Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 695-735-2 | CAS number: 68489-14-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Description of key information
The skin sensitizing potential of the test substance was evaluated in a combination of non-animal methods (in chemico and in vitro).
Three key events of skin sensitisation were evaluated: a) molecular interaction with skin proteins, b) inflammatory response in keratinocytes and c) activation of dendritic cells were addressed.
a) Molecular interaction with skin proteins
The protein reactivity of the test substance was investigated in a Direct Peptide Reactivity Assay (DPRA) (2017) performed according to OECD Guideline 442C and in compliance with GLP.
WS-5 was dissolved in acetonitrile at a concentration of 100 mM. The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours
in glass autosampler vials, protected from light and set at 25°C. Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with UV detection.
Cysteine and lysine peptide percent depletion (PPD) values were then calculated and used in a prediction model which allows assigning the test article to one of four reactivity classes used
to support the discrimination between sensitisers and non-sensitisers.
In presence of the test substance, WS-5, the percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%. All analytical acceptance criteria for each peptide run were met and the positive control proved the validity of the test.
The test article, WS-5, was therefore considered to have no or minimal reactivity and gave a negative prediction in the Direct Peptide Reactivity Assay.
b) Activation of keratinocytes
The activation of keratinocytes of the test substance, WS-5 was investigated in an ARE-Nrf2 Luciferase Test (2017) in the transgenic KeratinoSens™ cell line according to OECD Guideline 442D and in compliance
with GLP. For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.The study was conducted in two independent experiments.
Cells were incubated with test substance concentrations of 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 61.5, 125, 250, 500, 1000, 2000 µM in DMSO for 48 h at 37°C. After exposure cells were lysed and luciferase activity
was assessed by luminescence measurement.
In the first experiment a max luciferase activity (Imax) induction of 3.48 was determined at a test substance concentration of 2000µM (EC1.5). Cytotoxicity cell viability of 33.1% was observed at 2000 µM.
No dose response for luciferase Induction was noticed.
In the second experiment, an Imax induction of 1.25 was determined at test substance concentrations of 250 µM. The corresponding cell viability was >70%.No EC1.5 could be calculated as there no statistically
significant increases in induction.
Under the conditions of the ARE-Nrf2 Luciferase test the test substance, WS-5 did not induce luciferase activity in the transgenic KeratinoSens™ cell line.
c) activation of dendritic cells
Dendritic cell response of the test substance was investigated in a human Cell Line Activation Test (h-CLAT) according to OECD Guideline 442E and in compliance with GLP.
The aim of the study was to investigate the potential of WS‑5 to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes
in the expression of cell surface markers (CD86 and CD54).
A reactivity check was performed two weeks after thawing using the following compounds:
- 2,4-dinitrochlorobenzene(DNCB, CAS No. 97-00-7, ≥99% purity)
- nickel sulphate (CAS No. 10101-97-0, 99% purity)
- lactic acid (CAS No. 50-21-5, 85% purity).
Only cells which passed the reactivity check were used for the assay.
WS-5 was dissolved in DMSO to give working solutions at concentrations of 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The top two concentrations following dilution in culture medium (500 and 1000 µg/mL) produced oily emulsions and were not used for cell treatment. Therefore, the maximum attainable concentration was therefore 250 µg/mL.
No CV75 value was calculated, as there was no effect of the treatment on cell viability.
In the main experiment test substance concentrations of69.77, 83.72, 100.46, 100.46, 120.56, 144.67, 173.61, 208.33 and 250.00were administered to human THP-1 cells for 24 hours.
Thereafter the cells were stained with FITC-labeled anti-CD86, CD54 antibody or mouse IgG1 (isotype control) and relative fluorescence intensity of the surface markers was measured in a flow cytometer.
In the first Experiment, a stable dispersion was not formed during the dilution steps at concentrations of 208.33 and 250 µg/mL, therefore the maximum concentration treated was 173.61 µg/mL. The relative fluorescence intensity (RFI) values for the test article, WS-5 were calculated.
