5H-1,2-oxathiole 2,2-dioxide

Endpoint summary

Description of key information

Skin sensitisation (in vitro):

1. Direct Peptide Reactivity Assay (DPRA): Positive (OECD 442C/GLP)

2.ARE-Nrf2 luciferase test assay: Positive (OECD 442D/GLP)

The results of the in chemico and in vitro tests were both positve so OECD 442E was not performed; however the results did not allow discrimmination between Skin sensitiser category 1A and 1B. There were no potential source candidates with relaible data found for read-across so, as a last resort, in vivo testing (OECD 429) was performed.

Skin sensitisation (in vivo): Sensitising (OECD 429/GLP)

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

Study period:

20.06. 2019 - 19.08.2019

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))

Version / remarks:

2019

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: Shijiazhuang Suntec-chem Co.,Ltd; 190403
- Expiration date of the lot/batch: Apr 10, 2020
- Purity: 99.92%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place. Containers which are opened must be carefully resealed.
- Stability under test conditions: During the study the test item sample was stored in in original containers in a dry place.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 429 were also performed. The OECD 442C assay was run once, in triplicate.

This in chemico method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its reactivity towards a synthetic cysteine and lysine containing peptide, by measuring the depletion using high performance liquid chromatography (HPLC).

Solvent selection was performed according to DB-ALM (INVITTOX) Protocol No.154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing, 2012. According to this protocol, acetonitrile is the preferred solvent for test chemicals. An approximately 100 mM solution of 1,3-Propene Sultone in acetonitrile was prepared. Test item was dissolved completely, therefore acetonitrile was used for further tests.

Controls
Reference controls, co-elution controls, positive control and negative control were set up in parallel to the test item in order to confirm the validity of the test.

Reference control
Three types of "Reference Controls" were included in this study. Reference Control is a peptide solution where the test chemical is replaced by the solvent used to dissolve it.
• Reference Control A was made with acetonitrile and used to verify the accuracy of the calibration curve for peptide quantification.
• Reference Control B was made with acetonitrile and its replicates are injected in the beginning and in the end of the experimental run to verify the stability of the peptide over the analysis time.
• Reference Controls C was made with acetonitrile (solvent used to solubilise the test chemicals). They are used to verify that the solvent does not impact the Percent Peptide Depletion. The appropriate Reference Controls C for each chemical are used to calculate Percent Peptide Depletion.

Co-elution control
Co-elution control was constituted by the test chemical alone without the addition of peptides. Instead of the addition of peptides, the appropriate amount of buffer was added. The co-elution test serves to verify that the test item does not have the same retention time and does not absorb at 220 nm as the Cysteine and Lysine peptides.

Positive control
As a positive control, a 100 mM solution of Cinnamaldehyde was used. The amount required to prepare this solution was calculated according to the formula above. The weighed amount of Cinnamic aldehyd (approximately 40.13 mg) was dissolved in 3 ml of acetonitrile.

Negative control
As a negative control, a 100 mM solution of 1-butanol was used. The amount required to prepare this solution was calculated according to the formula above. The weighed amount of 1-butanol (approximately 22.7 mg) was dissolved in 3 ml of acetonitrile.

Peptides
The necessary amount of peptide was estimated. For each analysis with addition of peptide, 800 µl of stock solution is needed.
20 ml of the stock solution of Cysteine or Lysine peptide (0.667 mM) was prepared.
The appropriate amount of buffer to individual peptides was added just before testing itself.
Approximately 10.02 mg of the Cysteine peptide was added to 20 ml of phosphate buffer, pH 7.5. Approximately 10.36 mg of the Lysine peptide was added to 20 ml of ammonium acetate buffer, pH 10.2.

Dose Groups
Reference Control C (solvent control) undiluted

Test Item 100 mM stock solution

Positive Control 100 mM stock solution

Experimental Procedure
Incubation of the Test Item with the Cysteine and Lysine Peptide
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls, positive control as well as the negative control were set up in parallel.
Samples were visually inspected prior to the start of incubation and prior to HPLC analysis.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure

Preparation of the HPLC Standard Calibration Curve
Using serial dilution, standards were prepared of the peptide stock solutions covering the range from 0.534 – 0.0167 mM.
STD 1 (0.534 mM) was prepared by diluting 1600 µL of the peptide stock solution (0.667 mM) with 400 µl acetonitrile.
STD 2 (0.267 mM) was prepared by diluting 1.0 ml of standard STD1 with 1.0 ml of an appropriate dilution buffer (via. 3.4.6 ).
STD3 (0.1335 mM) was prepared by diluting 1.0 ml of standard STD2 with 1.0 ml of an appropriate dilution buffer.
STD4 (0.0667 mM) was prepared by diluting 1.0 ml of standard STD3 with 1.0 ml of an appropriate dilution buffer.
STD5 (0.0334 mM) was prepared by diluting 1.0 ml of standard STD4 with 1.0 ml of an appropriate dilution buffer.
STD6 (0.0167 mM) was prepared by diluting 1.0 ml of standard STD5 with 1.0 ml of an appropriate dilution buffer.
STD 7 (0 mM) contained only the dilution buffer as blank.
The vials were closed, mixed and placed in the HPLC autosampler (dark) at 25 °C for 24 hours.

