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EC number: 218-638-5 | CAS number: 2210-25-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Description of key information
In-silico assessment: Taking into account the prediction results on skin sensitizing, toxicokinetic and physicochemical properties there is an evidence that N-isopropylacryamide may be a low skin sensitizer.
Invitro-DPRA study: Solutions of N-isopropylacrylamide were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall mean result of 6.15% depletion places N-isopropylacrylamide in the reactivity class of “minimal” and hence it is predicted by DPRA not to be a skin sensitizer.
Invitro KeratinoSens study: In this study, N-isopropylacrylamide was classified as a Negative using the KeratinoSens prediction model.
Invitro h-CLAT study: For N-isopropylacrylamide the CV75 was not determined and based on 2/3 independent repetitions, the thresholds of CD54 and CD86 expression were not crossed and therefore, N-isopropylacrylamide was classified as Negative as per the prediction model.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation, other
- Remarks:
- Prediction and Evaluation of the Skin Sensitisation Potential of N isoprpylacrylamide (in silico assessment)
- Type of information:
- (Q)SAR
- Adequacy of study:
- key study
- Study period:
- 24 October 2017
- Reliability:
- 1 (reliable without restriction)
- Justification for type of information:
- A range of different software and models were used please review the results and discussion section for full justification.
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- Before to start any new testing an assessment of all available in vitro data, in vivo data, historical human data, data from valid (Q)SARs and data from structurally related substances (read-across approach) is requested. The screened databases are related with the substance identification, physicochemical and toxicological properties (ChemIDplus, Chemspider, SRC's Fate Pointer File, ECHA, EFSA and TOXLINE).
- GLP compliance:
- no
- Key result
- Run / experiment:
- other: Skin sensitization modelling of N-isopropylacrylamide (CAS: 2210-25-5)
- Parameter:
- other:
- Remarks on result:
- positive indication of skin sensitisation
- Interpretation of results:
- other: Results should be considered with other in-vitro studies to classify for skin sensitisation.
- Conclusions:
- Taking into account the prediction results on skin sensitizing, toxicokinetic and physicochemical properties there is an evidence that N-isopropylacryamide may be a low skin sensitizer.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 28th August 2018- 29th August 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: Human Cell Line Activation Test (h-CLAT) method as detailed in OECD TG 442E (adopted 27JUN18)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
- Justification for non-LLNA method:
- OECD TG 442E cites the h-CLAT model using THP-1 cells as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.
- Details on the study design:
- Skin sensitisers have been reported to induce the expression of cell membrane markers associated with Dendritic Cell (DC) activation. The h-CLAT method is an in vitro assay that quantifies these changes in cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells (a cell line that mimics DCs), following 24-hour exposure to the test item. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface maker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.
Solubility was first determined for the test item using either culture medium (RPMI 1640) or DMSO. Note that for this method, test items with a Log Kow (octanol/water partition coefficient) of up to 3.5 have been tested successfully. However, test items with a Log Kow of greater than 3.5 can still be tested at lower soluble concentrations. In such a case, a negative result (non-sensitiser) should be considered inconclusive, whereas a positive result (sensitiser) could still be considered valid.
THP-1 cells were pre-cultured for 72hrs. Following this, the cells were dosed with the test item over an 8 dose range and incubated for 24 ±0.5hrs. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The dose of test item that yields 75% cell viability (CV75) was calculated and taken forward for the next stage of testing. This dose finding assay was carried out over two independent runs.
THP-1 cells were pre-cultured 72hrs. Cell viability was not reduced below 75% by the test item during the CV75 assessment, and so a narrower dilution series was produced for the test item with the top concentration at the possible dose for the assay (1 mg/ml in DMSO). This dilution range was used to dose the cells again for 24 ±0.5hrs. The cells were then washed and stained with propidium iodide and also with antibodies that detect CD54 and CD86 expression as well as a negative control antibody. This allowed for discrimination of live/dead cells and also changes in CD54 and CD86 marker expression by flow cytometry. - Key result
- Run / experiment:
- other: 1
- Parameter:
- other: CD54/86 expression
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: 2
- Parameter:
- other: CD54/86 expression
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Run / experiment:
- other: 3
- Parameter:
- other: CD54/86 expression
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Other effects / acceptance of results:
- CV75 Acceptance Criteria
•Cell viability must be ≥ 75% at the lowest dose.
