N-isopropylacrylamide

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

N-isopropylacrylamide

EC Number:

218-638-5

EC Name:

N-isopropylacrylamide

Cas Number:

2210-25-5

Molecular formula:

C6H11NO

IUPAC Name:

N-isopropylacrylamide

Test material form:

solid: flakes

Details on test material:

Identification: N-isopropylacrylamide
Physical state/Appearance: White flakes
CAS Number: 2210-25-5
Batch: 2161004
Purity: 99.41%
Expiry Date: 31 January 2018
Storage Conditions: Room temperature in the dark

In vitro test system

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.

Results and discussion

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.