HPLC Preparation and Analysis
Using 1 ml autosampler vials as container, was prepare the sample by adding the reagents in the quantity and order listed in Table No. 1 and Table No. 2. Samples /controls were prepared in triplicate.
The vials were closed, mixed and placed in the HPLC autosampler (dark) at 25 °C for 24 hours. Then, samples were visually inspected prior to HPLC analysis. Test chemical was analysed in triplicate for both peptides.
For Cysteine peptide: All vials were placed in the autosampler at 10:50 hours, on 9.7.2019. The temperature of the autosampler was set to 25°C. All vials were removed from the autosampler and checked at 10:27 hours, on 10.7.2019. The temperature of the autosampler was 25 °C.
For Lysine peptide: All vials were placed in the autosampler at 11:05 hours, on 10.7.2019. The temperature of the autosampler was set to 25°C. All vials were removed from the autosampler and checked at 9:15 hours, on 11.7.2019. The temperature of the autosampler was 25 °C.

Analyses
The validated HPLC method was used, it is described in detail in the SOP M/98/1.

HPLC conditions
Liquid chromatograph: Shimadzu Nexera LC-30AC with DAD
Column: Agilent Zorbax SB-C18, 100x2.1 mm, 3.5µm
Precolumn: Phenomex security guard C18, 4.0 x 2.0 mm
Mobile phase A: 0.1% Trifluoracetic acid in water
Mobile phase B: 0.085% Trifluoracetic acid in acetonitrile
Time programmer: 0 min 10 % B
10 min 25 % B
11 min 90 % B
13 min 90 % B
13.5-20 min 10 % B
Column temperature: 30 °C
Sample temperature: 25°C
Flow rate: 0.35 mL/min
Injection volume: 10 μL
Detection: 220 nm

Data evaluation
The Cysteine and Lysine peptide concentrations were determined at a wavelength of 220 nm by evaluating the peak area of each sample and calculating from the linear calibration curve constructed from the calibration line.
For the positive (negative) controls and for test item were calculated the percent peptide depletion in each replicate from the peptide peak area of replicate injection and the mean peak area in the the relevant Reference controls C (in acetonitrile).

Positive control results:

Mean Percent Cysteine depletion of the positive control (Cinnamaldehyde) is 70.4%; mean Percent Lysine depletion is 50.4%.

Run / experiment:

other: other: other: Test item

Parameter:

other: cysteine depletion (migrated information)

Value:

99.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

70.4%

Run / experiment:

other: other: other: Test item

Parameter:

other: lysine depletion (migrated information)

Value:

32.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

50.4%

Run / experiment:

other: other: other: test item (Cysteine 1:10/Lysine 1:50 prediction model)

Parameter:

other: The mean of Cysteine and Lysine percent depletion values

Value:

66

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not specified

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Samples were visually inspected prior to the start of incubation and prior to HPLC analysis. Immediately after addition of the cysteine peptide to the test item solution, no turbidity or cloudiness was observed in the vial. There was no color change in the vial. After 24-hour incubation, a slight cloud of precipitation was formed in the vial. There was no color change in the vial. Immediately after addition of the lysine peptide to the test item solution, no turbidity or cloudiness was observed in the vial. There was no color change in the vial. After 24-hour incubation, the reaction solution was free of turbidity. There was no color change in the vial. Phase separation was not observed for individual peptides.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for controls: Cinnamaldehyde was used as positive control at a concentration of 100 mM in acetonitrile. As a negative control, 1-butanol at a concentration of 100 mM in acetonitrile was used. Samples were evaluated by peptide peak area at 220 nm for each control, peptide concentration (mM) and mean peptide concentration, SD and CV. The mean percent peptide depletion value of three replicates for Cinnamaldehyde and 1-butanol was calculated.

The results are shown in Table No. 5 and Table No. 6. Illustrative HPLC chromatograms of Cysteine and Lysine peptides in solutions of the positive and negative controls are shown in Figure No. 5 - Figure No. 8. Summary of results and criteria acceptance are shown in chapter 4.6

Interpretation of results:

other: As part of a tiered testing strategy, the substance may be considered as a “sensitiser”.

