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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance N1,N3-diallylpropane-1,3-diamine dihydrochloride was tested in an in vitro genotoxicity testing battery (OECD 471, 487 and 490, GLP) as required by Annex VIII of the REACH regulation (EC) 1907/2006.

N1,N3-diallylpropane-1,3-diamine dihydrochloride was tested negative in a bacterial reverse gene mutation test conducted according to OECD 471, in an in vitro micronucleus assay conducted according to OECD 487 and in a mammalian cell gene mutation assay conducted according to OECD 490. Based on the assessment in a weight of evidence approach, the test item was considered to not induce genetic toxicity.

Link to relevant study records

Reference

Reference 1

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

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

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.

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

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

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

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.

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-04-14 to 2020-06-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was dissolved in purified water and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: MOLTOX, INC., NC 28607, USA (TA98 and TA1535), Xenometrix AG, Switzerland (TA100 and TA1537)

MEDIA USED
- Type and identity of media: Nutrient medium: 8 g Nutrient Broth and 5 g NaCl per litre, plus 125 µL ampicillin (10 mg/mL) for TA98, TA100); Agar Plates: Vogel-Bonner Medium E agar plates contain per litre 15 g Agar Agar, 20 mL Vogel-Bonner salts and 50 mL glucose solution (40%); Overlay Agar: The overlay agar contains per litre: 7.0 g Agar Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H20 and 12.2 mg biotin

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: MOLTOX, INC., NC 28607, USA

MEDIA USED
- Type and identity of media: Luria Bertani: 10 g tryptone 10 g NaCl and 5 g yeast extract per litre, plus 125 µL ampicillin (10 mg/mL); Agar Plates: Vogel-Bonner Medium E agar plates contain per litre 15 g Agar Agar, 20 mL Vogel-Bonner salts and 50 mL glucose solution (40%); Overlay Agar: The overlay agar contains per litre: 7.0 g Agar Agar, 6.0 g NaCl, 10.2 mg tryptophan.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver microsomal fraction S9 mix

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment (see box "Any other information on materials and methods incl. tables"; Results: see box "Any other information on results incl tables", Table 2). Two independent main experiments were performed with the following concentrations: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:A. dest. (purified water), Eurofins Munich, Lot No. 200304, 200318, 200402
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100 and TA1535 (10 µg/plate), without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD)

Remarks:

TA98 (10 µg/plate) and TA1537 (40 µg/plate), without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA (pKM 101) (1 µL/plate), without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

All strains (2.5 µg/plate), except E.coli WP2 uvrA pKM 101 (10 µg/plate), with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, Experiment I), pre-incubation (Experiment II)

EXPERIMENTAL PERFORMANCE
- Experiment I:
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate: 100 μL of Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control), 500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation), 100 μL Bacteria suspension, 2000 μL Overlay agar.
- Experiment II:
For the pre-incubation method 100 µL of the test solution was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.

DURATION
- Pre-incubation period (Experiment II): 60 min at 37 °C
- Exposure duration: 48 h in the dark at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/concentration level including the controls

DETERMINATION OF CYTOTOXICITY
Cytotoxicity is considered either as a clearing or diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

EVALUATION OF MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if in at least one tester strain with or without metabolic activation:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM 101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher when compared to the reversion rate of the solvent control.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, and a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system

Evaluation criteria:

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA (pKM 101))
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2017 -2019 for all tester strains, except for E. coli WP2 uvrA (pKM 101)) (see box “Any other information on material and methods incl. tables”, Table 1)
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. No precipitation of the test item was observed in any tester strain used in experiments I and II (with and without metabolic activation). No cytotoxic effects of the test item were noted in any of the five tester strains used up to the highest concentration evaluated (with and without metabolic activation) in experiment I and II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Table 2: Results of the pre-experiment

Substance	Dose (µg/plate)	TA98		TA100
		Mutation Factor*		Mutation Factor*
		without S9	with S9	without S9	with S9
Solvent Control (A. dest)		1.0	1.0	1.0	1.0
4-NOPD	10.0	6.3	-	-	-
NaN3	10.0	-	-	4.7	-
2-AA	2.50	-	46.3	-	14.3
Test Item	3.16	0.8	0.9	1.0	0.9
	10.0	0.8	1.1	0.8	0.9
	31.6	1.0	0.9	0.9	0.9
	100	0.7	0.8	0.8	0.9
	316	0.7	0.9	0.9	0.8
	1000	0.8	1.0	0.9	1.0
	2500	0.5	1.1	0.9	0.8
	5000	0.9	1.1	1.0	0.8

*Mutation factor= mean revertants (test item, negative or positive control)/ mean revertants (solvent control)

Conclusions:

Under the experimental conditions reported, N1,N3-diallylpropane-1,3-diamine dihydrochloride did not cause gene mutations in an Ames Test conducted according to OECD 471. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse gene mutation assay.

