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EC number: 228-779-4 | CAS number: 6358-57-2
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Endpoint summary
Administrative data
Description of key information
Skin sensitising.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From September 16th to 26th, 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)
- Version / remarks:
- 2018
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Details on the study design:
- Formulation of test item
Possible solvents for the test item were dimethyl sulfoxide (DMSO) and sterile ultrapure water or exposure medium.
Test item was formulated and examined in the test as follows: first, solubility of the test item was evaluated in DMSO at the concentration of 200 mM. The test item could be properly dissolved in DMSO after approximately 5 minutes of vortexing, and formed a clear, dark red homogenous solution. In ultrapure water the test item could not be dissolved, but a cloudy suspension was gained.
Since the formulation with the preferred vehicle, DMSO fulfilled all requirements, it was chosen as the appropriate solvent of the test item in this study.
Positive control: trans-cinnamaldehyde
Solvent: DMSO. The final concentration of DMSO was 1 % in exposure medium on the plates treated with the test item, the negative (solvent) control and the positive control. This concentration in DMSO is known not to affect cell viability.
Preparation of solutions
EDTA solution 10 %, pH 8: ultrapure water supplemented with 10 (w/v) % EDTA, brought to pH 8 with sodium hydroxide (NaOH) or hydrogen chloride (HCl)
DPBS-EDTA: Dulbecco’s Phosphate Buffered Saline (DPBS) containing 0.05 % EDTA solution
MTT stock solution: MTT was dissolved in DPBS at the concentration of 5 mg/ml.
MTT working solution: 8.4-fold dilution of MTT stock solution in exposure medium
1× Lysis Buffer: 5-fold dilution of 5× Lysis Buffer in ultrapure water
Luciferase reagent: 1 vial Luciferase Assay Substrate dissolved in 10 ml (content of 1 vial) Luciferase Assay Buffer
Cell or Test System (KeratinoSens™ cell line)
KeratinoSens™ cell line is a transgenic cell line with a stable Luciferase construct insertion.
Preparation of media for KeratinoSens™ cells
Maintenance (culture) medium: DMEM supplemented with 9.0 (v/v) % fetal bovine serum (FBS) and ~ 500 µg/ml G418.
Thawing medium: DMEM containing 9.1 (v/v) % FBS without G418
Exposure medium: DMEM containing 1 (v/v) % FBS without G418
Procedure of the KeratinoSens™ method
0. Preincubation of cells; solubility assessment
1. Seeding of cells for testing - 24 h incubation
2. Preparation of the stock solution
3. Preparation of master plates
4. Exposure – 48 h incubation
5. Luciferase activation measurement
6. Cytotoxicity assessment
Principle of the KeratinoSens™ method
The KeratinoSens™ method is an in vitro assay that quantifies the extent of luciferase gene induction following 48 hours incubation time of the KeratinoSens™ reporter cells with the test items. Luciferase gene induction is measured in the cell lysates by luminescence detection using a light producing luciferase substrate (Luciferase Reagent). Cytotoxicity and the relative luminescence intensity of luciferase substrate in the lysates are measured and luciferase induction compared to solvent/vehicle control is calculated.
KeratinoSens™ cells were derived from HaCaT human keratinocytes and transfected with selectable plasmids containing luciferase gene under the transcriptional control of the AKR1C2 ARE gene sequence, upstream of the SV40 promoter. AKR1C2 is known to be one of the genes up-regulated upon contacting skin sensitisers in dendritic cells. Therefore, this method is able to mimic the activation of the Keap1-Nrf2-ARE regulatory pathway, and is relevant for the assessment of the skin sensitisation potential of test items. A prediction model is used, to support the discrimination between sensitisers and non-sensitisers.
Preparation of the master plate
Test item master solutions
Based on the test item stock solutions made of DMSO, 2-fold serial dilutions were made using the solvent to obtain twelve 100 × master concentrations of the test item creating a 100 × master plate. The 100 × master concentrations were further diluted 25-fold into exposure medium to obtain the 4 × master plate, by adding 10 µl of the 100 × master concentrations to 240 µl exposure medium.
