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

Endpoint summary

Description of key information

Skin sensitisation

For skin sensitisation two WoE studies are available, an in chemico Direct Peptide Reactive Assay (DPRA) and an in vitro human cell line activation test (h-CLAT). They have both been performed in accordance to their respective OECD TG and in compliance to GLP.

The two individual in vitro skin sensitisation assays are part of a test battery and need to be discussed together as they reflect two key events in the adverse outcome pathway (AOP) leading to skin sensitisation.

The mean depletion of both peptides in the DPRA study was 46.9 %. Based on the prediction model 1 the test item can be considered as sensitiser (high reactivity). In the h-CLAT study, the expression of both cell surface marker clearly exceeded the threshold in two independent experiments. In both studies it is concluded that the test substance has skin sensitising potential. Since a positive result was obtained for 2 key events of the AOP, it is concluded that the substance is skin sensitising.

Respiratory sensitisation

No data is available for respiratory sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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%.

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.

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

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

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

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).

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

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.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-06-17 to 2017-09-01

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:

yes

Remarks:

These deviations did not influence the quality or integrity of the present study.

Principles of method if other than guideline:

- Principle of test: DPRA is supposed to address the molecular initiating event to the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic model peptides containing either lysine or cysteine. The percentage of depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between sensitizers and non sensitizers.
- The method is design to predict and classify the skin sensitising potential of a chemical by assessment of its reactivity towards a synthetic cysteine and lysine containing peptide, by measuring the depletion using HPLC.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In accordance with Annex VII of REACH regulation

Details on the study design:

1. Preparation of Peptides:
- 18.12 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (33.52 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 21.3 mg lysine peptide with an amino acid sequence of Ac RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (39.76 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

2. Preparation of controls:
- Reference controls (RC):
- Reference control A (undiluted) was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run
- Reference control B (undiluted) was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run
- Reference control C (undiluted) was set up for the test item and the positive control. RC C for the test item was prepared using the respective solvent used to solubilize the test item. RC C for the positive control was prepared using acetonitrile. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

- Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution.. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
- Positive control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetronitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

3. Dose group:
- Reference Control C: undiluted
- Test item: 100 mM stock solution
- Positive control: 100 mM stock solution

4. Pre-experiment:
Solubiluty of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100mM).
The test item was dissolved in methanol.

5. Experimental procedure:
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide).
The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis.

Key result

Run / experiment:

other: 1

Parameter:

other: Mean peptide depletion

Remarks:

Prediction Model 1

Value:

46.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes

1. Pre-experiments:

Solubility of the test item was determined. The test item was not soluble in acetonitrile, dist water or isopropanol but completely soluble in methanol. No turbidity, precipitation and phase separation was observed for the test item solutions. All test item preparations of the main experiment were prepares using methanol.

2. Precipitation and Phase separations:

All test item solutions were freshly prepared immediately prior to use. No precipitation, turbidity or phase separation was observed for the samples of the test item when diluted with cysteine. For samples of the positive control and the solvent control of positive control (RC C) slight precipitation was noted after 24h incubation. Precipitation and turbidity was noted for the samples of the co-elution control of the positive control after 24h incubation (prior the HPLC analysis). However, since the acceptance criteria for the depletion of the positive control were fulfilled, the observed precipitation was regarded as insignificant.

3. Co-elution with the peptide peaks:

No co-elution of the test item with any of the peptides peaks was observed

4. Results calibration curve: Cysteine and Lysine values of the calibration curve:

Sample	Cysteine peptide		Lysine peptide
	Peak Area 220 nm	Peptide Concentration mM	Peak Area 220 nm	Peptide concentration mM
STD1	4946.3618	0.5340	4453.7617	0.5340
STD2	2473.8633	0.2670	2214.0920	0.2670
STD3	1198.9833	0.1335	1103.6776	0.1335
STD4	613.2764	0.0667	542.1525	0.0667
STD5	313.3556	0.0334	261.3623	0.0334
STD6	155.0795	0.0167	131.8409	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Cysteine Peptide Calibration Curve: y = 9265.42x-5.69 ; R² = 0.9999