•The highest test item concentration should produce cytotoxicity (< 90% cell viability) unless 5mg/ml in medium, 1mg/ml in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item.
CD54/CD86 Expression: Run acceptance criteria
•Cell viability of medium and DMSO controls should be greater than 90%
•In the positive control (Nickel Sulphate; 100µg/ml), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥ 150 and CD54 ≥ 200) and cell viability should be > 50%
•In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150 and CD54 ≥ 200)
•For both medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%
CD54/CD86 Expression: Data acceptance criteria
•For a test item resulting in a negative value (i.e. Non-Sensitiser), the cell viability at the 1.2 x CV75 value should be less than 90%
•For a test item resulting in a positive value (i.e. Sensitiser), a cell viability at the 1.2 x CV75 value of more than 90% is considered acceptable
•When test items in RPMI are tested at 5mg/ml or test items in DMSO are tested at 1mg/ml or the highest soluble concentration is used as the maximal dose instead of the CV75 based dose, the data for the test item are accepted regardless of the cell viability
•Cell viability of at least 4 doses in each assay should be > 50%.
Abnormal Values
•RFI values cannot be less than zero. Such values should be excluded from the prediction.
•If an abnormal value such as strongly induced CD54 or CD86 expression at only one non-cytotoxic concentration is observed, this should be checked to determine if there were any abnormal events/conditions in the run and the details recorded.
Prediction Model
Number of runs required for prediction
At least two independent runs need to be performed to derive a final prediction. If a more precisely derived EC value is required, three independent runs should be performed. If 2 runs are carried out the higher of the EC values is taken as the final value. If 3 runs are carried out, then the median EC value is taken as the final result. Up to 6 total runs, meeting the “Run acceptance criteria”, may be performed in order to reach a conclusion for each test item. The six runs may include runs for which the “Data acceptance criteria” are not met for this test item e.g. Run 1: invalid, 2: valid, 3: invalid, 4: invalid, 5: valid and 6: invalid. If no prediction can be made after the sixth run the result is inconclusive and the test item is to be classified accordingly.
Prediction Model Criteria
If the RFI of CD86 is equal to or greater than 150% at any tested dose (≥ 50% cell viability) in at least 2 independent runs, AND/OR the RFI of CD54 is equal to or greater than 200% at any tested dose (≥ 50% cell viability) in at least 2 independent runs, the test item prediction is considered Positive (Sensitiser). Otherwise the result is considered as Negative (Non-Sensitiser). In case the first 2 runs are not concordant, a third run needs to be performed and the final prediction will be based on the mode of the conclusions from the three individual runs (i.e. 2 out of 3).
Maximal Doses and the prediction model
When test items in RPMI are tested at 5mg/ml or test items in DMSO are tested at 1mg/ml or the highest soluble concentration is used as the maximal dose instead of the CV75 based dose and the data for the test item does not meet the positive criteria for the prediction model without affecting the cytotoxicity at all tested doses, the test item prediction should be considered as negative. - Interpretation of results:
- GHS criteria not met
- Conclusions:
- For N-isopropylacrylamide the CV75 was not determined and based on 2/3 independent repetitions, the thresholds of CD54 and CD86 expression were not crossed and therefore, N-isopropylacrylamide was classified as Negative as per the prediction model.
- Executive summary:
In this study, the skin sensitisation potential of N-isopropylacrylamide was assessed using theIn Vitrohuman Cell Line Activation Test (h-CLAT) method according to OECD Test Guideline 442E. After a 24h incubation with the test item the expression of cell surface markers CD54 and CD86 on THP-1 cells was measured by flow cytometry.
