Methylthiouracil

Endpoint summary

Description of key information

OECD 442C (WoE): The test item was concluded as positive or sensitiser with high reactivity in Direct Peptide Reactivity Assay (DPRA).

OECD 442D (WoE): The test item is a non-sensitiser in the in vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens assay) perfomed according to OECD 442D guideline.

QSAR (WoE): According to 3/4 of predictions of different sofwares, test item was scored as positive for skin sensitisation as a result of search from Danish (Q)SAR Database.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The correlation of protein reactivity with skin sensitisation potential is well established. Haptenation i.e. covalent binding of low molecular weight substances (Haptens) to proteins present in skin is considered a prominent mechanism through which chemicals or their metabolites become antigenic. Therefore, information inferred from the DRPA assay is relevant for assessment for skin sensitisation potential of substances.

Details on the study design:

On the day of test sample preparation, appropriate amount of cysteine and lysine peptide was weighed and dissolved in phosphate buffer and ammonium acetate buffer respectively. The stock concentration of cysteine peptide prepared was 0.501 mg/mL (0.667 mM) and lysine peptide was 0.518 mg/mL (0.667 mM). The preparations were done immediately before use.

Test item and positive control solutions were prepared at 100 mM concentration and used in the test sample preparation for analysis. Test item, positive control and reference control was added to cysteine and lysine peptides and incubated in dark at 25°C for 24±2 hours. Post incubation, the test samples were analysed using HPLC to measure the peptide depletion.


HPLC ANALYSIS :
1. Agilent Zorbax SB-C18 2.1 mm X 100 mm X 3.5 micron (or alternate column: Phenomenex Luna C18 (2) 2.0 mm X 100 mm X 3 micron) column was installed in the HPLC system.
2. HPLC Analysis was performed using a flow of 0.35 mL/min and a linear gradient from 10% to 25% Acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile to remove other materials.
3. Equal volumes of each standard, sample and control were injected onto the column. Injection volume range of 10 µL. Absorbance is monitored at 220 mm.
4. Column was re-equilibrated under initial conditions.

DATA ANALYSIS:
The concentration of cysteine or lysine peptides were photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

Positive control results:

Positive control cinnamaldehyde showed 74.96% mean cysteine depletion and 51.27% mean lysine peptide depletion.

Key result

Parameter:

cysteine depletion

Value:

90.93 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Parameter:

cysteine depletion

Value:

5.34 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1.    Mean and SD of percent peptide depletion for cysteine and lysine.

For Cysteine peptide
Sample ID	Peak area of cysteine peptide	% peptide depletion	Mean % Peptide Depletion	SD
Cinnamic aldehyde-replicate 1	954336.0	72.77	74.96	1.90
Cinnamic aldehyde-replicate 2	835304	76.17
Cinnamic aldehyde-replicate 3	843654	75.93
K010-01 replicate 1	309768.0	91.16	90.93	2.72
K010-01 replicate 2	226878.0	93.53
K010-01 replicate 3	417155	88.10

 

For Lysine peptide
Sample ID	Peak area of Lysine peptide	% peptide depletion	Mean % Peptide Depletion	SD
Cinnamic aldehyde-replicate 1	1257486	59.35	51.27	12.59
Cinnamic aldehyde-replicate 2	1308743	57.69
*Cinnamic aldehyde-replicate 3	1956255	36.76
K010-01 replicate 1	3287685	-6.29	5.34	10.09
K010-01 replicate 2	2727511	11.82
K010-01 replicate 3	2768916	10.49

K010-01:6-Methyl-2-Thiouracil, %: Percent, SD: Standard deviation,        

*: replicate 3 is an slight outlier

Table 2. Reactivity class classification of test item and positive control as per cysteine 1:10/lysine 1:50 prediction model.

Sample ID/ Code	Name of the chemical	Mean % of Cysteine Peptide Depletion	Mean % of Lysine Peptide Depletion	Mean % peptide depletion	Reactivity class	DPRA prediction
CA	Cinnamic aldehyde	74.96	51.27	63.12	High Reactivity	Positive
K010-01	6-Methyl-2-Thiouracil	90.93	5.34	48.14	High Reactivity	Positive

Interpretation of results:

other: Positive result. Can be used in assessing weight of evidence for skin sensitisation

Conclusions:

The mean of cysteine and lysine peptide depletion by the positive control (cinnamaldehyde) was 63.12% which shows that the positive control is a sensitizer with high reactivity confirming the sensitivity of the assay. The mean of cysteine and lysine peptide depletion by the test item [6-Methyl-2-Thiouracil] was 48.14% which shows that the test item has high reactivity on the skin sensitisation potential. Under the testing conditions, test item [6-Methyl-2-Thiouracil] was concluded as positive or sensitiser with high reactivity in Direct Peptide Reactivity Assay (DPRA).