Positive control
The positive control used was trans-cinnamaldehyde for which a series of five 100 × master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 200 mM stock solution) and diluted as described for the 4 × master solutions. The final concentration of the positive control on the treated plates ranged from 4 to 64 µM.
Negative control
The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as described for the master and working solution concentrations in 6.3.1, so that the final negative (solvent) control concentration was 1 % DMSO in exposure medium on the treated plates.
This DMSO concentration is known not to affect cell viability and corresponds to the same concentration of DMSO used in the tested chemical and in the positive control.
Preparation of cells
Cells were subcultured upon reaching 80 - 90 % confluence and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium and distributed into 96-well plates (10 000 cells/well) homogenously. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1 °C in the presence of 5 % CO2.
Exposure
After the 24-hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test item and control substances were added to each well in a way that an additional 4-fold dilution was achieved on the plate for the final concentrations to be established (50 µL of 4× master solution to 150 µL of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test item.
Luciferase activity measurements
After the 48-hour exposure time with the test item and control substances, cells were washed with DPBS (270 µl), and 1× lysis buffer (20 µl) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 µl) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.
Cytotoxicity
For the cell viability assay, medium was replaced after the 48-hour exposure time with MTT working solution (200 µl) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilised by the addition of isopropanol (50 µl). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer.
Acceptance criteria
For each test item and positive control substance, in order to derive a prediction, at least two independent tests, each containing three replicates for the luminescence measurements and one for viability measurement, were needed. In case of discordant results between the two independent tests, a third test should be performed. Each independent test was to be performed on a different day with fresh stock solution of test items and independently harvested cells. Cells may however have come from the same passage. KeratinoSens™ prediction should be considered in the framework of an IATA and in accordance with the limitations stated in the OECD test guideline.
The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations. The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility or between 7 μM and 30 μM (based on the validation dataset). In addition, the average induction in the parallel plates for Trans Cinnamaldehyde at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of Trans-Cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
Finally, the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO should be below 20 % in each test which consists of 6 wells tested in triplicate.
Furthermore, the cellular viability is measured at the lowest concentration leading to ≥ 1.5-fold luciferase induction. This concentration should induce no significant cytotoxic effects and be below the IC30 value. In addition, viability should be > 70 % in at least two consecutive concentrations, otherwise the concentration range needs adjustment.
Data evaluation
The following parameters (endpoint values) are calculated in the KeratinoSens™ test method:
- the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control;
- the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5-fold threshold (i.e. 50 % enhanced luciferase activity) was obtained;
- the IC50 and IC30 concentration values for 50 % and 30 % reduction of cellular viability.
Fold induction and maximal average fold induction
Fold luciferase activity induction is calculated by the equation below. Maximal fold induction is determined in each individual test, while the overall maximal fold induction (Imax) is calculated as the average of the individual tests.
fold induction = (Lsample – Lblank) / (Lsolvent – Lblank)
where:
Lsample is the luminescence reading in the test item well
Lblank is the luminescence reading in the blank well containing no cells or treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control
Determination of EC1.5
The concentrations of the test item needed for a 1.5-fold luciferase induction are calculated by linear interpolation according to the equation below, and the overall EC1.5 is calculated as the geometric mean of the individual tests.
EC1.5 = (Cb – Ca) × [(1.5 – Ia) / (Ib – Ia)] + Ca
where: Ca is the lowest concentration in μM with > 1.5-fold induction
Cb is the highest concentration in μM with < 1.5-fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5-fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5-fold induction (mean of three replicate wells)
Cytotoxicity (determination of IC50 and IC30)
Viability is calculated by the equation below:
viability = [(Vsample – Vblank) / (Vsolvent – Vblank)] × 100
where: Vsample is the MTT-absorbance reading in the test item well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control
IC50 and IC30 are calculated by linear interpolation according to the equation below, and the overall IC50 and IC30 are calculated as the geometric mean of the individual tests.
