LUMIPOD

Endpoint summary

Description of key information

Conclusion OECD 442 E:

Based on these results, the test item " L-Leucine, reaction products with 1,4:3,6-dianhydro-D glucitol, iso-Pr alc. and octanoyl chloride" demonstrated an in vitro sensitizing potential with a Minimum Induction Threshold (MIT) of 159 ug/mL under the conditions used during this study.
However, the low increase of the markers expression observed only at one concentration, without any dose response relationship, suggest a poor ability of the test item to activate dendritic cells.

 

Conclusion OECD 442 D:

Under the retained experimental conditions L-LEUCINE, REACTION PRODUCTS WITH 1,4:3,6 DIANHYDRO-D-GLUCITOL, ISO-PR ALC. AND OCTANOYL CHLORIDE code ID-20/02092 may be classified as not skin sensitizer.
The test method KeratinoSensTM is considered scientifically valid to be used as part of an integrated approaches to testing and assessment, to support the identification of the sensitization potential of test item for hazard classification and labeling purposes.

 

Conclusion Sens-IS test:

Under the experimental conditions of this study, the test product, L-Leucine, reaction products with 1,4:3,6- dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride was found an irritant and a non sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27/02/2020 - 14/04/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Specific details on test material used for the study:

*Test item:
Test item denomination: L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride
Batch number: 200122016662
Type of ingredient: Complex ingredient (UVCB)
Expiry date: 20 Jan 2022
Storage conditions: RT, protected from light
EUROSAFE code: 22835/01
Supplied form: Colorless gel
Purity: The test item was considered as 100% pure according to the sponsor's information


*Vehicle control:
As the maximal dose for this test item (500 mg/mL) was obtained in the DMSO (cf $ 5.2.), then, the vehicle control for all of the assays in this study was the DMSO.

Vehicle control denomination: DMSO
Supplier code: D5879-M (Sigma)
Batch number: SHBJ2421
Expiry date: 21 Mar 2025
Storage conditions: RT


*References items:
- Positive control:
Denomination: DNCB
Tested concentration: 4 µg/mL
CAS: 97-00-7
Molecular weight: 202.55
Supplier code: 237329 (Sigma)
Batch number: BCBS42011
Expiry date: 19 Jun 2021
Storage conditions: RT
Sample vehicle: DMSO
Sample preparation date: 19 Nov 2018
Sample expiry date: 19 Nov 2020
Sample storage conditions: -20°C

Denomination: NiSO4
Tested concentration: 100 µg/mL
CAS: 10101-97-0
Molecular weight: 262.85
Supplier code: N4882 (Sigma)
Batch number: SLBS9975
Expiry date: 07 Feb 2023
Storage conditions: RT
Sample vehicle: PBS
Sample preparation date: 11 Sep 2019
Sample expiry date: 11 Sep 2021
Sample storage conditions: -20°C

- Negative control:
Denomination: LA
Tested concentration: 1000 µg/mL
CAS: 50-21-5
Molecular weight: 90.08
Supplier code: 69775 (Sigma)
Batch number: BCBW0524
Expiry date: 05 Mar 2023
Storage conditions: RT
Sample vehicle: 0.9% NaCl
Sample preparation date: 19 Mar 2019
Sample expiry date: 19 Mar 2021
Sample storage conditions: -20°C

Details of test system:

THP-1 cell line [442E]

Details on the study design:

4.1 - Preliminary study: Cytotoxicity assays
The cytotoxicity of the test item was evaluated in order to select at least 4-5 concentrations able to induce cytotoxicity, around 50%, for the highest one. Assessment of cell toxicity was performed by determining cell viability on THP-1 cells, using the 7-AAD inclusion methods.

