N1,N3-diallylpropane-1,3-diamine dihydrochloride

Endpoint summary

Description of key information

The potential of N1,N3-diallylpropane-1,3-diamine dihydrochloride to induce skin sensitisation was evaluated in two in vitro tests conducted according to OECD 442D and OECD 442E and further supported by the results of an OECD QSAR Toolbox v4.4.1 prediction. Based on the results, the target substance can be considered as non-sensitiser. Therefore, classification is not warranted in accordance with CLP regulation 1272/2008.

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:

2020-04-22 to 2020-08-14

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)

Version / remarks:

adopted in June 2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment (immediately before treatment) N1,N3-diallylpropane-1,3-diamine dihydrochloride was dissolved or stably dispersed/suspended in DMSO to prepare a stock solution with a concentration of 2064 mM. Due to a technical error in the pre-experiment, the top dose was higher than required by the OECD Guideline 442D.

OTHER SPECIFICS
Partition coefficient (n-octanol/water): -4.78

Details on the study design:

Test System:
- The LuSens cell-line is an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The plasmid contains a luciferase gene (reporter gene) which is under transcriptional control of an antioxidant response element (ARE) of the rat NQO1 gene.The HaCaT human keratinocytes (provided by RWTH, Aachen, Germany) were genetically modified at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of Wruck). The LuSens cells were obtained by BASF and in 2019 a contract services agreement was established between ICCR-Roßdorf GmbH (Licensee) and Promega.

Cell Culture:
- Cultivation medium : Dulbecco´s Modified Eagle Medium (DMEM) culture medium with Penicillin/Streptomycin (1% v/v), FBS (10% v/v) and puromycin (0.005% v/v).
- Seeding medium: DMEM culture medium with FBS (10% v/v)
- Treatment medium: DMEM culture medium with FBS (1% v/v)

- LuSens Cell Cultures: Stocks of the LuSens cell line were stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH (aliquots of cells in freezing medium at 1.5 - 5×10^6 cells/cryovial) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. The stock cultures were thawed at 37 ± 1.5 °C in a water bath. The cells were sub-cultured twice weekly. The cell density should not exceed a cell density of 80 – 90%. The confluent cells were incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % CO2 (standard cell culture conditions). The LuSens cells can be used up to one and a half month after thawing (passage number should not exceed passage 15 for the main experiment and passage 20 for the dose range finder).The passage numbers of the used LuSens cells were 5 in the cytotoxicity test and 9/K1 and 9/K2 in the LuSens test for the main experiments 3 and 4, respectively.

Preparation and seeding of the cells:
Between 4 and 6×10^5 or 6 and 8×10^5 cells were seeded in 15 mL Cultivation Medium and pre-cultured at least twice a week in culture flasks (80 – 90% confluent). The cell density between approximately 80 and 90% should not be exceeded. After microscopic assessment of the cells, the cells were washed at least twice with 10 mL Ca2+/Mg2+ free DPBS including EDTA. Thereafter, the cells were trypsinated with 1 to 2 mL 0.05% Trypsin/EDTA for approximately 5 minutes at 37 ± 1.5 °C and 5.0 ± 0.5% CO2. The cells were resuspended in 10 mL Cultivation Medium to neutralise the trypsin.
For seeding of the cells, the Cultivation Medium was removed, and the cells were transferred into Seeding Medium. Each treatment well of a 96 well microtiter plate was seeded with 100 μL cell suspension (9000 to 11000 LuSens cells per well), except the well of the blank control. The cells were incubated for 24 hours ± 30 minutes under standard cell culture conditions.

Main Experiment (LuSens and MTT):
The test item was tested in four independent main experiments. The first two experiments were canceled due to observed contaminations. The main experiments were performed with independent cell cultures (cells are collected from different culture flasks). The test item was prepared separately for each run. For each main experiment one 96 well microtiter plate was prepared for MTT assay and one for the luciferase activity measurement.