ICx = (Cb – Ca) × [(100 – x) – Va) / (Vb – Va)] + Ca
where: x is the % reduction at the concentration to be calculated (50 and 30)
Ca is the lowest concentration in μM with > x % reduction in viability
Cb is the highest concentration in μM with < x % reduction in viability
Va is the % viability at the lowest concentration with > x % reduction in viability
Vb is the % viability at the highest concentration with < x % reduction in viability
For each concentration showing a luciferase activity induction equal or higher than 1.5-fold, statistical significance was determined (e.g. using a two-tailed Student’s t-test) by comparing the luminescence values of the three replicate samples with the luminescence values in the solvent/vehicle control wells (p < 0.05).
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 tests, otherwise the KeratinoSens™ prediction is considered negative:
- the Imax is equal or higher than 1.5-fold and statistically significantly different as compared to the negative/solvent control (as determined by a two-tailed, unpaired Student’s T-test);
- the cellular viability is higher than 70 % at the lowest concentration with induction of luciferase activity ≥ 1.5-fold;
- the EC1.5 value is less than 1000 μM
- there is an apparent overall dose-response for luciferase induction (or a biphasic response). - Positive control results:
- The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at several concentrations in both tests. The EC1.5 values of the positive control fell between 7 µM and 30 µM (15 µM and 7 µM in the first and second tests respectively).
The average inductions in the parallel plates for Trans Cinnamaldehyde at 64 μM were 11.65 fold and 6.68 fold in the first and second tests, respectively. Although the luciferase activity induction in the first test was is outside of the 2 – 8-fold induction range, there was a clear dose response relationship in the luciferase activity induction for the positive control, therefore it was accepted as valid. In any of the tests, there was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the tested concentrations. - Run / experiment:
- other: 1
- Parameter:
- other: IC30
- Remarks:
- measured in µg/mL
- Value:
- 12.46
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: 1
- Parameter:
- other: IC50
- Remarks:
- measured in µg/mL
- Value:
- 16.55
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: 1
- Parameter:
- other: EC1.5
- Remarks:
- measured in µg/mL
- Value:
- 5.02
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: 2
- Parameter:
- other: IC30
- Remarks:
- measured in µg/mL
- Value:
- 42.41
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: 2
- Parameter:
- other: IC50
- Remarks:
- measured in µg/mL
- Value:
- 53.57
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: 2
- Parameter:
- other: EC1.5
- Remarks:
- measured in µg/mL
- Value:
- 15.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- Test item
For the test item, twelve doses ranging from 2000 µM to 0.98 µM were used in two independent tests.
First test:
The test item induced cytotoxicity (viability < 70 %) compared to the solvent/vehicle control in KeratinoSens™ cells at 15.63 µM and over. The IC30 and IC50 values were 12.46 µM and 16.55 µM, respectively.
The luciferase activity induction exceeded the threshold of 1.5-fold compared to the respective negative control at the concentration of 7.81 and 15.63 µM. Thus, an EC1.5 value of 5.02 μM was calculated. The lower concentration (7.81 µM) leading to ≥ 1.5-fold luciferase induction (significant), induced no significant cytotoxic effects (viability of 95 %). The maximal fold induction (Imax) was 3.31 fold. A clear dose response could be observed.
No precipitation was observed at any point during the first test.
Test item in the first test
Criterion for a postive reponse
Statistically significant induction over 1.5-fold yes
Viability ≥ 70 % at lowest concentration with >1.5-fold yes
EC1.5 (µM) 5.02
Clear dose response yes
positive / negative positive
Based on the prediction model and the above described results, the test item was concluded positive in the first test, since the conditions for a positive result were met.
Second test:
The test item induced cytotoxicity (viability < 70 %) compared to the solvent/vehicle control at 62.50 µM and over in KeratinoSens™ cells. The IC30 and IC50 values were 42.41 µM and 53.57 µM, respectively.