Eight concentrations of test item were prepared by a two-fold serial dilution from a maximum final concentration of 1000 ug/mL or lower, depending on its solubility limit.
In the day of testing, cells harvested from culture flask were suspended with fresh culture medium at 2 x 106 cells/mL. Then, THP-1 cells were distributed into a 24 well flat-bottom plate with 500 uL (1 x 10° cells/well) with various concentrations of test item (1:1 ratio) for 24+0.5 hours at 37 °C under 5 % CO2. After treatment, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were resuspended with phosphate-buffered containing 0.1% (w/v) bovine serum albumin identified as Fb .Cells were stained with 7-AAD (5 ug/mL final concentration). Then cells were analysed with flow cytometry using GUAVA (Merck Millipore, France) and InCyte software to measure cell viability. The living cells (7-AAD) gate was set in the 7-AAD negative area. 104 7-AAD cells were counted as the living population.
Note: Before staining with 7-AAD, the two additional steps of washing the cells with Fb (recommended in the guidelines) is not performed in Eurosafe (validated in internal research).
In case of product cytotoxicity, the CV75 value is calculated by log-linear interpolation using the following equation:
Log CV75 = (75-c) x Log (b) – (75-a) x Log (d)
a-c a: minimum value of cell viability over 75% c: maximum value of cell viability below 75% band d are the concentrations showing the value of cell viability a and c respectively
According to the results the dose levels for the main study were selected.
4.2 - Main study: Activation test
In case of non-toxic concentration for the top dose used in preliminary test, the maximum concentration selected for activation test must not exceed 1000 ug/mL when the test item is dissolved or stably dispersed in Ethanol or DMSO, and 5000 ug/mL when the test item is dissolved in a saline vehicle.
concentrations are selected approximately
In case of product cytotoxicity, the eight final test item according to the calculated CV75.
The range of concentrations was adjusted for activation test 2 and 3 (if necessary) depending on results obtained in previous experiments, to calculate the minimum induction threshold.
Each activation test was performed on eight concentrations. THP-1 cells were plated at 1*109 cells/mL/well in 24 well plates and treated for 24+0.5 hours at 37+1°C under 5£1% CO2 with selected test item concentrations. After treatment cells were washed twice with Fb.
Note: After washing, blocking the cells with Fb buffer containing 0.01% (w/v) globulin (recommended in the guidelines) was not performed in Eurosafe, due to low expression of receptors FcR by the cells THP
Then cells were stained for 30 min at about 4°C with the following fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (mAbs): anti-human CD54, anti-human CD86; FITC labelled-mouse IgG1. Using the manufacturer's recommended dilutions, cells were incubated with above mAbs at 6 UL/3*10 cells 150ul for the anti-human CD86 mAb, and 3 UL/3*10° cells 150ul for the anti-human CD54 mAb. FITC labelled-mouse IgG1 was used as an isotype control at a dilution of 3 uL/3*109 cells 150uL. Then, the cells were stained also with 7-AAD for at least 30 min at about 4°C. After washing and resuspension with Fb, the fluorescence intensities of the THP-1 cell surface markers were then analysed by flow cytometry using GUAVA (Merck Millipore, France) and InCyte software, on 10000 living cells.
Note: Before staining with 7-AAD, the cells were washed once with Fb instead of twice as recommended in the guidelines (validated in internal research).

Vehicle / solvent control:

DMSO

Negative control:

other: LA

Positive control:

other: DNCB and NiSO4

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC150, CD86 [442E]

Value:

152 µg/mL

Cell viability:

Cell viability >90%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC200, CD54 [442E]

Value:

159 µg/mL

Cell viability:

Cell viability >90%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

DISCUSSION
1 - Cytotoxicity assays

Cytotoxicity was induced on THP-1 cells by L-Leucine, reaction products with 1,4:3,6-dianhydro-D glucitol, iso-Pr alc. and octanoyl chloride but was only observed from the first activation test (with the 285 µg/mL could be determined. In the following experiments, the doses-range was chosen between 97.7 and 350 µg/mL to cover a cytotoxic profile until 50% of dead cells.

2 - Activation test
Test system was validated as all acceptability criteria were fulfilled (Tables 1, 2,3,4,5 and 6).
Vehicle controls (DMSO) showed cell viability values acceptable regarding the acceptance criteria.
Positive controls showed an increase of CD54/86 expression (RFI >= 200/150 respectively) compared to the respective control (Tables 1,2,3,4,5, 6 and 10).
These results validated the experimental conditions. The test item " L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride” was tested in a medium containing 0.2% DMSO. Based on the solubility and cell toxicity assays, the maximum dose level selected for the main study was 500 ug/mL for the first activation test, then 350 ug/mL for the others tests.
On the three activation experiments, seven to eight concentrations could be analysed.
Under the assay conditions, a slight "increase" of the CD54 expression compared with the vehicle control at least for one dose-level of L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride was noticed.

In the first and the third experiment, a slight increase of expression for CD54 marker (fold increase 2.12 to 2.95) was observed at least for one concentration. None of the tested doses induced a 1.5 increase of CD86 expression compared to the vehicle control.

