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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 941-809-7 | CAS number: -
- 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
![](https://www.echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Endpoint summary
Administrative data
Description of key information
- in an in chemico study (DPRA) according to OECD test guideline 442C, GLP, Reliability 1, the test item was found not to react with the peptides and was thus considered as non-sensitiser.
- in an in vitro study conducted according to OECD test guideline 442D using the ARE-Nrf2 Luciferase Test Method, GLP, Reliability 1, the test item induced luciferase activity only at cytotoxic test item concentrations (viability <70 %). The test item was considered as non-sensitiser at non-cytotoxic concentrations.
- In an in vitro study according to the OECD Draft Proposal for a new test guideline, "in vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)", GLP, Reliability 1, the test item did upregulate one cell surface marker (CD54) in at least two independent experiments. Therefore, the test item is considered to be a sensitiser in accordance with UN GHS category 1. Cytotoxicity was observed (CV75: 174.92 ± 24.9 µg/mL).
- in a read-across study based on the mouse local lymphnode assay (LLNA) according to OECD test guideline 429, GLP, Reliability 2, the source substance Sophorolipid C18 unsaturated acid was found to be a non-sensitiser.
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:
- 2016-03-07 to 2016-04-13
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Version / remarks:
- adopted: February 04, 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
- Version / remarks:
- January 12, 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Justification for non-LLNA method:
- The Direct Peptide Reactivity Assay (DPRA) is accepted as a reliable and relevant replacement test for in vivo skin sensitisation testing.
- Specific details on test material used for the study:
- Preparation of the Test Item
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighted into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. - Details on the study design:
- The DPRA is supposed to address the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic model peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.
The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.
This test may be used for the hazard identification of sensitising chemicals in accordance with UN GHS “Category 1”. It does not allow the classification of chemicals to the subcategories 1A and 1B as defined by UN GHS nor predict potency for safety assessment decisions. Therefore, all substances giving a positive result in the DPRA will be classified into UN GHS “Category 1”.
For detailed information on experimental procedure, materials, methods, controls, prediction model and acceptance criteria please see section “any other details on materials and methods incl. tables”. - Positive control results:
- The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 62.90 %.
- Key result
- Run / experiment:
- other: Both Cysteine and Lysine
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 2.03
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Run / experiment:
- other: Cysteine
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 0
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- Values were set to zero due to negative depletion
- Run / experiment:
- other: Lysine
- Parameter:
- other: mean peptide depletion [%]
- Value:
- 4.07
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS: all criteria were fulfilled. For details, please see Tables 10 and 11 in section “any other details on results incl. tables”. - Interpretation of results:
- other: Expert judgement: no indication of sensitisation
- Conclusions:
- In this study under the given conditions the test item showed minimal reactivity towards the peptides. The test item can be considered as “non-sensitizer”. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and will be considered in the context of integrated approach.
- Executive summary:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential
of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP),
namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides
containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine
peptide is used to categorize a substance in one of four reactivity classes to support discrimination
between skin sensitiser and non-sensitisers.
In the present study, the test item was dissolved in water and a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC analysis.
After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine and lysine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1 and 2 of the cysteine run. No centrifugation was necessary to perform the HPLC analysis. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.
After the HPLC run samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1, 2 and 3 of the cysteine runs. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.
Since the turbidity noted for the test item samples was also observed for reference controls, positive controls and standard solutions it can be considered that it is related to the peptide and that it is not a precipitation of the test substance. Additionally, the turbidity did not change during the HPLC analysis period. Since stability of the cysteine peptide in the used acetonitrile batch was demonstrated successfully, the reactivity of the positive control towards the cysteine peptide and peptide depletion were identified correctly and the validity of the cysteine run was acceptable the precipitation was considered as not relevant.
No co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).
The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤6.38 % (2.03 %). Based on the prediction model 1 the test item can be considered as non-sensitiser.
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 62.90 %.
The controls confirmed the validity of the study for both, the cysteine and lysine run. For the cysteine run the coefficient of determination for the calibration curve was >0.99 (0.9995). The mean peptide depletion of the cysteine peptide by the positive control was between 60.8 % and 100 % (72.27 %).
The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and <0.55 mM (RC A: 0.4969 mM, RC C (acetonitrile): 0.4827 mM, RC C (water): 0.4584 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 2.38 %, RC C (acetonitrile): 0.85 %, RC C (water): 9.07 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <14.9 % (PC: 0.90 %; test item 0.00 %).
For the lysine run the coefficient of determination for the calibration curve was >0.99 (0.9996). The mean peptide depletion of the lysine peptide by the positive control was between 40.2 % and 69.0 % (53.53 %). The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and < 0.55 mM (RC A 0.5067 mM, RC C (acetonitrile): 0.5017 mM, RC C water): 0.4930 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 0.54 %, RC C (acetonitrile: 0.61 %, RC C water): 0.54 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <11.6 % (PC: 0.22 %; test item: 0.10 %).
The test item can be considered as non-sensitiser. The data generated with this test will be considered in the context of an integrated approached such as IATA, combining this result with other complementary information.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2016-06-13 to 2016-07-21
- 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:
- adopted: February 04, 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: KeratinoSensTM, EURL ECVAM DB-ALM Protocol No. 155
- Version / remarks:
- July 1st, 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Justification for non-LLNA method:
- The ARE-Nrf2 Luciferase Test Method is accepted as a reliable and relevant replacement test for in vivo skin sensitisation testing. This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as an IATA, combining them with other complementary information e.g., derived from in chemico or in vitro assays addressing other key events of the AOP.
- Details on the study design:
- Skin sensitisation (In vitro test system) ARE-Nrf-2 Luciferase Test Method (KeratinoSens™): The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers represents the second key event of the skin sensitisation process. This in vitro method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its potential to induce the Keap1-Nrf2-ARE signalling pathway by quantifying the luciferase gene expression using luminescence detection.
This test may be used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with UN GHS “Category 1”. It does not allow the classification of chemicals to the subcategories 1A and 1B as defined by UN GHS nor predict potency for safety assessment decisions. Therefore, all substances giving a positive result in the KeratinoSens™ will be classified into UN GHS “Category 1”.
For details on study design, experimental procedure and material and methods please see section “any other information on materials and methods incl. tables”. - Positive control results:
- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.98 (experiment 1), 5.07 (experiment 2); 4.36 (experiment 3)). The calculated EC 1.5 was between 7 and 30 µM (21.66 µM (experiment 1), 14.38 µM (experiment 2); 15.30 µM (experiment 3)). The positive control result is therefore considered valid.
- Run / experiment:
- other: mean of 3 experiments
- Parameter:
- Imax [442D]
- Value:
- 125 µM
- Cell viability:
- < 24%
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: 6.79-fold luciferase induction (average of 3 experiments). Induction of luciferase was observed only at cytotoxic test item concentrations (viability < 70 %) of 125 µM
- Run / experiment:
- other: mean of 3 experiments
- Parameter:
- EC 1.5 [442D]
- Value:
- 70.07 µM
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: induction of luciferase was observed only at cytotoxic test item concentrations (viability < 70 %) of 125 µM
- Run / experiment:
- other: mean of 3 experiments
- Parameter:
- IC30 [442D]
- Value:
- 32.27 µM
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Run / experiment:
- other: mean of 3 experiments
- Parameter:
- IC50 [442D]
- Value:
- 65.12 µM
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS: all criteria were fulfilled. For details, please see Table 7 in section “any other details on results incl. tables”. - Interpretation of results:
- other: expert statement: no indication of sensitisation
- Conclusions:
- In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs at non-cytotoxic concentrations. Therefore, the test item can be considered as non-sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and will be considered in the context of integrated approach.
