thiourea; thiocarbamide

Endpoint summary

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

Reference 1

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.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-05-25 to 2021-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The DPRA assay is part of an in vitro/in chemico testing strategy (OECD guidelines 442C-E) addressing The Adverse Outcome Pathway Key Event of covalent binding to proteins. Mechanistically-based in chemico and in vitro test methods addressing the first three key events of the skin sensitisation AOP have been adopted for contributing to the evaluation of the skin sensitisation hazard potential of chemicals.

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
Cysteine: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 11.6 mg of cysteine peptides in 23.15 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
Lysine: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.0 mg of lysine peptides in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes

- Preparation of the test chemical solutions: No correction for the purity/composition of the test item was performed. Solubility of the test item in acetonitrile was assessed before performing the DPRA. It dissolved the test item completely, i.e. by visual inspection the solution was not cloudy nor had noticeable precipitate. Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay, 33.57 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1803 μL ACN after vortex mixing to obtain a 100 mM solution. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

- Preparation of the positive controls, reference controls and co-elution controls:
Positive controls: The cinnamic aldehyde positive control samples (PC) were prepared with 750 μL Stock solution of 0.667 mM cysteine/lysine peptides and 250 μL Cinnamic aldehyde solution (100 mM in ACN)
Reference controls: Three 0.5 mM peptide reference control (RC) solutions (RCA, RCB and RCC) were prepared in amber vials by mixing 750 μL of the 0.667 mM cysteine/lysine peptide stock solution with 250 μL ACN.
Co-elution controls: The co-elution control (CC) samples were prepared with 750 μL Ammonium acetate buffer pH 10.2 and 250 μL test item solution (100 mM)

INCUBATION
- Incubation conditions: The samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.2 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.

- Precipitation noted: Prior to HPLC analysis the samples were visually inspected for precipitation. No precipitate or phase separation was observed in any of the samples.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys was prepared

DATA EVALUATION
- The concentration of SPCC or SPCL was spectrophotometrically determined by
High-Performance Liquid Chromatography (HPLC) with gradient elution and
spectrophotometric detection at 220 nm and 258 nm in each sample and measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 61.7% ± 1.2%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the
maximum (SD <11.6%)

Key result

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

2.4 %

At concentration:

0.5 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Lysine 1:50 prediction model

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

0 %

At concentration:

0.5 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Cysteine 1:10 prediction model

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, see "Attached background material"
- Acceptance criteria met for positive control: Yes, see "Attached background material"
- Acceptance criteria met for reference controls A to C: Yes, see "Attached background material"
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): Yes, see "Attached background material"
- Acceptance criteria met for variability between replicate measurements: Yes, see "Attached background material"

Table 1: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPLC depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction mode
Thiourea	0.0%	±0.0%	2.4%	±3.1%	1.2%	Negative: No or minimal reactivity

SD = Standard Deviation.

Interpretation of results:

other: Negative in peptide reactivity

Conclusions:

In conclusion, this DPRA test is valid. The DPRA with the test item Thiourea was considered to be negative and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-05-26 to 2021-06-11

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 KeratinoSens™ test method)

Version / remarks:

adopted June, 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

The KeratinoSens test (ARE-Nrf2 luciferase reporter assay) is part of an in vitro/in chemico testing strategy (OECD guidelines 442C-E) addressing The second Adverse Outcome Pathway Key Event of inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways. Mechanistically-based in chemico and in vitro test methods addressing the first three key events of the skin sensitisation AOP have been adopted for contributing to the evaluation of the skin sensitisation hazard potential of chemicals.

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution:
In the main experiments the test item was dissolved in Milli-Q water at 200 mM (clear colourless solution). From this stock 11 spike solutions in Milli-Q water were prepared (2-fold dilution series).

- Preparation of the test chemical serial dilutions:
The stock and spike solutions were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration Milli-Q water of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution.

- Preparation of the positive controls:
The positive control used in the case of KeratinoSens is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.4.1, so that the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate. The formulation of the positive control was used in studies performed concurrently

- Preparation of the vehicle and blank
The vehicle control was 1% DMSO in exposure medium for the positive control and 1% Milli-Q water in exposure medium for the test item. Eighteen wells were tested per plate. On each plate three blank wells were tested (no cells and no treatment).

DOSE RANGE FINDING ASSAY:
- Highest concentration used: yes, 200 mM
- Solubility in solvents:
A solubility test was performed. The test item was initially dissolved in DMSO to a final concentration of 200 mM (clear colourless solution). The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax showed no precipitation. This concentration was initially selected as highest concentration for the main assay (highest dose required in the current guideline).
At the start of the main study, a solubility assessment in Milli-Q water was performed due to the stability in Milli-Q water. In this solubility assessment, Milli-Q water proved to be soluble at 200 mM. Therefore, Milli-Q water was the preferred vehicle for the main experiment, being a stable solution at 200 mM (highest dose required in the current guideline).

- Solubility in incubation medium: yes
No precipitation was observed at the start and end of the incubation period in the 96-well plates

- Cytotoxicity assessment performed: yes
For the KeratinoSens cell viability assay, medium was replaced after the 48 hour exposure time with fresh DMEM Glutamax containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: triplicate
- Number of repetitions: 2 experiments
- Test chemical concentrations: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM
- Application procedure:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate
(lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates
were removed from the incubator and the medium is removed. Then 200 μL of the Steady-
Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was
added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates
with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess
the quantity of luciferase (integration time two seconds).
- Exposure time: 48 hours ± 1 h at 37±1.0 °C
- Study evaluation and decision criteria used: yes, see below
- Description on study acceptance criteria:
The KeratinoSens test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be statistically significant equal or above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM).
b) The EC1.5 of the positive control should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the doseresponse of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO and Milli-Q water should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded. If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test. If the variability is still higher, the results should be discarded.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.


SEEDING AND INCUBATION
- Seeding conditions: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

- Incubation conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 53 – 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.8 – 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
- Washing conditions
Before treatment, The medium (Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands) was removed and replaced with fresh exposure medium ((Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.)150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added.

- Precipitation noted: no

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer with demonstration of appropriate luminescence measurements based on control test: TECAN Infinite® M200 Pro Plate Reader
- Plate used: 96-well plates
- Lysate preparation:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady- Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature.

Vehicle / solvent control:

water

Negative control:

not applicable

Positive control:

other: Ethylene dimethacrylate glycol (CAS number: 97-90-5, Sigma, Zwijndrecht, The Netherlands)

Positive control results:

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).

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.3

Cell viability:

higher than 70 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Cell viability:

higher than 70%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Cell viability:

higher than 70 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Table 1: Overview EC1.5, Imax, IC30 and IC50 Values

	EC1.5 (μM)	Imax	IC30 (μM)	IC50 (μM)
Test item Experiment 1	NA	1.32	NA	NA
Test item Experiment 2	NA	1.28	NA	NA
Pos Control Experiment 1	7.9	4.25	NA	NA
Pos Control Experiment 2	34	3.02	NA	NA

 

Two independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 μM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

 

Experiment 1
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
• No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.32 and therefore no EC1.5 could be calculated.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.25 and the EC1.5 7.9 μM.

 

Experiment 2
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• The test item showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
• No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.28 and therefore no EC1.5 could be calculated.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.02 and the EC1.5 34 μM.

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.

Interpretation of results:

other: Negative in activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes

Conclusions:

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.

Executive summary:

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.

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.