Conclusions:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 429 were also performed. Under the experimental conditions of this study, the DPRA prediction is considered as positive and the test item 1,3-Propenesultone was considered to have high reactivity for both peptides.

Executive summary:

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA; 152-19-65), 1,3-Propenesultone (99.92%) in acetonitrile was evaluated by monitoring peptide depletion between the test item and synthetic cysteine and lysine peptides (24 ± 2 h at 25 ± 2.5 °C). Subsequently samples were analysed by HPLC. Reference controls (A, B, C (solvent control)), co-elution controls, positive control (Cinnamaldehyde in acetonitrile) and negative control (1-butanol in acetonitrile) were set up in parallel to the test item in order to confirm the validity of the test.

The acceptance criteria for the calibration curve samples, the reference and co-elution controls, as well as for the study samples were satisfied. The study was therefore considered to be valid. Immediately after addition of the Cysteine peptide to the test item solution, no turbidity or cloudiness was observed in the vial. There was no color change in the vial. After 24-hour incubation, a slight cloud of precipitation was formed in the vial. There was no color change in the vial. Immediately after addition of the Lysine peptide to the test item solution, no turbidity or cloudiness was observed in the vial. There was no color change in the vial. After 24-hour incubation, the reaction solution was free of turbidity. There was no color change in the vial. Phase separation was not observed for individual peptides. No co-elution of the test item with either peptide was demonstrated.

The positive control Cinnamaldehyde showed high reactivity towards the synthetic peptides. The percent cysteine depletion was 70.4 and the percent lysine depletion was 50.4. The mean Percent Cysteine depletion % of the test item was 99.4 and mean Percent Lysine depletion % was 32.5. The mean of Cysteine and Lysine percent depletion values of the test item was 66.0. Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used. The test item, 1,3-Propenesultone, was classified as positive (skin sensitizer) in the DPRA prediction; it was assigned to reactivity class – high reactivity, for both peptides.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 429 were also performed. The data generated with this test will be considered in the context of an integrated approache such as IATA, combining the result with other complementary information from the other 2 tests.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11/06/2019 - 30/09/2019

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: Shijiazhuang Suntec-chem Co.,Ltd.; 190403
- Expiration date of the lot/batch: Apr 10, 2020
- Purity: 99.92%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at room temperature. Keep container tightly closed in a dry and well-ventilated place. Containers which are opened must be carefully resealed.

Details on the study design:

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

This test is part of a tiered strategy for skin sensitization assessment. OECD 442C and OECD 429 were also performed.

Test System
The immortalized adherent cell line derived from HaCaT human keratinocytes transfected with a selectable plasmid (KeratinoSensTM) is used for testing. It is supplied by Givaudan, Schweiz. Cells are stored in liquid nitrogen. For the test, the cells are seeded in a 96-well plate. Original cell stock can be kept in culture for a maximum passage number of 25. For every study, the absence of mycoplasma contamination is verified.
The cell cycle of this cell line is about 24 hours and it is evaluated with every new lot of the cells or after one year.

Test item preparation:
The test item was dissolved in DMSO. DMSO is the first of choice solvent according OECD TG 442D.
Maximal concentration of stock solution was 200mM. A fresh stock solution of the test item was prepared before each experiment.

Preparation of cells:
The KerationoSens cells were cultivated and prepared for testing according to internal SOP M/101.

Sensitisation test:
Every concentration of the test item, NC and PC was tested in 2 independent experiments.
In the 1st and the 2nd experiment there was no precipitation observed and the highest test concentration was 2000 µM.
Each experiment included corresponding positive controls (PC = Cinnamic aldehyde), negative controls (NC = untreated wells with cells and solvent 1% DMSO) and blank of the negative controls (BL = untreated wells without cells with solvent 1% DMSO).
Two 96-well plates were used for each experiment (one plate for luminescence measurement and one plate for viability measurement).
Every concentration, NC, PC and BL was tested in tetraplicates.
The 1st and 2nd experiments, which fulfilled the acceptability criteria, were used for further evaluation.

Experimental Procedures
Preparation of the test item:
The test item was dissolved in DMSO at a stock concentration of 200 mM. The stock concentration was diluted into 12 concentrations in DMSO (100x concentrated solutions) in a two-fold range according procedure in OECD 442D. Then solutions of the test item were diluted in medium for exposure (DMEM with low glucose and supplemented with L-Glutamine + 1% FBS). In both experiments, the final concentrations were in the range 0.98 -2000 µM (0.98, 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM). The three highest concentrations (2000, 1000 and 500 µM) were cytotoxic. So the highest concentration used for the evaluation of results was 250 µM. Fresh stock solutions were prepared before each experiment in glass vials and 96-well plates.