- Executive summary:
In this study conducted according to OECD test guideline 442D (adopted February 04, 2015), transgenic keratinocytes constitutively expressing an ARE-reporter gene were incubated with Sophorolipids, fermentation products of glucose and fatty acids, at concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0 µM (solvent control) for 48 h at 37 °C. Afterwards the test substance containing medium was removed and the cells lysed and luminescence subsequently measured. Beside the luminescence the cell viability was measured using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method.
The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:
- Imax is > 1.5-fold increased and statistically significant (p < 0.05) compared to the negative control
- cell viability is > 70 % at the lowest concentration with an induction of luciferase activity > 1.5
- EC 1.5 value is < 1000 µM
- an apparent overall dose-response for luciferase induction
In the first experiment, a max luciferase activity (Imax) induction of 7.96 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 21.8 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC1.5 was < 1000 µM (67.67 µM).
In the second experiment, a max luciferase activity (Imax) induction of 4.63 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 24.0 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of I max . The calculated EC1.5 was < 1000 µM (73.70 µM).
In the third experiment, a max luciferase activity (Imax) induction of 7.78 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 18.0 %. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC1.5 was < 1000 µM (68.85 µM).
Since the induction of the luciferase was observed only at cytotoxic test item concentrations (viability < 70 %), the effect cannot be considered for sensitization evaluation. Additionally, no clear dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.98 (experiment 1), 5.07 (experiment 2); 4.36 (experiment 3)). The calculated EC1.5 was between 7 and 30 µM (21.66 µM (experiment 1), 14.38 µM (experiment 2); 15.30 µM (experiment 3)). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (13.9 % (experiment 1), (9.4 % (experiment 2), (14.0 % (experiment 3)).
Under the conditions of this study the test item is therefore considered as non-sensitiser at non-cytotoxic concentrations. The data generated with this method may be not sufficient to conclude definitely on the absence of skin sensitisation potential of the test item and will thus be considered in the context of integrated approach.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2016-03-07 to 2016-04-14
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- OECD draft proposal for a new test guideline for "h-CLAT"
- Qualifier:
- according to guideline
- Guideline:
- other: OECD guidelines for Testing of Chemicals, Draft Proposal for a new test guideline, "in vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)"
- Version / remarks:
- July 2014
- Qualifier:
- according to guideline
- Guideline:
- other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol Nr. 158
- Version / remarks:
- July 1st, 2015
- Principles of method if other than guideline:
- The h-CLAT is supposed to address the third key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the activation of dendritic cells (DC) typically accompanied by expression of specific cell surface markers, chemokines and cytokines. The h-CLAT quantifies the expression of the two surface markers CD86 and CD54 which are considered to be associated with the process of DC activation by using the human monocytic leukemia cell line THP-1 as a surrogate. The expression level of CD86 and CD54 following exposure to test chemicals are used for supporting the discrimination between sensitisers and non-sensitisers.
This test may be used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with UN GHS “Category 1”. It does not allow the classification of chemicals to the subcategories 1A and 1B as defined by UN GHS nor predict potency for safety assessment decisions. Therefore, all substances giving a positive result in the h-CLAT will be classified into UN GHS “Category 1”.
For detailed information on experimental procedure, materials, methods, controls, prediction model and acceptance criteria please see section “any other details on materials and methods incl. tables”. - GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
- Justification for non-LLNA method:
- The correlation of upregulation of immunological relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event in the skin sensitisation process as described by the AOP. This method that measures the markers of DC activation, based on DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals.
This test method is able to detect chemicals that cause skin sensitisation and allows hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP. - Positive control results:
- The positive control (DCNB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150 % for CD86 (252 % in experiment 1 and 351 % in experiment 2) and 200 % for CD54 (431 % in experiment 1 and 772 % in experiment 2) were clearly exceeded.
- Key result
- Run / experiment:
- other: 174.93 µg/mL (1)
- Parameter:
- other: upregulation of CD54 [% control]
- Value:
- 348
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- Corresponding CV was 90.1 %
- Key result
- Run / experiment:
- other: 174.93 µg/mL (2)
- Parameter:
- other: upregulation of CD54 [% control]
- Value:
- 446
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- Corresponding CV was 84.1 %
- Key result
- Run / experiment:
- other: both experiments
- Parameter:
- other: upregulation of CD86 [% control]
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Remarks:
- no dose response observed.
- Run / experiment:
- other: 209.91 µg/mL (1)
- Parameter:
- other: upregulation of CD54 [% control]
- Value:
- 1 476
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: Corresponding CV was 52.5 %
- Remarks:
- cytotoxic effects observed
- Run / experiment:
- other: 209.91 µg/mL (2)
- Parameter:
- other: upregulation of CD54 [% control]
- Value:
- 2 198
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: Corresponding CV was 48.0 %
- Remarks:
- cytotoxic effects observed
- Run / experiment:
- other: mean of two experiments (dose range finding]
- Parameter:
- other: CV75 [µg/mL]
- Value:
- 174.92
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Remarks:
- Lactic acid
- Positive controls validity:
- valid
- Remarks on result:
- other: cytotoxicity was observed
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS: all criteria were fulfilled. For details, please see section “any other details on results incl. tables”. - Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Remarks:
- the result must be considered in the context of an integrated approach
- Conclusions:
- The test item did upregulate one cell surface marker in at least two independent experiments. Therefore, the test item is considered to be a sensitiser in accordance with UN GHS category 1. The data generated in this test should be considered in the context of an integrated approach combining the result with other complementary information derived from other in vitro studies addressing the key events of the skin sensitisation AOP.
- Executive summary:
In an in vitro study according to the OECD Draft Proposal for a new test guideline, "in vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)", the test item was dissolved in 0.9 % NaCl. A CV75 of 174.92 ± 24.9 µg/mL was derived in a dose range finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 209.91, 174.93, 145.77, 121.48, 101.23, 84.36, 70.30 and 58.58 µg/mL.
Cells were incubated with the test item for 24 h at 37 °C. After exposure, cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 52.3 % (CD86), 52.5 % (CD54) and 53.1 (isotype IgG1 control) in the first experiment and to 47.9 % (CD86), 48.0 % (CD54) and 46.2 % (isotype IgG1 control) in the second experiment.
The expression of cell surface marker CD86 was not upregulated above the threshold of 150 % in both independent experiments. No dose response was observed for the induction of CD86.
The expression of the cell surface marker CD54 was upregulated at two concentration steps in the first experiment. The relative expression of CD54 was found to be 1476 % at a concentration of 209.91 µg/mL and 348 % at a concentration of 174.93 µg/mL. The corresponding cell viability was 52.5 % and 90.1 %, respectively, indicating cytotoxic effects triggered by the test item at the highest concentration. Therefore, the upregulation of CD54 at the highest test item concentration should be considered with care.