In the CV75 assessment, the cell viability was not reduced below 75% by the test item and therefore the highest possible dose (1mg/ml) was taken forward as the top dose for the CD54/86 expression measurements. RFI values did not cross the sensitisation threshold for either CD54 or CD86 in 2/3 repetitions and therefore the response for N-isopropylacrylamide was classified as Negative (Non-Sensitiser) as per the prediction model.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 27 March 2018 - 04 April 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- direct peptide reactivity assay (DPRA)
- Details on the study design:
- Skin sensitisation (In vitro test system) - Details on study design:
The DPRA is a chemistry-based assay (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) exploiting the fact that most chemical allergens have electrophilic properties and are therefore able to react with the nucleophilic sidechains of amino acids to form covalent bonds. The underlying rationale of the assay is that if a chemical is capable of reacting with proteins then it has the potential to act as a sensitizer. The endpoint measured in the assay is the percentage depletion over time of two synthetic peptides (containing respectively a cysteine and a lysine amino acid) from the peptide mixtures following an approximate 24 hour (22-26 hours) incubation with the test item. The percentage of peptide depletion is calculated by High Performance Liquid Chromatography using ultra-violet detection.
The DPRA test allows quantification of a chemical’s reactivity and is used to categorize a substance in one of four classes of reactivity to allow discriminating between skin sensitizing and non-sensitizing chemicals and thus assesses their sensitization potential.
Assessment of Test Item Solubility
The solubility of N-isopropylacrylamide was assessed at a concentration of 100 mM in a selection of solvents.
Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).
Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.
Preparation of Stability Controls and Precision Controls
Stability controls (Reference Control B) and precision controls (Reference control A) of both peptides were prepared at a concentration of 0.5 mM.
Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of N-isopropylacrylamide was prepared in acetonitrile.
Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Accurate volume aliquots of N-isopropylacrylamide and the positive control were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of either N-isopropylacrylamide or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.
Preparation of Positive Control and Lysine Peptide Depletion Samples and Co elution Controls
Accurate volume aliquots of N-isopropylacrylamide and the positive control were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of either N-isopropylacrylamide or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.
Incubation
The appearance of the N-isopropylacrylamide and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection of the samples as part of analytical run. Before initiation of the run the appearance of the samples in the vials was assessed and documented again.
Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of
N-isopropylacrylamide and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section. - Positive control results:
- Acceptance criteria for positive control Positive control depletion (%):
Cysteine - 60.8-100 (SD <14.9%)
Lysine - 40.2-69.0 (SD <11.6%)
Achieved results (%):
Cysteine - 72.0 (SD, 0.11%, n=3)
Lysine - 56.1 (SD, 0.64%, n=3) - Key result
- Run / experiment:
- mean
- Parameter:
- other: Cysteine peptide depletion (%)
- Value:
- 9.94
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: Lysine
- Parameter:
- other: Lysine peptide depletion (%)
- Value:
- 2.36
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- mean
- Parameter:
- other: Overall mean depletion (%)
- Value:
- 6.15
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- 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 specified
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not measured
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: n/a - Interpretation of results:
- GHS criteria not met
- Conclusions:
- Solutions of N-isopropylacrylamide were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall mean result of 6.15% depletion places N-isopropylacrylamide in the reactivity class of “minimal” and hence it is predicted by DPRA not to be a skin sensitizer.
- Executive summary:
The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of N-isopropylacrylamide.
Solutions of N-isopropylacrylamide were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The mean depletion result of 6.15% places N-isopropylacrylamide in the reactivity class of minimal and therefore it is predicted by DPRA to be a non-skin sensitizer.
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 23rd April 2018-18th May 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
- Justification for non-LLNA method:
- Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). The KeratinoSensTM test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSensTM test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by the UN GHS, for authorities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework a positive result may be used on its own to classify a chemical into UN GHS category 1.
- Specific details on test material used for the study:
- Batch: 2161004
Purity: 99.41%
Storage: Romm temperature - Details on the study design:
- Description of the test system
The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.
Method of administration of test item
Per plate, a single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of 1% DMSO. The top concentration was previously determined by solubility testing.
Method of administration of reference items
Per plate, a single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of 1% DMSO and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).
Exposure times of test items and reference items
Cells were incubated with the test or reference item for 48 ± 2h prior to endpoint measurements.
Number of repetitions
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence (n=9 overall) and 2 x 96-well plates for MTT (n=6 overall). The validity of each repetition was assessed following acceptance criteria described in section 12.1.