In the second experiment, a slight increase of expression for CD54 marker (fold increase 2.20) was observed at least for one concentration. A slight increase of expression for CD86 marker (fold increase 1.64 to 1.76) was observed for three concentrations but without really dose-dependent.

Conclusions:

Based on these results, the test item " L-Leucine, reaction products with 1,4:3,6-dianhydro-D glucitol, iso-Pr alc. and octanoyl chloride" demonstrated an in vitro sensitizing potential with a Minimum Induction Threshold (MIT) of 159 ug/mL under the conditions used during this study.
However, the low increase of the markers expression observed only at one concentration, without any
dose response relationship, suggest a poor ability of the test item to activate dendritic cells.

Executive summary:

AIM OF THE STUDY – PRINCIPLE

The objective of this study was to evaluate the in vitro intrinsic sensitizing potential of the test item L Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride (Complex ingredient). The method used for this study was the human cell line activation test (h-CLAT). 

The study was performed at EUROSAFE according to the guidelines for the Testing of Chemicals based on key events, Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT) from OECD 442E (2017) based to the following publications: Ashikaga et al. (2006), Sakaguchi et al. (2006, 2009 and 2010) and to the h-CLAT DB-ALM protocol N°158. 

 

SUMMARY

Cytotoxicity was induced on THP-1 cells by L-Leucine, reaction products with 1,4:3,6-dianhydro-D glucitol, iso-Pr alc. and octanoyl chloride but was only observed from the first activation test (with the maximum tested concentration of 500 ug/mL). According to this cytotoxic profile, CV75 value of 285 ug/mL could be determined. In the following experiments, the doses-range was chosen between 97.7 and 350 ug/mL to cover a cytotoxic profile until 50% of dead cells. 

Under the assay conditions, a slight "increase" of the CD54 expression compared with the vehicle control at least for one dose-level of L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride was noticed. 

In the first and the third experiment, a slight increase of expression for CD54 marker (fold increase 2.12 to 2.95) was observed at least for one concentration. None of the tested doses induced a 1.5 increase of CD86 expression compared to the vehicle control. 

In the second experiment, a slight increase of expression for CD54 marker (fold increase 2.20) was observed at least for one concentration. A slight increase of expression for CD86 marker (fold increase 1.64 to 1.76) was observed for three concentrations but without really dose-dependent. 

Based on these results, the test item“ L-Leucine, reaction products with 1,4:3,6-dianhydro-D glucitol, iso-Pr alc. and octanoyl chloride " demonstrated an in vitro sensitizing potential with a Minimum Induction Threshold (MIT) of 159 ug/mL under the conditions used during this study. However, the low increase of the markers expression observed only at one concentration, without any dose response relationship, suggest a poor ability of the test Item to activate dendritic cells.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09/03/2018 - 20/03/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

Preparation of the 100 X plate A 100-fold concentrated dilutions series was prepared in 96-well plate.
Test item The test item was placed in one of the rows B to G. 100 ul of DMSO were distributed from columns 1 to 11. 200 ul of the 40 mg/ml stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 ul from column 12 to column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
Positive control 100 ul of DMSO were distributed in row F (repetition 1, plate 1 RLU, and repetition 2) and in row G (repetiton 1, plates RLU 2/3) from columns 7 to 10.200 ul of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 ul from column 11 to column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
Negative control
100 ul of DMSO were distributed in row F (repetition 1, plate 1 RLU, and repetition 2) and in row G (repetiton 1, plates RLU 2/3) columns 1 to 6 and 12 and in the well H12.
Preparation of the 4 X dilution plate The 100 X DMSO plate was diluted 25 fold in a new plate (4 X) in treatment medium.
9.3. Contact between the cells and the test and reference items (second day)
In the 5 seeded plates, the medium was aspirated and replaced with 150 ul of treatment medium. Then the 4 X plate was replicated 5 times: 50 ul from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours + 1 hour (37°C, 5% CO2).

Vehicle / solvent control:

DMSO

Negative control:

other: Treatment culture medium, 1% DMSO, 1% Non-heat inactivated foetal calf serum:

Positive control:

cinnamic aldehyde [442D]

Positive control results:

Mean EC1.5 = 11.62 µg/mL
Mean Imax = 2.94

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

EC1.5>400µg/ml (max dose tested)

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.02

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Group:

test chemical

Run / experiment:

mean

Parameter:

other: IC70µg/ml

Value:

268.43 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Results calculation and interpretation
1. Expression of results
Two parameters are measured, the luciferase induction and the cytotoxicity.