For the second experiment, upregulated expression of CD54 was observed at a concentration of 209.91 µg/mL (2198 %) and at a concentration of 174.93 µg/mL (446 %). The corresponding cell viability was 48.0 % and 84.1 %, respectively. For the second experiment, the cell viability of the highest tested dose (209.91 µg/mL) should not be considered for evaluation with respect to a sensitising potential since cytotoxicity was below the threshold of ≤50 %. Irrespective of that, the expression of CD54 exceeded the threshold of 200 % in both independent experiments at a concentration of 174.93µg/mL.
The positive control DNCB led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150 % for CD86 (252 % experiment 1; 351 % experiment 2) and 200 % for CD54 (431 % experiment 1; 772 % experiment 2) were clearly exceeded.
In this study, the test item did upregulate one cell surface marker in at least two independent experiments. Therefore, the test item is considered to be a sensitiser in accordance with UN GHS category 1.
The data generated in this test should be considered in the context of an integrated approach combining the result with other complementary information derived from other in vitro studies addressing the key events of the skin sensitisation AOP.
Referenceopen allclose all
Pre-Experiments
Solubility of the test item was determined prior to the main experiment. All test item solutions were freshly prepared immediately prior to use. The test item was not soluble in acetonitrile but completely soluble in water. No turbidity, precipitation and phase separation were observed for the test item solutions. All test item preparations of the main experiment were prepared using water.
Precipitation and Phase Separation
All test item solutions were freshly prepared immediately prior to use.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution.
After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, positive control, RC A, RC B, RC C and for STD 1 and 2. Samples were not centrifuged prior to the HPLC analysis.
After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the any test item samples. Slight phase separation was observed for the positive control and the respective co-elution control.
After the HPLC run samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, positive control, RC A, RC B, RC C and for STD 1, 2 and 3.
After the HPLC run samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the any test item samples. Slight phase separation was observed for the positive control and the respective co-elution control.
Co-elution with the peptide peak
No co-elution of the test item with any of the peptide peaks was observed.
Results Calibration Curve
Table 6: Cysteine and Lysine Values of the Calibration Curve
Sample |
Cysteine Peptide |
Lysine Peptide |
||
Peak Area |
Peptide Concentration [mM] |
Peak Area |
Peptide Concentration [mM] |
|
STD1 |
3495.8242 |
0.5340 |
3675.5410 |
0.5340 |
STD2 |
1805.4337 |
0.2670 |
1867.7395 |
0.2670 |
STD3 |
944.5201 |
0.1335 |
985.2802 |
0.1335 |
STD4 |
466.7887 |
0.0667 |
516.4465 |
0.0667 |
STD5 |
237.0339 |
0.0334 |
282.1445 |
0.0334 |
STD6 |
116.4341 |
0.0167 |
179.8809 |
0.0167 |
STD7 |
0.0000 |
0.0000 |
0.0000 |
0.0000 |
Based on these results, linear regression was performed and the following calibration curves were determined:
Cysteine Peptide Calibration Curve : y = 6546.17x + 26.29 ; R2= 0.9995
Lysine Peptide Calibration Curve : y = 6805.79x + 50.30 ; R2= 0.9996
Results of the Cysteine Peptide Depletion
Table 7: Depletion of the Cysteine Peptide. * Values were set to zero due to negative depletion.
Cysteine Peptide |
||||||
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
906.7542 |
0.1345 |
71.54 |
72.27 |
0.90 |
0.01 |
892.3105 |
0.1323 |
71.99 |
||||
851.7282 |
0.1261 |
73.27 |
||||
Test Item |
3217.3611 |
0.4875 |
0.00* |
0.00 |
0.00 |
- |
3224.2549 |
0.4885 |
0.00* |
||||
3206.4368 |
0.4858 |
0.00* |
Results of the Lysine Peptide Depletion
Table 8: Depletion of the Lysine Peptide
Lysine Peptide |
||||||
Sample |
Peak Area |
Peptide Conc. [mM] |
Peptide Depletion [%] |
Mean Peptide Depletion [%] |
SD of Peptide Depletion [%] |
CV of Peptide Depletion [%] |
Positive Control |
1608.7321 |
0.2290 |
53.57 |
53.53 |
0.22 |
0.00 |
1618.3588 |
0.2304 |
53.29 |
||||
1603.4867 |
0.2282 |
53.72 |
||||
Test Item |
3266.0764 |
0.4725 |
4.10 |
4.07 |
0.10 |
0.02 |
3271.2881 |
0.4733 |
3.95 |
||||
3264.7769 |
0.4723 |
4.14 |
Detailed results about the reference controls can be found in Table 15.
Categorization of the Test Item
Based on the results of the peptide depletion, categorization according to the prediction model was performed. In case that no co-elution was detected, the prediction model based on the combination of cysteine and lysine peptide should be used. Since no co-elution was observed the prediction model of cysteine and lysine was used.
Table 9: Categorization of the Test Item
Predicition Model |
Prediction Model 1 |
Prediction Model 2 |
||||
Test Substance |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Mean Peptide Depletion [%] |
Reactivity Category |
Prediction |
Test Item |
2.03 |
Minimal reactivity |
No sensitiser |
0.00 |
Minimal reactivity |
No sensitiser |
Positive Control |
62.90 |
High reactivity |
sensitizer |
72.27 |
Moderate reactivity |
sensitizer |
Acceptance Criteria
Table 10: Acceptance Criteria for Cysteine Peptide
Cysteine Peptide Run |
|||
Acceptance Criterion |
Range |
Value |
pass/fail |
coefficient of determination |
R2> 0.99 |
0.9995 |
pass |
mean peptide concentration of RC A |
0.45 ≤ x ≤ 0.55 mM |
0.4969 |
pass |
mean peptide concentration of RC C (PC) |
0.45 ≤ x ≤ 0.55 mM |
0.4827 |
pass |
mean peptide concentration of RC C (TI) |
0.45 ≤ x ≤ 0.55 mM |
0.4584 |
pass |
CV of the peak area of RC B |
< 15 % |
2.38 |
pass |
CV of the peak area of RC C (PC) |
< 15 % |
0.85 |
pass |
CV of the peak area of RC C (TI) |
< 15 % |
9.07 |
pass |
mean peptide depletion of the PC |
60.8 % < x < 100 % |
72.27 |
pass |
