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

Endpoint summary

Description of key information

SENS-IS

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.

QSAR

In conformity with Article 47 of REACH, a QSAR approach was performed to predict the skin sensitisation potential of the substance "Esterification products of 1,4:3,6-dianhydro-D-glucitol with sunflower oil fatty acids". The OECD ToolBox version 4.2 was used on the 3 main constituents of the registered substance, i.e. the Linoleic diester of isosorbide, the Oleic diester of isosorbide and the Linoleic/oleic diester of isosorbide. The predicted endpoint is skin sensitisation from experimental data coming from well identifiable test systems. The model used is an unambiguous algorithm as the prediction takes the highest mode value from the nearest 5 neighbours. All three constituents of Esterification products of 1,4:3,6-dianhydro-D-glucitol with sunflower oil fatty acids are predicted to be skin sensitisation negative. Thus, the target substance Esterification products of 1,4:3,6-dianhydro-D-glucitol with sunflower oil fatty acids is predicted to be negative for skin sensitisation.

In vitro study

In order to estimate the skin seinsitisation potency of the test substance, 3 in vitro standard studies were considered :

•       DPRA.

•       h-CLAT

•        Keratinosens

The test conditions of the DPRA were not evaluated since the test item is a UVCB and does fall within the applicability domain of this assay.

After performing solubility assays using vehicles suitable for KeratinoSens and h-CLAT tests at key concentrations for these assays, it was not possible to obtain a solution nor a stable dispersion.

Consequently, the test item LAB 4448 cannot be tested in the DPRA, the KeratinoSens nor the h-CLAT.

An in vitro test SENSIS is under progress and will complete soon information to predict the skin sensitisation potency.

Human data

A non-GLP compliant study was perform in order to investigate a potential irritation property or sensitizing property of the HydraSynol IDL on human based on an Human Repeated Insult Patch Test method (HRIPT).

54 subjects were used in this study. 0.2 ML of the diluted test item (10% in corn oil)  was dispensed onto the occlusive, hypoallergenic patch. The patch was applied directly to the skin of the infracapsular regions of the back to the right or left of the midline and the subject was dismissed with instructions not to wet or expose this area to direct sunlight. After 24 hours the patch was removed by the panelist at home. This procedure was repeated until a series of nine consecutive 24 hours exposures have been made for every Monday, Wednesday and Friday for three consecutive weeks. In the event of adverse reaction, the area of erythema and edema is measured. Subject were then given 10-14 day rest period after which a challenge or retest dose was applied once to a previously unexposed site. The retest dose is equivalent to any one of the original nine exposures. Reactions are scored 24 and 48 hours after application. Comparison was made between the nine inductive responses and the retest dose. No adverse reactions of any kind were noted during the course of this study. Under the experimental conditions of the study, the test item HydraSynol was considered as a Non-Primary Irritant and a Non-Primary Sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

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

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

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.

Positive control results:

see tables in section any other information on tabels and 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

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).

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Current data available indicated an absence of skin sensitisation potency for the test substance "Esterification products of 1,4:3,6 -dianhydro-D-glucitol with sunflower oil fatty acids", with a SENSIS Test and QSAR evaluation.