1.1. Luciferase induction
I_max, maximal fold induction of luciferase activity value observed at any concentration of the test item and positive control. The induction value I is calculated according to the following formula:

I = (Luminescence_{Test item} - Luminescence_Blank) / (Luminescence_{Negative control} - Luminescence_Blank)

The I_max of an item is the average of the I_max calculated for each of the repetitions.
EC1.5, value representing the concentration for which induction of luciferase activity is above 1.5 threshold, is obtained according to the following equation:

EC1.5 = (C_b - C_a) x [(1.5 - I_a) / (I_b - I_a)] + C_a

Where:

C_a = the lowest concentration with more than 1.5 fold the induction

C_b = the highest concentration with less than 1.5 fold the induction

I_a = induction factor for the lowest concentration with more than 1.5 fold the induction.

I_b = induction factor for the highest concentration with less than 1.5 fold the induction

The EC1.5 of a test item is the geometric average of the EC1.5 calculated for each of the repetitions.

1.2. Cytotoxicity
The viability V is calculated according to the following formula:

V= (Absorbance_{Test item} - Absorbance_Blank) / (Absorbance_{Negative control} - Absorbance_Blank) x 100

IC₇₀, concentration for which we obtained 70% cell viability:

ICx = (C_b - C_a) x [ (x - V_a) / (V_b - V_a)] + C_a

where x is the % viability at the concentration to be calculated (70)

C_a is the lowest concentration for which the % viability is lower than X%

C_b is the highest concentration for which the % viability is higher than X%

V_a is the % viability at the lowest concentration for which the % viability is lower than X%

V_b is the % viability at the highest concentration for which the % viability is higher than X%

The data processing is carried out by a locked Excel sheet provided by Givaudan. The raw data generated from the reading of the plates are directly introduced into the dedicated fields, and a data processing is performed automatically.
A graph showing the gene induction and the cytotoxicity of each element, the I_max and EC1.5 value are automatically generated. The treatment of the data generated by the matrix Excel is carried out according to the current working instruction IL 04.

 

2. Test validation / bistorical data
To validate the test, it is essential to check the validity criteria for the test:

 

Positive Control:
- the gene induction must be statistically significant above the threshold of 1.5 in at least one dose,
- the EC1.5 value should be between IDEA Lab historical data: mean EC1.5 value +/- 2 SD and the average induction, in each repetition, for cinnamaldehyde at 64 um should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.

Follow-up of positive control: Cinnamaldehyde (CAS N°104-55-2):

Updated on 20/12/2019 - N = 404

	Imax	EC1.5	IC70
Mean value	5.10	10.98 µM	> 64 µM
Standard deviation	2.44	4.28	-
Mean EC1.5 value #2 SD	-	2.4 µM =< EC1.5 =< 20 µM	-

OECD validation dataset: between 7 µM and 30 µM.

Negative Control:

For each repetition, the coefficient of variation of the solvent controls (3 x 6 wells) must be less than 20%.

If for one repetition the validity criteria are not met, a third repetition should be considered. The validation of the results is carried out by the Study Director in accordance with the current working instruction IL 04.

 

3. Interpretation
The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or in 2 of 3 repetitions. Otherwise the Keratinosens^TM prediction is considered as negative:

- the I_max is strictly 1.5 fold higher of the basal luciferase activity* statistically significantly to the value obtained for the negative control (as determined by a two-tailed, unpaired Student's t-test on the raw RLU values),

*lf the lour is exactly equal 10 1.5. the test item is rated as negative and no EC1.5 value is calculated

- the EC1.5 value is strictly below 200 µg/ml,

- at the lowest concentration with a gene induction above 1.5, the cell viability must be strictly above 70% (i.e. EC1.5 <IC70),

- there is an apparent overall dose-response for luciferase induction, which is similar between the
repetitions.

In rare cases, the test items which induce the gene activity at a concentration very close to the cytotoxic levels, are positive in some repetitions at non-cytotoxic levels, and in other repetitions only at cytotoxic levels. In this case, the test item must be tested again with a narrower range using a dilution factor of 4/3 instead of 2.

Test items that only induce the gene activity at cytotoxic levels are not rated as positive, as it is the case for some non-sensitizing skin irritants.

