Sodium 3-chloro-2-hydroxypropanesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

yes

Remarks:

Identity, strength, purity and composition (other characteristics) defining assay controls, and stability of test article/controls under storage conditions at testing facility and under actual test conditions were not determined by the testing facility.

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-chloro-2-hydroxypropanesulphonate

EC Number:

204-807-0

EC Name:

Sodium 3-chloro-2-hydroxypropanesulphonate

Cas Number:

126-83-0

Molecular formula:

C3H7ClO4S.Na

IUPAC Name:

sodium 3-chloro-2-hydroxypropanesulphonate

Test material form:

solid

Specific details on test material used for the study:

- Purity: 99.1%

In vitro test system

Details on the study design:

Cell Thawing Procedure: A cryovial of the cryopreserved KeratinoSens cells was thawed in a water bath at approximately 37°C (in a beaker containing 70% ethanol). Once thawed, the cryovial was immediately decontaminated with 70% ethanol and placed in a laminar flow hood. The cells were added to a sterile conical tube and diluted by slowly adding 9 mL pre-warmed Assay Medium. The cells were then centrifuged at approximately 200 x g for 5 minutes at room temperature. The supernatant was aspirated, the pellet was resuspended in approximately 15 mL of fresh prewarmed Assay Medium, and then transferred into a T75 tissue-culture flask. The flask was then incubated at 37 ± 1°C, 90 ± 10% humidity, and 5.0 ± 1% C02 in air (standard culture conditions), until the KeratinoSens cells reached approximately 60% to 90% confluence.

Culturing of the KeratinoSens Cells: When the cultures reached approximately 60 to 90% confluence, they were removed from the flask by trypsinization. The medium was aspirated and the cell sheet rinsed twice with approximately 10 mL of CMF-DPBS containing 0.05% EDT A. One mL of trypsin/EDT A was added to cover the cell sheet. The flask was then placed into the incubator and incubated at standard culture conditions for 6-8 minutes, or until the cells became dislodged. When more than 50% of the cells became dislodged, the flask was rapped sharply against the palm of the hand. Then approximately 7 mL of Maintenance Medium was added to each T75 flask to obtain a single cell suspension and cells passaged at appropriate densities. The KeratinoSens cells were routinely passaged every 2-4 days.

Subculture of KeratinoSens Cells into 96-Well Plates: The KeratinoSens cells were subcultured into transparent Costar 96-well plates or white-walled Perkin Elmer plates when the flasks were approximately 60 to 90% confluent. The flasks were rinsed and trypsinized as previously described. The cells were resuspended in 5 mL of Assay Medium per flask. The concentration of cells in suspension was determined using a Coulter Counter. A cell suspension of 1.0E5 cells/mL in Assay Medium was prepared. One hundred μL of the cell suspension was added to all but one well designated as a blank well. The stock cell suspension was mixed often to ensure a uniform distribution of cells into each well. Three white-walled and one transparent plate were seeded for the test article replicate set for each assay. The plates were incubated for approximately 24 hours at standard culture conditions. The plate cultures seeded into the clear plates were examined under a phase contrast microscope and evaluated for uniform seeding and confluence prior to treating the cells with the test article or positive control.

MTT Direct Reduction Test: The ability of the test article to directly reduce MTT was assessed at the same time of test article treatment in the definitive assays. A 1.0 mg/mL MTT solution was prepared. Approximately 100 μL of a 100X (200000 μM) test article concentration in DMSO was added to 1 mL of the MTT solution and then incubated in the dark at 37°C for one to three hours. One hundred μL of a negative control (e.g. DMSO) was tested concurrently. If the MTT solution colour turned blue/purple, the test article was presumed to have reduced the MTT. The test article did not directly reduce MTT.

Definitive Assay: The test article was tested in six definitive assays. The first 3 definitive assays (Trials 1-3) were discontinued prior to completion because contamination was visually identified in the 96-well plates. Only the results of the second set of definitive assays (Trials 4-6) with acceptable controls were considered valid and were presented. Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). Each plate tested a range of 12 dosing concentrations for the test article. Each plate also included 5 wells designated for the positive control (tested over a range of 5 dosing concentrations), 6 wells designated as the DMSO solvent control, and 1 well which was left blank. The positive control, cinnamic aldehyde was tested at concentrations of 64, 32, 16, 8, and 4 μM. The solvent control for the test article and the positive control was 1% DMSO in the dilution solvent (1% DMEM). Each definitive assay was performed independently but in parallel on the same day. After approximately 24 hours of incubation, the Assay Medium was removed from the cells. The plates were decanted and gently blotted on sterile paper towels. 150 μL of fresh pre-warmed 1% DMEM were added to all wells, including the blank. The plates were returned to the incubator until the dosing was initiated. For the test article, 12 decreasing doses were selected for the assay (test article was diluted up to a final concentration of 200000 μM in DMSO). For the positive control, 5 decreasing doses were prepared. For each experiment, the positive control (5 doses), and the solvent control, a 100x DMSO master plate was made, followed by a 4x Master Plate. When added to the 150 μL of 1% DMEM already in each well, the addition of the 50 μL 4x dose brought the final dose on the plates to 1x. The blank well received 50 μL of 1% DMEM. When all of the plates were dosed, the plates were sealed with a plate sealer to avoid evaporation of volatile compounds and to avoid cross-contamination between wells. The plates were then incubated at standard culture conditions for 48±1 hours. After approximately 48 hours of post-treatment incubation, visual observations of the cultures were performed for the cytotoxicity plate and recorded.