Controls:
A stock solution (200 mM) of cinnamic aldehyde was prepared fresh by dilution in DMSO before each experiment or stored in the freezer in glass vials (no older than 1 month). The stock solution was diluted in DMSO and subsequently in exposure medium to the final concentrations. The final concentrations of cinnamic aldehyde (= PC) in the wells were 4, 8, 16, 32 and 64 µM.

Cell cycle length determination:
The cell cycle length of KeratinoSensTM cells was evaluated by a doubling time experiment: on the first day, cells in twelve Petri dishes (60 mm) were seeded at three concentrations: 2000, 4200 and 9000 cells/mL. Each subsequent day, cells were counted in 1 Petri dish at each concentration.
Proliferation curves were constructed for each concentration (average number of counted cells in three Petri dishes vs. time in hours) and cell cycle length was determined. Results of the experiment are archived in the laboratory.

Manipulation with cell line:
Passage number of the cells used for seeding in experiments was 23 in both experiments. The level of confluence was about 80 90%. Cells used in experiments were without contamination of Mycoplasma (see chapter 4. Study Results and chapter 3.11. Deviations).
The density of the cells for calculation of cell concentration for experiments was evaluated microscopically by Cellometer Auto T4.

Demonstration of the proficiency of the luminescence measurement:
For the luminescence measurement, the luminometer Synergy HTX Biotek, made by Biotek, USA, was used. The luminescence was measured without any filter.
For the measurements, the Luciferase glow substrate manufactured by Promega was used (ONE Glo Ex Cat. No. E8110).

Treatment and cultivation:
The KeratinoSensTM cells were seeded into two 96-well plates at a density of 80 000 cells per well (125 µL/well) and incubated for 24 ± 2 hours. After that, the cultivation medium was aspirated and 150 µL of exposure medium and 50 µL of prepared test item solutions were added to each well of the plates. Treatments were carried out in a laminar flow hood at room temperature in aseptic conditions. Cultures were treated with the test item for 48 hours ± 30 minutes in an incubator (5% CO2, 37 ± 1°C, 95% humidity). After exposure, the medium was aspirated from the second plate and cells were mixed with the luciferase substrate (50 µL per well substrate + 50 µL per well medium for exposure) and after 3 - 5 minutes shaking, the luminescence was measured (luminometer Synergy HTX Biotek). The medium from the first plate was aspirated, fresh medium (200 µL/well) was added and the cells were incubated with MTT solution (concentration 5 mg/mL – 27 µL/well) for 4 hours. After incubation, the MTT was extracted by isopropanol (200 µL/well) and absorbance was measured (600 nm) by spectrophotometer BioTek EPOCH.

Results Evaluation
From the measured values the following parameters are calculated:
- the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested item and positive control
- the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 threshold (i.e. 50% enhanced luciferase activity)
- the IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability

For each concentration showing > 1.5 fold luciferase activity induction, statistical significance will be calculated (Student´s t-test), comparing the luminescence values for the samples with luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p < 0.05).
The dose-response curves were constructed.

Acceptability of Experimental Results
The results are accepted if:
The luciferase activity induction obtained with the positive control (Cinnamic aldehyde) is statistically significant above the threshold 1.5 in at least one of the tested concentration (4 – 64 µM).

EC1.5 value of the positive control is in two standard deviation of the historical control mean (4.4 – 30.28 µM from 13 measurements).

The average coefficient of variation of the luminescence reading for the negative control (DMSO) is below 20%. If the variability is higher, results should be discarded.

Interpretation of Results and Prediction Model
The prediction of test item sensitisation potential is considered positive in the KeratinoSensTM method, if the following 4 conditions are all met (in 2 of 2 or in the 2 of 3 repetitions):
the Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control
the cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold
the EC1.5 value is less than (<) 1000 µM
there is an apparent overall dose-response for luciferase induction
If these 4 conditions are not met, the prediction is considered negative.
If the highest test item concentration is < 1000 µM:
if the test item induces luciferase at a lower, non cytotoxic soluble concentration, this positive result can be accepted to rate the chemical as positive
if the test item does induce cytotoxicity (viability < 70 %) at the maximal soluble concentration, this result can be accepted to rate a test item as negative
if the test item does not lead to cytotoxicity or luciferase induction at a maximal tested soluble concentration (< 1000 µM), the test item is rated as inconclusive
If the result is inconclusive (see OECD TG 442D par. 32), further testing may be required.

Positive control results:

The luciferase activity induction obtained with the positive control (Cinnamic aldehyde) are statistically significant above the threshold 1.5 in 3 of the 5 tested concentrations in run 1 and 3 of 5 tested concentrations in run 2.

