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Description of key information

The dermal sensitization potential of thiourea was assessed by means of a weight of evidence approach using two in vitro studies of the skin sensitization battery: DPRA (OECD guideline 442C) and KeratinoSens (OECD guideline 442D) as well as a GPMT study (OECD guideline 406). All studies conclude that thiourea does not exhibit skin sensitzing properties.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
no information on use of 10% SDS
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Test was performed before LLNA was made mandatory
Species:
guinea pig
Strain:
other: pirbright white
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lippische Versuchstierzucht, Hagemann GmbH & Co, D-4923 Exertal 1
- Age at study initiation: 21-28 Tage
- Weight at study initiation: 250-300 g
- Housing: two animals in one cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/-1°C
- Humidity (%): 60% +/- 5%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal
Vehicle:
no data
Concentration / amount:
0.5-10% induction; 20 % challenge
Route:
other: percutaneous
Vehicle:
no data
Concentration / amount:
0.5-10% induction; 20 % challenge
No. of animals per dose:
20 male animals were tested altogether; in the positive control additional 20 animals were tested
Details on study design:
RANGE FINDING TESTS: yes; 2-3 animals; estimation of the probable sentising effects; determination of dose to induce after intradermal treatment an irritation; percutaneous treat did not lead to irritation

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Exposure period: 24 h
- Test groups: 0.5-10% substance; 0.5%-10% substance + mixture containing of Freund`s adjuvant Complete and vehicle (1:1)
- Control group: Freund`s adjuvant Complete and vehicle (1:1)
- Site:at the back
- Frequency of applications: once
- Duration: 24 h
- Concentrations: 0.5-10%
Reading after 1 h and 24 h

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 7 days after first exposure
- Exposure period: 48 h (occlusive)
- Test groups: 2 ml 20% solution of test substance
- Control group: not reported
- Site: at the back between front and rear extremities
- Concentrations: 20 % solution
- Evaluation (hr after challenge): 72 h

OTHER: positive control: 20 animals treated with Penicllin G-Na
Positive control substance(s):
yes
Remarks:
Penicllin G-Na
Positive control results:
valid; 100% sensibilization
Key result
Reading:
rechallenge
Hours after challenge:
72
Group:
test chemical
Dose level:
20 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
not reported
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
72
Group:
positive control
Dose level:
5000 IE/ 0.1ml
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
not reported
Remarks on result:
other: Reading: other: 3rd. Hours after challenge: 72.0. Group: positive control. Dose level: 5000 IE/ 0.1ml. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: not reported.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no adverse effect
Interpretation of results:
GHS criteria not met
Conclusions:
In this study thiourea is not sensitising.
Executive summary:

In a dermal sensitization study with Thiourea (0.5-10%) male guinea pigs (pirbright white) were tested using the method of Magnusson and Kligman. Induction was performed intradermal. Penicillin G-Na was used as positive control.


In this study Thiourea is not a dermal sensitizer.

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

The chemical and biological mechanisms associated with skin sensitisation are summarised in the form of an Adverse Outcome Pathway (AOP). This AOP includes the following key events:


1) The molecular initiating event is the covalent binding of electrophilic substances to nucleophilic centres in skin proteins.


2) The inflammatory responses and gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways in the keratinocytes.


3) The activation of dendritic cells, typically assessed by expression of specific cell surface markers, chemokines and cytokines.


4) The T-cell proliferation


The adverse outcome is the presentation of allergic contact dermatitis.


Studies addressing one of the key events can be used as part of a testing battery based on the OECD adverse outcome pathway (AOP) for the assessment of the skin sensitisation potential of chemicals. According to the OECD TG 497, the "2 out of 3" (2o3) defined approach for skin sensitisation hazard identification was applied. For thiourea, Key event 1 is addressed by the DPRA assay (OECD 442C). The second key event is addressed by the KeratinoSens test (OECD 442D). Additionaly, the adverse outcome is represtented in a GPMT-study (OECD 406). All assays were evaluated in terms of a weight-of-evidence approach. As the results of both in vitro tests were negative, no further testing is required to fulfil the information requirements according to Annex VII, section 8.3.1.


 


1. Skin Sensitisation in chemico (DPRA)


The objective of this study was to determine the reactivity of Thiourea towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and spectrophotometric detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which assigns the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. The study procedures described in this report were based on the most recent OECD 442C guideline.
Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.
The validation parameters, i.e., calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA as stated in the OECD 442C guideline.
Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples. An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 0.0% SPCC depletion while in the lysine reactivity assay the test item showed 2.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.2% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction.


In conclusion, since all acceptability criteria were met, this DPRA is considered to be valid. Thiourea was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.


 


2. Skin sensitisation in vitro (ARE-Nrf2 KeratinoSens Test Method)


The objective of this study was to evaluate the ability of Thiourea to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.
The study procedures described in this report were based on the most recent OECD guideline. Batch 021701 of the test item was a white solid. The test item was dissolved in Milli-Q water at 200 mM. From this stock 11 spike solutions in Milli-Q water were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed. Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (7.9 μM and 34 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (4.25 fold and 3.02-fold in experiment 1 and 2, respectively).
• The average coefficient of variation of the luminescence reading for the vehicle (negative) control Milli-Q water was below 20% (9.6% and 5.3% in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (5.4% and 6.2% in experiment 1 and 2, respectively).


Overall it is concluded that the test conditions were adequate and that the test system functioned properly. The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.32-fold and 1.28-fold in experiment 1 and 2 respectively. The test item is classified as nonsensitizer in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.


In conclusion, Thiourea is classified as non-sensitizer (no activation of the antioxidant/electrophile responsive element (ARE)- dependent pathway in keratinocytes) under the experimental conditions described in this report.


 


3. Guinea Pig Maximisataion Method (GPMT)


In a an additional dermal sensitization study with 0.1 mL thiourea (0.5–10 %) male guinea pigs were tested using the method of Magnusson and Kligman. In this study Thiourea is not a dermal sensitizer. Several case reports on human exposure are available indication a low degree of sensitization. The number of reports are not considered to represent a "substantial number" as required for a classification under CLP. In addition it is not clear whether or not they are attributable to Thiourea derivatives. In several cases thiourea derivatives and not thiourea as such were reported to cause contact dermatitis.


 


 


Based on the experimental outcomes of all conducted assays adressing key event 1 and 2 of the AOP as well as the adverse outcome outlined above, it can be concluded that thiourea is non-sensitizing to skin.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008


Based on the available studies, which are considered reliable for this assessment, it is concluded that the test item is not a skin sensitiser and should not be classified under EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No 1272/2008, as amended for seventeenth time in Regulation (EU) No 2021/849.

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