Diallyl hexahydrophthalate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 December 2017 to 19 January 2018 (Experimental start to experimental completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline No. 442E: "In vitro skin sensitization: human Cell Line Activation Test (h-CLAT)", 29 July 2016.

Deviations:

yes

Remarks:

Main test: Run B, treated plates incubated for 24h 57 min, not 24h ±30 min. Acceptance criteria: Run B (18.1.18), positive control cells did not show RFI-CD86 & RFI-CD54 above threshold. Cells from (Run A, 25.1.18) responded. No impact to study..

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Human Cell Line Activation Test

Test material

Test material information

Constituent 1

Reference substance name:

Diallyl hexahydrophthalate

EC Number:

237-580-1

EC Name:

Diallyl hexahydrophthalate

Cas Number:

13846-31-6

Molecular formula:

C14H20O4

IUPAC Name:

1,2-bis(prop-2-en-1-yl) cyclohexane-1,2-dicarboxylate

Test material form:

liquid

Remarks:

Colourless

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Osaka Soda Co., Ltd
- Batch No.of test material: 40201
- Expiration date of the batch: 26 January 2019
- Purity test date: 7 October 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protcected from light
- Stability under test conditions:assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO (upto 500 mg/mL), stability of the test item in DMSO not determined.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

The study was divided in two successive phases.
1), a Dose-Range Finding assay (DRF) was performed to assess test item toxicity and, etermine the CV75 ( test item concentration resulting in 75% cell viability compared to the vehicle control).
2) based on cytotoxicity data obtained from the DRF, a concentration series was tested in successive runs in the main test.
At each phase, all information relating to test item concentrations and run identification were given by the Study Director in the study files and no study plan amendment was issued for that purpose.

Dose-Range Finding assay (DRF)

The DRF consisted in two separated assays.
Treatments of DRF assays were performed at the following concentrations (final concentrations): 7.81, 15.63, 31.25, 62.50, 125, 250, 500 and 1000 μg/mL.
Each assay was performed as described here below.
Test item stock solutions were prepared at 8 different concentrations by 2-fold dilutions using the selected vehicle (DMSO). These stock formulations were then diluted 250-fold into cRPMI to obtain working solutions.
The working solutions were finally used for exposure by adding 500 μL of working solutions to the volume of THP-1 cell suspension in the plate (500 μL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.
The treated plates were incubated for 24 hours ± 30 minutes in a humidified incubator set at 37°C and 5% CO2.
At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation. The supernatants were discarded and the remaining cells were resuspended with 600 μL of FACS buffer. Finally, cells were resuspended in 200 μL FACS buffer and the plate was positioned into the plate-reader of the flow cytometer. A volume of 10 μL of Propidium Iodide (PI) solution at 12.5 μg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μg/mL per well.

Main test

The main test consisted in two separated runs being performed as described here below.
Test item stock solutions were prepared at 8 different concentrations by 1.2-fold dilutions using the selected vehicle. The highest concentration corresponded to 1.2-fold the mean CV75. The maximum concentration in the plates was 676.33 μg/mL.
All stock formulations were then 250-fold diluted into cRPMI to obtain working solutions.
In parallel, the working solutions of positive controls DNCB and NiSO4 and vehicle control were prepared..
All working solutions were finally used for exposure by adding 500 μL of working solutions to the volume of THP-1 cell suspension in the plate (500 μL) to achieve a further 2-fold dilution. In order to avoid evaporation of volatile chemicals and cross-contamination between wells, a sealer was placed on each 24-well plate just after treatment, before putting the plastic lids back on each plate.
The treated plates were then incubated for 24 hours ± 30 minutes (see § Study plan adherence) in a humidified incubator set at 37°C and 5% CO2.
During the main test, treatments were performed at the following concentrations (final concentrations in the wells): 188.75, 226.5, 271.8, 326.16, 391.39, 469.67, 563.61 and 676.33 μg/mL.
At the end of the treatment phase, cells were transferred into sample tubes and collected by centrifugation, washed twice with 1 mL FACS buffer and blocked with 600 μL of blocking solution and incubated at 4°C for 15 minutes (± 1 minute). After blocking, cells were split in three aliquots of 180 μL into a 96-well round bottom plate and centrifuged before staining with antibodies. A volume of 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies prepared in FACS buffer was added to each aliquot before incubation for 30 minutes (± 2 minutes) at 4°C.Finally, cells were washed with 150 μL FACS buffer 2 times and re-suspended in 200 μL FACS buffer. The plate was then positioned into the plate-reader of the flow cytometer. A volume of 10 μL of PI solution at 12.5 μg/mL was added automatically by the flow cytometer before acquisition of a sample to obtain a final PI concentration of 0.625 μg/mL per well.

Results and discussion

Positive control results:

Reference chemicals were correctly classfied in a maximum of three validated runs using both working cell banks.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Run A

Parameter:

other: RFI CD86

Value:

391.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Run A

Parameter:

other: RFI CD86

Value:

271.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: Run A

Parameter:

other: RFI CD54

Value:

226.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 4

Key result

Run / experiment:

other: Run B

Parameter:

other: RFI CD54

Value:

676.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Run B: RFI CD86 did not exceed the positivity thresholds at any tested concentration.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Part of the tiered strategy for skin sensitization assessment

Conclusions:

Under the experimental conditions of this study, the test item, MDAC, was found to be positive in the h-CLAT test method

Executive summary:

The objective of the study was to determine the ability of the test item to induce an increase in cell surface markers expression in THP-1 cells using the h-CLAT test method.

A solubility assessment was first performed in 0.9% NaCl and DMSO to select the vehicle and highest concentration to be used for test item formulation preparations. Following the solubility assays, the cytotoxic potential was assessed in a Dose-Range Finding assay in order to select sub-toxic concentrations for testing in the main test. The skin sensitizing potential of the test item was then tested in the main test in two successive runs.

In each run, the test item formulations were applied to THP-1 cells and cultured in a 24-well plate for 24h ± 30 minutes at 37°C, 5% CO2 in a humidified incubator. A set of control wells was also added in each plate to guarantee the validity of each run. At the end of the incubation period, cells from each well were distributed to three wells of 96-well plate: the first well was labeled with IgG1-FITC antibodies, the second one was labeled with CD86-FITC antibodies and the third one was labelled with CD54-FITC antibodies. Then, just before flow cytometry analysis of CD86 and CD54 expression, all cells were dyed with Propidium Iodide for viability discrimination.

For each run, the Mean Fluoresence Intensity (MFI) obtained for each test sample was corrected by the isotype control IgG1 MFI value to obtain the corrected MFI. Corrected MFI value from the corresponding vehicle control was set to 100% CD54 and CD86 expression by default. Then, corrected MFI values from each test sample were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index for CD86 and CD54 expression for each tested concentration (RFI CD86 and RFI CD54).

Results showed that the test item was found soluble in DMSO at the concentration of 500 mg/mL The test item induced a decrease in cell viability < 75% in both DRF runs and the calculated mean CV75 value was: 563.61 μg/mL. The highest concentration tested in the main test was therefore 676.33 μg/mL (i.e. 1.2-fold the mean CV75).

Under the experimental conditions of this study, the test item, MDAC, was found to be positive in the h-CLAT test method.