N-isopropylacrylamide

Endpoint summary

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

Reference

Reference 1

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

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

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.

Reference 2

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.

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.

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.

Reference 3

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

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 concentration^1(µg/mL)	Peptide Depletion^2(%)	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 concentration^1(µg/mL)	Peptide Depletion^2(%)	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)

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. 

Reference 4

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

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

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 KeratinoSens^TM 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.

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.