The RFI of CD86 was ≥150% at concentrations of 120.56, 144.67 and 173.61 µg/mL in the first Experiment, with viability ≥50%. The RFI of CD54 was≥200% at concentrations of 144.67 and 173.61 µg/mL, with viability ≥50%.
In second Experiment, the RFI of CD86 was ≥150% at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61µg/mL, with viability≥50%. The RFI of CD54 was ≥200% at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61µg/mL, with viability ≥50%.
The assay acceptance criteria required for acceptance of results in the test were satisfied according to OECD Test Guideline 422E.
Based on the results of this study performed according to OECD TG 442, the test article, WS‑5, was considered to be positive in the human Cell Line Activation Test.
Conclusion:
Based on a weight of evidence approach considering one positive and two negative results in the in vitro test battery according to the Adverse Outcome Pathway defined for skin sensitization, the test substance, WS-5 is not considered to be a skin sensitizer.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in chemico
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017
- 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))
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of study:
- direct peptide reactivity assay (DPRA)
- Justification for non-LLNA method:
- The OECD TG 442 C may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: white crystalline powder, WS-5, batch no.: 80100039
- Expiration date of the batch: 16 January 2019
- Purity test date: 99.57%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry ambient temperature
The test article, WS-5 produced a visually clear solution at a concentration of 100 mM acetonitrile, which was the first of the listed vehicles specified in the protocol.
Formulations were prepared shortly before testing. - Details on the study design:
- Skin sensitisation (In chemico test system) - Details on study design:
Test Article Incubation:
Each test solution was prepared at ratios of 1:10 and 1:50 with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25°C for 24±2 hours, in the dark. At the end of the incubation period the samples were visually inspected for precipitate formation. Samples were centrifuged at 400 g for 5
minutes.
Analytical Method:
The remaining concentration of cysteine- or lysine-containing peptides following the 24-hour incubation period was measured by high performance liquid chromatography
(HPLC) with gradient elution and UV detection at 220 nm.
Reference and Co-elution Controls:
Reference controls were prepared for each peptide.
Reference Control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference Control B (six replicates) was used to verify the
stability of the reference controls over time and Reference Control C (in triplicate) was used to verify that acetonitrile did not impact the percent peptide depletion.
Co-elution controls were prepared to detect possible co-elution of the test article with the peptides.
Calibration Curves for Peptides:
Calibration curves were prepared for each peptide using concentrations of 0.0167, 0.0334, 0.0667, 0.1335, 0.267 and 0.534 mM (Standards 1 to 6) - Positive control results:
- Cinnamaldehyde (CAS No. 104-55-2, batch number MKBT8955V, purity 98.5%, expiry 29 February 2020) was used as the positive control. The positive control was dissolved in acetonitrile at a concentration of 100 mM. For results, please see table 2 and 3.
- Key result
- Run / experiment:
- other: 3
- Parameter:
- other: The mean of cysteine and lysine percentage depletion
- Value:
- 3.73
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: DPRA prediction: negative (no or minimal reactivity) according to OECD 442C
- Remarks:
- Negative prediction in the Direct Peptide Reactivity Assay
- Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
The mean percent cysteine and percent lysine depletion value was calculated. Negative depletion was considered as “0” when calculating the mean. By using the
cysteine 1:10/lysine 1:50 prediction model below, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an IATA.
The percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%.
ACCEPTANCE OF RESULTS:
The following criteria should be met for a run to be considered valid:
- The standard calibration curve should have a r2 >0.99.
The r value for the standard calibration curve was 1.000 and as 0.9963 for lysine and cyteine depletion, respectively.
- The mean peptide concentration for reference controls A should be 0.50±0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference
controls B and C should be <15.0%.
