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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-03-10 to 2020-07-01

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 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

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: LWDDP00072
- Colour: white
- Purity (GC): 100%
- Expiry date: 2020-08-11
- Storage conditions: room temperature
- Stability: stable under recommended storage conditions

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A solubility test was performed. The test item was dissolved and diluted in cell culture medium within 1 hour prior to treatment. The solvent was compatible with the survival of the cells and the S9 activity.

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections were carried out before freezing.
For the experiments thawed cultures were set up in 75 cm² cell culture plastic flasks at 37 °C in a 5% carbon dioxide atmosphere (95% air). 5 x 10^5 cells per flask were seeded in 15 mL of MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum) and subcultures were made every 3-4 days.

MEDIA USED:
MEM medium supplemented with:
- 10% fetal bovine serum (FBS)
- 100 U/100 µg/mL penicillin/streptomycin solution
- 2 mM L-glutamine
- 2.5 µg/mL amphotericin
- 25 mM HEPES

Treatment Medium (short-term exposure):
- Complete culture medium without FBS.

After Treatment Medium / Treatment Medium (long-term exposure):
- Complete culture medium with 10% FBS and 1.5 µg/mL cytochalasin B.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany. Male Sprague Dawley rats were induced with phenobarbital / β-naphthoflavone. The following quality control determinations were performed by Trinova Biochem GmbH: a) Alkoxyresorufin-0-dealkylase activities, b) Test for the presence of adventitious agents, c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene), A stock of the supernatant containing the microsomes was frozen in aliquots of 5 mL and stored at ≤ -75 °C. The protein concentration in the S9 preparation (Lot: 4180) was 39.2 mg/mL.

- method of preparation of S9 mix: An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice. The final concentration of S9 mix in the cultures is 5%.

Test concentrations with justification for top dose:

The concentrations evaluated in the main experiment were based on the results obtained in the pre-experiment:

Experiment 1: short term, 4h
- Without metabolic activation: 50, 100, 500, 1000, 1500 and 2000 µg/mL; 50, 100, 500 and 2000 µg/mL were selected for the microscopic analysis of micronuclei.
- With metabolic activation: 10, 50, 125, 250, 500, 1000, 1500 and 1800 µg/mL; 10, 50, 125, 500 and 1800 µg/mL were selected for the microscopic analysis of micronuclei.

Experiment 2: long-term, 24 h
- Without metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL; 10, 25, 50 and 100 µg/mL were selected for the microscopic analysis of micronuclei.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Cell culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

clastogenic control; without metabolic activation, final concentration: 20 µg/mL

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Cell culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

clastogenic control, with metabolic activation, final concentration: 2.5 µg/mL

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Cell culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Colchicine

Remarks:

aneugenic control, without metabolic activation, final concentration: 1.5 and 0.08 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Three or four-day-old stock cultures (in exponential growth), more than 50% confluent, were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment. Cells subsequently were trypsinised with a solution of 0.2% trypsin in Ca-Mg-free PBS at 37 °C for 5 min. By adding complete culture medium the detachment was stopped and a single cell suspension was prepared.

Experiment 1:
Exponentially growing V79 cells were seeded into 25 cm² cell culture flasks (two flasks per test group). Approx. 50000 cells were seeded per cell culture flask, containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS). After an attachment period of approx. 48 h, the complete culture medium was removed and subsequently the test item was added to the treatment medium in appropriate concentrations. The cells were incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 µg/mL cytochalasin B for 20 h at 37 °C.
Experiment 2:
If negative or equivocal results are obtained, they should be confirmed using continuous treatment (long-term treatment) without metabolic activation. Approx. 50 000 exponentially growing V79 cells were seeded in 25 cm² cell culture flasks in absence of metabolic activation. After an attachment period of approx. 48 h the test item was added in complete culture medium. 1 h later 1.5 µg/mL cytochalasin B were added and the cells were incubated for 23 h at 37 °C. At the end of the treatment the cell culture medium was removed and the cells were prepared for microscopic analysis.

Number of Cultures:
Duplicate cultures were performed at each concentration level except for the pre-experiment.

Preparation of the Cultures:
At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 mL complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. Prior to this an aliquot of each culture was removed to determine the cell count by a cell counter (AL-Systems). After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.

Analysis of Micronuclei:
At least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to criteria of Fenech, i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmatic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.

As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells according to the following formula:
CBPI= (c1 x 1) + (c2 x 2) + (cx x 3)/n

c1: mononucleate cells
c2: binucleate cells
cx: multinucleate cells
n: total number of cells

The CBPI can be used to calculate the % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures: % Cytostasis= 100 – 100 x ((CBPIT – 1) / (CBPIC – 1))

CBPIT: Cytokinesis Block proliferation index of treated cultures
CBPIC: Cytokinesis Block proliferation index of control cultures

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria:
- The concurrent negative/solvent control is considered acceptable for addition to the laboratory historical negative/solvent control database.
- Concurrent positive controls should induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative/solvent control.
- Cell proliferation criteria in the negative/solvent control according to OECD 487 [4] should be fulfilled.
- All experimental conditions are tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analysable.
- Criteria for the selection of top concentration are fulfilled.