SD of peptide depletion of the PC replicates |
< 14.9 % |
0.90 |
pass |
SD of peptide depletion of the TI replicates |
< 14.9 % |
0.00 |
pass |
Table 11: Acceptance Criteria for Lysine Peptide
Lysine Peptide Run |
|||
Acceptance Criterion |
Range |
Value |
pass/fail |
coefficient of determination |
R² > 0.99 |
0.9996 |
pass |
mean peptide concentration of RC A |
0.45 ≤ x ≤ 0.55 mM |
0.5067 |
pass |
mean peptide concentration of RC C (PC) |
0.45 ≤ x ≤ 0.55 mM |
0.5017 |
pass |
mean peptide concentration of RC C (TI) |
0.45 ≤ x ≤ 0.55 mM |
0.4930 |
pass |
CV of the peak area of RC B |
< 15 % |
0.54 |
pass |
CV of the peak area of RC C (PC) |
< 15 % |
0.61 |
pass |
CV of the peak area of RC C (TI) |
< 15 % |
0.54 |
pass |
mean peptide depletion of the PC |
40.2 % < x < 69.0 % |
53.53 |
pass |
SD of peptide depletion of the PC replicates |
< 11.6 % |
0.22 |
pass |
SD of peptide depletion of the TI replicates |
< 11.6 % |
0.00 |
pass |
Table 12: Historical Data Cysteine Peptide
Cysteine Peptide |
|||
|
mean |
SD |
N |
linearity of the calibration curve |
0.9991 |
0.0006 |
8 |
mean peptide concentration of reference A [mM] |
0.52 |
0.00 |
8 |
mean peptide concentration of reference C [mM] |
0.50 |
0.00 |
10 |
CV of the peak area of control B [%] |
2.10 |
0.34 |
8 |
CV of the peak area of control C [%] |
1.60 |
0.85 |
10 |
mean peptide depletion of the PC [%] |
74.67 |
2.32 |
8 |
SD of peptide depletion of the PC replicates [%] |
0.84 |
0.72 |
8 |
SD of peptide depletion of the test items [%] |
4.60 |
14.80 |
20 |
Table 13: Historical Data Lysine Peptide
Lysine Peptide |
|||
|
mean |
SD |
N |
linearity of the calibration curve |
0.9998 |
0.0001 |
7 |
mean peptide concentration of reference A [mM] |
0.49 |
0.02 |
7 |
mean peptide concentration of reference C [mM] |
0.49 |
0.24 |
9 |
CV of the peak area of control B [%] |
1.26 |
0.24 |
7 |
CV of the peak area of control C [%] |
0.81 |
0.89 |
9 |
mean peptide depletion of the PC [%] |
59.54 |
6.09 |
7 |
SD of peptide depletion of the PC replicates [%] |
2.58 |
1.90 |
7 |
SD of peptide depletion of the test items [%] |
1.02 |
1.08 |
21 |
Table 14: Exemplary Analysis Sequence
Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7 Run 8 Run 9 Run 10 Run 11 |
STD1 STD2 STD3 STD4 STD5 STD6 SDT7 (DB) Reference Control A, replicate 1 Reference Control A, replicate 2 Reference Control A, replicate 3 |
Run 12 Run 13 |
Co-Elution Control Positive Control Co-Elution Test Item 1 |
Run 14 Run 15 Run 16 |
Reference Control B, replicate 1 Reference Control B, replicate 2 Reference Control B, replicate 3 |
Run 17 Run 18 Run 19 |
Reference Control C, replicate 1 Positive Control, replicate 1 Test Item 1, replicate 1 |
Run 20 Run 21 Run 22 |
Reference Control C, replicate 2 Positive Control, replicate 2 Test Item 1, replicate 2 |
Run 23 Run 24 Run 25 |
Reference Control C, replicate 3 Positive Control, replicate 3 Test Item 1, replicate 3 |
Run 26 Run 27 Run 28 |
Reference Control B, replicate 4 Reference Control B, replicate 5 Reference Control B, replicate 6 |
Table 15: Results of the Reference Controls for the Cysteine Peptide
Cysteine Peptide Run |
|||||||||
Sample |
Peptide Peak Area |
Peptide Concentration [mM] |
|||||||
PA |
Mean |
SD |
CV [%] |
Peptide Concentration |
Mean |
SD |
CV [%] |
||
Reference A 1 |
3295.67 |
3278.9512 |
26.0108 |
0.79 |
0.4994 |
0.4969 |
0.0040 |
0.80 |
|
Reference A 2 |
3292.20 |
0.4989 |
|||||||
Reference A 3 |
3248.98 |
0.4923 |
|||||||
Reference B 1 |
3211.87 |
3162.2388 |
74.5299 |
2.36 |
0.4866 |
0.4791 |
0.0114 |
2.38 |
|
Reference B 2 |
3253.24 |
0.4930 |
|||||||
Reference B 3 |
3222.13 |
0.4882 |
|||||||
Reference B 4 |
3097.92 |
0.4692 |
|||||||
Reference B 5 |
3090.32 |
0.4681 |
|||||||
Reference B 6 |
3097.95 |
0.4692 |
|||||||
Reference C 1 (PC solvent) |
3208.51 |
3186.06 |
26.9566 |
0.85 |
0.4861 |
0.4827 |
0.0041 |
0.85 |
|
Reference C 2 (PC solvent) |
3193.52 |
0.4838 |
|||||||
Reference C 3 (PC solvent) |
3156.16 |
0.4781 |
|||||||
Reference C 1 (TI solvent) |
3205.63 |
3027.02 |
272.2805 |
8.99 |
0.4857 |
0.4584 |
0.0416 |
9.07 |
|
Reference C 2 (TI solvent) |
2713.64 |
0.4105 |
|||||||
Reference C 3 (TI solvent) |
3161.80 |
0.4790 |
Table 16: Results of the Reference Controls for the Lysine Peptide
Lysine Peptide Run |
|||||||||
Sample |
Peptide Peak Area |
Peptide Concentration [mM] |
|||||||
PA |
Mean |
SD |
CV [%] |
Peptide Concentration |
Mean |
SD |
CV [%] |
||
Reference A 1 |
3484.17 |
3498.5079 |
12.6544 |
0.36 |
0.5046 |
0.5067 |
0.0019 |
0.37 |
|
Reference A 2 |
3508.10 |
0.5081 |
|||||||
Reference A 3 |
3503.26 |
0.5074 |
|||||||
Reference B 1 |
3472.14 |
3482.0649 |
18.3933 |
0.53 |
0.5028 |
0.5042 |
0.0027 |
0.54 |
|
Reference B 2 |
3506.05 |
0.5078 |
|||||||
Reference B 3 |
3497.27 |
0.5065 |
|||||||
Reference B 4 |
3473.01 |
0.5029 |
|||||||
Reference B 5 |
3487.77 |
0.5051 |
|||||||
Reference B 6 |
3456.15 |
0.5004 |
|||||||
Reference C 1 (PC solvent) |
3445.89 |
3465.01 |
20.6789 |
0.60 |
0.4989 |
0.5017 |
0.0030 |
0.61 |
|
Reference C 2 (PC solvent) |
3462.18 |
0.5013 |
|||||||
Reference C 3 (PC solvent) |
3486.96 |
0.5050 |
|||||||
Reference C 1 (TI solvent) |
3426.42 |
3405.85 |
18.0704 |
0.53 |
0.4961 |
0.4930 |
0.0027 |
0.54 |
|
Reference C 2 (TI solvent) |
3392.54 |
0.4911 |
|||||||
Reference C 3 (TI solvent) |
3398.60 |
0.4920 |
In the first experiment, a max luciferase activity (Imax) induction of 7.96 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 21.8 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC 1.5 was < 1000 µM (67.67 µM).
In the second experiment, a max luciferase activity (Imax) induction of 4.63 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 24.0 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of I max . The calculated EC 1.5 was < 1000 µM (73.70 µM).
In the third experiment, a max luciferase activity (Imax) induction of 7.78 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 18.0%. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC 1.5 was < 1000 µM (68.85 µM).