If, in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, in case of a test item with poor solubility tested at a concentration lower than 200 ug/ml, a negative result obtained should also be considered as inconclusive.

 

Negative results should be interpreted with caution as substances with an exclusive reactivity towards lysine-residues can be detected as negative by the test method. Furthermore, because of the limited metabolic capability of the cell line used and because of the experimental conditions, pro-haptens (i.e.cheinicals requiring enzymatic activation for example via P450 enzymes) and pre-haptens (i.e, chemicals activated by auto-oxidation) in particular with a slow oxidation rate may also provide negative results. Test chemicals that do not act as a sensitizer but are nevertheless chemical stressors may lead on the other hand to false positive results. Furthermore, highly cytotoxic test chemicals cannot always be reliably assessed. Finally, test chemicals that interfere with the luciferase enzyme can confound the activity of luciferase in cell-based assays causing either apparent inhibition or increased luminescence.

 

 

Results
1. Reference item

Cinnamaldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1.15	1.28	1.72	2.06	3.12	11.97	3.12
Rep 2	1.20	1.38	1.67	2.07	2.75	11.27	2.75
Mean	1.18	1.33	1.70	2.06	2.94	11.62*	2.94

*geometric mean

Control solvent	CV % control solvent
Rep 1	8.0
Rep 2	11.0

Since all validity criteria are fulfilled, study is considered valid.

 

2.Test item

ID20-02092	VIABILITY	INDUCTION
	IC70 µg/ml	Imax	Linear EC1.5 µg/ml	EC1.5 Lin/Log µg/ml
Rep 1	243.72	1.02	-	-
Rep 2	295.65	1.01	-	-
Mean	-	1.02	-	-
Geometric mean	268.43	-	-	-

I_max is less than 1.5, the EC1.5 is not determined.

Conclusions:

Under the retained experimental conditions L-LEUCINE, REACTION PRODUCTS WITH 1,4:3,6 DIANHYDRO-D-GLUCITOL, ISO-PR ALC. AND OCTANOYL CHLORIDE code ID-20/02092 may be classified as not skin sensitizer.
The test method KeratinoSensTM is considered scientifically valid to be used as part of an integrated approaches to testing and assessment, to support the identification of the sensitization potential of test item for hazard classification and labeling purposes.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30/04/2020

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:

no guideline followed

Principles of method if other than guideline:

3.1 Purpose of the method
Application of irritant and/or sensitizer onto the skin induces the reversible destruction of tissue as well as the activation of the innate and adaptative immune system. These biological changes are induced by the specific expression (or repression) of distinct set of genes with a kinetic dependent of the type of reaction observed.
The SENS-IS test is based on the analysis by RT-PCR (reverse transcription polymerase chain reaction) of two set of genes, one specifically reflecting the irritant potential of a chemical after application to the skin (“IRRITATION” set of genes) and the other set of genes correlating with the sensitization potential (splited in 2 subsets of genes namely “SENS_IS” and “REDOX”). By measuring the level of expression of these two separate sets of genes at a given time point after application and by comparison with internal negative, irritant and sensitizer positive control, the SENS-IS test can determine the irritant and sensitizer potential of a chemical.

3.2 Brief experimental procedure
The test product L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride was tested pure or diluted in DMSO at 40°C at 50% v/v and 10% v/v.
30 µl (26.3µl/cm2) of each dilution was applied on the top of each reconstituted epidermis (Episkin model), using a positive displacement pipette. The test product was gently spread on the epidermis surfaces to ensure it covers all the surface.
After 15 mn exposure, the Episkin were rinsed with PBS. Epidermis were then incubated at 37°C for 6 hours.
After incubation, the complete epidermis was collected, placed in a RNAzol solution and the total RNA was isolated by homogenization of the skin. After reverse transcription, quantitative gene expression was measured by RT-PCR using a sybr green buffer using the LC480 Roche’s apparatus and
specific biomarkers primers defined for the Sens-IS test.
For each analysis three negative controls (PBS, olive oil and DMSO treated skins), a positive irritation control (5% SLS) and two positive sensitization controls (HCA at 50% and TNBS at 1%) were performed.
The test product and the controls are tested in at least 2 experiments (using different batches of Episkin models). Further experiments can be conducted if invalid results are obtained in the previous experiments.