A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.

A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

The nonparametric Chi-Quadrat Test was performed to verify the results of the experiment. No statistically significant enhancement (p< 0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment I and II.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I, short term: A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment II, long-term: A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment II without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range (pH 7.4).
- Precipitation: No precipitate of the test item was noted in any concentration group evaluated in experiment I and II.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to 50 µg/mL. At 100 µg/mL 34%, at 500 µg/mL 37% and at 2000 µg/mL a cytostasis of 46% was noted.
In experiment I with metabolic activation no increase of the cytostasis above 30% was noted up to 10 µg/mL. At 50 µg/mL 43%, at 124 µg/mL 48%, at 500 µg/mL 33% and at 1800 µg/mL a cytostasis of 55% was noted

In experiment II without metabolic activation no increase of the cytostasis above 30% was noted at 10 µg/mL and 100 µg/mL. At 25 µg/mL and 50 µg/mL a cytostasis of 31% and 36%, respectively, was observed. At concentrations higher than 100 µg/mL the cytostasis further increased and viable cell numbers were strongly decreased. The decrease in cell numbers was so pronounced that evaluation of the proliferation index or micronuclei frequencies was not possible. It seems that the test item caused pronounced cell death while surviving cells showed normal proliferation capacity thus mimicking relatively high values of the proliferation index. Thus, a concentration of 100 µg/mL was used as highest concentration for evaluation of micronuclei frequencies.

Micronuclei Analysis:
In experiment I without metabolic activation the micronucleated cell frequency of the negative control (0.90%) was within the historical control limits of the negative control (0.32% - 1.44%). The mean values of micronucleated cells found after treatment with the test item were 0.60% (50 µg/mL), 0.65% (100 µg/mL), 0.55% (500 µg/mL) and 0.90% (2000 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.
In experiment I with metabolic activation the micronucleated cell frequency of the negative control (1.05%) was within the historical control limits of the negative control (0.45% – 1.68%). The mean values of micronucleated cells found after treatment with the test item were 0.70% (10 µg/mL), 0.75% (50 µg/mL), 0.70% (125 µg/mL), 1.30% (500 µg/mL) and 0.65% (1800 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.
In experiment II without metabolic activation the micronucleated cell frequency of the negative control (0.78%) was within the historical control limits of the negative control (0.32% – 1.44%). The mean values of micronucleated cells found after treatment with the test item were 0.80% (10 µg/mL), 1.08% (25 µg/mL), 0.80 (50 µg/mL) and 0.85% (100 µg/mL). The numbers of micronucleated cells were within the historical control limits of the negative control and did not show a biologically relevant increase compared to the concurrent negative control.

The nonparametric x² Test was performed to verify the results in both experiments. No statistically significant enhancement (p< 0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II with and without metabolic activation.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, N1,N3-diallylpropane-1,3-diamine dihydrochloride did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In an in vitro mammalian micronucleus assay (OECD 487), V79 cells cultured in vitro were exposed to N1,N3-diallylpropane-1,3-diamine dihydrochloride (100% purity) in cell culture medium in experiment 1 (short-term exposure, 4 h) at concentrations of 50, 100, 500, 2000 µg/mL (without metabolic activation) and at 10, 50, 125, 500, 1800 µg/mL (with metabolic activation). In experiment 2 (long-term exposure 24 h, without metabolic activation), concentration of 10, 25, 50, 100 µg/mL were used.

 In experiment 1 without metabolic activation the mean values of micronucleated cells found after treatment with the test item were 0.60% (50 µg/mL), 0.65% (100 µg/mL), 0.55% (500 µg/mL) and 0.90% (2000 µg/mL). In experiment 1 with metabolic activation the mean values of micronucleated cells found after treatment with the test item were 0.70% (10 µg/mL), 0.75% (50 µg/mL), 0.70% (125 µg/mL), 1.30% (500 µg/mL) and 0.65% (1800 µg/mL). In experiment 2 without metabolic activation the mean values of micronucleated cells found after treatment with the test item were 0.80% (10 µg/mL), 1.08% (25 µg/mL), 0.80 (50 µg/mL) and 0.85% (100 µg/mL). No statistically significant enhancement (p< 0.05) of cells with micronuclei was noted in the concentration groups of the test item evaluated in experiment 1 and 2.

The positive controls did induce the appropriate response. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

 

This study is classified as acceptable and satisfies the requirements for Test Guideline OECD 487 for in vitro mutagenicity data.