EC1.5 of the positive control in the 1st experiment was 10.2 µM and in the 2nd experiment 11.1 µM, which in two standard deviation of the historical control mean

Run / experiment:

other: other: 1st experiment_test item

Parameter:

other: Imax (250 µM)

Value:

783.87

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: viability of cells at 15.6 µM was 136.4 %; EC1.5 = 8.5 µM

Run / experiment:

other: other: 2nd experiment_test item

Parameter:

other: Imax(250 µM)

Value:

465.37

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: viability of cells at 15.6 µM was 120.5%; EC1.5 = 9.3 µM

Run / experiment:

mean

Parameter:

other: Imax (250 µM)

Value:

624.62

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Imax SD=225.21; IC50=420.97; IC30=388

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The average coefficient of variation of the luminescence reading for the negative control (DMSO) should be below 20%. If the variability is higher, results should be discarded.
In the 1st experiment, the coefficient of variation was 14.6 % and in the 2nd experiment the coefficient of variation was 14.9 % (see Tab. No. 4).

- Acceptance criteria met for positive control:
I) The luciferase activity induction obtained with the positive control (Cinnamic aldehyde) should be statistically significant above the threshold 1.5 in at least one of the tested concentration (4 – 64 µM).
In the 1st experiment, the values of Imax were 1.58 at 16 µM, 2.84 at 32 µM and 24.81 at 64 µM (see Tab. No. 2, 9).
In the 2nd experiment, the values of Imax were 1.67 at 16 µM, 5.17 at 32 µM and 58.36 at 64 µM (see Tab. No. 2 and 12).
All these values were statistically significantly above the threshold 1.5.
II) EC1.5 value of the positive control should be in two standard deviation of the historical control mean (4.4 – 30.28 µM from 13 measurements).
EC1.5 in the 1st experiment was 10.2 µM and in the 2nd experiment 11.1 µM, which in two standard deviation of the historical control mean (see Tab. No. 3)).

Interpretation of results:

other: As part of a tiered testing strategy, the substance may be considered as a “sensitiser”.

Conclusions:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442C and OECD 429 were also performed. Under the experimental condition of this study, the luciferase induction at the maximum non-cytotoxic concentration (250 µM) is higher than 1.5 threshold in both experiments. Therefore the test item, 1,3-Propenesultone, had sensitisation potential predicted by the ARE-Nrf2 Luciferase KeratinoSens™ Test Method.

Executive summary:

In an in vitro skin sensitisation: ARE-Nrf2 luciferase test method assay; (152-19-66), 1,3-Propenesultone (99.92%) was evaluated for its potential to activate the Nrf2 transcription factor in KeratinoSens cells. 1,3-Propene Sultone in DMSO was applied to KeratinoSens cells at concentrations from 0.98 to 2000 µM for 48 hours ± 30 minutes at 37°C. Luciferase production was measured by flash luminescence. Cytotoxicity was measured using an MTT assay. Two independent experiments were performed and cells were assayed for viability and luciferase activity in each experiment. The positive control was cinnamic aldehyde and the negative control was untreated cells and 1% DMSO.

In the first experiment, the max luciferase activity (Imax) at the highest non-cytotoxic concentration (250 µM) was 783.87. The viability of cells at 15.6 µM was 136.4 %. The EC1.5 was 8.5 µM. In the second experiment, the Imax at the highest non-cytotoxic concentration (250 µM) was 465.37. The viability of cells at 15.6 µM was 120.5%. The EC1.5 was 9.3 µM. There was clear dose-response for luciferase induction in both experiments. The controls confirmed the validity of the study.

Under the condition of this study, the luciferase induction at the maximum non-cytotoxic concentration (250 µM) is higher than 1.5 threshold in both experiments. Therefore, the test item 1,3-Propenesultone is considered as a sensitiser.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442C and OECD 429 were also performed. The data generated with this test will be considered in the context of integrated approached such as IATA, combining the result with other complementary information from the other 2 tests.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.09.2019 - 02.12.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Shijiazhuang Suntec-chem Co., Ltd.; 190403
- Expiration date of the lot/batch: Apr 10, 2020
- Purity: 99.92%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store at room temperature. Keep container tightly closed in a dry and well-ventilated place. Containers which are opened must be carefully resealed.

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Lysolajské údolí 15/53, 165 00 Prague 6, Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Microbiological status of animals, when known: Microbiologically defined background according to internal SOP
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: In pilot experiment: 16.67-17.54 g; In main test: 16.10 - 19.63 g
- Housing: Animals in groups in macrolon cages with sterilized softwood shavings
- Diet: Pelleted standard diet for experimental animals ad libitum (Altromin, manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany) ad libitum
- Water: Drinking tap water ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: During clinical observations the examination of skin irritation at application site was carried out.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am

Vehicle:

other: DAE 433

Remarks:

Mixture of 40 % dimethylacetamide, 30 % acetone and 30 % ethanol. This vehicle (1) is used in our laboratory for approximately 10 years and it elicited a consistent response over whole period. (1) Ehling et al.. (2005), Toxicol., 212, 60-68.

