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Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23 May 2018 - 01 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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:
direct peptide reactivity assay (DPRA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 55507861
- Expiration date of the lot/batch: 04 January 2020
- Purity: 99.4%
- Appearance: White solid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15°C to 25°C) in the dark
Positive control results:
In the case of Cysteine Peptide Depletion: 73.3 %
In the case of Lysine Peptide Depletion: 57.8 %
Key result
Run / experiment:
other: Mean of 3 runs
Parameter:
other: Cysteine peptide depletion
Value:
1.12 %
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Run / experiment:
other: The mean of 3 runs
Parameter:
other: Lysine peptide depletion
Value:
3.43 %
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Interpretation of results:
study cannot be used for classification
Conclusions:
Solutions of Semicarbazide HCl were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. There was minimal reactivity in both peptides therefore Semicarbazide HCl is placed in the reactivity class of “no or minimal” and hence it is predicted by DPRA to be negative in terms being a potential skin sensitizer.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 May 2018 - 07 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 442E
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 55507861
- Expiration date of the lot/batch: 04 January 2020
- Purity: 99.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the refrigerator
- Stability of the test substance in the solvent/vehicle: Stable in water
- Solubility in water: 100 g/L

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Semicarbazide hydrochloride was dissolved in culture medium.
- Final dilution of a dissolved solid, stock liquid or gel: For the XTT test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 5000 μg/mL.

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

Experimental Design and Procedures of h-CLAT
The test item was tested in two independent runs.

Treatment of the Cells
For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.
Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.

Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
Preparation of the acquisition
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.
Acquisition
Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).
Positive control results:
The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.
Key result
Run / experiment:
other: First run
Parameter:
other: CD 54 Antibody
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: positive in 1 out of 8 concentrations
Key result
Run / experiment:
other: First run
Parameter:
other: CD 86 Antibody
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: positive in 1 out of 8 concentrations
Key result
Run / experiment:
other: Second run
Parameter:
other: CD 54 Antibody
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Negative in all concentrations
Key result
Run / experiment:
other: Second run
Parameter:
other: CD 86 Antibody
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: positive in 1 out of 8 concentrations
Interpretation of results:
study cannot be used for classification
Conclusions:
The test item Semicarbazide hydrochloride with a log Pow of -2.75 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04 Jul 2018 - 13 Jul 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 Test Method)
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 55507861
- Expiration date of the lot/batch: 04 Jan 2018
- Purity: 99.3 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, dark
Details on the study design:
Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Cell seeding (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells
per well, passage number 20.
Day 2: 24 hours after seeding, the test and control items were applied and plates were incubated at
37oC, 5% CO2, ≥ 95% relative humidity for 48 ± 2 hours.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing
(2 plates)
Positive control results:
Positive Control (PC) (Cinnamic aldehyde) induction >1.5-fold in
at least one concentration
Key result
Run / experiment:
other: 1st run
Parameter:
other: I MAX
Value:
0.993
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: Negaitive at all concentrations
Key result
Run / experiment:
other: 2nd run
Parameter:
other: I MAX
Value:
0.754
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: Positive in at least 1 concentration
Key result
Run / experiment:
other: 3rd run
Parameter:
other: I MAX
Value:
0.861
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: Negative in all concentrations
Interpretation of results:
study cannot be used for classification
Conclusions:
Semicarbazide-Hydrochloride was classified as negative according to the KeratinoSens prediction model.
Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
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:
Refer to the attached report for model justification and validation criteria.
Qualifier:
no guideline required
Principles of method if other than guideline:
QSAR prediction
Due to the complexity of the skin sensitisation endpoint, a combination of alternative test methods (e.g. in silico, in chemico and in vitro) in a weight of evidence approach needs to be considered to increase confidence in the final assessment of skin sensitisation.
GLP compliance:
no
Run / experiment:
other: TOPKAT
Parameter:
other: QSAR prediction for skin sensitisation
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Remarks:
the prediction was low reliable due to the following uncertainty of the prediction: low structural similarity of the four structural analogues, low concordance of analogues with the predicted value and moderate accuracy (one out of four structural similar compounds was predicted as false-negative).
Run / experiment:
other: CAESAR of Vega
Parameter:
other: QSAR prediction for skin sensitisation
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Remarks:
CAESAR predicted Semicarbazide to be sensitizer with low reliability (ADI: 0.461) and indicated that the query structure is outside applicability domain of the model.
Run / experiment:
other: Derek
Parameter:
other: QSAR prediction for skin sensitisation
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
not determinable
Remarks:
DEREK prediction was based also on the cationic part of the formula: Semicarbazide. The model triggered for Semicarbazide an alert described as hydazine or precursor. The reasoning level of the prediction was equivocal, defined as equal weight of evidence for and against the proposition.
Run / experiment:
other: Toxtree
Parameter:
other: QSAR prediction for skin sensitisation
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: OECD QSAR Toolbox
Parameter:
other: QSAR prediction for skin sensitisation
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no indication of skin sensitisation

TOPKAT predicted Semicarbazide hydrochloride to be sensitizer; however, the prediction was low reliable due to the following uncertainty of the prediction: low structural similarity of the four structural analogues, low concordance of analogues with the predicted value and moderate accuracy (one out of four structural similar compounds was predicted as false-negative). In addition, the Bayesian score was close to the best split value and probability of 0.69 indicated

that likelihood of positive response in an experimental assay was within indeterminate range (0.30 - 0.70). Furhermore, there were two unknown fingerprint features of the query molecule fragments which were found in the training set (see TOPKAT QPRF and printout).