Lysine Peptide Calibration Curve: y = 8353.22x-10.69 ; R² = 1.0000

5. Results of Cysteine Peptide Depletion:

Sample	Peak Area 220 nm	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV of Peptide Depletion (%)
Positive Control	1367.6532	0.1482	70.48	70.90	0.44	0.62
	1350.3103	0.1464	70.86
	1327.2146	0.1439	71.36
Test Item	4793.5825	0.5180	0.00	0.00	0.00	n.a.
	4837.5991	0.5227	0.00
	4784.8184	0.5170	0.00

6. Results of Lysine Peptide Depletion:

Sample	Peak Area 220 nm	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV of Peptide Depletion (%)
Positive Control	1562.8215	0.1884	62.85	62.34	0.45	0.72
	1598.4821	0.1926	62.00
	1591.5383	0.1918	62.17
Test Item	277.3393	0.0345	93.42	93.80	0.38	0.41
	260.5554	0.0325	93.81
	245.2688	0.0306	94.18

7. Categorization of the Test item

Predicition Model	Prediction Model 1 (Cysteine and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	46.90	High Reactivity	sensitiser	0.00	Minimal Reactivity	no sensitiser
Positive Control	66.62	High Reactivity	sensitiser	70.90	Moderate Reactivity	sensitiser

Interpretation of results:

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

Conclusions:

The test item showed moderate reactivity towards the peptides. Based on these results the test item is considered to have a sensitising potential (PPD 46.90%).

Executive summary:

In this in chemico study Direct Peptide Reactive Assay (DPRA), the skin sensitisation potential of the test item was assessed according to the OECD 442C and under GLP without signiticant deviations.

Following a pre-experiment to determine the solubility of the test item in different solvent (dissolved in methanol), the test item (in triplicate) was incubating at a concentration of 100 mM (14.516 mg/mL) with the peptides containing either cystiene or lysine for 24 ± 2h at 25 ± °C. After the incubation periode, the samples were analysed by HPLC in order to quantifyed the percentage of the mean peptide depletion.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C) (prediction model 1).

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of positive control on both peptides was 66.62%. The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 46.9 %. Based on the prediction model 1 the test item can be considered as sensitiser (high reactivity).

In conlusion, in this study under the given conditions the test item showed high reactivity towards the peptides. Therefore, the test item is considered to have skin sensitising potential. 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisastion DPRA

In this in chemico study Direct Peptide Reactive Assay (DPRA), the skin sensitisation potential of the test item was assessed according to the OECD 442C and under GLP. The test item (in triplicate) was incubating at a concentration of 100 mM (14.516 mg/mL) with the peptides containing either cystiene or lysine for 24 ± 2h at 25 ± °C. After the incubation periode, the samples were analysed by HPLC in order to quantifyed the percentage of the mean peptide depletion.

The sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control (prediction model 1). The mean depletion of both peptides was 46.9 %. Based on the prediction model 1 the test item can be considered as sensitiser (high reactivity).

Skin sensitisation h-CLAT

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 measuring 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.

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.

The expression of the cell surface marker CD86 was upregulated to 219% and 452% in both independent experiments, respectively. The expression of the cell surface marker CD54 was upregulated to 246% and 220% in one experiment (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.

Individual results generated with the two above described methods are not sufficient to conclude on the skin sensitisation potential of chemicals, therefore the data has to be considered in a weight of evidence approach.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation

Regarding the classification of the substance for skin sensitisation the results of the two individual assays of the in vitro skin sensitisation test battery need to be taken together as they reflect 2 key events in the adverse outcome pathway (AOP) leading to skin sensitisation. For this reason, a weight of evidence approach is applied for the test battery stating that: 'any two of the three tests determine the overall results, i.e. any two positive results drive the prediction of a sensitizer, while any two negative results drive the prediction of a test substance to be a non-sensitizer'.

Positive results were obtained both in the DPRA and in the h-CLAT studies, leading to the conclusion that the substance is probably skin sensitizer, category 1. The data are not sufficient for further sub-categorization.