The luciferase activity induction exceeded the threshold of 1.5-fold compared to the respective negative control at the concentration of 15.63 and 31.25 µM. Thus, an EC1.5 value of 15.50 μM was calculated. Both concentrations (15.63 and 31.25 µM) leading to ≥ 1.5-fold luciferase induction (significant), induced no significant cytotoxic effects and these concentrations are below the calculated IC30 of 42.41 and IC50 of 53.57 values, meeting the evaluation criteria for a positive result. The maximal fold induction (Imax) was 2.19 fold. A clear dose response could be observed.
No precipitation was observed at any point during the second test.
Test item in the second test
Criterion for a postive reponse
Statistically significant induction over 1.5-fold yes
Viability ≥ 70 % at lowest concentration with >1.5-fold yes
EC1.5 (µM) 15.50
Clear dose response yes
positive / negative positive
Based on the prediction model and the above described results, the test item was concluded positive in the second test, since conditions for a positive result were met.
Based on the prediction model and the above described results, the test item was concluded positive, since the above-mentioned conditions for a positive result were all met in both tests.
The coefficient of variation (CV%) of the luminescence reading for the negative control DMSO was below 20 % in both tests (7.83 % and 15.50 % respectively). - Interpretation of results:
- other: decision on classification within the CLP Regulation (EC 1272/2008) is based on an evaluation of all available data.
- Conclusions:
- Test item was concluded positive under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).
- Executive summary:
The skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method) according to OECD guideline 442D.
For the test item and positive control substance, in order to derive a prediction two independent tests were conducted, in which the concluded results were concordant.
The luciferase activity induction obtained with the test item was statistically significant above the threshold of 1.5 at the concentration of 7.81 and 15.63 µM in the first test, meeting all acceptance criteria and the criteria for a positive response.
The luciferase activity induction obtained with the test item was statistically significant above the threshold of 1.5 at the concentration of 15.63 and 31.25 µM in the second test, also meeting all acceptance criteria and the criteria for a positive response.
Since the results of the two tests were concordant, two out of the two valid tests were concluded positive for luciferase gene induction and met the acceptance criteria, no repeat tests were needed.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From August 16 to September 06, 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome pathway for Skin Sensitisation)
- Version / remarks:
- adopted in 25 June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
- Details on the study design:
- FORMULATION OF THE TEST ITEM
First, the test item was tried to be formulated in sodium chloride solution (saline) since saline or medium is the first solvent/vehicle options according to the test guideline. Moreover, the Certificate of Analysis of the Test Item states that is soluble in water.
At the concentration of 100 mg/ml partial dissolution of the test item was observed with undissolved particles floating in the solution. Thus a less concentrated solution was tried by going down two-fold. At the concentration of 50 mg/ml the test item formed a clear and homogenous solution with saline.
Thus saline was chosen as the appropriate solvent of the test item. The highest soluble concentration was determined as 50 mg/ml by visual inspection.
CELL OR TEST SYSTEM
- Cell: the THP-1 cell line is an immortalized human monocytic leukemia cell line.
- Supplier: ATCC
- Subculturing: the original cells (TIB-202) were subcultured into prepared cell lines (master cultures-MCs) in the testing laboratory.
DOUBLING TIME AND REACTIVITY CHECK
Prior to using the master cells culture for testing, they were qualified by conducting a doubling time and reactivity check (approximately two weeks after thawing).
Doubling time was monitored and the measured doubling time of approximately 45 hours fell into reference range of the guideline. The generated data was introduced into a historical control database.
Reactivity check of the cells was performed using the positive controls 1 chloro 2,4 dinitrobenzene (DNCB), nickel sulphate (NiSO4) and the negative control lactic acid (LA). The positive controls produced a positive response for both CD86 and CD54 marker expression, while the negative control produced a negative response for both CD86 and CD54 marker expression.
PRELIMINARY TEST
- Preparation of the cells
THP-1 cells were seeded at a density of either 0.1 × 10^6 cells/ml or 0.2 × 10^6 cells/ml and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintance medium at 2 × 10^6 cells/ml. Then, cells were distributed into 24 well flat-bottom plate with 500 µl / wells (1 × 10^6 cells/well).