Since the induction of the luciferase was observed only at cytotoxic test item concentrations (viability < 70 %), the effect cannot be considered for sensitization evaluation. Additionally, no clear dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
Controls
The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64µM was between 2 and 8 (2.98 (experiment 1), 5.07 (experiment 2); 4.36 (experiment 3)). The calculated EC1.5 was between 7 and 30 µM (21.66 µM (experiment 1), 14.38 µM (experiment 2); 15.30 µM (experiment 3)). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (13.9 % (experiment 1), (9.4 % (experiment 2), (14.0 % (experiment 3)).
Cytotoxicity
Table 1: Results of the Cytotoxicity Measurement
|
Concentration [µM] |
Cell viability [%] |
|||
|
|
Experiment 1 |
Experiment 2 |
Mean |
SD |
Solvent control |
- |
100 |
100 |
100 |
0.0 |
Positive control |
4.00 |
98.1 |
115.2 |
102.3 |
11.3 |
8.00 |
192.2 |
130.5 |
137.7 |
51.3 |
|
16.00 |
98.9 |
136.9 |
112.2 |
21.4 |
|
32.00 |
103.0 |
141.3 |
120.7 |
19.3 |
|
64.00 |
95.8 |
141.8 |
115.8 |
23.6 |
|
Test item |
0.98 |
72.3 |
61.0 |
69.6 |
7.7 |
1.95 |
103.1 |
55.1 |
71.5 |
27.4 |
|
3.91 |
100.2 |
58.7 |
70.5 |
25.9 |
|
7.81 |
102.9 |
56.4 |
70.0 |
28.6 |
|
15.63 |
91.5 |
54.9 |
65.6 |
22.5 |
|
31.25 |
90.5 |
57.1 |
63.7 |
24.3 |
|
62.50 |
70.6 |
70.3 |
64.9 |
9.6 |
|
125.00 |
21.8 |
24.0 |
21.3 |
3.0 |
|
250.00 |
0.2 |
1.7 |
0.7 |
0.9 |
|
500.00 |
0.4 |
0.6 |
0.4 |
0.2 |
|
1000.00 |
0.1 |
0.2 |
0.1 |
0.1 |
|
2000.00 |
0.1 |
0.4 |
0.3 |
0.1 |
Luciferase induction
Table 2: Induction of Luciferase Activity Experiment 1. * = significant induction according to Student’s t-test, p < 0.05.
Experiment 1 |
Concentration [µM] |
Fold induction |
|||||
|
|
Rep. 1 |
Rep. 2 |
Rep. 3 |
Mean |
SD |
|
Solvent control |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.0 |
|
Positive control |
4.00 |
1.23 |
1.19 |
1.00 |
1.14 |
0.12 |
|
8.00 |
1.26 |
1.16 |
1.21 |
1.21 |
0.05 |
|
|
16.00 |
1.48 |
1.40 |
1.36 |
1.36 |
0.14 |
|
|
32.00 |
1.88 |
1.73 |
1.75 |
1.75 * |
0.12 |
|
|
64.00 |
3.38 |
2.52 |
2.98 |
2.98 * |
0.43 |
|
|
Test item |
0.98 |
0.83 |
0.94 |
0.90 |
0.90 |
0.06 |
|
1.95 |
1.14 |
1.10 |
1.09 |
1.09 |
0.05 |
|
|
3.91 |
1.10 |
1.07 |
1.10 |
1.10 |
0.03 |
|
|
7.81 |
1.23 |
1.06 |
1.10 |
1.10 |
0.11 |
|
|
15.63 |
1.06 |
1.41 |
1.16 |
1.16 |
0.22 |
|
|
31.25 |
0.99 |
1.13 |
1.08 |
1.08 |
0.08 |
|
|
62.50 |
0.98 |
0.69 |
0.92 |
0.92 |
0.20 |
|
|
125.00 |
4.95 |
6.85 |
12.08 |
7.96 * |
3.96 |
|
|
250.00 |
0 |
0 |
0 |
0 |
0 |
|
|
500.00 |
0 |
0 |
0 |
0 |
0 |
|
|
1000.00 |
0 |
0 |
0 |
0 |
0 |
|
|
2000.00 |
0 |
0 |
0 |
0 |
0 |
|
Table 3: Induction of Luciferase Activity Experiment 2. * = significant induction according to Student’s t-test, p < 0.05.
Experiment 1 |
Concentration [µM] |
Fold induction |
|||||
|
|
Rep. 1 |
Rep. 2 |
Rep. 3 |
Mean |
SD |
|
Solvent control |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.0 |
|
Positive control |
4.00 |
1.09 |
1.27 |
1.11 |
1.15 |
0.10 |
|
8.00 |
1.34 |
1.37 |
1.20 |
1.30 |
0.09 |
|
|
16.00 |
1.59 |
1.62 |
1.44 |
1.55 * |
0.09 |
|
|
32.00 |
2.50 |
2.31 |
2.05 |
2.29 * |
0.22 |
|
|
64.00 |
5.44 |
5.49 |
4.28 |
5.07 * |
0.68 |
|
|
Test item |
0.98 |
0.90 |
1.15 |
1.02 |
1.02 |
0.13 |
|
1.95 |
1.02 |
1.11 |
0.98 |
1.04 |
0.07 |
|
|
3.91 |
0.89 |
1.02 |
1.00 |
0.97 |
0.07 |
|
|
7.81 |
0.91 |
1.06 |
1.07 |
1.01 |
0.09 |
|
|
15.63 |
0.95 |
1.09 |
0.99 |
1.01 |
0.08 |
|
|
31.25 |
0.85 |
0.99 |
0.92 |
0.92 |
0.07 |
|
|
62.50 |
0.75 |
0.94 |
0.77 |
0.82 |
0.10 |
|
|
125.00 |
4.33 |
5.41 |
4.14 |
4.63 * |
0.68 |
|
|
250.00 |
0 |
0 |
0 |
0 |
0 |
|
|
500.00 |
0 |
0 |
0 |
0 |
0 |
|
|
1000.00 |
0 |
0 |
0 |
0 |
0 |
|
|
2000.00 |
0 |
0 |
0 |
0 |
0 |
|
Table 4: Induction of Luciferase Activity Experiment 3. * = significant induction according to Student’s t-test, p < 0.05.
Experiment 1 |
Concentration [µM] |
Fold induction |
|||||
|
|
Rep. 1 |
Rep. 2 |
Rep. 3 |
Mean |
SD |
|
Solvent control |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.0 |
|
Positive control |
4.00 |
1.07 |
1.17 |
1.15 |
1.13 |
0.05 |
|
8.00 |
1.58 |
1.13 |
1.06 |
1.26 |
0.28 |
|
|
16.00 |
1.57 |
1.70 |
1.30 |
1.52 * |
0.20 |
|
|
32.00 |
1.98 |
2.29 |
1.82 |
2.03 * |
0.24 |
|
|
64.00 |
4.45 |
4.95 |
3.67 |
4.36 * |
0.64 |
|
|
Test item |
0.98 |
0.79 |
1.06 |
1.17 |
1.01 |
0.19 |
|
1.95 |
0.82 |
0.94 |
0.33 |
0.70 |
0.32 |
|
|
3.91 |
0.92 |
1.12 |
0.82 |
0.95 |
0.15 |
|
|
7.81 |
0.82 |
1.09 |
1.03 |
0.98 |
0.14 |
|
|
15.63 |
0.80 |
1.99 |
1.30 |
1.36 |
0.60 |
|
|
31.25 |
0.67 |
0.97 |
0.93 |
0.86 |
0.17 |
|
|
62.50 |
0.70 |
0.83 |
0.84 |
0.79 |
0.08 |
|
|
125.00 |
10.47 |
8.16 |
4.70 |
7.78 * |
2.90 |
|
|
250.00 |
0 |
0 |
0 |
0 |
0 |
|
|
500.00 |
0 |
0 |
0 |
0 |
0 |
|
|
1000.00 |
0 |
0 |
0 |
0 |
0 |
|
|
2000.00 |
0 |
0 |
0 |
0 |
0 |
|
Table 5: Induction of Luciferase Activity - Overall Induction. * = significant induction according to Student’s t-test, p < 0.05.