For lysine and cysteine depletion, the mean Peptide Concentration (mM) for the Reference Controls A and C were as follows:
Reference Controls A - 0.50
- The mean PPD value of the three replicates for the positive control and maximum standard deviation (SD) must fall within the ranges:
Peptide Mean PPD Values (%) Lower Bound Mean PPD Values (%) Upper Bound SD
Cysteine 60.8 100 <14.9
Lysine 40.2 69.0 <11.6
- The maximum standard deviation for the test article replicates should be <14.9 for the percent cysteine depletion and <11.6 for the percent lysine depletion.
The SD for Mean PPD for lysine and cysteine depletion were 0.81 and 2.72 , respectively. - Interpretation of results:
- other: negative prediction for skin sensitisation
- Conclusions:
- The percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%. The test article, WS-5, was therefore considered to have no or minimal reactivity and gave a negative prediction in the Direct Peptide Reactivity Assay, performed according to OECD Guidelines for Testing of Chemicals Method 442C.
- Executive summary:
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptides following incubation with the test article.
The test article, WS-5 was dissolved in acetonitrile at a concentration of 100 mM. The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours in glass autosampler vials, protected from light and set at 25°C.
Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with UV detection.
Cysteine and lysine peptide percent depletion (PPD) values were then calculated and used in a prediction model which allows assigning the test article to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.
In presence of the test substance, WS-5, the percentage peptide depletion for cysteine was 7.16% and the percentage peptide depletion for lysine was 0.3%. The mean of cysteine and lysine percentage depletion was therefore 3.73%.
All analytical acceptance criteria for each peptide run were met and the positive control proved the validity of the test.
Conclusion:
The test article, WS-5, was considered to have no or minimal reactivity and gave a negative prediction in the Direct Peptide Reactivity Assay according to OECD TG 442 C.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 05 September 2017 - 22 September 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)
- Deviations:
- yes
- Remarks:
- see any other information on materials and methods including tables
- GLP compliance:
- yes
- Type of study:
- activation of keratinocytes
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: Symrise
- batch No.of test material: 80100039
- Expiration date of the lot/batch: 16 January 2019
- Purity test date: 99.57%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Analysis for stability, achieved concentration and homogeneity of test article formulations was not conducted as part of this study as it was not a requirement of the Test Guideline.
- Storage: 15 to 25°C, protected from light
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: WS-5 was dissolved in dimethyl sulfoxide (DMSO) to the final concentration of 200 mM. Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM). - Details on the study design:
- Skin sensitisation (In vitro test system) - Details on study design:
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
Aliquots of 50 µL of each of the final concentrations of WS-5 were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.
After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2.
The MTT medium was then removed and SDS (at 10% w/v) was added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e (Molecular Devices, LLC).
After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well.
The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate reader and read.
The negative control was diluted into culture medium containing serum so that the final concentration was 1%.
The positive control was prepared at a concentration of 6.4 mM in DMSO. Five master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 6.4 mM stock solution). The master concentrations were then further diluted 25 fold into culture medium containing serum and finally used for treatment with a further 4 fold dilution factor so that the final concentrations range from 4 to 64 µM. - Positive control results:
- Cinnamic aldehyde (CAS No. 14371-10-9), supplied by Sigma Aldrich Chemical Co. Ltd. was used as the positive control.
