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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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).

Test material

Test material information

Constituent 1

Reference substance name:

(prop-2-en-1-yl)({3-[(prop-2-en-1-yl)amino]propyl})amine dihydrochloride

EC Number:

812-745-6

Cas Number:

205041-15-2

Molecular formula:

C9 H20 Cl2 N2

IUPAC Name:

(prop-2-en-1-yl)({3-[(prop-2-en-1-yl)amino]propyl})amine dihydrochloride

Test material form:

solid

Details on test material:

- CAS: 205041-15-2
- Batch: LWDDP00087
- Colour: white
- Purity (GC): 99%
- Expiry date: 2020-09-25

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

In vitro test system

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

Results and discussion

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%).

In vitro / in chemico

Results

Results 1

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

Results 2

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%.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).