Treatment of the Cells:
The solvent (twenty-four replicates), positive (five replicates) and negative (six replicates) controls as well as the test item concentrations (three replicates for each concentration) were diluted 1:25 in Treatment Medium. After incubation of the LuSens cells, Seeding Medium was removed and 150 µL of Treatment Medium was distributed in each well. Thereafter, 50 µL of the test item and control dilutions and the medium control (twelve replicates) were added into the corresponding wells. At the end of
the incubation period of 48 ± 1 hours under standard cell culture conditions, the cell cultures were microscopically evaluated for morphological alterations, precipitation or phase separation.

MTT assay (cell viability):
At the end of the incubation period, Treatment Medium was removed from the wells and the cells were washed at least twice with 200 µL DPBS including Ca2+/Mg2+. Thereafter, 200 µL of the MTT working solution were added to each treatment well and the cells were incubated for 3 hours ± 30 minutes under standard cell culture conditions. After rinsing the MTT working solution, the cells of each well were treated with 100 µL MTT lysis agent (Isopropanol with 0.04 N HCl) for at least 30 minutes, while gently shaking. Thereafter the microplate was transferred to a microplate reader equipped with a 570 nm filter to measure the absorbance (reference wavelength 690 nm).

Luciferase activity measurement:
- The Steady-Glo® Mix was be prepared by adding Steady-Glo® buffer to one bottle of Steady-Glo® Substrate and mixing by inversion until the substrate was dissolved. The Steady-Glo® working solution was prepared by mixing one part of DPBS (without Ca^2+/Mg^2+) with one part of Steady-Glo®-Mix.
At the end of the incubation period, the Treatment Medium was removed from the wells and the cells were washed at least twice with 200 μL DPBS including Ca^2+/Mg^2+. Thereafter, 200 μL of the Steady-Glo® working solution was added in each well. After slowly shaking of the microtiter plate for at least 10 min in the dark, the plate was transferred to a microplate reader and the luminescence was measured for 2 seconds per well.


Dose groups:
- Medium control: Treatment medium
- Solvent control: DMSO (final concentration 1% (v/v) in Treatment Medium), purity: ≥ 99%
- Negative control: Lactic acid (final concentration 5000 μM), CAS 50-21-5, purity: ~ 90%
- Positive Control: Ethylene glycol dimethylacrylate (EGDMA) (final concentration 120 μM), CAS 97-90-5, purity: ≥ 97.5%
- Test Item, main experiments: 178, 214, 257, 308, 370, 444 μM

Positive control results:

The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (main experiment (ME) 3: 6.878; ME 4: 5.603) and statistically significant. The positive control also had a relative cell viability ≥ 70% as compared to the solvent control (ME 3: 88.87%; ME 4: 114.00%).

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: highest luciferase induction with a cell viability > 70% (at 178 µM)

Value:

1.466

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 4

Parameter:

other: highest luciferase induction with a cell viability > 70% (at 370 µM)

Value:

1.4

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:

The acceptance criteria were met:
- The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 3: 6.878; ME 4: 5.603) and statistically significant.
- The positive control had a relative cell viability ≥ 70% as compared to the solvent control (ME 3: 88.87%; ME 4: 114.00%).
- The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 3: 0.872; ME 4: 0.744).
- The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) should be below 20% in each main experiment (ME 3: 13.0%; ME 4: 8.8%).
- At least three test concentrations had a cell viability of at least 70% relative to the solvent controls. At least one concentration was cytotoxic, i.e. had a cell viability < 70%.

Table 2: Luminescence induction and cell viability of the test item in Experiment 3