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 µM in Experiments 1 and 2. The EC1.5 values for the positive control were 8.26 and 11.54 µM in Experiments 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM were 10.96 and 3.81 in Experiments 1 and 2, respectively. - Key result
- Run / experiment:
- other: 3 runs / Experiment 1
- Parameter:
- other: maximum luciferase activity induction (I max)
- Value:
- 3.48
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: negative in the KeratinoSens™ prediction model
- Remarks:
- Corresponding cytotoxicity cell viability of 33.1%. EC1.5 at 2000 µM. No dose response for luciferase Induction
- Key result
- Run / experiment:
- other: 3 runs / Experiment 2
- Parameter:
- other: maximum luciferase activity induction (I max)
- Value:
- 1.25
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: negative in the KeratinoSens™ prediction model
- Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prediction model:
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens™ prediction is considered negative:
1.The Imax is >1.5 fold and statistically significantly different when compared to the solvent (negative) control (se determined by a two-tailed, unpaired Student’s T-test);
2.The cell viability is >70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration);
3.The EC1.5 value is <1000 µM (or <200 µg/mL for test articles with no defined MW);
4.There is an apparent overall dose response for luciferase induction or if the dose response curve is biphasic. - Interpretation of results:
- other: negative prediction using the KeratinoSens™ prediction model
- Conclusions:
- Under the conditions of the ARE-Nrf2 Luciferase test, the test substance, WS-5 did not induce luciferase activity in the transgenic KeratinoSens™ cell line.
- Executive summary:
This study according to OECD TG 442D was conducted to investigate the potential ofWS-5to induce genes that are regulated by the antioxidant response element (ARE).
The applied ARE-Nrf2 Luciferase Test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.The study was conducted in two independent experiments.
Cells were incubated with test substance concentrations of 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 61.5, 125, 250, 500, 1000, 2000 µM in DMSO for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.
In the first experiment a max luciferase activity (Imax) induction of 3.48 was determined at a test substance concentration of 2000µM (EC1.5). Cytotoxicity cell viability of 33.1% was observed at 2000 µM. No dose response for luciferase Induction was noticed.
In the second experiment, an Imax induction of 1.25 was determined at test substance concentrations of 250 µM. The corresponding cell viability was >70%.No EC1.5 could be calculated as there no statistically significant increases in induction.
Conculsions:
Under the conditions of the ARE-Nrf2 Luciferase test the test substance, WS-5 did not induce luciferase activity in the transgenic KeratinoSens™ cell line.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09 October 2017 - 27 October 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guidelines for Testing of Chemicals Method 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of study:
- other: human Cell Line Activation Test (h - CLAT)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: Symrise
- batch No.of test material: 80100039
- Expiration date of the lot/batch: 16 January 2019
- Purity test date: 99.57%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Analysis for stability, achieved concentration and homogeneity of test article formulations was not conducted as part of this study as it was not a requirement of the Test Guideline.
- Storage: 15 to 25°C, protected from light
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: the test article, WS-5, was dissolved at 500 mg/mL in DMSO. Eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent.
The stock solutions were further diluted 250-fold in culture medium to give working solutions at concentrations of 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL.
The top two concentrations following dilution in culture medium (500 and 1000 µg/mL) produced oily emulsions and were not used for cell treatment. Therefore, the maximum attainable concentration was therefore 250 µg/mL. - Details on the study design:
- Skin sensitisation (In vitro test system) - Details on study design:
In a performed Dose Finding Assay, an oily emulsion was observed during the dilution steps at concentrations of 500 and 1000 µg/mL, therefore the maximum attainable concentration was 250 µg/mL. No CV75 value was calculated, as there was no effect of the treatment on cell viability.
CD86/CD54 Expression procedure:
Eight stock solutions of WS-5 were prepared by 1.2-fold serial dilutions using DMSO to give eight concentrations ranging from 34.9 to 125 mg/mL. These stock solutions were then diluted 250-fold into the culture medium (working solutions).
Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 cells per well. Each plate was sealed using a plate sealer and then incubated at 37±1C, 5% (v/v) CO2 in air, in a humidified environment for 24±0.5 hours.
After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4C for 15 minutes.
After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate (or sample tubes), centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4C for 30 minutes.
The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry. - Positive control results:
- For the positive control, 2,4-dinitrochlorobenzene (DNCB, CAS No. 97-00-7, ≥99% purity), RFI values were ≥150% for CD86 and ≥200% for CD54 in all each independent runs. Cell viability was >50% in each independent run.