- Preparation master and working solutions
Master solutions (MS) were prepared with saline as follows: Eight master solutions (eight concentrations) were prepared of the test item stock solution, by two-fold serial dilutions using saline. These master solutions were then further diluted 50 fold into culture medium to obtain the working solutions (WS).
The working solutions were finally used for exposure by adding an equal volume of working solution (500 µl) to the volume of THP-1 cell suspension (500 µl) in the 24-well plate to achieve a further two-fold dilution as the final concentration of the test item.
- Solvent/vehicle control
The solvent/vehicle control used for the test item was maintenance medium.
- Test item exposure
The culture medium or working solutions described above were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well. The treated plates were then incubated for 24 ± 0.5 hours at 37 °C under 5 % CO2. The plates were sealed with breathable microplate covers prior to the incubation to avoid evaporation of test item.
- PI Staining
After 24±0.5 hours of exposure, cells were transferred into sample tubes and 600 μl of FACS buffer was added to each sample. Cells were then collected by centrifugation (250 g, 5 min, 4 ºC). The supernatants were discarded and the remaining cells were washed again with 600 μl of FACS buffer. Finally, cells were resuspended in 400 μl of FACS buffer and 20 μl of 1 × PI solution was added for each sample.
- Cytotoxicity measurement by flow cytometry and estimation of CV75 value
The PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of minimum 10000 living cells (PI negative) were acquired. When the cell viability was low, up to 30000 cells including dead cells were acquired or data of one minute after the initiation of the analysis. Cell viability was analyzed by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.
MAIN TEST
- Test item dilutions
Saline was used to dissolve the test item for the stock solution (SS) in the main tests, as well. The test item was first diluted to the concentration corresponding to the highest soluble concentration determined in the dose finding assay. For the master solutions (MS), 1.2-fold serial dilutions were made from the stock solution using saline (eight concentrations). The master solutions were then further diluted 50-fold into the culture medium to obtain the working solutions (WS). These working solutions were finally used for exposure with a further final two-fold dilution factor in the plate.
- Solvent/vehicle controls
The solvent/vehicle control was prepared as in the dose finding test. Also, in the h CLAT method DMSO is tested as a solvent control for the positive control at a single final concentration in the plate of 0.2 %, so it underwent the same dilution as described for the working solutions.
- Positive control
DNCB was used as the positive control for CD86/CD54 expression measurement at a final nominal concentration of 4.0 μg/ml in the plate. To obtain a 4.0 μg/ml concentration a 2 mg/ml stock solution of DNCB in DMSO were prepared and further diluted 250-fold with culture medium to a 8 μg/ml working solution. The working solution then was diluted 2-fold when added to the cells.
- Application of test item and control substances
Test item and control substances prepared as working solutions (500 μl) were mixed with 500 μl of suspended cells (1 × 10^6 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24±0.5 hours as described for the preliminary test.
- FITC staining
After 24 ± 0.5 hours of exposure, cells were transferred from 24-well plate into sample tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by centrifugation (250 g, 5 min, 4 ºC). The washing step was repeated once more with 1 mL of FACS buffer. After washing, cells were blocked with 600 μl of 1 × blocking solution and incubated at 4°C for 15 min. After blocking, cells were split in three aliquots of 200 μl into sample tubes. After centrifugation (250 g, 5 min, 4 ºC), cells were stained with 50 μl of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at 4 °C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol 158° were used. After washing twice with 150 μl of FACS buffer, cells were resuspended in 400 μl of FACS buffer and 20 μl of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.
- Flow cytometry measurement and RFI determination
The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1. A total of minimum 10000 living cells (PI negative) were acquired. When the cell viability was low, up to 30000 cells including dead cells were acquired or data of one minute after the initiation of the analysis.
The calculated cell viabilities from the isotype control (ctrl) cells (which are stained with mouse IgG1 isotype antibodies) were also noted.
The PI uptake was analysed on channel FL-3. Cell viability was determined by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.
