1-imino-1H-isoindol-3-amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

201707-05 to 2017-09-12

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: OECD guideline 442E (In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

1-imino-1H-isoindol-3-amine

EC Number:

222-426-8

EC Name:

1-imino-1H-isoindol-3-amine

Cas Number:

3468-11-9

Molecular formula:

C8H7N3

IUPAC Name:

1-imino-1H-isoindol-3-amine

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line (ATCC TIB-202)

TEST-SUBSTANCE PREPARATION
- Concentrations: experiment 1 and 2: 33.18; 27.65; 23.04; 19.20; 16.00; 13.33; 11.11 and 9.26 µg/mL
- Stock: diluting the highest soluble concentration seven times
- Vehicle: DMSO

CONTROLS
- Solvent control: 0.2% DMSO (v/v) in cell culture medium
- Positive control: 1-chloro-2-4-dinitrobenzene (DNCB) at a final conc. of 4.0 μg/mL

MEDIUM
- Culture medium: RPMI 1640: with 2 mM L-glutamine, 25mM HEPES + 10% FBS + 100 U/ml Penicillin/100 µg/mL Streptomycin + 0.05 mM 2-mercaptoethanol
- FACS Buffer: Phosphate Buffered Saline (DPBS) + 0.1% BSA
- Blocking buffer: FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction)
- Reagent for cytotoxicity test: Propidium iodide (Sigma)

EXPERIMENTAL PROCEDURE
- Replicates: 1
- Experiments: 2
- Exposure period: 24 ± 0.5 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, streptomycin and 2 -mercaptethanol

ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 ± 0.5 hours after exposure

DATA EVALUATION
- CV75 calculation: Relative survival rate is calculated by linear extrapolation. This value is the substance concentration at which cell viability is 75% compared to the vehicle control.
- Relative cell viability: % relative cell viability = (number of living cells / total number of aquired cells)* 100
- Relative fluorescence intensity: RFI (%) = ( MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) / (MFI of vehicle control cells - MFI of vehicle isotype control cells) * 100

EVALUATION RESULTS
- Positive result: A test is considered to be positive when the dendritic cells are activated meaning that CD86 expression is increased ≥ 150% and/or CD54 expression increased ≥ 200% in relation to vehicle control in at least 2 independent experiments (cell vialbility ≥ 50%)
- Negative result: A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%.

ACCEPTANCE CRITERIA
The test meets acceptance criteria if:
- the cell viability of the solvent controls is > 90%,
- the cell viability of at least four tested doses of the test item in each run is > 50%,
- the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥ 200% for CD54 at a cell viability of > 50%,
- the RFI values of the solvent control is not ≥150% for CD86 and not ≥ 200% for CD54,
- the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is > 105%.

Results and discussion

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments.
The threshold of 150% for CD86 (319% experiment 1; 274% experiment 2) and 200% for CD54 (252% experiment 1; 334% experiment 2) were clearly exceeded.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: experiment 1

Parameter:

other: CD86 expression (%)

Value:

219

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: experiment 2

Parameter:

other: CD86 expression (%)

Value:

452

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: experiment 1

Parameter:

other: CD54 expression (%)

Value:

134

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Run / experiment:

other: experiment 2

Parameter:

other: CD54 expression (%)

Value:

246

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

The controls confirmed the validity of the study.
The viability of the solvent control was > 90% (95.9-97.1% experiment 1; 95.1-95.9% experiment 2).
The number of tested test item concentrations with cell viability > 50% was ≥ 4 (7 in experiment 1 and 8 in experiment 2).
The RFI for CD86 and CD54 of cells treated with the solvent DMSO was ≤ 150% (115% experiment 1; 115% experiment 2) and ≤ 200% (127% experiment 1; 104% experiment 2).
The MFI ratio of the medium control and isotype IgG1control was ≥ 105% for CD86 (366% experiment 1; 148% experiment 2) and CD54 (204% experiment 1; 137% experiment 2).
The MFI ratio of the solvent control (DMSO) and isotype IgG1 control was ≥ 105% for CD86 (408% experiment 1; 156% experiment 2) and CD54 (233% experiment 1; 139% experiment 2).

Any other information on results incl. tables

CD54 and CD86 Expression Experiment 1

Sample	Conc (mg/ml)	Cell Viability (%) CD86	Cell Viability (%) CD54	RFI (%) CD 86	RFI (%) CD54
Medium control		95.9	95.9	87	79
Solvent Control	0.20%	96.8	96.3	100	100
DNCB	4	88.8	86.6	319	252
Test item	33.18	31.3	30.9	85	66
	27.65	60.3	60.9	94	55
	23.04	68.7	68.1	108	83
	19.2	73.8	74.5	140	102
	16	77.1	75.3	175	124
	13.33	76.4	78.7	213	134
	11.11	78.5	80.6	219	128
	9.26	85.3	85.1	201	125

CD54 and CD86 Expression Experiment 2

Sample	Conc (mg/ml)	Cell Viability (%) CD86	Cell Viability (%) CD54	RFI (%) CD 86	RFI (%) CD54
Medium control		95.7	95.9	87	96
Solvent Control	0.20%	95.9	95.9	100	100
DNCB	4	89	89.7	274	334
test item	33.18	78.4	80.4	368	246
	27.65	85.7	85.1	452	220
	23.04	87.7	87.7	415	183
	19.2	90.6	90.6	140	174
	16	94.2	93.8	194	101
	13.33	94	94.8	281	119
	11.11	94.9	95	202	152
	9.26	95.8	95.5	145	144

Applicant's summary and conclusion

Interpretation of results:

other: The study alone cannot be used for the classification purpose but in a WoE approach

Conclusions:

The test item did upregulate the cell surface marker in at least two independant runs. Based on these results the test item is considered to have a sensitising potential

Executive summary:

In the current study the skin sensitisation potential of the test item was assessed according to the new OECD 442E TG, h-CLAT and in compliance to GLP.

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

The test item was dissolved in DMSO. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. A CV75 of 27.65±1.65 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 33.18; 27.65; 23.04; 19.20; 16.00; 13.33; 11.11 and 9.26 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.

In the first experiment clear cytotoxic effects were observed for the cells treated with the test item, whereas in the second experiment only very slight cytotoxic effects were observed. Relative cell viability at the highest test item concentration was reduced to 31.3% (CD86), 30.9% (CD54) and 27.7% (isotype IgG1 control) in the first experiment and to 78.4% (CD86), 80.4% (CD54) and 79.9% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to 219% and 452% in both independent experiments, respectively. An upregulation above the threshold of 150% was observed from 9.26 up to 16.0 µg/mL in experiment 1 and at all concentrations except 9.26 and 19.20 µg/mL in experiment 2. The expression of the cell surface marker CD54 was upregulated to 246% and 220% in one experiment (experiment 2). The upregulation above the threshold of 200% was observed at the two highest concentrations in experiment 2.

Since the expression of both cell surface marker clearly exceeded the threshold in two independent experiments the test item considered to be a skin sensitiser.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (319% experiment 1; 274% experiment 2) and 200% for CD54 (252% experiment 1; 334% experiment 2) were clearly exceeded.

In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item might be considered as sensitiser. The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in a weight of evidence approach in which minimally 2 key events of the adverse outcome pathway (AOP) are tested.