Treatment Group	Concentration					Luminescence						Mean Luminescence	SD Luminescence	Mean Luminescence blank corr.	Fold Induction	Cell viability [%]
						Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank						185
Solvent control												282.3	36.8	97.3	1.00	100.0
Medium control												249.9	19.4	64.9	0.667	81.66
Positive Control		120		µM		1057	724	1256	650	584		854.2	289.1	669.2	6.878	88.87
Negative Control		5000		µM		266	259	266	325	244	259	269.8	28.2	84.8	0.872	79.73
Test Item	C1	178		µM		429	281	273				327.7	87.8	142.7	1.466	90.14
	C2	214		µM		318	296	251				288.3	34.2	103.3	1.062	71.70
	C3	257		µM		340	244	229				271.0	60.2	86.0	0.884	74.50
	C4	308		µM		303	281	259				281.0	22.0	96.0	0.987	77.03
	C5	370		µM		288	273	251				270.7	18.6	85.7	0.881	62.35
	C6	444		µM		288	281	281				283.3	4.0	98.3	1.011	42.61

Table 3: Luminescence induction and cell viability of the test item in Experiment 4

Treatment Group	Concentration					Luminescence						Mean Luminescence	SD Luminescence	Mean Luminescence blank corr.	Fold Induction	Cell viability [%]
						Well 1	Well 2	Well 3	Well 4	Well 5	Well 6
Blank						207
Solvent control												282.7	24.8	75.7	1.00	100.0
Medium control												290.0	19.0	83.0	1.096	123.35
Positive Control		120		µM		636	643	591	665	621		631.2	27.5	424.2	5.603	114.00
Negative Control		5000		µM		266	273	244	273	273	251	263.3	12.8	56.3	0.744	114.25
Test Item	C1	178		µM		340	273	259				290.7	43.3	83.7	1.105	92.32
	C2	214		µM		281	273	236				263.3	24.0	56.3	0.744	96.44
	C3	257		µM		273	340	266				293.0	40.9	86.0	1.136	80.83
	C4	308		µM		281	251	259				263.7	15.5	56.7	0.748	93.42
	C5	370		µM		325	318	296				313.0	15.1	106.0	1.400	84.66
	C6	444		µM		362	355	281				332.7	44.9	125.7	1.660	57.79

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the test item N1,N3-diallylpropane-1,3-diamine dihydrochloride did not activate the LuSens cells up to a concentration of 444 µM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

In an in vitro skin sensitisation study conducted according to OECD 442D with N1,N3-diallylpropane-1,3-diamine dihydrochloride (99% purity) in DMSO, the sensitisation potential of the test item was assessed in the LuSens keratinocyte cell line as changes in the expression of genes associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization AOP). The cells were incubated with the test item for 48 h at concentrations of up to 444 µM in two independent experiments. All acceptance criteria were met showing the validity of the study. After treatment with the test item, the luciferase induction was not above or equal to (≥) 1.5-fold compared to the solvent control in at least 2 consecutive non-cytotoxic tested concentrations. Therefore, the test item can be considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-04-16 to 2020-08-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (human Cell Line Activation Test (h-CLAT))

Version / remarks:

adopted June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as a surrogate for human myeloid dendritic cells, since these cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.
This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
On the day of the experiment (prior to start) N1,N3-diallylpropane-1,3-diamine dihydrochloride was dissolved in culture medium. As tested by a solubility test, 5160 µg/mL in culture medium (the OECD 442E guideline recommended maximal to be tested test item concentration is 5000 µg/mL, but due to too high adjustment of the purity the top dose was 5160 µg/mL) was used as highest test item concentration in the cytotoxicity test. For the cytotoxicity test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions.

OTHER SPECIFICS
Partition coefficient (n-octanol/water): -4.78

Details on the study design:

Test System:
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as a surrogate for human myeloid dendritic cells, because the THP-1 cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.

Controls:
- Positive Control (h-CLAT): DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7) final concentration: 3 and 4 μg/mL, Purity ≥ 99%), Solvent: DMSO (final concentration 0.2%)
- Solvent Control for the Positive Control (h-CLAT): DMSO (Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium, final concentration 0.2%, Purity ≥ 99%
- Medium Control and solvent control for the test item: Culture Medium

TEST SYSTEM AND SUPPORTING INFORMATION
- THP-1 Cell Cultures:
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of ICCR-Roßdorf GmbH (aliquots of cells in freezing medium at 1 × 10^6 to 2 × 10^6 cells/mL) allowing the repeated use of the same working cell stock in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured at least twice a week. The cell density should not exceed 1 × 10^6 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30). The passage numbers of the used THP-1 cells were 12/K1 and 12/K2 in the cytotoxicity tests and 14/K1 and 14/K2 in the h-CLAT for runs 1 and 2, respectively.
- Culture Medium: RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 - 8 °C and used within one month.
- Preparation and Seeding of THP-1 Cells: On the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of the cells, a volume of 500 μL with a cell density of 1.8 - 2 × 10^6 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.

