be 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.
- Short description of test conditions:
30 µL of test item was deposited on the 3 D reconstituted epidermis surface (EpiskinTM) during 15 minutes. After incubation, the epidermis was removed and RNA were extracted, bocked with bromochloropropane, and purified.
- Parameters analysed / observed:
After reverse transcription, quantitative gene expression was measured by qRT-PCR using SYBRGreen® buffer and specific primers defined for the SENS-IS test. The housekeeping genes (glucuronidase β, β2 microglobuline, and nono « non-POU domain containing octamer-binding ») were analyzed in parallel. The overexpresion of genes lead to different conclusion :
- irritant if at least 16/23 genes of the « IRRITATION » group are significantly over expressed
- sensitizer if at least 7/17 genes of the « ARE » groupe or 7/21 genes in the « SENS-IS » group are significantly overexpressed.

GLP compliance:

yes

Type of study:

other: SENS-IS assay

Justification for non-LLNA method:

according to the requirement of the Annex VII of REACh regulation, and in order to avoid animal testing this in-viitro method, SENS-IS lead to a skin sensitistaion data

Test material

Test material information

1

Reference substance name:

Esterification products of 1,4:3,6-dianhydro-D-glucitol with sunflower oil fatty acids

Cas Number:

1818326-42-9

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

Esterification products of 1,4:3,6-dianhydro-D-glucitol with sunflower oil fatty acids

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
LAB 4448- dilinoléate d'isosorbide batch number E8057
- Expiration date of the lot/batch:
retest december 2019
- Purity test date: 99.8% - the test item was considered as 100%pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle : the solubility of the test item was assessed in phosphate buffered saline (PBS), olive oil (OO) and dimethylsulfoxide (DMSO) at a concentration of 10% and 50%, at room temperature. The test item “LAB 4448 – Dilinoléate d’isosorbide” was soluble at 10 and 50% (v/v) in olive oil and in DMSO. It was not soluble at 10 and 50% (v/v) in PBS.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : neat (100%), 50% in olive oil, 10% in olive oil, 1% in olive oil, 10% in DMSO

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
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 Episkin™ was rinsed with PBS and then incubated at 37°C for 6 hours.
In experiment 1: the test item was tested in solution : 50%, 10%, 1% in olive oil, and 10 % in DMSO
In experiment 2: the test item was tested pure and in a solution : 50% olive oil.
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. Epidermis 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 (14 µL of each sample) 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. The amplification program consisted of one cycle at 95 °C with a 10 min hold, followed by 45 amplification cycles (95 °C for 10 s, annealing at 60 °C for 10 s, 72°C for 10 s) and completed by three steps for generating a melting curve and a cooling phase.
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 expression 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 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 (E > 1.25). 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 negative results are observed at 100% and at all other analyzed concentrations.
At least two independent experiments (repetitions) are performed in order to obtain two concordant conclusions. If three repetitions should be performed for a given concentration, a majority of positive results (2 out of 3) must be obtained so that the final outcome is positive, otherwise the final outcome is negative.

Results and discussion

Positive control results:

see tables in section any other information on tabels and results

In vitro / in chemico

Results

Key result

Run / experiment:

other: number of overexpressed genes

Parameter:

other: number of overexpressed genes in the "SENS-IS" and "ARE" when it was tested at 1, 10 and 50% (v/v) in olive oil and at 10% (v/v) in DMSO and at 100%

Remarks:

number of overexpressed genes in the "SENS-IS" and "ARE" when it was tested at 1, 10 and 50% (v/v) in olive oil and at 10% (v/v) in DMSO and at 100%

Value:

7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Analysis of positive and negative controls

Experiment 1 (2SI19004):

	Number of overexpressed genes
Gene group	SLS 5% Positive control for irritation and negative control for sensitization	TNBS 1M Positive control for  sensitization	DMSO 100% Negative control for            irritation & sensitization
IRRITATION	21	5	6
SENS-IS	5	4	3
ARE	2	14	0
Conclusion
IRRITATION	POSITIVE	NEGATIVE	NEGATIVE
SENSITIZATION	NEGATIVE	POSITIVE	NEGATIVE

Experiment 2 (2SI19005):

	Number of overexpressed genes
Gene group	SLS 5% Positive control for irritation and negative control for sensitization	TNBS 1M Positive control for  sensitization	DMSO 100% Negative control for            irritation & sensitization
IRRITATION	21	4	11
SENS-IS	4	2	3
ARE	4	13	0
Conclusion
IRRITATION	POSITIVE	NEGATIVE	NEGATIVE
SENSITIZATION	NEGATIVE	POSITIVE	NEGATIVE

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.

Analysis of test item

 

Experiment 1 (2SI19004):

	Number of overexpressed genes
Gene group	50% Oliveoil	10% Oliveoil	1% Oliveoil	10% DMSO
IRRITATION	2	1	2	5
SENS-IS	3	2	1	0
ARE	2	1	1	1
Cp value - HSP90AA1	17.5	17.5	17.7	18.1
Conclusion
SENSITIZATION	NEGATIVE	NEGATIVE	NEGATIVE	NEGATIVE

 

Experiment 2 (2SI19005):

	Number of overexpressed genes
Gene group	100%	50% Oliveoil
IRRITATION	0	1
SENS-IS	3	2
ARE	1	0
Cp value - HSP90AA1	17.9	17.9
Conclusion
SENSITIZATION	NEGATIVE	NEGATIVE

In the first experiment, the test item “LAB 4448 –Dilinoléated’isosorbide” induced less than 7 genes in the “SENS-IS” and “ARE” gene groups when tested at 1, 10 and 50% (v/v) in olive oil and at 10% in DMSO.

In the second experiment, the test item “LAB 4448 –Dilinoléated’isosorbide” induced less than 7 genes in the “SENS-IS” and “ARE” gene groups when tested at 50% (v/v) in olive oil and at 100% (not diluted).

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained for the positive and negative controls were within acceptance criteria defined in the Study Plan.
Considering the number of over-expressed gene in the “SENS-IS” and “ARE” gene groups, the test item “LAB 4448 – Dilinoléate d’isosorbide” gave negative result (less than 7 genes induced) when it was tested at 1, 10 and 50% (v/v) in olive oil and at 10% (v/v) in DMSO. Moreover, negative results were obtained when the test item was tested at 100% (not diluted).
In conclusion, under the experimental conditions of this SENS-IS assay, the test item “LAB 4448 – Dilinoléate d’isosorbide” can be classified as a non- skin sensitizer under CLP criteria.

Executive summary:

The objective of this study was to evaluate the capacity of the test item “LAB 4448 – Dilinoléate d’isosorbide” to induce the expression of specific irritation and sensitization biomarkers in a 3D-reconstructed epidermis model, according to SENS-IS assay.

The results obtained for the positive and negative controls were within acceptance criteria defined in the Study Plan.

Considering the number of over-expressed gene in the “SENS-IS” and “ARE” gene groups, the test item “LAB 4448 – Dilinoléate d’isosorbide” gave negative result (less than 7 genes induced) when it was tested at 1, 10 and 50% (v/v) in olive oil and at 10% (v/v) in DMSO. Moreover, negative results were obtained when the test item was tested at 100% (not diluted).

In conclusion, under the experimental conditions of this SENS-IS assay, the test item “LAB 4448 – Dilinoléate d’isosorbide” can be classified as a non- skin sensitizer under CLP criteria.