Overall induction |
Concentration [µM] |
Fold induction
|
||||
|
|
Experiment 1 |
Experiment 2 |
Experiment 3 |
Mean |
SD |
Solvent control |
- |
1.00 |
1.00 |
1.00 |
1.00 |
0.00 |
Positive control |
4.00 |
1.14 |
1.15 |
1.13 |
1.14 |
0.01 |
8.00 |
1.21 |
1.30 |
1.26 |
1.26 |
0.05 |
|
16.00 |
1.36 |
1.55 |
1.52 |
1.48 |
0.10 |
|
32.00 |
1.75 |
2.29 |
2.03 |
2.02 |
0.27 |
|
64.00 |
2.98 |
5.07 |
4.36 |
4.14 |
1.06 |
|
Test item |
0.98 |
0.90 |
1.02 |
1.01 |
0.98 |
0.07 |
1.95 |
1.09 |
1.04 |
0.70 |
0.94 |
0.21 |
|
3.91 |
1.10 |
0.97 |
0.95 |
1.01 |
0.08 |
|
7.81 |
1.10 |
1.01 |
0.98 |
1.03 |
0.06 |
|
15.63 |
1.16 |
1.01 |
1.36 |
1.18 |
0.18 |
|
31.25 |
1.08 |
0.92 |
0.86 |
0.95 |
0.11 |
|
62.50 |
0.92 |
0.82 |
0.79 |
0.84 |
0.07 |
|
125.00 |
7.96 |
4.63 |
7.78 |
6.79 |
1.87 |
|
250.00 |
0 |
0 |
0 |
0 |
0 |
|
500.00 |
0 |
0 |
0 |
0 |
0 |
|
1000.00 |
0 |
0 |
0 |
0 |
0 |
|
2000.00 |
0 |
0 |
0 |
0 |
0 |
Table 6: Additional Parameters; n.a. = not applicable
Parameter |
Experiment 1 |
Experiment 2 |
Experiment 3 |
Mean |
SD |
EC1.5 |
67.67 |
73.70 |
68.85 |
70.07 |
3.19 |
Imax |
7.96 |
4.63 |
7.78 |
6.79 |
1.87 |
IC30 |
63.29 |
n.a. |
1.26 |
32.27 |
43.86 |
IC50 |
88.88 |
89.91 |
16.58 |
65.12 |
42.04 |
Table 7: Acceptance criteria
Criterion |
Range |
Experiment 1 |
Pass/fail |
Experiment 2 |
Pass/fail |
Experiment 3 |
Pass/fail |
CV Solvent Control |
< 20 % |
13.9 |
pass |
9.4 |
pass |
14.0 |
pass |
No. of positive control concentration steps with significant luciferase activity induction > 1.5 |
≥1 |
2.0 |
pass |
3.0 |
pass |
3.0 |
pass |
EC1.5 PC [µM] |
7 < x < 30µM |
21.66 |
pass |
14.38 |
pass |
15.30 |
pass |
Induction PC at 64 µM |
2 < x < 8 |
2.98 |
pass |
5.07 |
pass |
4.36 |
pass |
Table 8: Historical data
Acceptance criterion |
Range |
Mean |
SD |
n |
CV Solvent Control |
< 20 % |
11.5 |
2.4 |
15 |
No. of positive control concentration steps with significant luciferase activity induction > 1.5 |
≥1 |
2.5 |
0.5 |
15 |
EC1.5 PC [µM] |
7 < x < 30µM |
16.96 |
5.56 |
15 |
Induction PC at 64 µM |
2 < x < 8 |
3.98 |
1.02 |
15 |
Reactivity Check of the Cell Stock
Doubling time of the cells was monitored and found to be 40.07 h which is within the doubling time range specified by the manufacturer (35 - 50 h).
Table 1: Results of the Cell Batch Activation Test
Sample |
Concentration |
CD86 |
CD54 |
Activated |
||
Cell Viability [%] |
RFI |
Cell Viability [%] |
RFI |
yes/no |
||
DNCB |
4 µg/mL |
85.1 |
298 |
86.3 |
385 |
Yes |
NiSO4 |
100 µg/mL |
90.5 |
219 |
91.2 |
260 |
Yes |
LA |
1000 µg/mL |
98.2 |
68 |
98.1 |
65 |
No |
Solvent finding
All test item solutions were freshly prepared immediately prior to use. The test item was soluble in 0.9 % NaCl solution (Braun, Lot No.: 140696, 140698, 152418002) at a concentration of 100 mg/mL.
Dose finding assay
The dose finding assay was performed using stock solutions with a concentration of 100 mg/mL.
Table 2: Results of the Dose Finding Assay
|
Experiment 1 |
Experiment 2 |
||
Sample |
Concentration applied [µg/ml] |
Cell Viability [%] |
Concentration applied [µg/ml] |
Cell Viability [%] |
Medium Control (= solvent control) |
0.00 |
98.20 |
0.00 |
97.30 |
Test item |
7.81 |
98.20 |
7.81 |
96.50 |
15.63 |
97.40 |
15.63 |
96.60 |
|
31.25 |
97.20 |
31.25 |
96.90 |
|
62.50 |
97.10 |
62.50 |
96.80 |
|
125.00 |
96.50 |
125.00 |
95.60 |
|
250.00 |
62.00 |
250.00 |
33.50 |
|
500.00 |
0.60 |
500.00 |
0.80 |
|
1000.00 |
11.00 |
1000.00 |
7.20 |
|
Calculated CV75 [µg/mL] |
192.53 |
157.31 |
||
Mean CV75 [µg/mL] |
174.92 |
|||
SD CV75 [µg/mL] |
24.90 |
The mean CV75 was derived from two single runs and was found to be 174.92±24.9µg/mL. Based on the mean CV75, the main experiment was performed covering a concentration range from 209.91 – 58.58µg/mL (20.99 – 5.86 mg/mL stock solution).
Results CD54 and CD86 Expression
For determination of the cell surface markers CD54 and CD86 two independent experiments were performed using separate cultivated cells at passage 18 (first experiment) and passage 19 (second experiment). For each experiment separately weighted samples and preparations were used.