EXPERIMENTAL DESIGN AND PROCEDURES OF H-CLAT:
The test item was tested in two independent runs. The tests were performed on different days. The test item was prepared separately for each run.
- Treatment of the Cells: For the test item exposure, the highest dose solution calculated from the cytotoxicity test was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1)
- Staining of the Cells: The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution (0.01% (w/v) Globulins Chon -fraction II, III human) at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures. The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
- Sample Preparation for Measurement: After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD (5 μL or 0.25 μg/1x10^6 cells) solution were added.
- Data Analysis and Interpretation: The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:
RFI [%] = ((MFI of test item treated cells - MFI of test item treated isotype control cells)/(MFI of solvent control cells - MFI of solvent isotype control cells)) × 100
MFI = Geometric Mean Fluorescence Intensity (GeoMean)
The cell viability from the isotype control cells, CD54 and CD86 cells is calculated according to the following equation: Cell Viability [%]= (Number of living cells/Total Number of acquired cells) × 100
Where only the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies) are used for the cell viability evaluation.

- Prediction model: For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE

- Acceptance criteria: According to OECD Guideline 442E

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was > 50%.
For details on results see Section "Other effects/acceptance of results" and "Any other information on results incl. tables".

Key result

Run / experiment:

other: Experiment I

Parameter:

other: highest RFI of CD86 with cell viability >50%

Value:

796.9

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 II

Parameter:

other: highest RFI of CD86 with cell viability >50%

Value:

688.8

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 I

Parameter:

other: highest RFI of CD54 with cell viability >50%

Value:

751.3

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 II

Parameter:

other: highest RFI of CD84 with cell viability >50%

Value:

728.9

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:

For details on results see Section "Any other information on results incl. tables".

OTHER EFFECTS:
- Visible damage on test system: not described

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The technical proficiency of the h-CLAT with the OECD 442E guideline recommended proficiency substances was demonstrated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes,
Cell viability of medium/DMSO control first run: 96.92%/97.30%; Cell viability of medium/DMSO control second run: 97.72%/97.77%;
DMSO First run: RFI (CD54) = 114.5%, RFI (CD86) = 116.1%; DMSO Second run RFI (CD54) = 99.0%, RFI (CD86) = 93.5%.
- Acceptance criteria met for positive control: yes,
First run: RFI (CD54) 3 µg/ml DNCB = 404.6%, RFI (CD86) 3 µg/ml DNCB = 442.0%; RFI (CD54) 4 µg/ml DNCB = 659.8%, RFI (CD86) 4 µg/ml DNCB = 631.7%;
Second run: RFI (CD54) 3 µg/ml DNCB = 309.4%, RFI (CD86) 3 µg/ml DNCB = 421.7%; RFI (CD54) 4 µg/ml DNCB = 445.8%, RFI (CD86) 4 µg/ml DNCB = 528.7%;
First run MFI ratio of Medium CD54 and CD86: 132.8% and 183.2%; First run MFI ratio of DMSO CD54 and CD86: 138.2% and 198.2%
Second run MFI ratio of Medium CD54 and CD86: 143.5% and 223.8%; Second run MFI ratio of DMSO CD54 and CD86: 144.7% and 220.0%

Table 1: Results of the first Cytotoxicity Test for the Test Item N1,N3-diallylpropane-1,3-diamine dihydrochloride

Test Group	Concentration [µg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium Control	-	no	98.36
Test Item	40.4	no	97.97
	80.6	no	96.13
	161	no	89.47
	323	yes	82.04
	645	yes	65.85
	1290	yes	47.29
	2580	yes	11.55
	5160	yes	4.17