- Optional Effective Concentration determination
For the test items predicted as positive, two Effective Concentration (EC) values were determined: the EC150 for CD86 and EC200 for CD54, the concentrations at which the test items induced a RFI of 150 or 200. These EC values potentially could contribute to the assessment of sensitising potency when used in integrated approaches such as IATA.
ACCEPTANCE CRITERIA
- Requirements for qualified testing
The cell viabilities of medium and solvent/vehicle controls should be higher than 90 %;
In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and cell viability should be more than 50 %;
In the solvent controls, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 > 150 % and CD54 > 200 %) and cell viability should be more than 50 %;
For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105 %.
- Abnormal values
RFI values cannot be less than zero. Regardless of the reason, such values should be omitted from the prediction;
If an abnormal value is observed, check whether there are abnormal conditions in the run and record them in the reporting section.
- Requirement for data acceptance:
For the test item resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90 % (when HSC is used instead of CV75, the data for the test chemical is accepted regardless the cell viability at the highest dose;
For the test item, the cell viability should be more than 50 % in at least four tested concentrations in each run. - Run / experiment:
- other: 3/3
- Parameter:
- other: CD86 expression (%)
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: 3/3
- Parameter:
- other: CD54 expression (%)
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- For the test item, the relative fluorescence intensity (RFI) of CD86 was consequently lower than 150 % at all tested doses (with cell viability > 50 %) in all the three runs.
However the HSC was used and CV 75 could not be determined in the preliminary tests, cell viabilities at the highest doses were lower than 90 %.
The RFI values for CD54 expression were higher than 200 % at the higher tested concentrations in all three independent runs. Cell viability at the highest dose in the third run was lower than 50 % therefore the corresponding RFI value was not considered valid and was excluded from the prediction. All other cell viabilities complied with the acceptance criteria.
Since 3 out of 3 runs were negative for CD86 marker expression and 3 out of 3 runs were positive for CD54 marker expression the overall outcome of the study was positive.
NEGATIVE AND POSITIVE CONTROLS
The positive control gave expected results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50 % in each run.
The DMSO controls had negative outcomes compared to the medium control for both markers in all runs, meaning that the RFI values of both CD86 and CD54 expression was never over the positive criteria.
The cell viabilities of medium and DMSO controls were higher than 90 % in all runs (taken cell viabilities of the IgG1 isotypic control). For medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control was over 105 %.
PRELIMINARY TESTS (DOSE FINDING ASSAYS)
Cell viability did not reach 75 % at any tested concentrations in either of the runs. Since not even the highest soluble concentration (HSC) caused significant reduction in cell viability in either of the runs, no CV75 value could be determined. Therefore, the highest soluble concentration 50 mg/ml, was used for setting the dose-range for measuring CD86 and CD54 expression in the main test.
Eight final concentrations were used for the test item tested of the main test: 1 × HSC (500 µg/ml), 1/1.2 × HSC (417 µg/ml), 1/1.22 × HSC (347 µg/ml), 1/1.23 × HSC (289 µg/ml), 1/1.24 × HSC (241 µg/ml), 1/1.25 × HSC (201 µg/ml), 1/1.26 × HSC (168 µg/ml) and 1/1.27 × HSC (140 µg/ml).
In the second CV75 determination the autofluorescence of the test item was addressed by analyzing the cells without PI staining. It could be concluded that PI staining led to the proper differentiation of living and dead cells and that the extent of the test item autofluorescence did not interfere the cell viability determination. - Interpretation of results:
- other: decision on classification within the CLP Regulation (EC 1272/2008) is based on an evaluation of all available data.
- Conclusions:
- The test item demonstrated an in vitro sensitizing potential under the experimental conditions of human Cell Line Activation Test.
- Executive summary:
The skin sensitization potential of test item was investigate by in vitro test system. The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. CV75 could not be determined, since there was no test item concentration that resulted in 75 % cell viability compared to the solvent/vehicle control. Thus, the highest soluble concentration (50 mg/ml) was used for setting the dose-range for measuring CD86 and CD54 expression in the main test. Eight doses were used in three independent runs between 500 –139 µg/ml.
