DL-thyroxin

Endpoint summary

Description of key information

In order to investigate the Skin sensitisation of Levothyroxine Acid, it has been attempted an In Chemico Determination of Skin Sensitisation (Direct Peptide Reactivity Assay – DPRA). Due to the intrinsic properties of the substance, the test could not be performed.

The investigation of the related structural domains for Skin sensitisation carried out with TOXTREE did not identified any alert for skin sensitisation, furthermore the experience during many years of manufacture and further processing of the intermediates did not reveal any sensitisation effect.

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

Nevertheless according to the Allergic Contact Dermatitis, Guinea Pig and Human - Danish QSAR DB CASE, as well as Ultra model together with the Allergic Contact Dermatitis, Guinea Pig and Human - Danish QSAR DB SciQSAR model the value predicted for Skin sensitisation is POSITIVE (The target chemical FALLS within the applicability domains).

For this reason it has been decided to classify the substance as sensitizing class 1.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Remarks:

human Cell Line Activation Test (h-CLAT)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

01 June 2020 - 08 July 2021

Reliability:

1 (reliable without restriction)

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)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Principle of the h-CLAT Method
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.
Demonstration of proficiency
Prior to routine use of the test method described in Annex II of the Test Guideline 442E, TOXI-COOP ZRT. demonstrated technical proficiency by correctly obtaining the expected h-CLAT prediction for the 10 proficiency substances recommended and listed in APPENDIX II of the OECD TG 442E and by obtaining CV75, EC150 and EC200 values that fell within the respective reference ranges (Study Number: 392-442-5369). Moreover, a historical database of data generated with the reactivity checks and with the positive and solvent/vehicle controls is maintained over time to confirm the reproducibility of the test method in the laboratory.
Procedure of the h-CLAT Method
0. Doubling time and reactivity check Solubility assessment of test chemicals (formulation)
1. Preliminary tests (CV75 determination)
2. Main Tests (CD86 and CD54 expression determination).

Doubling time and Reactivity check
Prior to using the master cell culture for testing, the cells were qualified by conducting a doubling time and reactivity check (approximately two weeks after thawing). Doubling time of the used master cell culture was monitored following the instructions of the relevant SOP and the measured doubling time of approximately 42 hours fell into reference range of the EURL ECVAM DB-ALM protocol (30 h – 55 h). The generated data were introduced into a historical control database.
Reactivity check of the cells was performed using the positive controls:
1-Chloro-2,4-dinitrobenzene (DNCB), Nickel(II) sulphate hexahydrate (NiSO4) and the negative control Lactic acid solution (LA). The positive controls produced a positive response for both CD86 and CD54 marker expression, while the negative control produced a negative response for both CD86 and CD54 marker expression in the tested master cell culture.
THP-1 (ATCC) MC1 20201211-20210426 master cell culture passed the reactivity check and concluded to be competent for the dose finding and main tests.

Preliminary test
Preparation of the cells
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)

Preparation master and working solutions
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 500.1 mg/mL in the first run, by two-fold serial dilutions using DMSO. In order to be able to determine CV75 value more precisely the concentration range was lowered and the master solutions were prepared of the test item stock solution at the concentration of 125.4 mg/mL
in the second run and 125.0 mg/mL in the third run. Moreover, 1.2-fold serial dilution with DMSO was used in the last two preliminary runs. 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.

Solvent/vehicle control
The solvent/vehicle control used in the h-CLAT method was DMSO tested at a single finalconcentration in the plate of 0.2 % and culture medium. DMSO underwent the same dilution as described for the working solutions.

Test item exposure
The culture medium or working solutions described above were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. The treated plates were then incubated for 24 ± 0.5 hours at 37° C under 5 % CO2. The plates were sealed with microplate covers prior to the incubation to avoid evaporation of test item.

Propidium iodide (PI) Staining
After 24 ± 0.5 hours of exposure, cells were transferred into sample tubes and 600 μL of FACS buffer was added to each sample. Cells were then collected by centrifugation (300 g, 5 min, 4 ºC). The supernatants were discarded and the remaining cells were washed again with 600 μL of FACS buffer. Finally, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added for each sample.

Cytotoxicity measurement by flow cytometry and estimation of CV75 value
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.

Main Test (CD86 and CD54 expression)
Test item dilutions
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 CV75 × 1.2 value (128.1 mg/mL) determined in the 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.

Solvent/vehicle controls
DMSO is tested as a solvent control for the test item and also for the positive control at a single final concentration in the plate of 0.2 %, so it underwent the same dilution as described for the working solutions. Culture medium was used as another negative control to assess the impact of DMSO. For the test item the relative fluorescence intensity of surface markers was calculated
compared to its relevant solvent/vehicle control.

Positive control
DNCB was used as the positive control for CD86/CD54 expression measurement at a final nominal concentration of 4.0 μg/mL in the plate. To obtain a 4.0 μg/mL concentration a 2 mg/mL stock solution of DNCB in DMSO were prepared and further diluted 250-fold with culture medium to an 8.0 μg/mL working solution. The working solution then was diluted 2-fold when added to the cells.

Application of test item and control substances
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.

Fluorescein Isothiocyanate (FITC) staining
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.

Flow cytometry measurement and relative fluorescence intensity (RFI) determination
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 (when the cell viability was low, up to 30,000 cells including dead cells could be acquired). Alternatively, data could be acquired for one minute after the initiation of the analysis. 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. The cell viability from the isotype control (ctrl) cells (which are stained with mouse IgG1 (isotype) antibodies) was also noted.

Requirements for qualified testing
- 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 %.

Abnormal values
- 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.

Requirement for data acceptance
- 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.