Study Design
Details of materials, reagents and equipment used are recorded in the study data. - Positive control results:
- Cinnamic aldehyde (mean of 3 repetitions) acceptance criteria:
Induction ≥ 1.5 fold in at least concentration? --> Yes
Average induction of Cinnamic aldehyde at 32 µM is 1.6-3.0? --> Yes (2.938)
EC1.5 value is 6-39µM? --> Yes (13.569)
CV% of blank values < 20%? --> Yes (15.736) - Key result
- Run / experiment:
- other: Repetition 1
- Parameter:
- other:
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this study, N-isopropylacrylamide was classified as a Negative using the KeratinoSens prediction model.
- Executive summary:
The human skin sensitisation potential of N-isopropylacrylamide was assessed using the validated in vitro method, the KeratinoSensTM assay, adapted to animal product-free conditions by XCellR8,and validated in-house to determine keratinocyte activation.
After 48h exposure of cells with 12 concentrations of N-isopropylacrylamide, Luciferase measurements and MTT viability testing were performed.
N-isopropylacrylamide was classified as Negative according to the KeratinoSens prediction model.
Referenceopen allclose all
Skin sensitization modelling of N-isopropylacrylamide (CAS: 2210-25-5)
Model |
Prediction result |
Reliability (model statistics) |
TOPKAT |
pos |
Low (probability: 0.74) |
CAESAR (VEGA) |
pos |
Moderate (AD index: 0.749) |
DEREK |
alfa,beta-unsaturated amide |
Equivocal |
Toxtree |
alert for Michael acceptor |
Restricted on 5 skin protein binding principles |
Danish (Q)SAR Database |
|
|
CASE Ultra |
pos |
Outside applicability domain |
Leadscope |
neg |
Outside applicability domain |
SciQSAR |
inc |
Outside applicability domain |
OECD QSAR Toolbox |
|
|
Protein binding alerts for skin sensitization by OASIS v1.4 |
Michael addition |
Restricted on 11 mechanistic domains |
Protein binding potency h-CLAT |
no alert |
Third key event for skin sensitization: dendritic cell activation |
Read-across |
weak sensitizer |
p-value: 0.00107 |
Acceptance criteria for all controls and the test item were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression.
CV75 Determination |
|||
Criterion |
Run 1 |
Run 2 |
Outcome |
Cell viability must be ≥ 75% at the lowest dose. |
97.87 |
98.13 |
PASS |
The highest test item concentration should produce cytotoxicity (< 90% cell viability) unless5mg/ml in medium, 1mg/ml in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item. |
96.94 |
97.08 |
PASS *Accepted because 1 mg/ml DMSO was used |
|
Measurement of CD54 and CD86 Expression |
||||
Criterion |
Run 1 |
Run 2 |
Run 3 |
Outcome |
|
Cell viabilities of medium and solvent controls should be higher than 90% |
Medium: 96.94 DMSO: 97.90 |
Medium: 98.69 DMSO: 98.62 |
Medium: 98.45 DMSO: 98.40 |
PASS |
|
In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) compared to the medium control |
CD54: 95 CD86: 90 |
CD54: 88 CD86: 99 |
CD54: 90 CD86: 88 |
PASS |
|
For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105% |
Medium: CD54: 171.69 % CD86: 173.22 % DMSO: CD54: 168.35 % CD86: 166.20 % |
Medium: CD54: 126.51 % CD86: 119.78 % DMSO: CD54: 123.13 % CD86: 119.52 % |
Medium: CD54: 131.93 % CD86: 135.08 % DMSO: CD54: 126.74 % CD86: 128.57 % |
PASS |
|
In the positive control (Nickel Sulphate), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be greater than 50%. |
CD54 RFI: 209 CD86 RFI: 368 CD54 Via: 90.43 % CD86 Via: 90.36 % |