Luciferase Induction Determination: After 48±1 hours of exposure, each white-walled culture plate was removed from the incubator and allowed to equilibrated to room temperature for at least 30 minutes. Once at room temperature, the treatment medium was decanted from each plate. The cultures were rinsed with 200 μL of CMF-DPBS 9room temperature), the CMF-DPBS rinsate was decanted from the wells, and the plates were gently blotted onto paper towels. CMF-DPBS (50 μL) was added to each well followed by 50 μL of ONE-Glo™ Reagent. The plates remained at room temperature in the dark for at least 5 minutes before being read by the luminometer. The plates were read within 30 minutes of addition of the ONE-Glo™ Reagent. The luminescence determination of each plate was performed by a Berthold Detection Systems luminometer initiated from an IBM-PC hosting the Windows-based Simplicity™ software. The light intensity in each well was measured at 565 nm in the form of relative light units (RLUs).

Cytotoxicity Using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) Endpoint: A 0.59 mg/mL MTT solution was prepared in 1% DMEM and used within 2 hours. After 48±1 hours, the clear 96-well plates designated for the MTT endpoint were decanted and gently blotted on paper towels. No rinsing was performed. 1% DMEM containing 0.59 mg/mL MTT (200 μL) was added to each well. The plate was incubated with a plate seal at standard culture conditions for approximately 4 hours. The MTT solution was then decanted, the plate was blotted, and 200 μL of 10% SLS was added to each well. The plate was covered with a plate seal and incubated at standard culture conditions overnight. After the overnight incubation, each plate was placed on a plate shaker and shaken for at least 20 minutes at room temperature. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader.

Data Analysis: For each set of up to seven test articles, a copy of the standard data file, provided by Givaudan, was made (i.e. 3 trials, each with 4 plates per trial-3 plates for the luciferase induction and l plate for MTT). Raw data from the luminometer was transferred directly from the luminometer software into the designated Excel® spreadsheet for luminescence analyses. Raw data from the Vmax was transferred into the designated Excel® spreadsheet for cytotoxicity analyses. The data file automatically calculated the gene induction and the wells with statistically significant induction over a given threshold (default value set to 1.5 = 50% enhanced gene activity). Furthermore, the maximal induction (Imax), the concentration for maximal gene induction (CImax) and the EC1.5 value (concentration for induction above threshold), both with linear and log-linear extrapolation, was calculated similar to the LLNA (Local Lymph Node Assay). Relative survival (viability) was obtained by comparing the amount of MIT conversion by test article treated groups compared to the associated solvent treated group on the same plate. The Givaudan Excel file calculates an IC50 value for each test article. The IC50 is determined by averaging the viability percentage of each concentration for the 3 definitive assays and then calculating by linear interpolation the IC50 concentration which results in 50% reduced cell viability using the concentration and viability percentage below and above 50% viability. A summary of the results from the 3 definitive assays was calculated. The luciferase induction and cytotoxicity data were plotted on the graphs. For the luciferase induction, the fold gene induction (as compared to the solvent controls) was plotted over the test article concentration. For the cytotoxicity, the % viability (as compared to the solvent controls) was plotted over the test article concentration.

Criteria for Valid Definitive Assay: The KeratinoSens assay was accepted when the positive control (cinnamic aldehyde) caused an EC1.5 value that fell within two standard deviations of the historical mean. Additionally, the results of the three definitive trials for each plate were assessed using similar criteria outlined in the validation ring trial. Those acceptance criteria included: 1) variability in DMSO solvent control wells for each definitive assay was <20%; and 2) the positive control produced a statistically significant induction above 1.5 fold below 64 μM in each definitive assay.

Evaluation of Test Results: A test article was predicted to have sensitization potential if 1) The EC1.5 value fell below 1000 μM in at least 2 of 3 repetitions; 2) At the lowest concentration with a gene induction above 1.5, cellular viability was greater than 70%; and 3) There was apparent overall dose response which was similar between repetitions.

Results and discussion

Positive control results:

EC1.5: 16.42 µM
IC50: >64 µM

EC1.5 of the positive control fell within the acceptable historical range (0.0-22.08 µM).

In vitro / in chemico

Results

Key result

Parameter:

other: EC1.5 (µM)

Value:

2 000

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / 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

The mean Imax and mean CImax of the test substance were 1.08 and 500 μM, respectively. The test article did not directly reduce MTT.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was predicted to be a non-sensitizer

Executive summary:

The induction of Antioxidant-Response-Element Dependent Gene Activity in the Keratinocyte ARE-Reporter Cell Line KeratinoSens skin sensitization assay is a high-throughput cell-based in vitro test to screen for the skin sensitization potential of chemicals. Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). The test substance was tested at 12 concentrations ranging from 0.977 to 2000 µM. The positive control, Cinnamic aldehyde, was tested at 5 concentrations ranging from 4 to 64 µM in all plates. EC1.5 of the positive control fell within the acceptable historical range (0.0-22.08 µM).

A test article was predicted to have sensitization potential if: 1) The ECI.5 value fell below 1000 μM in at least 2 of 3 repetitions; 2) At the lowest concentration with a gene induction above 1.5, cellular viability was greater than 70%; and 3) There was an apparent overall dose response which was similar between the three definitive assays. According to the current prediction model, with a mean EC1.5 of >2000 µM, the test article was predicted to be a non-sensitizer.