The increase in CD86 marker expression (RFI) was not equal to or greater than 150 % at any tested dose (with > 50 % of cell viability) compared to the respective negative controls in any of the independent runs. Therefore, all three runs were negative for CD86 marker expression.
The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at several higher concentrations (with > 50 % of cell viability) in all three independent runs. Based on the concordant results of the three individual runs for CD54 expression, prediction was concluded as positive. Thus, the effective concentration for CD54 expression (EC200) was determined by linear interpolation. The EC200 value for CD54 was 207.9 µg/ml.
Since CD54 gave positive result, the overall h-CLAT prediction was concluded positive.
Conclusion
The test item demonstrated anin vitros ensitizing potential under the experimental conditions of human Cell Line Activation Test.
Referenceopen allclose all
Conc. (μg/ml) | Corrected MFI | RFI CD86 | RFI CD54 | Viability | |||
CD86 | CD54 | IgG | CD86 | CD54 | |||
EXPERIMENT I | |||||||
Control | 770 | 243 | 100 | 100 | 93.7 | 95.7 | 95.5 |
DMSO, 0.2 % | 1113 | 342 | 145 | 141 | 92.5 | 94.8 | 95.1 |
DNCB, 4.4 µg/ml | 8267 | 5087 | 743 | 1487 | 62.6 | 64.2 | 65.0 |
Test item, 498 µg/ml | 1033 | 608 | 134 | 250 | 69.6 | 76.8 | 80.4 |
Test item, 415 µg/ml | 930 | 639 | 121 | 263 | 84.5 | 87.9 | 87.5 |
Test item, 346 µg/ml | 819 | 600 | 106 | 247 | 85.6 | 89.6 | 90.0 |
Test item, 288 µg/ml | 885 | 542 | 115 | 223 | 90.1 | 91.6 | 92.6 |
Test item, 240 µg/ml | 942 | 573 | 122 | 236 | 89.3 | 92.3 | 93.9 |
Test item, 200 µg/ml | 930 | 465 | 121 | 191 | 88.3 | 91.7 | 92.0 |
Test item, 167 µg/ml | 846 | 314 | 110 | 129 | 87.8 | 91.9 | 92.9 |
Test item, 139 µg/ml | 863 | 359 | 112 | 148 | 88.2 | 91.6 | 92.9 |
EXPERIMENT II | |||||||
Control | 1409 | 257 | 100 | 100 | 93.2 | 95.5 | 95.2 |
DMSO, 0.2 % | 1348 | 328 | 96 | 128 | 93.8 | 94.9 | 95.7 |
DNCB, 4.2 µg/ml | 8375 | 3567 | 621 | 1088 | 65.9 | 66.7 | 68.2 |
Test item, 499 µg/ml | 639 | 957 | 45 | 372 | 71.5 | 71.5 | 81.9 |
Test item, 416 µg/ml | 628 | 617 | 45 | 240 | 80.6 | 80.3 | 84.2 |
Test item, 347 µg/ml | 627 | 911 | 45 | 354 | 72.0 | 72.6 | 75.9 |
Test item, 289 µg/ml | 893 | 623 | 63 | 242 | 77.9 | 80.8 | 81.8 |
Test item, 241 µg/ml | 942 | 645 | 67 | 251 | 77.7 | 81.8 | 82.4 |
Test item, 201 µg/ml | 1072 | 525 | 76 | 204 | 86.6 | 86.0 | 88.0 |
Test item, 167 µg/ml | 1011 | 498 | 72 | 194 | 86.1 | 88.5 | 89.2 |
Test item, 139 µg/ml | 1032 | 384 | 73 | 149 | 81.6 | 86.4 | 89.5 |
EXPERIMENT III | |||||||
Control | 941 | 935 | 100 | 100 | 96.4 | 94.7 | 96.6 |
DMSO, 0.2 % | 976 | 594 | 104 | 64 | 94.2 | 92.1 | 95.3 |
DNCB, 4.2 µg/ml | 7837 | 2054 | 803 | 346 | 81.7 | 83.0 | 83.3 |
Test item, 499 µg/ml | 375 | 2011 | 40 | 215 | 53.3 | 48.4 | 67.4 |
Test item, 416 µg/ml | 339 | 2012 | 36 | 215 | 57.1 | 56.5 | 74.5 |
Test item, 347 µg/ml | 650 | 1064 | 69 | 114 | 63.6 | 59.4 | 75.4 |
Test item, 289 µg/ml | 653 | 1153 | 69 | 123 | 66.3 | 64.2 | 75.1 |
Test item, 241 µg/ml | 665 | 1334 | 71 | 143 | 65.2 | 64.8 | 76.5 |
Test item, 201 µg/ml | 820 | 1276 | 87 | 136 | 67.7 | 59.6 | 75.2 |
Test item, 167 µg/ml | 896 | 1238 | 95 | 132 | 67.7 | 65.3 | 72.7 |