CD54 RFI: 645 CD86 RFI: 1923 CD54 Via: 90.09 % CD86 Via: 88.94 % |
CD54 RFI: 377 CD86 RFI: 747 CD54 Via: 92.58 % CD86 Via: 92.73 % |
PASS |
|
For each test item, the cell viability should be greater than 50% in at least four tested concentrations in each run |
8/8 |
8/8 |
8/8 |
PASS |
|
Negative results are acceptable only for test items exhibiting a cell viability of less than 90% at the highest concentration tested, unless 5mg/ml in medium, 1mg/ml in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item. |
96.64 % 1 mg/ml in DMSO was the highest concentration tested |
97.28 % 1 mg/ml in DMSO was the highest concentration tested |
96.55 % mg/ml in DMSO was the highest concentration tested |
PASS |
Results Summary
The CV75 dose informs the dosing range selected for the CD54/86 expression assay. Cell viability was not reduced below 75% in the CV75 determination assay and therefore the top dose for the CD54/86 expression assay (main test) is 1 mg/ml (1000 µg/ml). The following tables show the expression of CD54 and CD86 against test item dose with concurrent cytotoxicity measurement:
Run 1 (Valid): Result = Negative
Test Item Dose (µg/ml) |
Cell Viability (%) |
Average Cell Viability (%) |
CD54 RFI |
CD86 RFI |
||
Isotype |
CD54 |
CD86 |
||||
1000.00 |
96.85 |
96.60 |
96.48 |
96.64 |
63 |
80 |
833.33 |
97.13 |
96.83 |
96.61 |
96.86 |
49 |
54 |
694.44 |
97.24 |
96.87 |
96.51 |
96.87 |
54 |
76 |
578.70 |
96.98 |
96.94 |
96.48 |
96.80 |
59 |
78 |
482.25 |
97.48 |
97.23 |
96.78 |
97.16 |
57 |
67 |
401.88 |
97.76 |
97.56 |
97.61 |
97.64 |
42 |
57 |
334.90 |
97.78 |
97.62 |
97.30 |
97.57 |
40 |
52 |
279.08 |
97.72 |
97.17 |
97.24 |
97.38 |
62 |
54 |
Run 2 (Valid): Result = Positive
Test Item Dose (µg/ml) |
Cell Viability (%) |
Average Cell Viability (%) |
CD54 RFI |
CD86 RFI |
||
Isotype |
CD54 |
CD86 |
||||
1000.00 |
97.14 |
97.44 |
97.25 |
97.28 |
124 |
134 |
833.33 |
97.55 |
97.54 |
97.43 |
97.51 |
119 |
154 |
694.44 |
97.90 |
97.69 |
97.88 |
97.82 |
129 |
135 |
578.70 |
97.97 |
97.96 |
98.05 |
97.99 |
105 |
61 |
482.25 |
97.69 |
97.87 |
97.98 |
97.85 |
163 |
126 |
401.88 |
98.41 |
98.10 |
97.73 |
98.08 |
173 |
120 |
334.90 |
97.37 |
97.75 |
97.93 |
97.69 |
154 |
168 |
279.08 |
97.56 |
97.35 |
98.00 |
97.64 |
135 |
106 |
Red font: crossed the expression threshold.
Run 3 (Valid): Result = Negative
Test Item Dose (µg/ml) |
Cell Viability (%) |
Average Cell Viability (%) |
CD54 RFI |
CD86 RFI |
||
Isotype |
CD54 |
CD86 |
||||
1000.00 |
97.01 |
96.34 |
96.30 |
96.55 |
104 |
116 |
833.33 |
97.90 |
97.41 |
97.19 |
97.50 |
162 |
128 |
694.44 |
97.96 |
97.69 |
96.60 |
97.42 |
74 |
111 |
578.70 |
97.86 |
97.54 |
96.94 |
97.45 |
105 |
134 |
482.25 |
97.61 |
97.50 |
97.17 |
97.43 |
88 |
136 |
401.88 |
97.48 |
97.56 |
97.07 |
97.37 |
63 |
117 |
334.90 |
97.44 |
97.30 |
96.75 |
97.16 |
54 |
97 |
279.08 |
96.94 |
97.08 |
96.63 |
96.88 |
-15 |
40 |
Red font = excluded from prediction because RFI values cannot be below zero
As can be seen from the data, the expression of CD54 as measured by the RFI did not cross the threshold (RFI ≥200) at any of the doses tested. The expression of CD86 as measured by the RFI crossed (RFI ≥150) at the following doses in repetition 2 alone; 833.33 µg/ml and 334.90 µg/ml. As the CD54/CD86 expression only crossed the threshold in one repetition out of three, the test item is classified as Negative. Cell viability did not fall below 50% at any of the test item concentrations and therefore the result is deemed to be valid.