Table 2: Results of the second Cytotoxicity Test for the Test Item N1,N3-diallylpropane-1,3-diamine dihydrochloride

Test Group	Concentration [µg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium Control	-	no	98.3
Test Item	40.4	no	98.08
	80.6	no	94.72
	161	no	83.93
	323	yes	69.39
	645	yes	52.11
	1290	yes	46.91
	2580	yes	21.72
	5160	yes	4.85

The mean CV75 value of both Cytotoxicity Tests: 341.61 µg/mL

Table 3: Results of the first h-CLAT run for the Test Item N1,N3-diallylpropane-1,3-diamine dihydrochloride

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	96.92
DMSO Control	-	100.0	100.0	97.30
Positive Control (DNCB)	3.0	404.6*	442.0*	88.26
	4.0	659.8*	631.7*	77.69
Test Item	114	434.2*	469.9*	78.37
	137	517.1*	464.2*	72.66
	165	655.3*	587.0*	61.52
	198	617.1*	626.4*	67.73
	237	640.8*	628.5*	61.21
	285	751.3*	796.9*	51.62
	342	893.4*	1010.4*	47.07
	410	586.8*	859.1*	43.62

Table 4: Results of the second h-CLAT run for the Test Item N1,N3-diallylpropane-1,3-diamine dihydrochloride

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	97.72
DMSO Control	-	100.0	100.0	97.77
Positive Control (DNCB)	3.0	309.4*	421.7*	88.54
	4.0	445.8*	528.7*	85.31
Test Item	114	522.7*	413.0*	71.44
	137	685.6*	480.1*	62.88
	165	693.8*	531.2*	61.26
	198	641.2*	506.2*	62.83
	237	633.0*	585.9*	54.08
	285	728.9*	688.8*	54.29
	342	741.2*	718.1*	47.14
	410	711.3*	657.6*	48.98

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test item N1,N3-diallylpropane-1,3-diamine dihydrochloride with a log Pow of -4.78 activated THP-1 cells under the test conditions of this study. The RFI values for CD54 were >200% and the RFI values for CD86 were >150% in both independent runs. Therefore, the test item is considered positive for the third key event (activation of dendritic cells) of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

In an in vitro skin sensitisation study conducted according to OECD 442E with N1,N3-diallylpropane-1,3-diamine dihydrochloride (99% purity) in culture medium, the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells using the THP-1 cell line in two independent experimental runs. Based on the results from a pre-experiment, the following concentrations of the test item were used in the main experiment: 114, 137, 165, 198, 237, 285, 342 and 410 µg/mL.

Cells were incubated with the test item for 24 ± 0.5 hours and later checked for cell viability and expression of CD86 and CD54 cell surface markers. Both experiments passed the acceptance criteria.

The relative fluorescence intensity (RFI) of CD86 and/or CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration with a related cell viability of greater than 50% in both independent runs. In conclusion, the test item N1,N3-diallylpropane-1,3-diamine dihydrochloride with a log Pow of -4.78 activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

in silico, OECD QSAR Toolbox, profiling of structural alerts for skin sensitisation

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a (Q)SAR model, with limited documentation / justification, but validity of model and reliability of prediction considered adequate based on a generally acknowledged source

Justification for type of information:

1. SOFTWARE
OECD QSAR Toolbox

2. MODEL (incl. version number)
version 4.4.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Cl.Cl.C=CCNCCCNCC=C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Name of profilers:
- Protein binding potency Lys (DPRA 13%)
- Protein binding by OASIS
- Protein binding by OECD
- Protein binding potency Cys (DPRA 13%)
- Protein binding potency GSH
- Respiratory sensitisation
- Protein binding alerts for skin sensitization according to GHS
- Protein Binding Potency h-CLAT
- Keratinocyte gene expression
- Protein binding alerts for skin sensitization by OASIS

5. APPLICABILITY DOMAIN
Target chemical within the applicability domain.

6. ADEQUACY OF THE RESULT
These results are used in a weight-of-evidence approach to assess the skin sensitising potential of the target chemical. The selected profilers detect structural alerts, which are associated with the potential to bind to or interact with proteins. The binding to skin proteins is the first step of the Adverse Outcome Pathway for skin sensitisation i.e the molecular initiating event leading to the skin sensitisation.