Executive summary:

Skin sensitisation test by in chemico test method, Direct Peptide Reactivity Assay (DPRA) was carried out using the nucleophile containing synthetic cysteine and lysine peptides to evaluate the skin sensitisation potential of the test item, 6-Methyl-2-Thiouracil.

Solubility check was performed for the test item before test sample analysis. The test item, 6-Methyl-2-Thiouracil, was insoluble in Acetonitrile, acetonitrile: water (1:1), Isopropanol, Acetone, Acetone: Acetonitrile (1:1), 10% DMSO at 100 mM concentrations. Test item was found soluble in 50% DMSO (with 50% Acetonitrile) at 100 mM concentration and formed a homogenous solution. Hence, 50% DMSO (with 50% Acetonitrile) was selected as solvent.

On the day of test sample preparation, appropriate amount of cysteine and lysine peptide was weighed and dissolved in phosphate buffer and ammonium acetate buffer respectively. The stock concentration of cysteine peptide prepared was 0.501 mg/mL (0.667 mM) and lysine peptide was 0.518 mg/mL (0.667 mM). The preparations were done immediately before use.  

Test item and positive control solutions were prepared at 100 mM concentration and used in the test sample preparation for analysis. Test item, positive control and reference control was added to cysteine and lysine peptides and incubated in dark at 25°C for 24±2 hours. Post incubation, the test samples were analysed using HPLC as per section 9 to measure the peptide depletion.

Cysteine and lysine peptide percent depletion values are then calculated from peptide peak areas obtained from the HPLC analysis. The test item 6-Methyl-2-Thiouracil showed 90.93% mean cysteine peptide depletion and 5.34% mean lysine peptide depletion. Under the same conditions positive control cinnamaldehyde showed 74.96% mean cysteine depletion and 51.27% mean lysine peptide depletion.

To classify test item as sensitizers and non- sensitizers, the cysteine 1:10/lysine 1:50 prediction was used. The mean of cysteine and lysine peptide depletion by the positive control (cinnamaldehyde) was 63.12% which shows that the positive control is a sensitizer with high reactivity confirming the sensitivity of the assay. The mean of cysteine and lysine peptide depletion by the test item [6-Methyl-2-Thiouracil] was 48.14% which shows that the test item has high reactivity on the skin sensitisation potential. Under the testing conditions, test item [6-Methyl-2-Thiouracil] was concluded as positive or sensitiser with high reactivity in Direct Peptide Reactivity Assay (DPRA).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 March- 16 April 2017

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

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The ARE-Nrf2 Luciferase test method using KeratinoSens™ is an in vitro method which quantifies luciferase gene induction following 48 h treatment with test chemicals. Contact sensitizers which activate Nrf2-ARE regulatory pathways in cells, up-regulate the luciferase gene induction in KeratinoSens™ which are stably transfected with Luciferase gene under transcriptional control of promoter fused with ARE element. Luciferase gene induction is measured in the cell lysates by luminescence detection using a well-established light producing luciferase substrate. Fold induction of Luciferase activity is then calculated and used in a prediction model which allows assigning the test chemical to discriminate between sensitizers and non-sensitizers.

Details on the study design:

Culture and Maintenance of Keratinosens Cells

KeratinoSens cells were cultured and maintained in DMEM containing Glutamax supplemented with 9.1 % fetal bovine serum (FBS) and 500 μg/ml Geneticin at 37 ± 1ºC in the presence of 5% Co2. Cells were harvested for experiment when they reach 80-90% confluency. Cells were washed twice with DPBS (5 mL / flask), then 2 mL TrypLE Express Enzyme solution was added and flask were kept back into the incubator. After cells were detached (approx. 15 mins), they were resuspended in maintenance medium and splitted at a ratio of 1:3 - 1:12 in fresh medium and grown to 80-90% confluency.


