LUMIPOD

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride

Molecular formula:

Not applicable

IUPAC Name:

L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride

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

In vitro test system

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

Results and discussion

In vitro / in chemico

Results

Results 1

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

Results 2

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]

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.