Overall Achieved Depletion Values
Test item |
Cysteine peptide depletion (%) |
Lysine peptide depletion (%) |
Overall mean depletion (%) |
Reactivity class |
DPRA prediction |
||||||
N-isopropylacrylamide |
9.94 |
2.36 |
6.15 |
Minimal |
Negative |
Cysteine Peptide Depletion
Sample |
Peak area (µV.sec) |
Peptide concentration1(µg/mL) |
Peptide Depletion2(%) |
Mean Depletion (%) |
SD |
Positive control |
217738 |
106 |
71.9 |
72.0 |
0.11 |
217546 |
106 |
71.9 |
|||
216182 |
105 |
72.1 |
|||
N-isopropylacrylamide |
700176 |
341 |
9.66 |
9.94 |
0.36 |
699049 |
341 |
9.81 |
|||
694931 |
339 |
10.3 |
SD Standard Deviation
1 Samples prepared at a nominal concentration of 376 µg/mL (0.5 mM)
2 Calculated against a mean Reference Control area of 775080 µV.sec(n=6)
Lysine Peptide Depletion
Sample |
Peak area (µV.sec) |
Peptide concentration1(µg/mL) |
Peptide Depletion2(%) |
Mean Depletion (%) |
SD |
Positive control |
361084 |
172 |
55.7 |
56.1 |
0.64 |
360359 |
171 |
55.8 |
|||
351693 |
167 |
56.8 |
|||
N-isopropylacrylamide |
794066 |
379 |
2.57 |
2.36 |
0.48 |
793103 |
379 |
2.69 |
|||
800257 |
382 |
1.81 |
SD Standard Deviation
1 Samples prepared at a nominal concentration of 388 µg/mL (0.5 mM)
2 Calculated against a mean Reference Control area of 815020 µV.sec(n=6)
Rep 1 |
Test item concentration (µM) |
|||||||||||
|
0.977 |
1.953 |
3.906 |
7.813 |
15.625 |
31.250 |
62.5 |
125 |
250 |
500 |
1000 |
2000 |
Mean fold induction |
1.162 |
1.080 |
0.956 |
0.999 |
1.038 |
0.989 |
1.247 |
1.164 |
1.237 |
1.274 |
1.317 |
2.084 |
Viability % |
101.614 |
90.612 |
90.822 |
101.887 |
91.183 |
95.532 |
95.806 |
92.090 |
94.307 |
100.705 |
97.452 |
113.183 |
T-test |
1.52E-01 |
2.82E-01 |
5.88E-01 |
4.62E-01 |
3.66E-01 |
4.91E-01 |
6.62E-02 |
1.41E-01 |
7.54E-02 |
5.26E-02 |
3.29E-02 |
1.25E-06 |
SD |
0.335 |
0.339 |
0.167 |
0.169 |
0.230 |
0.157 |
0.044 |
0.142 |
0.214 |
0.210 |
0.104 |
0.484 |
IMAX |
2.084 at 2000 µM |
|||||||||||
EC1.5 |
1238.592 µM |
|||||||||||
IC30 |
N/A- the cell viability did not fall below 70% |
|||||||||||
IC50 |
N/A- the cell viability did not fall below 50% |
Rep 2 |
Test item concentration (µM) |
|||||||||||
|
0.977 |
1.953 |
3.906 |
7.813 |
15.625 |
31.250 |
62.500 |
125.000 |
250.000 |
500.000 |
1000.000 |
2000.000 |
Mean fold induction |
1.160 |
0.931 |
1.026 |
1.068 |
1.099 |
1.254 |
1.123 |
1.140 |
1.091 |
1.239 |
1.257 |
1.458 |
Viability % |
103.479 |
81.015 |
79.165 |
96.475 |
98.791 |
95.066 |
94.366 |
80.155 |
84.659 |
90.180 |
118.235 |
129.196 |
T-test |
1.47E-01 |
6.68E-01 |
3.91E-01 |
2.96E-01 |
2.38E-01 |
6.23E-02 |
1.97E-01 |
1.74E-01 |
2.52E-01 |
7.22E-02 |
6.09E-02 |
6.80E-03 |
SD |
0.184 |
0.092 |
0.071 |
0.113 |
0.177 |
0.075 |
0.124 |
0.160 |
0.149 |
0.088 |
0.137 |
0.214 |
IMAX |
1.458 at 2000 µM |
|||||||||||
EC1.5 |
N/A - threshold of induction was not crossed at any test item concentration |
|||||||||||
IC30 |
N/A- the cell viability did not fall below 70% |
|||||||||||
IC50 |
N/A- the cell viability did not fall below 50% |
Rep 3 |