GLP compliance:

no

Remarks:

The Sens-Is test is not an OECD test, it is the first step in our strategy to determine the sensitization of the substance, it is a screening test. The test was negative, so we performed 2 more OECD test with GLP compliance.

Type of study:

other: Evaluation of sensitization potential using the Sens-IS test

Details of test system:

other: Episkin large is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum provided by SkinEthic Laboratories.

Details on the study design:

*OBJECTIVE:
The study assessed the potential of the test products to specifically induce the expression of irritation and/or sensitization biomarkers in an in vitro 3D skin model.

1. Purpose of the method:
Application of irritant and/or sensitizer onto the skin induces the reversible destruction of tissue as well as the activation of the innate and adaptative immune system. These biological changes are induced by the specific expression (or repression) of distinct set of genes with a kinetic dependent of the type of reaction observed.
The SENS-IS test is based on the analysis by RT-PCR (reverse transcription polymerase chain reaction) of two set of genes, one specifically reflecting the irritant potential of a chemical after application to the skin (“IRRITATION” set of genes) and the other set of genes correlating with the sensitization potential (splited in 2 subsets of genes namely “SENS_IS” and “REDOX”). By measuring the level of expression of these two separate sets of genes at a given time point after application and by comparison with internal negative, irritant and sensitizer positive control, the SENS-IS test can determine the irritant and sensitizer potential of a chemical.

2. Brief experimental procedure:
The test product L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride was tested pure or diluted in DMSO at 40°C at 50% v/v and 10% v/v.
30 µl (26.3µl/cm2) of each dilution was applied on the top of each reconstituted epidermis (Episkin model), using a positive displacement pipette. The test product was gently spread on the epidermis surfaces to ensure it covers all the surface.
After 15 mn exposure, the Episkin were rinsed with PBS. Epidermis were then incubated at 37°C for 6 hours.
After incubation, the complete epidermis was collected, placed in a RNAzol solution and the total RNA was isolated by homogenization of the skin. After reverse transcription, quantitative gene expression was measured by RT-PCR using a sybr green buffer using the LC480 Roche’s apparatus and
specific biomarkers primers defined for the Sens-IS test.
For each analysis three negative controls (PBS, olive oil and DMSO treated skins), a positive irritation control (5% SLS) and two positive sensitization controls (HCA at 50% and TNBS at 1%) were performed.
The test product and the controls are tested in at least 2 experiments (using different batches of Episkin models). Further experiments can be conducted if invalid results are obtained in the previous experiments.

Known limitations of the method:
Although studies performed in our laboratory have shown a very good correlation between SENS-IS test results and sensitization potency for a number of tested chemicals, it has to be noted that the test has not been thoroughly evaluated by competent authorities yet.


*TEST SYSTEM:
Episkin large is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum provided by SkinEthic Laboratories.
The EpiSkin models are obtained by culturing adult human keratinocytes on a collagen substrate in conditions which permit their terminal differentiation and the reconstruction of an epidermis with a functional horny layer. After 3 days of immersed culture conditions, the epidermis is airlifted during 10 days allowing differentiation and formation of a horny layer. The human keratinocytes come from mammary samples obtained from healthy consenting donors during plastic surgery. HIV 1 & 2, B and C hepatitis tests are carried out on the donor bloods as well as verification of the bacteriological & fungal sterility of the cells and absence of mycoplasma.
The reconstructed human epidermis expresses the major differentiation markers (filaggrin and involucrin in granular cell layers, transglutaminase I and keratin 10 in supra basal cell layers and loricrin in upper granular cell layers), as well as expressing the basement membrane markers (type IV collagen; integrin alpha 6, integrin beta 4, antigen BP, laminin I and laminin V).
Free fatty acids and ceramides are detected in the lipid profile. The ultra-structural features show secretion and normal arrangement of bi-layered lipid content into the intercellular spaces of the cornified cell layers (formation of normal permeability barrier).
Some quality controls are performed by the provider of the test and must fulfill the following criteria: Histology scoring (HES stained vertical paraffin sections, n=6) ≥ 19.5
IC50 determination (SLS concentration, MTT test, n=14) ≥ 1 mg/ml


*EVALUATION OF THE RESULTS
1. Acceptance criteria
The first acceptance criteria is the IC50 value of SLS on Episkin that must be ≥ 1.2 mg/ml (ImmunoSearch defined specification).
Tissue destruction or over irritation induced by the chemical at a given concentration are then analyzed as follow :
- Tissue destruction is measured by the expression of the gene HSPAA1. The cycle threshold (CT) value of HSPAA1 gene for the sample must not be above 10% of that of the olive oil or PBS controls (data not shown).
- Over irritation due to cell stress is measured by the number of irritation biomarker genes over expressed. If more than 20 genes are overexpressed for a given concentration of the product, the sample is not accepted and the substance is analysed at a lower concentration.