- Key result
- Run / experiment:
- other: Experiment 1
- Parameter:
- other: relative fluorescence intensity (RFI)
- Remarks:
- RFI of CD86
- Value:
- 150
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: activation of dendritic cells positive according to OECD 442E at concentrations of 120.56, 144.67 and 173.61 µg/mL
- Remarks:
- viability ≥50%.
- Key result
- Run / experiment:
- other: Experiment 2
- Parameter:
- other: relative fluorescence intensity (RFI)
- Remarks:
- RFI of CD86
- Value:
- 150
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: activation of dendritic cells positive according to OECD 442E at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61 µg/mL
- Remarks:
- viability ≥50%
- Key result
- Run / experiment:
- other: Experiment 1
- Parameter:
- other: relative fluorescence intensity (RFI)
- Remarks:
- RFI of CD54
- Value:
- 200
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: activation of dendritic cells positive according to OECD 442E at concentrations of 144.67 and 173.61 µg/mL
- Remarks:
- viability ≥50%
- Key result
- Run / experiment:
- other: Experiment 2
- Parameter:
- other: relative fluorescence intensity (RFI) RFI of CD54
- Remarks:
- RFI of CD54
- Value:
- 200
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: activation of dendritic cells positive according to OECD 442E at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61 µg/mL
- Remarks:
- viability ≥50%
- Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
Assay Acceptance Criteria
- The cell viabilities of medium and solvent control should be higher than 90%.
- In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability should be more than 50%.
- For the test article, the cell viability should be more than 50% in at least four tested doses in each run.
ACCEPTANCE OF RESULTS:
- The cell viabilities of medium and solvent control were higher than 90% in each independent run.
- In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%). The assay was therefore considered valid as this did not impact on the results obtained with the test article, WS-5.
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
- For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54 in all each independent runs. Cell viability was >50% in each independent run.
- For the test article, WS-5, the cell viability was more than 50% in all tested concentrations in each independent run. - Interpretation of results:
- other: positive prediction of skin sensitisation in the human Cell Line Activation Test.
- Conclusions:
- Based on the results of this study performed according to OECD TG 442, the test article,WS-5, was considered to be positive in the human Cell Line Activation Test.
- Executive summary:
Dendritic cell response of the test substance was investigated in a human Cell Line Activation Test (h-CLAT) according to OECD Guideline 442E and in compliance with GLP. The aim of the study was to investigate the potential of WS‑5 to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54).
A reactivity check was performed two weeks after thawing using the following compounds:
- 2,4-dinitrochlorobenzene (DNCB, CAS No. 97-00-7, ≥99% purity)
- nickel sulphate (CAS No. 10101-97-0, 99% purity)
- lactic acid (CAS No. 50-21-5, 85% purity).
Only cells which passed the reactivity check were used for the assay.
WS-5 was dissolved in DMSO to give working solutions at concentrations of 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The top two concentrations following dilution in culture medium (500 and 1000 µg/mL) produced oily emulsions and were not used for cell treatment. Therefore, the maximum attainable concentration was therefore 250 µg/mL.No CV75 value was calculated, as there was no effect of the treatment on cell viability.
In the main experiment, the test substance concentrations of 69.77, 83.72, 100.46, 100.46, 120.56, 144.67, 173.61, 208.33 and 250.00 were administered to human THP-1 cells for 24 hours. Thereafter the cells were stained with FITC-labeled anti-CD86, CD54 antibody or mouse IgG1 (isotype control) and relative fluorescence intensity of the surface markers was measured in a flow cytometer.
In the first Experiment, a stable dispersion was not formed during the dilution steps at concentrations of 208.33 and 250 µg/mL, therefore the maximum concentration treated was 173.61 µg/mL. The relative fluorescence intensity (RFI) values for the test article, WS-5 were calculated.