Test item, 139 µg/ml | 917 | 1386 | 97 | 148 | 58.5 | 61.7 | 67.5 |
Positive and negative control data
sample | Conc. (μg/ml) | MFI (geom mean) | Viability (IgG1) |
MFI ratio of marker to isotype control is >105 % | |||
CD86 | CD54 | isotype | IgG | CD86 | CD54 | ||
control | - | 3113 | 2586 | 2343 | 93.7 | yes | yes |
DMSO | 0.2 % | 3321 | 2550 | 2208 | 92.5 | yes | yes |
control | - | 3664 | 2512 | 2255 | 93.2 | yes | yes |
DMSO | 0.2 % | 3534 | 2514 | 2186 | 93.8 | yes | yes |
control | - | 3435 | 3429 | 2494 | 96.4 | yes | yes |
DMSO | 0.2 % | 3357 | 2975 | 2381 | 94.2 | yes | yes |
Dose finding results
Test dose (µg/ml) | 3.9 | 7.9 | 16 | 31 | 63 | 126 | 252 | 503 |
Viability (%) | 96.8 | 96.2 | 95.4 | 95.1 | 93.1 | 88.2 | 80.2 | 78.5 |
Test dose (µg/ml) | 3.9 | 7.8 | 16 | 31 | 63 | 125 | 250 | 500 |
Viability (%) | 95.6 | 96.7 | 95.7 | 95.6 | 93.3 | 94.3 | 83.3 | 80.3 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
- Additional information:
The evaluation of the skin sensitising potential of test item relies on in vitro studies, evaluated in a weight of evidence approach.
The current knowledge of the chemical and biological mechanisms associated with skin sensitisation has been summarised as an Adverse Outcome Pathway (AOP), starting with the molecular initiating event through intermediate events to the adverse effect, namely allergic contact dermatitis. This AOP focuses on chemicals that react with thiol (i.e.cysteine) and primary amines (i.e. lysine) such as organic chemicals. In this instance, the molecular initiating event (i.e. the first key event) is the covalent binding of electrophilic substances to nucleophilic centres in skin proteins. The second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways. The third key event is the activation of dendritic cells, typically assessed by expression of specific cell surface markers, chemokines and cytokines. The fourth key event is T-cell proliferation. There is currently no validated in vitro study to assess the fourth key event.
An in vitro study was performed according to OECD guideline 442D, to assess the second key event of skin sensitisation. A positive response was found.
A second in vitro study, performed according to OECD guideline 442E, was run to assess the third key event of skin sentisitisation and was found to be positive.
Based on 2 positive responses in in vitro studies, it was deemed as not necessary to run a study to assess the key event 1. Indeed, irrespective of the response in such study, a skin sensitising potential of test substance could not be reasonably excluded.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
According to the CLP Regulation (EC 1272/2008), a skin sensitiser is a substance that leads to an allergic response following repeated skin contact.
Based on positive responses in in vitro studies assessing different key events of skin sensitisation, a skin sensitising potential of test substance could not be excluded.
Accordingly, a classification as skin sensitising, cat. 1 H317, is applied. Based on available data, no categorisation into category 1A or 1B could be done.
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