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Profiling of structural alerts for skin sensitisation was performed with the QSAR Toolbox Version 4.4.1.

Specific details on test material used for the study:

- other information: Smiles code Cl.Cl.C=CCNCCCNCC=C

Parameter:

other: OECD QSAR Toolbox_alerts for skin sensitisation

Remarks on result:

no indication of skin sensitisation

Remarks:

no alerts found by profilers

Table 1: N1,N3-diallylpropane-1,3-diamine dihydrochloride profiling for structural alerts for skin sensitisation sensitisation:

PROFILERS		RESULTS
Protein binding potency Lys (DPRA 13%)		DPRA less than 9% (DPRA 13%) DPRA less than 9% (DPRA 13%) >> No protein binding alert
Protein binding by OASIS		No alert found
Protein binding by OECD		No alert found
Protein binding potency Cys (DPRA 13%)		DPRA less than 9% (DPRA 13%) DPRA less than 9% (DPRA 13%) >> No protein binding alert
Protein binding potency GSH		Not possible to classify according to these rules (GSH)
Endpoint Specific
Respiratory sensitisation		No alert found
Protein binding alerts for skin sensitization according to GHS		No alert found
Protein Binding Potency h-CLAT		No alert found
Keratinocyte gene expression		Not possible to classify according to these rules
Protein binding alerts for skin sensitization by OASIS		No alert found

Interpretation of results:

other: No protein bindings alerts found for skin sensitization

Conclusions:

The OECD QSAR Toolbox, version 4.4.1. was used to predict potential protein binding alerts of the test item for inducing skin sensitization. No protein-binding alerts for skin sensitization were found by the ten different profilers used.

Executive summary:

Covalent binding to proteins is the first key event of the Adverse Outcome Pathway (AOP) for skin sensitisation. The OECD QSAR toolbox, version 4.4.1 was used to predict if the test item exhibits any protein binding alerts for inducing skin sensitisation. In total, ten different profilers were used for the prediction and none of them showed any protein binding alerts.

Thus, based on these results it can be concluded that the test item is negative for the first key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The potential of N1,N3-diallylpropane-1,3-diamine dihydrochloride to induce skin sensitisation was evaluated in two suitable in vitro test methods conducted according to OECD 442D and OECD 442E and additionally by an OECD QSAR Toolbox v4.4.1 prediction.

In the first study conducted according to OECD 442D, the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes using the LuSens keratinocyte cell line. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity. Under the given conditions the test item did not induce the luciferase activity in the transgenic LuSens cell line in two independent experiment runs. Therefore, test item can be considered to be a non-sensitizer for the second key event of skin sensitisation Adverse Outcome Pathway (AOP).

In the second study, conducted according to OECD 442E, the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells in vitro using the human cell line activation test (h-CLAT). Cells were incubated with the test item for 24 h at 37 °C and later checked for cell viability and expression of CD86 and CD54 cell surface markers. Based on the results, the test item was tested to be positive for the third event of the skin sensitisation AOP.

Sensitizing potential of the test item was also predicted from the OECD QSAR Toolbox, version 4.4.1., which was used to predict potential protein binding alerts of the test item for inducing skin sensitization. No protein-binding alerts for skin sensitization were found by the ten different profilers used. Therefore, test item was concluded negative for the first key event (covalent binding to proteins) of the skin sensitisation AOP.

Based on assessing the results in weight-of-evidence approach, the target substance can be considered as non-sensitiser to the skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

By assessing the available data in a weight-of-evidence approach, the target substance can be considered as non-sensitizer. Thus, in accordance with CLP Regulation 1272/2008 no classification for skin sensitization is warranted.