(2S)-2-amino-3-[3,5-diiodo-4-(4-methoxyphenoxy)phenyl]propanoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Remarks:

human Cell Line Activation Test (h-CLAT)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 June 2020 - 08 July 2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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)

Version / remarks:

25 June 2018

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:

(2S)-2-amino-3-[3,5-diiodo-4-(4-methoxyphenoxy)phenyl]propanoic acid

EC Number:

619-013-3

Cas Number:

94345-95-6

Molecular formula:

C16H15I2NO4

IUPAC Name:

(2S)-2-amino-3-[3,5-diiodo-4-(4-methoxyphenoxy)phenyl]propanoic acid

Test material form:

solid: particulate/powder

Details on test material:

Name: O-METHYL-L-DIIODOTHYRONIN
Batch No.: B488840
Manufacturing date: 25 September 2018
Expiry date: 24 September 2020
Storage condition: at room temperature, protected from light

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemicals. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.
Master solutions (MS) were prepared with DMSO as follows: Eight master solutions (eight concentrations) were prepared of the test item stock solution at the concentration of 62.6 mg/mL in the first run and 62.8 mg/mL in the second run, by two-fold serial dilutions using DMSO. These master solutions were then further diluted 250-fold into culture medium to obtain the working solutions (WS). The working solutions were finally used for exposure by adding an equal volume of working solution (500 µL) to the volume of THP-1 cell suspension (500 µL) in the 24-well plate to achieve a further two-fold dilution as the final concentration of the test item. The solvent/vehicle control used in the h-CLAT method was DMSO tested at a single final concentration in the plate of 0.2 % and culture medium. DMSO underwent the same dilution as described for the working solutions.
DMSO was used to dissolve the test item for the stock solution (SS) in the runs of main test, as well. The test item was first diluted to the concentration corresponding to the highest soluble concentration (HSC – 125.0 µg/mL) determined in the solubility and dose finding assay. For the master solutions (MS), 1.2-fold serial dilutions were made from the stock solution using DMSO (eight concentrations). The master solutions were then further diluted 250-fold into the culture medium to obtain the working solutions (WS). These working solutions were finally used for exposure with a further final two-fold dilution factor in the plate.
Highest concentration used: 125.2 µg/mL.
The PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 living cells (PI negative) were acquired.
Cell viability was analysed by the CytExpert Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.
- Final concentration range two independent runs for the dose finding assay were performed to determine the test item concentration that results in 75 % cell viability (CV75) compared to the solvent/vehicle control chosen in the trial formulation. In the first run the highest final test item concentration on the plate was 125.2 µg/mL and a 2-fold dilution was used when preparing the master solutions. Since no cytotoxicity was observed, the run was repeated with the same tested concentrations. Therefore, in the second run the highest final test item concentration on the plate was 125.6 µg/mL and a 2-fold dilution was used again.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 8
- Number of repetitions: 3
-Test item and control substances prepared as working solutions (500 μL) were mixed with 500 μL of suspended cells (1 × 106 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24±0.5 hours. After 24 ± 0.5 hours of exposure, cells were transferred from the 24-well plate into sample
tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by centrifugation (300 g, 5 min, 4 ºC). The washing step was repeated once more with 1 mL of FACS buffer. After washing, cells were blocked with 600 μL of 1 × blocking solution and incubated at approximately 4 °C for 15 min. After blocking, cells were split in three aliquots of 200 μL into sample tubes. After centrifugation (300 g, 5 min, 4 ºC), cells were stained with 50 μL of FITC-labelled antiCD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at approximately 4 °C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol 158° were used. After washing twice with 150 μL of FACS buffer, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry. The cell viabilities of medium and solvent/vehicle controls should be higher than 90%. In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %) and cell viability should be more than 50 %.In the solvent/vehicle controls, RFI values compared to the medium control of both CD86
and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150 % and
CD54 RFI ≥ 200 %). For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105 %. RFI values cannot be less than zero. Regardless of the reason, such values should be omitted
from the prediction. If an abnormal value is observed, check whether there are abnormal conditions in the run and record them in the reporting section. For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run. For the test chemical resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90%. When the test item is tested 5000 µg/mL in saline (maintenance medium alternatively) or 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration, a negative result is acceptable even if the cell viability is above 90 %. For the test chemical resulting in positive outcome, the cell viability at the 1.2 x CV75 is more than 90 %, the data is acceptable.