Table 3: CD54 and CD86 Expression Experiment 1
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
CD86 |
CD54 |
||
Medium Control |
- |
97.8 |
97.8 |
97.7 |
1817 |
809 |
546 |
1271 |
263 |
100 |
100 |
333 |
148 |
DMSO Control |
0.20 % |
97.6 |
97.9 |
97.8 |
2041 |
829 |
484 |
1557 |
345 |
123 |
131 |
422 |
171 |
DNCB |
4.00 |
84.7 |
84.6 |
83.4 |
4555 |
2114 |
627 |
3928 |
1487 |
252 |
431 |
726 |
337 |
Test item |
209.91 |
52.3 |
52.5 |
53.1 |
1803 |
4546 |
665 |
1138 |
3881 |
90 |
1476 |
271 |
684 |
174.93 |
90.4 |
90.1 |
92.3 |
1856 |
1462 |
548 |
1308 |
914 |
103 |
348 |
339 |
267 |
|
145.77 |
95.0 |
95.0 |
94.9 |
1616 |
948 |
523 |
1093 |
425 |
86 |
162 |
309 |
181 |
|
121.48 |
96.6 |
96.7 |
96.8 |
1534 |
833 |
526 |
1008 |
307 |
79 |
117 |
292 |
158 |
|
101.23 |
96.8 |
96.1 |
96.8 |
1370 |
763 |
514 |
856 |
249 |
67 |
95 |
267 |
148 |
|
84.36 |
97.4 |
97.3 |
97.4 |
1400 |
777 |
521 |
879 |
256 |
69 |
97 |
269 |
149 |
|
70.30 |
97.3 |
97.2 |
96.9 |
1429 |
820 |
525 |
904 |
295 |
71 |
112 |
272 |
156 |
|
58.58 |
97.7 |
97.2 |
97.5 |
1444 |
795 |
537 |
907 |
258 |
71 |
98 |
269 |
148 |
Table 4: CD54 and CD86 Expression Experiment 2
Sample |
Conc. |
Cell Viability [%] |
Mean Fluorescence Intensity |
corrected Mean Fluorescence Intensity |
Relative Flourescence Intensity (RFI) |
Ratio Isotype IgG1 to [%] |
|||||||
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
Isotype IgG1 |
CD86 |
CD54 |
CD86 |
CD54 |
C86 |
CD54 |
||
Medium Control |
- |
98.1 |
98.1 |
98.4 |
1117 |
617 |
498 |
619 |
119 |
100 |
100 |
224 |
124 |
DMSO Control |
0.20 % |
98.1 |
98.3 |
98.3 |
1190 |
633 |
483 |
707 |
150 |
114 |
126 |
246 |
131 |
DNCB |
4.0 |
73.4 |
72.3 |
72.9 |
3097 |
1770 |
612 |
2485 |
1158 |
351 |
772 |
506 |
289 |
Test item |
209.91 |
47.9 |
48.0 |
46.2 |
1183 |
3316 |
700 |
483 |
2616 |
78 |
2198 |
169 |
474 |
174.93 |
84.1 |
84.1 |
83.9 |
1140 |
1064 |
533 |
607 |
531 |
98 |
446 |
214 |
200 |
|
145.77 |
94.1 |
94.3 |
94.4 |
1013 |
689 |
478 |
535 |
211 |
86 |
177 |
212 |
144 |
|
121.48 |
95.4 |
95.4 |
96.0 |
875 |
634 |
476 |
399 |
158 |
64 |
133 |
184 |
133 |
|
101.23 |
97.6 |
97.2 |
97.2 |
960 |
617 |
491 |
469 |
126 |
76 |
106 |
196 |
126 |
|
84.36 |
97.4 |
97.4 |
97.8 |
930 |
620 |
484 |
446 |
135 |
72 |
114 |
192 |
128 |
|
70.30 |
97.7 |
97.9 |
97.8 |
932 |
615 |
489 |
443 |
126 |
72 |
106 |
191 |
126 |
|
58.58 |
98.0 |
98.0 |
97.9 |
953 |
622 |
487 |
466 |
135 |
75 |
113 |
196 |
128 |
Table 5: Acceptance criteria
Acceptance criterion |
range |
Experiment 1 |
pass/fail |
Experiment 2 |
pass/fail |
||||
cell viability solvent controls [%] |
>90 |
97.6 – 97.9 |
|
|
pass |
|
98.1 – 98.4 |
|
pass |
number of test dosed with viability >50% CD86 |
≥4 |
8 |
pass |
7 |
pass |
||||
number of test dosed with viability >50% CD54 |
≥4 |
8 |
pass |
7 |
pass |
||||
number of test dosed with viability >50% IgG1 |
≥4 |
8 |
pass |
7 |
pass |
||||
RFI of positive control of CD86 |
≥150 |
252 |
pass |
351 |
pass |
||||
RFI of positive control of CD54 |
≥200 |
431 |
pass |
772 |
pass |
||||
RFI of solvent control of CD86 |
<150 |
123 |
pass |
114 |
pass |
||||
RFI of solvent control of CD54 |
<200 |
131 |
pass |
126 |
pass |
||||
MFI ratio IgG1/CD86 for medium control [%] |
>105 |
333 |
pass |
224 |
pass |
||||
MFI ratio IgG1/CD86 for solvent control [%] |
>105 |
422 |
pass |
246 |
pass |
||||
MFI ratio IgG1/CD54 for medium control [%] |
>105 |
148 |
pass |
124 |
pass |
||||
MFI ratio IgG1/CD54 for solvent control [%] |
>105 |
171 |
pass |
|
pass |
||||
Table 6: Historical data
Criterion |
mean |
SD |
N |
cell viability solvent controls [%] |
94.7 |
3.2 |
70 |
number of test doses with viability >50 % |
- |
- |
242 |
RFI of positive control of CD86 |
292.8 |
136.2 |
11 |
RFI of positive control of CD54 |
386.0 |
75.4 |
11 |
RFI of solvent control of CD86 |
109.3 |
16.2 |
10 |
RFI of solvent control of CD54 |
121.8 |
15.2 |
10 |
MFI ratio IgG1/CD86 for medium control [%] |
224.0 |
77.9 |
12 |
MFI ratio IgG1/CD86 for DMSO control [%] |
146.3 |
12.9 |
12 |
MFI ratio IgG1/CD54 for medium control [%] |
242.5 |
89.6 |
12 |
MFI ratio IgG1/CD54 for DMSO control [%] |
156.4 |
14.2 |
12 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
DPRA
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitiser and non-sensitisers.
In the present study, the test item was dissolved in water and a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC analysis.
After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine and lysine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1 and 2 of the cysteine run. No centrifugation was necessary to perform the HPLC analysis. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.
After the HPLC run samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1, 2 and 3 of the cysteine runs. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.
Since the turbidity noted for the test item samples was also observed for reference controls, positive controls and standard solutions it can be considered that it is related to the peptide and that it is not a precipitation of the test substance. Additionally, the turbidity did not change during the HPLC analysis period. Since stability of the cysteine peptide in the used acetonitrile batch was demonstrated successfully, the reactivity of the positive control towards the cysteine peptide and peptide depletion were identified correctly and the validity of the cysteine run was acceptable the precipitation was considered as not relevant.
No co-elution of test item with the peptide peaks was observed. Sensitizing potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).
The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤6.38 % (2.03 %). Based on the prediction model 1 the test item can be considered as non-sensitiser.
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 62.90 %.
The controls confirmed the validity of the study for both, the cysteine and lysine run. For the cysteine run the coefficient of determination for the calibration curve was >0.99 (0.9995). The mean peptide depletion of the cysteine peptide by the positive control was between 60.8 % and 100 % (72.27 %).