Cell Seeding
• On the days of experiment i, e 19.03.18, 21.03.18, 23.03.18 and 24.03.18, the media were replaced with fresh medium without geneticin in the morning. Cells harvested for experiment were 80 – 90 % confluent on the day of seeding and never grown to full confluency.
• The cells were washed twice with DPBS (5 mL / flask), then 2 ml TrypLE Express Enzyme solution was added and flask kept back in to the incubator. After cells have detached (approx. 15 mins), cells were re-suspended in maintenance medium without Geneticin and centrifuged at 1000 rpm for 5 mins. Then the medium was aspirated and pellet was resuspended in fresh medium and the cells were adjusted to a density of 80,000 cells / mL in the maintenance medium.
• A volume of 125 μl of the cell suspension (containing 10,000 cells) was added to the wells F1-F12 and H1-H11 of 96 well plates. Treatment medium not containing cells was added in H12 well of 96 well plate and kept as blank control.
• For each experiment four parallel plates were prepared: Three white 96 well plates (assay plates for Luciferase activity) and one transparent 96 well plate (cell viability assay plate for MTT assay).
• The Plates were incubated at 37 ± 1ºC in the presence of 5% Co2 for 24 ± 1h.
7.6 Test Item Exposure
• After the incubation period the medium was removed by aspiration and replaced with 150 μL DMEM-medium containing 1% FBS without Geneticin (Treatment medium).
• A volume of 50 μL of each concentration from the intermediate stock was added to the respective prelabelled wells (F1-F12 test item concentration ranging from 0.095 μM to 2000 μM, H1-H6 DMSO control, H&-H11 positive concentration ranging from 4 μM to 64 μM, H12 only medium without cells) and mixed.
• All the plates were covered with a sealing tape to avoid evaporation of volatile compounds and to avoid cross-contamination between wells by volatile compounds.
• The plates were incubated at 37±1°C in presence of 5 % CO2 for 48 ± 2 h in the CO2 incubator.


Test Item Exposure
• After the incubation period the medium was removed by aspiration and replaced with 150 μL DMEM-medium containing 1% FBS without Geneticin (Treatment medium).
• A volume of 50 μL of each concentration from the intermediate stock was added to the respective prelabelled wells (F1-F12 test item concentration ranging from 0.095 μM to 2000 μM, H1-H6 DMSO control, H&-H11 positive concentration ranging from 4 μM to 64 μM, H12 only medium without cells) and mixed.
• All the plates were covered with a sealing tape to avoid evaporation of volatile compounds and to avoid cross-contamination between wells by volatile compounds.
• The plates were incubated at 37±1°C in presence of 5 % CO2 for 48 ± 2 h in the CO2 incubator.

Luciferase Activity
• After the treatment exposure time, the supernatant was aspirated from the white assay plates and discarded.
• The cells wers washed by adding 100 μL of DPBS and aspirated.
• To each well, 20 μL of 1x passive lysis buffer was added (care was taken to avoid formation of foam) and the cells were incubated at Room temperature for 20 mins.
• After incubation at room temperature, 50 μL of Luciferase substrate was added manually to the plates containing cell lysates and mixed. The plates with the cell lysate and the luciferase substrate were placed in the luminometer for reading.
• The luminometer was programmed to integrate the luciferase activity for 1500 ms (1.5 Seconds). The luminescence reading was saved as ‘pda’ and ‘pdf’ files for further analysis.

Viability Measurement (MTT Assay)
• For the cell viability measurement, the medium was aspirated and discarded from the transparent assay plates. A volume of 200 μL of treatment medium was added to the wells.
• In addition, a volume of 27 μl of a MTT solution (5mg/mL in DPBS) was added to the respective well of the transparent 96-well plate. The plate was protected from light while adding MTT solution.
• The plates were incubated at 37 ± 1oC in presence of 5% Co2 for 4 hours.
• After incubation, the MTT medium was removed and the cells were lysed by adding 200 μL of 10 % SDS to the respective wells and incubated overnight.
• After overnight incubation, the plates were shaken for 10 mins and absorbance was read at 600 nm using photometer. The absorbance reading was saved as ‘pda’ and ‘pdf’ files for further analysis.

Positive control results:

Cinnamic aldehyde induced the luciferase gene in all runs with a EC 1.5 value of 15.41, 10.78 and 16.07 and the EC1.5 value was between 7 and 30 μM in all experiments. All runs were below the target of maximal 20% variability, with an % standard deviation of 16.60, 13.09 and 15.05 in the three experiments respectively.

Key result

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.83 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

The following acceptance criteria were met while using the KeratinoSensTM test method.
a) The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 µM).
b) The average induction in the three replicates for cinnamic aldehyde at 64 μM was 4.33, 3.88 and 2.86 (Range 2 and 8).
c) The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was 16.6, 13.0 and 15.0 (Should be below 20%) in each repetition which consists of 6 wells tested in triplicate.