Test item concentration (µM) |
|||||||||||
|
0.977 |
1.953 |
3.906 |
7.813 |
15.625 |
31.250 |
62.5 |
125 |
250 |
500 |
1000 |
2000 |
Mean fold induction |
1.222 |
1.414 |
1.368 |
1.588 |
1.278 |
1.601 |
1.476 |
1.424 |
1.593 |
1.832 |
1.561 |
1.877 |
Viability % |
114.962 |
109.394 |
106.128 |
102.112 |
101.628 |
93.702 |
100.269 |
93.302 |
95.727 |
99.678 |
111.021 |
124.489 |
T-test |
9.81E-02 |
1.14E-02 |
1.87E-02 |
1.91E-03 |
4.97E-02 |
1.13E-03 |
5.49E-03 |
1.07E-02 |
1.15E-03 |
5.45E-05 |
2.03E-03 |
1.94E-05 |
SD |
0.487 |
0.179 |
0.083 |
0.517 |
0.153 |
0.287 |
0.219 |
0.285 |
0.177 |
0.441 |
0.327 |
0.279 |
IMAX |
1.877 at 2000 µM |
|||||||||||
EC1.5 |
6.248 µM |
|||||||||||
IC30 |
N/A- the cell viability did not fall below 70% |
|||||||||||
IC50 |
N/A- the cell viability did not fall below 50% |
Determination criteria for the skin sensitisation potential of the test item |
|||
|
REP1 |
REP2 |
REP3 |
Does at least one concentration of Test Item induce luciferase activity > 1.5-fold: |
Yes |
No |
Yes |
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%: |
Yes |
No |
Yes |
Is p value < 0.05 for at least one concentration that yielded>1.5-fold induction with viability above 70% |
Yes |
No |
Yes |
Does EC1.5value occur at a concentration <1000µM (or <200µg/ml) |
No |
No |
Yes |
Does the test item induce the luciferase in a dose-dependent manner |
Yes |
No |
No |
Classification |
Negative |
Negative |
Inconclusive |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
Justification for classification or non-classification
This document addresses the requirements of conducting skin sensitization assessment for N‑isopropylacrylamide according to Annex VII section 8.3 of the REACH Regulation (Regulation (EC) No 1907/2006 and Commission Regulation (EU) 2016/1688).
The assessment of physico-chemical properties on N‑isopropylacrylamide showed that substance may have a low dermal uptake due to very high water solubility. However, due to mild irritating property of N-isopropylacrylamide, the uptake via stratum corneum may be possible.
No of specified conditions in column 2 of Annex VII section 8.3 of the REACH Regulation were fulfilled to exclude the substance from the skin sensitization testing.
The assessment in silico suggested weak skin sensitizing properties of N-isopropylacrylamide. In addition, one of in silico simulated skin metabolites of N-isopropylacralamide, acrylamide was reported in experimental animals as a weak sensitizer.
However, all recommended for skin sensitization assessment in chemico/in vitro tests DPRA (OECD TG 442C, 2015), KeratinoSens™ (OECD TG 442D, 2015) and h-CLAT (OECD TG 442E, 2017) were negative. This battery of tests supported the weight of evidence that N‑isopropylacrylamide triggers no skin sensitization properties and therefore non-classification was recommended.
The full skin sensitisation assessment document and classification justification document is attached in section 13.2 of this dossier.
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.