SEEDING AND INCUBATION
For testing, THP-1 cells were seeded at a density of either 0.1 × 106 cells/mL or 0.2 × 106 cells/mL, and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintenance medium at 2 × 106 cells/mL. Then, cells were distributed into 24 well flat-bottom plate with 500 µL cell suspension / well (1 × 106 cells/well)

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1. Cytotoxicity was checked based on the PI uptake as in the preliminary tests and was analysed with the acquisition channel FL-3.
A total of 10,000 living cells (PI negative) were acquired. The cell viability was automatically calculated by the cytometer analysis program.
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 marked positive control (ctrl) and chemical-treated cells were calculated

DATA EVALUATION
If the RFI of CD86 is equal to or greater than 150% at any tested dose (>50 % of cell viability) in at least 2 independent runs, AND/OR if the RFI of CD54 is equal to or greater than 200 % at any tested dose (>50 % of cell viability) in at least 2 independent runs, the test item prediction is considered as positive. Otherwise it is considered as a negative.
In case the first two independent runs are not concordant a third run needs to be performed and the final prediction will be based on the mode of conclusions from the three individual runs.
When the item is tested at 5000 µg/mL in saline, 1000 µg/ml in DMSO, or highest soluble dose as the maximal test concentration instead of CV75-based dose and does not meet the positive criteria above without affecting cytotoxicity at all tested doses, the test item prediction should be considered as negative.
Since EC150 and EC200 values are just optional for test items that are found to be sensitisers, the values are calculated only in cases where a firm dose response curve can be constructed.
Up to six runs, meeting requirements for qualified testing, are permitted to reach a conclusion for each test item. The six runs may include runs for which the data adoption criteria are not met for this test item. If no prediction can be made after the sixth run, the result is inconclusive.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

Positive control results:

The positive control gave expected results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50 % in each run.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC200, CD54 [442E]

Value:

108.3 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC200, CD54 [442E]

Value:

132.2 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on these results and the h-CLAT prediction model, the test item “O-METHYLL-DIIODTHYRONIN” is concluded positive for skin sensitisation potential under the experimental conditions of human Cell Line Activation Test.

Executive summary:

In the course of this study the skin sensitisation potential of “O-METHYL-LDIIODTHYRONIN” was examined.
The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. The maximal final concentrations used on the plates for the test item previously dissolved in DMSO were 125.2 µg/mL (first run) and 125.6 µg/mL (second run). Since no cytotoxicity was observed, no CV75 value was determined. Based on the results of the two dose finding tests, eight doses between 125.0 µg/mL - 34.9 µg/mL (nominal concentrations) were used for the main test in three independent runs and all three runs were concluded valid.
The increase in CD86 marker expression (RFI) was lower than 150 % at all tested doses (with > 50 % of cell viability) compared to the respective negative controls in two out of three independent valid runs. Based on the two out of three valid negative results, CD86 marker expression was concluded to be negative. Therefore, effective concentration for CD86 expression (EC150) was not determined.
The increase of CD54 marker expression (RFI) was greater than 200 % compared to the respective negative controls at the highest tested concentration (with > 50 % of cell viability) in two out of three independent valid runs. Based on the two out of three valid positive runs, CD54 marker expression was concluded to be positive. Thus, the effective concentration for CD54 expression (EC200) was determined by using linear interpolation, the EC200 value for CD54 expression was 123.2 µg/mL.
Despite of the fact that CD86 marker expression was concluded to be negative, since two out of three valid independent runs were positive for CD54 marker expressions, the overall h-CLAT prediction was concluded to be positive.

Table 1. Summary of the h-CLAT results for the test item

Obtained CV75 value (µg/mL)	h-CLAT result for CD86 (positive/negative	h-CLAT result for CD54 (positive/negative)	h-CLAT result obtained (sensitiser/ non-sensitiser)
-	negative	positive (123.2 µg/mL)	sensitiser

Based on these results and the h-CLAT prediction model, the test item “O-METHYLL-DIIODTHYRONIN” is concluded positive for skin sensitisation potential under the experimental conditions of human Cell Line Activation Test.