Concentration:

50%, 5.0%, 0.5% w/v in pilot experiment.
20%, 2.0%, 0.2% w/v in main study.

No. of animals per dose:

1 female per group (pilot)
5 females per group (main test)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Three concentrations were used in pilot experiment: 50%, 5%, 0.5% concentrations in the form of suspension in DAE 433, one mouse per concentration.
- Irritation: The ear weights in pilot experiment were relatively balanced.
- Systemic toxicity: Reduction of body weight after treatment was recorded in all animals during the pilot experiment, however, the weight fluctuations are common in mice.
- Ear thickness measurements: The thickness of ears in all animals during pilot experiment was slightly increased.
- Erythema scores: In treated animals, no erythema and skin reaction were observed.
During pathological examination, the auricular lymph nodes enlargement was not detected in all animals.

According to the specification of the study monitor, the following doses (20%, 2%, 0.2%) were determined for the main study.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The response towards the test item is considered positive, if the stimulation index (SI) is ≥ 3, and the response increases in dose-related manner (dose-response relationship).

TREATMENT PREPARATION AND ADMINISTRATION:
In the application period, the test item was administered in the form of a suspension in DAE 433. The volume of the application form was constant at all groups of animals - 25 μL of the appropriate suspension to the dorsum of each ear every morning for 3 consecutive days. The application was performed very slowly by micropipette. The application forms of the test item (suspensions) were prepared immediately before administration.
Day 4 and day 5 were no treatment.
In the day 6, the weight of animals was recorded. Injection 250 μL of phosphate-buffered saline (PBS) containing 7.43 x105 Bq of 3H-methyl thymidine into all test and control mice via the tail vein. Five hours later, the animals were killed and draining auricular lymph nodes were collected. btained lymph nodes were processed with PBS for each treatment group.
Incorporation of 3H-methyl thymidine was measured by β-scintillation counting as disintegrations per minute (DPM).

Positive control substance(s):

other: Dinitrochlorobenzene (DNCB, CAS No 97-00-7)

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed by applying the parametric test for testing whether all group samples originate from the same distribution and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for two-group comparisons.

Positive control results:

The positive control item, Dinitrochlorobenzene (DNCB), as a known contact allergen (0.5% (w/v)) elicited the expected reaction pattern with a significant increase in the Stimulation Index (24.70) and ear weight.

Parameter:

SI

Value:

1.09

Test group / Remarks:

0.2%

Parameter:

SI

Value:

2.06

Test group / Remarks:

2%

Parameter:

SI

Value:

3.12

Test group / Remarks:

20%

Parameter:

EC3

Value:

17.96

Test group / Remarks:

EC3 = [(3-d)/(b-d)] x (a-c) + c; a=20, b=3.12, c=2, d=2.06

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: The value of the DPM and SI for the positive control group (DNCB) was statistically significantly increased. The SI was ≥ 3 (24.70) – the LLNA was efficient (see Table 9).
The SI for the 2% (2.06) and 0.2% (1.09) test item groups was below the threshold, and the stimulation index (SI) is < 3. The SI for the 20% test item group is above the threshold (3.12), and the stimulation index (SI) is ≥3. The value of the DPM at dose levels 20% and 2% was statistically significantly increased compared to the negative control.

DETAILS ON STIMULATION INDEX CALCULATION: Mean values and standard deviations of incorporation of 3H-methyl thymidine were computed for the test item groups and for the positive as well as the vehicle control group. Stimulation Index (for incorporation of 3H-methyl thymidine) was calculated by dividing mean values from exposed groups and the positive control group by the corresponding mean value of the vehicle control group. The index for the vehicle control group was set at 1 by definition.

EC3 CALCULATION: The EC3 value was derived by interpolating between two points on the Stimulation Index (SI) axis, one immediately above and the other immediately below the SI value of 3 (vehicle-treated control values [SI=1] not being used for the latter). Where the data points lying immediately above and below the SI value of three have the co-ordinates a (the concentration giving the SI immediately above 3), b (the SI of a), c (the concentration giving the SI immediately below 3) and d (the SI of c), the EC3 value was calculated using the following equation: EC3 = [(3-d)/(b-d)] x (a-c) + c.