The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and <0.55 mM (RC A: 0.4969 mM, RC C (acetonitrile): 0.4827 mM, RC C (water): 0.4584 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 2.38 %, RC C (acetonitrile): 0.85 %, RC C (water): 9.07 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <14.9 % (PC: 0.90 %; test item 0.00 %).
For the lysine run the coefficient of determination for the calibration curve was >0.99 (0.9996). The mean peptide depletion of the lysine peptide by the positive control was between 40.2 % and 69.0 % (53.53 %). The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and < 0.55 mM (RC A 0.5067 mM, RC C (acetonitrile): 0.5017 mM, RC C water): 0.4930 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 0.54 %, RC C (acetonitrile: 0.61 %, RC C water): 0.54 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <11.6 % (PC: 0.22 %; test item: 0.10 %).
The test item can be considered as non-sensitiser in the DPRA.
KeratinoSens assay
In a study conducted according to OECD test guideline 442D (adopted February 04, 2015), transgenic keratinocytes constitutively expressing an ARE-reporter gene were incubated with Sophorolipids, fermentation products of glucose and fatty acids, at concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0 µM (solvent control) for 48 h at 37 °C. Afterwards the test substance containing medium was removed and the cells lysed and luminescence subsequently measured. Beside the luminescence the cell viability was measured using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method.
The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:
- Imax is > 1.5-fold increased and statistically significant (p < 0.05) compared to the negative control
- cell viability is > 70 % at the lowest concentration with an induction of luciferase activity > 1.5
- EC 1.5 value is < 1000 µM
- an apparent overall dose-response for luciferase induction
In the first experiment, a max luciferase activity (Imax) induction of 7.96 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 21.8 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC1.5 was < 1000 µM (67.67 µM).
In the second experiment, a max luciferase activity (Imax) induction of 4.63 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 24.0 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of I max. The calculated EC1.5 was < 1000 µM (73.70 µM).
In the third experiment, a max luciferase activity (Imax) induction of 7.78 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 18.0 %. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC1.5 was < 1000 µM (68.85 µM).
Since the induction of the luciferase was observed only at cytotoxic test item concentrations (viability < 70 %), the effect cannot be considered for sensitization evaluation. Additionally, no clear dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.98 (experiment 1), 5.07 (experiment 2); 4.36 (experiment 3)). The calculated EC1.5 was between 7 and 30 µM (21.66 µM (experiment 1), 14.38 µM (experiment 2); 15.30 µM (experiment 3)). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (13.9 % (experiment 1), (9.4 % (experiment 2), (14.0 % (experiment 3)).
Under the conditions of this study the test item is therefore considered as non-sensitiser at non-cytotoxic concentrations.
h-CLAT
In an in vitro study according to the OECD Draft Proposal for a new test guideline, "in vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)", the test item was dissolved in 0.9 % NaCl. A CV75 of 174.92 ± 24.9 µg/mL was derived in a dose range finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 209.91, 174.93, 145.77, 121.48, 101.23, 84.36, 70.30 and 58.58 µg/mL.
Cells were incubated with the test item for 24 h at 37 °C. After exposure, cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.
Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 52.3 % (CD86), 52.5 % (CD54) and 53.1 (isotype IgG1 control) in the first experiment and to 47.9 % (CD86), 48.0 % (CD54) and 46.2 % (isotype IgG1 control) in the second experiment.
The expression of cell surface marker CD86 was not upregulated above the threshold of 150 % in both independent experiments. No dose response was observed for the induction of CD86.
The expression of the cell surface marker CD54 was upregulated at two concentration steps in the first experiment. The relative expression of CD54 was found to be 1476 % at a concentration of 209.91 µg/mL and 348 % at a concentration of 174.93 µg/mL. The corresponding cell viability was 52.5 % and 90.1 %, respectively, indicating cytotoxic effects triggered by the test item at the highest concentration. Therefore, the upregulation of CD54 at the highest test item concentration should be considered with care.
For the second experiment, upregulated expression of CD54 was observed at a concentration of 209.91 µg/mL (2198 %) and at a concentration of 174.93 µg/mL (446 %). The corresponding cell viability was 48.0 % and 84.1 %, respectively. For the second experiment, the cell viability of the highest tested dose (209.91 µg/mL) should not be considered for evaluation with respect to a sensitising potential since cytotoxicity was below the threshold of ≤50 %. Irrespective of that, the expression of CD54 exceeded the threshold of 200 % in both independent experiments at a concentration of 174.93 µg/mL.
The positive control DNCB led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150 % for CD86 (252 % experiment 1; 351 % experiment 2) and 200 % for CD54 (431 % experiment 1; 772 % experiment 2) were clearly exceeded.
In this study, the test item did upregulate one cell surface marker in at least two independent experiments. Therefore, the test item is considered to be a sensitiser in accordance with UN GHS category 1.
Further supporting data are available. In a dermal sensitisation study with a source substance, groups of 4 female CBA mice were tested using the LLNA method according to OECD Guideline 429, 24 April 2002. The source substance was considered to be a non-sensitiser under the conditions of the test.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
- Additional information:
There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfiled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account.
Justification for classification or non-classification
The test item “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed”, is considered to be a non-sensitiser. This decision is based on the following experimental approach:
Two out of three reliable in chemico and in vitro studies gave no indication of a sensitising potential of “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed”. Additionally, in a read-across study based on the mouse local lymphnode assay (LLNA), the source substance Sophorolipid C18 unsaturated acid was found to be a non-sensitiser.
The DPRA as well as the ARE-Nrf2 Luciferase Test Method both clearly lead to the result that the test item is a non-sensitiser. In the third study (h-CLAT), the test item did upregulate one cell surface marker in at least two independent experiments and was considered to be a sensitiser in accordance with UN GHS category 1. Cytotoxicity of the test item was observed (CV75: 174.92 ± 24.9 µg/mL).
The Direct Peptide Reactivity Assay (DPRA), the ARE-Nrf2 Luciferase Test Method and the in vitro human cell line activation test (h-CLAT) are validated test methods for the assessment of skin sensitisation which have not been developed as stand-alone test methods, but to be used in a Weight-of-Evidence approach. When used in an AOP-based IATA, the outcome of these studies targets key events along the defined toxicity pathway and the results enable a regulatory decision. The AOP “2 out of 3” approach predicts skin sensitization hazard by sequential testing in up to three internationally accepted non-animal methods that map to key events 1-3 of the AOP (ENV/JM/MONO(2016)67, ENV/JM/MONO(2016)29). The overall result is based on the two concordant findings.
In this AOP-based IATA, no indication of skin sensitisation was found regarding key event 1 and 2 tested with the DPRA and the ARE-Nrf2 Luciferase Method. Even though the third assay (h-CLAT) lead to a positive result, the criteria of the “2 out of 3” approach are fulfilled, and a regulatory decision can be made. Besides, it must be taken into consideration that the test item showed pronounced cytotoxicity in the h-CLAT assay which may render these results less reliable. Furthermore, related scientific literature supports the higher relevance of DPRA results- which were clearly negative here- for exploration of the AOP as compared to other tests (Asturiol et al. 2016; Benigni et al. 2016).
Taken together, the test item “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed” is considered to be a non-sensitiser according to the AOP “2 out of 3” approach and a supporting read-across study.
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.
![ECHA](/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/echa_logo.png)