The maximal gene induction (Imax) shows the dynamic range of gene induction by the test item and positive control. As the prediction model rates any chemical positive with an Imax, which is statistically significant above solvent control and above the threshold of 1.5, the absolute value of the Imax is not very important for the prediction.

Interpretation of results:

GHS criteria not met

Conclusions:

Methylthiouracil is non-sensitiser in the in vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens assay) perfomed according to OECD 442D guideline.

Executive summary:

The potential of the test item to cause skin sensitisation was evaluated in ARE-Nrf2 Luciferase (Keratinosens Assay) Test KeratinoSens assay is an in vitro cell based assay in which the keratinosens cells were exposed to the test item for 48 hours and subsequently measured for luciferase activity using a luminometer. The assay was run in 96 well plates, and test item was tested at 12 concentrations ranging form 0.98 μM to 2000 μM reference compound cinnamic aldehyde ranging from 4 μM to 64 μM. 

Each plate was tested in parallel in triplicate for analysis of luciferase induction and one additional replicate plate was used for cytotoxicity assessment. The full test in triplicate analysis was independently conducted three times.

The cells were exposed to the test concentrations for 48 ± 2 hours at 37 °C. After the exposure time luciferase substrate was added and the luminescence activity at each concentration was integrated for 1500 ms (1.5 Seconds).

The viability assay plates containing the cells were treated with treatment medium containing MTT solution. After overnight incubation, the absorbance was read at 600 nm using a photometer.

The luminescence reading and the absorbance reading obtained were excel sheet provided by Givaduan. The test item did not show any statistical significance induction above the threshold of 1.5 or 50% till 1000 uM concentration. At 2000 uM concentration statistically significant induction was observed in two out of three repetitions. Under the same circumstances the postive control cinnamaldehy showed statistifically significant induction over the solvent control confirming the senstitivity of the assay.

Based on the results it is concluded that the test item is non-sensitiser in the in vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens assay).

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

QSAR prediction

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

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:

QSAR prediction is used as a complementrary information for in vitro senstitisation studies.

Qualifier:

according to guideline

Guideline:

other: REACH guidance on QSARs (R.6)

Key result

Run / experiment:

other: Battery

Parameter:

other: Prediction

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: CASE Ultra

Parameter:

other: Prediction

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Leadscope

Parameter:

other: Prediction

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

not determinable

Key result

Run / experiment:

other: SciQSAR

Parameter:

other: Prediction

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

According to 3/4 of predictions of different sofwares, test item was scored as positive for skin sensitisation as a result of search from Danish (Q)SAR Database.

Executive summary:

Prediction for skin sensitisation according to Danish (Q)SAR Database:

Allergic contact dermatitis in guinea pig and human: Battery: POS_IN, CASE Ultra: POS_IN, Leadscope: POS_OUT, SciQSAR: POS_IN.

IN: inside applicability domain

OUT: outside applicability domain

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Skin sensitisation test by in chemico test method, Direct Peptide Reactivity Assay (DPRA) was carried out using the nucleophile containing synthetic cysteine and lysine peptides to evaluate the skin sensitisation potential of the test item, 6-Methyl-2-Thiouracil. The mean of cysteine and lysine peptide depletion by the positive control (cinnamaldehyde) was 63.12% which shows that the positive control is a sensitizer with high reactivity confirming the sensitivity of the assay. The mean of cysteine and lysine peptide depletion by the test item [6-Methyl-2-Thiouracil] was 48.14% which shows that the test item has high reactivity on the skin sensitisation potential. Under the testing conditions, test item [6-Methyl-2-Thiouracil] was concluded as positive or sensitiser with high reactivity in Direct Peptide Reactivity Assay (DPRA).

 

Methythiouracol was evaluated in ARE-Nrf2 Luciferase (Keratinosens Assay) Test KeratinoSens assay is an in vitro cell based assay in which the keratinosens cells were exposed to the test item for 48 hours and subsequently measured for luciferase activity using a luminometer. The test item is non-sensitiser in the in vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens assay).

 

Methylthiouracil was scored as positive for skin sensitisation as a result of search from Danish (Q)SAR Database.

 

Taken together, based on positive results in Direct Peptide Reactivity Assay (DRPA) and the outcome of search from Danish (Q)SAR Database methylthiouracil is skin sensitising.

Justification for classification or non-classification

The substance is classified for Skin Sens. 1 according to the CLP Regulation No.1272/2008.