For the test item, a=20, b=3.12, c=2, d=2.06. Therefore the EC3 value = [(3-2.06)/(3.12-2.06)] x (20-2) + 2 = 17.96%

CLINICAL OBSERVATIONS: During the pilot experiment no clinical symptoms of systemic toxicity were observed. In treated animals, no erythema and skin reaction were observed.
The thickness of ears in all animals during pilot experiment was slightly increased. During pathological examination, the auricular lymph nodes enlargement was not detected in all animals.
No animals died during the main study. All animals from the 20% concentration group of the test item showed hyperemia of the auricle from the second day. No symptoms of toxicity and no erythema on the application site were observed in all animals from the 2% and 0.2% test item groups. All animals in the positive control group showed symptoms caused by the application of DNCB: hyperemia of auricle, clonic spasm and increased response to stimuli (Table 8).

BODY WEIGHTS: Pilot study - Individual body weight of animals before administration was similar. Reduction of body weight after treatment was recorded in all animals during the pilot experiment, however, the weight fluctuations are common in mice (Table 3). Main study - The body weights of treated groups and the positive control group were slightly decreased compared to the negative control animals during the whole study. Body weight increment was calculated from values of day 6 just before necropsy and day 1 before first application. Body weight increments were negative at all dose levels (Table 6, 7).

Ear Weights/Redness

In the main test, the ear weight of the positive control was statistically significantly increased compared to the negative control group (Table 10). Ear weight for the 20% test item group was statistically significantly increased compared to the negative control group. No statistically significantly changes of ear weight were recorded in the 2% and 0.2% test item groups.

No erythema of the skin was observed during the clinical observation at 2% and 0.2% dose levels (Table 11). Very slight erythema was observed during the clinical observations at the 20% dose level. Very slight erythema and well-defined erythema in animals of the positive control group were recorded during the study (see Table 11).

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Remarks:

Category 1B based on CLP criteria

Conclusions:

In an in vivo LLNA test in Balb/c female mice, 1,3-Propenesultone, demonstrated a positive sensitising response in the LLNA assay. The SI at the 20% dose level was ≥ 3.

Executive summary:

In a dermal sensitization study (152/19/6) with 0, 0.2, 2 and 20% w/v 1,3-Propenesultone (99.92%) in DAE 433 (40 % dimethylacetamide, 30 % acetone and 30 % ethanol), young adult female BALB/c mice (5/group) were tested using the local lymph node assay. The reliability of the test system was confirmed by the concurrent testing of the positive control, Dinitrochlorobenzene (DNCB).

The pilot study with 0.5, 5 and 50% w/v 1,3-Propenesultone in DAE433 established the doses for the main study. The positive control item, Dinitrochlorobenzene (DNCB), as a known contact allergen (0.5% (w/v)) elicited the expected reaction pattern with a significant increase in the Stimulation Index (SI; 24.70) and ear weight.

The test item 1,3-Propenesultone, caused an increase in ear weight at the 20% dose level only, compared to the negative control. The animals exposed to 20% 1,3-Propenesultone showed very slight erythema and hyperemia of auricles during the clinical observations. The animals exposed to 2% and 0.2% 1,3 -Propenesultone showed no skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. The DPM values were statistically significantly increased in animals dosed at 20% and 2% 1,3 - Propenesultone compared to the negative control. The value of the SI for the group treated at the 20% dose level was over the threshold of ≥3 (3.12). The value of the SI for groups treated at the 2% and 0.2% dose levels was below the threshold (2.06 and 1.09, respectively). The EC3 was calculated to be 17.96%. Under the given test conditions, the test item, 1,3 - Propenesultone, demonstrated a positive sensitising response in the LLNA assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A tiered strategy for skin sensitization assessment was used. OECD 442C, OECD 442D and OECD 429 were performed. The data generated with these tests was considered in the context of an integrated approach, and the final result was decided by combining the results from all tests.

Skin sensitisation (in vitro):

Two in vitro skin sensitisation assays are available: Direct Peptide Reactivity Assay (DPRA) and ARE-Nrf2 luciferase test assay.

Direct Peptide Reactivity Assay (DPRA)