The RFI of CD86 was ≥150% at concentrations of 120.56, 144.67 and 173.61 µg/mL in the first Experiment, with viability ≥50%. The RFI of CD54 was≥200% at concentrations of 144.67 and 173.61 µg/mL, with viability ≥50%.
In second experiment, the RFI of CD86 was ≥150% at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61µg/mL, with viability ≥50%. The RFI of CD54 was ≥200% at concentrations of 69.77, 83.72, 100.46, 120.56, 144.67 and 173.61µg/mL, with viability ≥50%.
The assay acceptance criteria required for acceptance of results in the test were satisfied according to the OECD Test Guideline 422E.
Conclusions:
Based on the results of this study performed according to OECD TG 442, the test article, WS‑5, was considered to be positive in the human Cell Line Activation Test.
Referenceopen allclose all
Table 1. Cysteine 1:10/lysine 1:50 Prediction Model used in this testing to discriminate between skin sensitisers and non-sensitisers in the framework of an IATA
Mean of Cysteine and Lysine % Depletion |
Reactivity Class |
DPRA Prediction |
0% ≤ mean % depletion ≤6.38% |
No or minimal reactivity |
Negative |
6.38% < mean % depletion ≤22.62% |
Low reactivity |
Positive |
22.62% < mean % depletion ≤42.47% |
Moderate reactivity |
|
42.47% < mean % depletion ≤100% |
High reactivity |
Table 2. The percentage peptide depletion (PPD) for lysine
Substance |
Replicate Peptide Peak Areas |
Reference Control C |
PPD |
Mean PPD |
SD |
WS-5 Test Article |
37.49 |
37.26 |
-0.62 # |
0.30 |
0.81 |
37.41 |
-0.40 # |
||||
36.93 |
0.89 |
||||
Cinnamaldehyde Positive Control |
17.63 |
37.26 |
52.68 |
50.35 |
4.61 |
17.39 |
53.33 |
||||
20.48 |
45.03 |
#negative value was considered as “0” when calculating the mean
Table 3. The percentage peptide depletion (PPD) for cysteine
Substance |
Replicate Peptide Peak Areas |
Reference Control C |
PPD |
Mean PPD |
SD |
WS-5 Test Article |
22.13 |
23.06 |
4.03 |
7.16 |
2.72 |
20.99 |
8.98 |
||||
21.11 |
8.46 |
||||
Cinnamaldehyde Positive Control |
8.15 |
23.06 |
64.66 |
65.50 |
0.74 |
7.89 |
65.78 |
||||
7.83 |
66.05 |
Table1. Luminescence Readingsfor WS-5 - Experiment 1 |
|||||||||||||
Substance |
Concentration (µM) |
||||||||||||
0.98 |
1.95 |
3.91 |
7.81 |
15.63 |
31.25 |
62.5 |
125 |
250 |
500 |
1000 # |
2000 |
||
Test Article |
Plate 1 |
1133240 |
843755 |
898692 |
1050636 |
786297 |
718257 |
847774 |
1019132 |
995454 |
1002721 |
0 |
2538667 |
Plate 2 |
957910 |
983945 |
957037 |
746169 |
898212 |
938561 |
995668 |
1078826 |
1324627 |
1037682 |
0 |
2944623 |
|
Plate 3 |
799640 |
871633 |
753495 |
860062 |
719102 |
716173 |
809385 |
898619 |
890782 |
1247988 |
0 |
3350900 |
|
Mean Fold Induction |
1.14 |
1.06 |
1.02 |
1.06 |
0.94 |