Prediction model
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]

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.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

CV75 [442D and 442E]

Value:

213.5 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on these results and the h-CLAT prediction model, the test item
“LEVOTHYROXINE ACID” is concluded negative 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 “LEVOTHYROXINE ACID” was examined.
The extent of cytotoxicity induced on THP-1 cells by the test item was studied in three dose finding tests. Based on their results, eight doses between 256.2 µg/mL and 71.5 µg/mL (nominal concentrations) were used for the main test in two independent runs which were concluded valid.
CD86 and CD54 marker expression was concluded to be negative (based on two negative valid runs).
Since both CD86 and CD54 marker expressions gave negative results, the overall h-CLAT prediction was also concluded to be negative.

Table. 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)
213,5	negative	negative	non-sensitiser

Based on these results and the h-CLAT prediction model, the test item “LEVOTHYROXINE ACID” is concluded negative for skin sensitisation potential under the experimental conditions of human Cell Line Activation Test

 

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

QSAR Toolbox prediction

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

Allergic Contact Dermatitis, Guinea Pig and Human - Danish QSAR DB
battery model

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Version / remarks:

Allergic Contact Dermatitis, Guinea Pig and Human - Danish QSAR DB battery
model (v.1.0)

Specific details on test material used for the study:

SMILES: NC(Cc1cc(I)c(Oc2cc(I)c(O)c(I)c2)c(I)c1)C(O)=O

Run / experiment:

other: other: Allergic Contact Dermatitis, Guinea Pig and Human - Danish QSAR DB battery model (v.1.0)

Parameter:

other: Skin sensitisation II (ECETOC)

Remarks:

POSITEVE

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

According to the Allergic Contact Dermatitis, Guinea Pig and Human - Danish QSAR DB CASE
Ultra model together with the Allergic Contact Dermatitis, Guinea Pig and Human - Danish QSAR DB SciQSAR model the value predicted for Skin sensitisation is POSITIVE (The target chemical FALLS within the applicability domains)

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 April 2019 - 4 december 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay – DPRA

Details on the study design:

The DPRA is proposed to address the molecular initiating event of the skin sensitization Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of the test chemical towards model synthetic peptides containing either cysteine or lysine. Cysteine and lysine peptide depletion values will be then used to categorize the test substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

Remarks on result:

not determinable because of methodological limitations

Other effects / acceptance of results:

The solubility of Levothyroxine Acid was assessed in a way that all acceptable solvents were evaluated (noted in the OECD 442C guideline and DB-ALM protocol 153°). Only acetonitrile dissolved the test item completely. This formulation was added to acetate buffer and phosphate buffer but unfortunately both mixtures appeared to be inhomogeneousand separate phases could be observed. Therefore no formulation was found which is suitable for HPLC analysis.

Interpretation of results:

study cannot be used for classification

Conclusions:

Due to the intrinsic properties of the test item, the test could not be performed.

Executive summary:

The Chemico Determination of Skin Sensitisation (Direct Peptide Reactivity Assay – DPRA) of LEVOTHYROXINE ACID is not a suitable testing for the investigation of Skinsensitisation endpoint.

Reference 4

Endpoint:

skin sensitisation, other

Remarks:

Experience with the chemical class of the substance and from observations of workers.

Type of information:

other: Experience with the chemical class of the substance and from observations of workers.

Adequacy of study:

other information

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Experience with the chemical class of the substance and from observations of workers.

Qualifier:

no guideline followed

GLP compliance:

no

Type of study:

other: Experience with the chemical class of the substance and from observations of workers.

Species:

other: humans

Sex:

male/female

Interpretation of results:

study cannot be used for classification

Conclusions:

According to the Company Medical Officer experience the intermediates of the levothyroxine production never shoved cutaneous rushes as well as allergic outcome

Reference 5

Endpoint:

skin sensitisation, other

Type of information:

calculation (if not (Q)SAR)

Remarks:

Tox Tree calulation

Adequacy of study:

supporting study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

1. SOFTWARE

Toxtree (Estimation of Toxic Hazard - A Decision Tree Approach)
www.ideaconsult.net
2. Version: 3.1.0-1851-1525442531402

3. SMILES USED AS INPUT FOR THE MODEL: NC(Cc1ccc(O)cc1)C(=O)O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Identification of mechanisms of toxic action for skin sensitisation using a SMARTS pattern based approach
http://toxtree.sourceforge.net/skinsensitisation.html
Vendor: Procter&Gamble

References:
Enoch SJ, Madden JC, Cronin MT,Identification of mechanisms of toxic action for skin sensitisation using a SMARTS pattern based approach.,SAR QSAR Environ Res. 2008;19(5-6):555-78

5. APPLICABILITY DOMAIN
- Structural and mechanistic domains:
QSNAR.SNAr-Nucleophilic Aromatic Substitution 
QSB.Schiff Base Formation 
QMA.Michael Acceptor  
Qacyl.Acyl Transfer Agents 
QSN2.SN2-Nucleophilic Aliphatic Substitution 

6. ADEQUACY OF THE RESULT
Identification of at least one alert for skin sensitisation

Qualifier:

no guideline available

Specific details on test material used for the study:

SMILES USED: NC(Cc1ccc(O)cc1)C(=O)O

Parameter:

other: No Class No skin sensitisation reactivity domains alerts identified.

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

study cannot be used for classification

Conclusions:

The software verifies the structural alerts for Skin sensitisation reactivity domains in NC(Cc1ccc(O)cc1)C(=O)O: No skin sensitisation reactivity domains alerts identified.

Executive summary:

ToxTree vefifed no structural alerts for Skin sensitisation reactivity domains in O- LEVOTHYROXINE ACID, the substance do not falls into skin sensitisation reactivity domain.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)