2. Evaluation criteria
The results for a test substance are considered to be valid if the controls are correctly classified based on the number of gene overexpressed.
A substance is considered irritant if it induces the overexpression of at least 15 genes among a group of 23 genes named “IRRITATION”.
A test substance is considered a sensitizer if it induces the over expression (fold value >1.25) (compared to the mean of the PBS and olive oil controls) of at least 7 genes among two groups of respectively 21 and 17 genes named “SENS-IS” and “REDOX”. Both groups of genes are involved in skin sensitization but are related to different pathways: the “REDOX” group gathers genes involved in the oxidative stress responses, whereas the “SENS-IS” group gathers genes biomarkers of skin sensitization but not involved in the oxidative stress response.
The sensitizing response is analyzed under different concentration to measure the concentration at which no positive response is induced. The lowest test item concentration fulfilling the prediction model criteria is then used for potency classification as follow: A chemical is classified as an extreme, a strong, a moderate or a weak sensitizer if found positive at 0.1%, 1%, 10% or 50% respectively.

Negative control:

other: PBS - Olive oil - DMSO

Positive control:

other: sodium lauryl sulfate (SLS) for irritation and 2, 4,6-trinitrobenzene sulfonic acid (TNBS) for sensitization

Positive control results:

In all experiments BM298, BM299, BM300 and BM301, SLS at 5% revealed as an irritant (number of overexpressed irritant genes ≥ 15) and a non sensitizer, the number of genes in both the SENS-IS or the REDOX group being below 7.
In all experiments BM298, BM299, BM300 and BM301, TNBS at 1% revealed as a sensitizer, the number of genes induced in at least one of in the SENS-IS and/or the REDOX group being at least 7.
In conclusion the results of the positive control reached the acceptance criteria in experiments BM298, BM299, BM300 and BM301, which were therefore considered to be valid.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Number of overexpressed genes for sensitization parameter (REDOX) - Product pure

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Number of overexpressed genes for sensitization parameter (REDOX) - 50 % Product in DMSO

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Number of overexpressed genes for sensitization parameter (REDOX) - 10% product in DMSO

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Number of overexpressed genes for sensitization parameter (Sens-IS) - 10% product in DSMO

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Number of overexpressed genes for sensitization parameter (Sens-IS) - 50% product in DMSO

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Number of overexpressed genes for sensitization parameter (Sens-IS) - Product pure

Value:

5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Number of overexpressed genes for iritation parameter - 10% product in DMSO

Value:

14

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of skin irritation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Number of overexpressed genes for iritation parameter - Product pure

Value:

21

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive indication for skin irritation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Number of overexpressed genes for irritation parameter - 50% product in DMSO

Value:

21

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Positive indication for skin irritation

Outcome of the prediction model:

other: Product is irritant and a non sensitizer

Conclusions:

Under the experimental conditions of this study, the test product, L-Leucine, reaction products with 1,4:3,6- dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride was found an irritant and a non sensitizer.

Executive summary:

For the SENS-IS assay, the ingredient L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride was tested pure or diluted in DMSO at 40°C at 50% v/v and 10% v/v. The product was a pipetable gel at all concentrations tested. The diluted ingredient was placed onto a 3D reconstituted epidermis model (Episkin model). After 15 min the epidermis were rinsed and post incubated for 6 hrs. The expression of biomarkers was analyzed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Biomarkers are grouped into genes involved in irritation, anti-oxidant responsive element or SENS-IS. The fold increase expression (over controls treated epidermis) is determined and when at least 7 genes in the respective groups are overexpressed at a given concentration of the ingredient, the test is considered positive.

 

Results :

When tested pure and diluted in DMSO at 40°C at 50% v/v and 10% v/v., the ingredient L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride  did not induced more than 7 genes in either the SENS-IS or the ARE groups of genes. It is therefore classified as a non sensitizer.

Conclusions :

Under the experimental conditions of this study, the test product, L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride  was found an irritant and a non sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)