0.93 |
1.04 |
1.18 |
1.25 |
1.30 |
0.00 |
3.48 |
# no luminescence readings obtained due to the technical error
Table 2. Luminescence Readings forNegative Control- Experiment 1 |
|||||||
Substance |
Individual Values |
||||||
Negative Control |
Plate 1 |
744394 |
817814 |
829305 |
811138 |
840569 |
737785 |
Plate 2 |
784779 |
1045144 |
1048312 |
887335 |
958900 |
1010644 |
|
Plate 3 |
738233 |
775860 |
880078 |
798360 |
928753 |
684907 |
Table 3. Luminescence Readings forPositive Control- Experiment 1 |
||||||
Substance |
Concentration (µM) |
|||||
4 |
8 |
16 |
32 |
64 |
||
Positive Control |
Plate 1 |
1209862 |
1164304 |
1988218 |
3053730 |
6682098 |
Plate 2 |
1155343 |
1500502 |
1891800 |
2592479 |
12724356 |
|
Plate 3 |
1043222 |
1127017 |
1493561 |
2641184 |
8953845 |
|
Mean Fold Induction |
1.34 |
1.48 |
2.11 |
3.28 |
10.96 |
Table 4. Luminescence Readings for WS-5 - Experiment 2 |
|||||||||||||
Substance |
Concentration (µM) |
||||||||||||
0.98 |
1.95 |
3.91 |
7.81 |
15.63 |
31.25 |
62.5 |
125 |
250 |
500 |
1000 |
2000 |
||
Test Article |
Plate 1 |
1152178 |
975059 |
926744 |
883957 |
884997 |
866903 |
906172 |
999005 |
1044621 |
1086959 |
17853 |
-297 |
Plate 2 |
1041039 |
921073 |
936655 |
915797 |
870204 |
900632 |
853558 |
981584 |
1048706 |
1174808 |
21617 |
-2 |
|
Plate 3 |
963458 |
842209 |
891649 |
885147 |
879821 |
811156 |
884135 |
962980 |
983201 |
1083250 |
908870 |
68 |
|
Mean Fold Induction |
1.18 |
1.02 |
1.03 |
1.01 |
0.99 |
0.96 |
0.99 |
1.10 |
1.15 |
1.25 |
0.37 |
0.00 |
Table 5. Luminescence Readings for Negative Control - Experiment 2 |
|||||||
Substance |
Individual Values |
||||||
Negative Control |
Plate 1 |
980930 |
932116 |
843684 |
915855 |
910491 |
945191 |
Plate 2 |
974030 |
847451 |
858485 |
887633 |
845526 |
924225 |
|
Plate 3 |
895280 |
843147 |
760858 |
867474 |
912730 |
890969 |
Table 6. Luminescence Readings for Positive Control - Experiment 2 |
||||||
Substance |
Concentration (µM) |
|||||
4 |
8 |
16 |
32 |
64 |
||
Positive Control |
Plate 1 |
1053652 |
1258489 |
1341586 |
2106567 |
3757925 |
Plate 2 |
1038281 |
1213899 |
1523531 |
1989432 |
3401688 |
|
Plate 3 |
1078725 |
1320105 |
1405587 |
2034596 |
3037362 |
|
Mean Fold Induction |
1.19 |
1.42 |
1.60 |
2.29 |
3.81 |
Table 1. Dose Finding Assay: Cell Viability |
||||||
Run |
Cell Viability (%) at Concentration (µg/mL) |
|||||
7.8 |
15.6 |
31.3 |
62.5 |
125.0 |
250.0 |
|
1 |
99.7 |
99.7 |
99.5 |
99.2 |
96.6 |
95.2 |
2 |
99.6 |
99.4 |
99.2 |
98.6 |
96.3 |
96.7 |
Table 2. Experiment 1 - MFI and Cell Viability Values |
||||||||
WS-5 Concentration (µg/mL) |
MFI (Geo Mean) |
Corrected MFI |
Cell Viability |
|||||
CD86 |
CD54 |
Isotype |
CD86 |
CD54 |
IgG |
CD86 |
CD54 |