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA; 152-19-65), 1,3-Propenesultone (99.92%) in acetonitrile was evaluated by monitoring peptide depletion between the test item and synthetic cysteine and lysine peptides (24 ± 2 h at 25 ± 2.5 °C). Subsequently samples were analysed by HPLC. Reference controls (A, B, C (solvent control)), co-elution controls, positive control (Cinnamaldehyde in acetonitrile) and negative control (1-butanol in acetonitrile) were set up in parallel to the test item in order to confirm the validity of the test. The acceptance criteria for the calibration curve samples, the reference and co-elution controls, as well as for the study samples were satisfied. The study was therefore considered to be valid. Immediately after addition of the Cysteine peptide to the test item solution, no turbidity or cloudiness was observed in the vial. There was no color change in the vial. After 24-hour incubation, a slight cloud of precipitation was formed in the vial. There was no color change in the vial. Immediately after addition of the Lysine peptide to the test item solution, no turbidity or cloudiness was observed in the vial. There was no color change in the vial. After 24-hour incubation, the reaction solution was free of turbidity. There was no color change in the vial. Phase separation was not observed for individual peptides. No co-elution of the test item with either peptide was demonstrated. The positive control Cinnamaldehyde showed high reactivity towards the synthetic peptides. The percent cysteine depletion was 70.4 and the percent lysine depletion was 50.4. The mean Percent Cysteine depletion % of the test item was 99.4 and mean Percent Lysine depletion % was 32.5. The mean of Cysteine and Lysine percent depletion values of the test item was 66.0. Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used. The test item, 1,3-Propenesultone, was classified as positive (skin sensitizer) in the DPRA prediction; it was assigned to reactivity class – high reactivity, for both peptides.

ARE-Nrf2 luciferase test assay

In an in vitro skin sensitisation: ARE-Nrf2 luciferase test method assay; (152-19-66), 1,3-Propene Sultone was evaluated for its potential to activate the Nrf2 transcription factor in KeratinoSens cells. 1,3-Propene Sultone (dissolved in DMSO at a stock concentration of 200 mM) was applied to KeratinoSens cells at concentrations from 0.98 to 2000 µM for 48 hours ± 30 minutes at an incubator (5% CO2, 37 ± 1°C, 95% humidity). Luciferase production was measured by flash luminescence. Cytotoxicity was measured using an MTT assay. Two independent experiments were performed and cells were assayed for viability and luciferase activity in each experiment. The positive control was cinnamic aldehyde and the negative control was untreated wells with cells and 1% DMSO. In the first experiment, the max luciferase activity (Imax) at the highest non-cytotoxic concentration (250 µM) was 783.87. The viability of cells at 15.6 µM was 136.4 %. The EC1.5 was 8.5 µM.In the second experiment, the Imax at the highest non-cytotoxic concentration (250 µM) was 465.37. The viability of cells at 15.6 µM was 120.5%. The EC1.5 was 9.3 µM. There was clear dose-response for luciferase induction in both experiments. The controls confirmed the validity of the study. Under the condition of this study, the luciferase induction at the maximum non-cytotoxic concentration (250 µM) is higher than 1.5 threshold in both experiments. Therefore the test item 1,3-Propene Sultone is considered as a sensitiser.

The results of the in chemico and in vitro tests were both positve so OECD 442E was not performed; however the results did not allow discrimmination between Skin sensitiser category 1A and 1B. There were no potential source candidates found for read-across so, as a last resort, in vivo testing (OECD 429) was performed.

Skin sensitisation (in vivo):

There is one in vivo LLNA test in mice available.

In a dermal sensitization study (OECD 429/GLP) with 0, 0.2, 2 and 20% w/v 1,3 Propenesultone (99.92%) in DAE 433 (40 % dimethylacetamide, 30 % acetone and 30 % ethanol), young adult female BALB/c mice (5/group) were tested using the local lymph node assay. The reliability of the test system was confirmed by the concurrent testing of the positive control, Dinitrochlorobenzene (DNCB). The pilot study with 0.5, 5 and 50% w/v 1,3 Propene Sultone in DAE433 established the doses for the main study. The positive control item, Dinitrochlorobenzene (DNCB), as a known contact allergen (0.5% (w/v)) elicited the expected reaction pattern with a significant increase in the Stimulation Index (SI; 24.70) and ear weight. The test item 1,3 - Propene Sultone, caused an increase in ear weight at the 20% dose level only, compared to the negative control. The animals exposed to 20% 1,3 - Propene Sultone showed very slight erythema and hyperemia of auricles during the clinical observations. The animals exposed to 2% and 0.2% 1,3 - Propene Sultone showed no skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. The DPM values were statistically significantly increased in animals dosed at 20% and 2% 1,3-Propenesultone compared to the negative control. The value of the SI for the group treated at the 20% dose level was over the threshold of ≥3 (3.12). The value of the SI for groups treated at the 2% and 0.2% dose levels was below the threshold (2.06 and 1.09, respectively). The EC3 was calculated to be 17.96%. Under the given test conditions, the test item, 1,3 -Propenesultone, demonstrated a positive sensitising response in the LLNA assay.

According to Annex I of 1272/2008/EC and Annex I of 286/2011/EC, as the EC3 value is >2%, 1,3-Propenesultone is classified as Skin Sensitiser Category 1B.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information in the dossier, the substance 1,3-Propenesultone (CAS No. 21806-61-1) is classified as Skin Sensitiser Category 1B when the criteria outlined in Annex I of 1272/2008/EC and Annex I of 286/2011/EC are applied.