CAESAR of Vega was one of models, not able to predict the query compound in the salt form. Therefore, the prediction was based on the cationic part of the formula: Semicarbazide (CAS: 57-56-7). CAESAR predicted Semicarbazide to be sensitizer with low reliability (ADI: 0.461) and indicated that the query structure is outside applicability domain of the model. In addition, one unknown fragment and two infrequent fragments of the query structure have been found in

the compounds of the training set (see CAESAR QPRF and printout).

DEREK prediction was based also on the cationic part of the formula: Semicarbazide. The model triggered for Semicarbazide an alert described as hydazine or precursor. The reasoning level of the prediction was equivocal, defined as equal weight of evidence for and against the proposition. Semicarbazide (CAS: 57-56-7). According to DEREK, for Semicarbazide the potential skin sensitization mechanism was suggested via pre-hapten producing an electrophile and/or generation a free radical. In the section EC3 Result for Derek EC3 model, two structural similar compounds edaravone (similarity 5.2%) and 2-(3,4-dimethylphenyl)-5-methyl-2,4-dihydro-3H-pyrazol-3-one (similarity 4.5%) were reported to be moderate and extreme sensitizers, respectively. Since of

low analogue number, there was insufficient data to calculate an EC3 value. In addition, the alert by DEREK demonstrated weak predictive performance as described in validation comments. There was no compounds activating this alert in (1) Cronin and Basketter and (3) Contact Dermatitis databases. Only (2) Gerberick database showed one compound, which activates this alert and reported as positive (see DEREK QPRF and printout).

Toxtree identified no alert in respect to skin sensitization reactivity domains for Semicarbazide hydrochloride (see Toxtree printout).

No alerts were triggered for Semicarbazide hydrochloride in the protein binding alerts for skin sensitization by OASIS and according to GHS v1.4 in OECD QSAR Toolbox profiler.

The autoxidation, skin metabolism and neutral hydrolysis simulators of OECD QSAR Toolbox generated four, five and two metabolites for Semicarbazide hydrochloride, respectively. However, no of them were profiled for the protein binding alerts for skin sensitization (see OECD QSAR Toolbox printouts). Therefore, the presence of pre- and pro-haptens may be excluded.

Model Prediction result Reliability (model statistics)
TOPKAT pos Low (probability: 0.69)
CAESAR (VEGA)* pos Low (AD index: 0.461)
DEREK* alert for hydrazine or precursor Equivocal (equivocal or above)
Toxtree no alert Restricted on 5 skin protein binding principles
OECD QSAR Toolbox     
Protein binding alerts for skin sensitization by OASIS no alert Restricted on 11 mechanistic domains
Protein binding alerts for skin sensitization according to GHS no alert The absence of a protein binding alert shouldnot be taken as an absence of toxicity
Autoxidation simulator: 4 metabolites    
Protein binding alerts for skin sensitization by OASIS no alert Restricted on 11 mechanistic domains
Protein binding alerts for skin sensitization according to GHS no alert The absence of a protein binding alert shouldnot be taken as an absence of toxicity
Skin metabolism simulator: 5 metabolites    
Protein binding alerts for skin sensitization according to GHS no alert Restricted on 11 mechanistic domains
Protein binding alerts for skin sensitization according to GHS no alert The absence of a protein binding alert shouldnot be taken as an absence of toxicity
Hydrolysis simulator (neutral): 2 metabolites    
Protein binding alerts for skin sensitization according to GHS no alert Restricted on 11 mechanistic domains
Protein binding alerts for skin sensitization according to GHS no alert The absence of a protein binding alert shouldnot be taken as an absence of toxicity
*not salt form
Interpretation of results:
GHS criteria not met
Conclusions:

Taking into account that TOPKAT and CAESAR positive predictions were low reliable and the alert for hydrazine or precursor based on pre-hapten reactivity in DEREK was not supported with the OECD QSAR Toolbox profiler for skin sensitization alerts of simulated metabolites, Semicarbazide hydrochloride is likely not a skin sensitizer.
Executive summary:

The present in silico assessment was performed with five computational tools: TOPKAT, CAESAR of Vega, Derek, Toxtree and OECD QSAR Toolbox.

Weight of evidence of this in silico assessment suggested that Semicarbazide hydrochloride is likely not a skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

Taking into account that TOPKAT and CAESAR positive predictions were low reliable, and the alert for hydrazine or precursor based on pre-hapten reactivity in DEREK was not supported with the OECD QSAR Toolbox profiler for skin sensitization alerts of simulated metabolites, Semicarbazide hydrochloride is likely not a skin sensitizer.

The results of the h-CLAT test were positive whereas the KeratinoSens and DPRA tests were negative. Therefore, applying the '2 out of 3' interpretation, the substance does not require classification.

Overall, weight of evidence shows the substance to be a non-sensitiser.