|
69.77 |
1677 |
878 |
608 |
1069 |
270 |
97.0 |
96.5 |
97.2 |
83.72 |
1714 |
866 |
628 |
1086 |
238 |
96.9 |
96.1 |
96.8 |
100.46 |
1844 |
917 |
629 |
1215 |
288 |
95.4 |
94.8 |
94.8 |
120.56 |
2100 |
955 |
629 |
1471 |
326 |
90.9 |
89.6 |
91.0 |
144.67 |
2142 |
982 |
635 |
1507 |
347 |
91.0 |
89.4 |
87.5 |
173.61 |
2273 |
1117 |
652 |
1621 |
465 |
83.0 |
82.6 |
80.5 |
Medium |
1352 |
731 |
594 |
758 |
137 |
98.8 |
98.6 |
98.9 |
DMSO |
1486 |
750 |
586 |
900 |
164 |
98.8 |
98.4 |
98.7 |
DNCB |
2291 |
1269 |
759 |
1532 |
510 |
86.5 |
86.8 |
88.8 |
MFI (Geo Mean) Geometric mean fluorescence intensity
Corrected MFI Corrected mean fluorescence intensity
Table 3. Experiment 2 -MFI and Cell Viability Values |
|||||||||
WS-5 Concentration (µg/mL) |
MFI (Geo Mean) |
Corrected MFI |
Viability |
||||||
CD86 |
CD54 |
Isotype |
CD86 |
CD54 |
Isotype |
CD86 |
CD54 |
||
69.77 |
1848 |
906 |
637 |
1211 |
269 |
93.8 |
91.9 |
91.6 |
|
83.72 |
1959 |
1003 |
649 |
1310 |
354 |
91.9 |
88.8 |
88.9 |
|
100.46 |
2097 |
1020 |
658 |
1439 |
362 |
90.7 |
88.3 |
87.5 |
|
120.56 |
2336 |
1136 |
684 |
1652 |
452 |
83.9 |
77.8 |
77.9 |
|
144.67 |
2240 |
1176 |
708 |
1532 |
468 |
78.0 |
74.0 |
72.8 |
|
173.61 |
2278 |
1259 |
741 |
1537 |
518 |
70.0 |
63.5 |
62.1 |
|
208.33 |
2210 |
1256 |
836 |
1374 |
420 |
40.9 |
40.7 |
41.3 |
|
250.00 |
2620 |
1261 |
919 |
1701 |
342 |
24.6 |
21.8 |
24.0 |
|
Medium |
1086 |
694 |
578 |
508 |
116 |
99.2 |
99.1 |
98.7 |
|
DMSO |
1102 |
690 |
571 |
531 |
119 |
99.0 |
98.8 |
98.8 |
|
DNCB |
2281 |
1341 |
750 |
1531 |
591 |
84.6 |
82.2 |
79.7 |
MFI (Geo Mean) Geometric mean fluorescence intensity
Corrected MFI Corrected mean fluorescence intensity
Table 4. The relative fluorescence intensity (RFI) values for WS-5 |
||||
Concentration (µg/mL) |
RFI (CD86) |
RFI (CD54) |
||
Exp 1 |
Exp 2 |
Exp 1 |
Exp 2 |
|
69.77 |
119 |
228 |
165 |
226 |
83.72 |
121 |
247 |
145 |
297 |
100.46 |
135 |
271 |
176 |
304 |
120.56 |
163 |
311 |
199 |
380 |
144.67 |
167 |
289 |
212 |
393 |
173.61 |
180 |
289 |
284 |
435 |
208.33 |
- |
259 |
- |
353 |
250.00 |
- |
320 |
- |
287 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Skin sensitisation results:
Skin sensitisation (OECD 442C and 442D): negative
Skin sensitisation (OECD 442E): positive
Conclusion:
Based on a weight of evidence approach considering one positive and two negative results in the in vitro test battery according to the Adverse Outcome Pathway defined for skin sensitisation, the test substance, WS-5 is not considered to be a skin sensitiser.
The available data on skin sensitisation of the test substance do not meet the criteria for classification according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.