Executive summary:

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, S. typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM 101) were exposed to N1,N3 -diallylpropane-1,3-diamine dihydrochloride (100% purity) in purified water at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains and both experiments (plate incorporation and pre-incubation). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-03-30 to 2020-07-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Based on the pre-experiment for solubility, performed for the in vitro Mammalian Micronucleus Assay (Eurofins Munich Study No. STUGC20AA0354-3), RPMI cell culture medium (+ 5% HS) was used as solvent and 2 mg/mL was used as the highest concentration. The solvent was compatible with the survival of the cells and the S9 activity. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4).

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins Munich stock culture
- Cell cycle length, doubling time or proliferation index: 10-12 hours doubling time, cloning efficiency >50%
- Methods for maintenance in cell culture if applicable: Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Complete Culture Medium: RPMI 1640 medium supplemented with:
10% HS, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B,
Treatment Medium: RPMI 1640 medium supplemented with:
5% HS, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B,
Selective Medium: RPMI 1640 medium supplemented with:
20% HS, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B, 5 µg/mL TFT


RPMI complete 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: yes, by culturing in RPMI 1640 supplemented with 9.0 µg/mL hypoxanthine, 15.0 µg/mL thymidine, 22.5 µg/mL glycine, 0.1 µg/mL methotrexate

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

without metabolic activation: 1, 5, 10, 50, 175, 500 and 1000 µg/mL
with metabolic activation: 50, 250, 500, 1000, 1200, 1500 and 1600 µg/mL

The selection of the concentrations used in the main experiment was based on data from the pre-experiment. The following concentrations were tested in the pre-experiment: 50, 150, 500, 100, 1500 and 2000 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 treatment medium
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity. The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

Experimental procedure:
For a short-term exposure experiment 1 x 10^7 cells were suspended in 11 mL RPMI medium with 5% horse serum (25 cm² flasks) and exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h the test item was removed by centrifugation (200 x g, 7 min) and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an
expression and growth period of 2 days in total at 37 °C in 5% CO2/95% humidified air. The cell density was determined each day and adjusted to 3 x 10^5 cells/mL in a total culture volume of 20 mL, if necessary.
After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 7 days at 37 °C in a humidified atmosphere with 5% CO2. Analysis of the results was based on the number of cultures with cell growth (positive wells, PW) and those without cell growth (negative wells, NW) compared to the total number of cultures seeded (total wells, TW). Additionally, cultures were
seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µL selective medium with TFT. The plates were scored after an incubation period of about 12 days at 37 °C in 5% CO2/95% humidified air.
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. Based on the null hypothesis of the Poisson distribution, the probable number of clones/well (P) is equal to -ln(negative wells/total wells) and the plating efficiency (PE) equals P/(number of cells plated per well).
Mutant frequency then was calculated as MF = (PE(cultures in selective medium) / PE(cultures in non-selective medium)).
Suspension growth (SG) of the cell cultures reflects the number of times the cell number increases from the starting cell density. When carrying out a short-term treatment (4 h) a 2-day growth period was considered. The relative total growth (RTG) is the product of the relative suspension growth (RSG; calculated by comparing the SG of the dose groups with the SG of the control) and the relative cloning efficiency (RCE) for each culture: RTG = RSG x RCE / 100.
The mutant frequencies obtained from the experiments were compared with the Global Evaluation Factor (GEF). To arrive at a GEF, the workgroup (IWGT MLA Workgroup) analysed distributions of negative/vehicle mutant frequencies of the MLA that they gathered from ten laboratories. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation. Applying this definition to the collected data, the GEF arrived to be 126 mutants/10^6 cells for the microwell method.

Evaluation criteria:

Test item is considered mutagenic if the following criteria are met:
- the induced mutant frequency meets or exceeds the Global Evaluation Factor (GEF) of 126 mutants per 1x10^6 cells
- a concentration-dependent increase in mutant frequency is detected
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
A test item is considered to be negative if the induced mutant frequency is below the GEF or the trend of the test is negative.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative controls. Mutant frequencies of the negative controls were used as reference. Statistical significance at the 5% level (p< 0.05) was evaluated.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The relative total growth (RTG) was 15.2 and 6.7% (without metabolic activation) and 10.6 and 13.7% (with metabolic activation) for the highest concentrations evaluated.

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 7.0 ± 0.4).
- Precipitation: In the main experiment no precipitation was observed.

RANGE-FINDING/SCREENING STUDIES: The selection of the concentrations used in the main experiment was based on data from the pre-experiment according to the OECD guideline 490.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): All mutant frequencies for negative, and positive controls were found within the historical range of the test facility Eurofins Munich.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative suspension growth

For individual results see tables 1 to 10 in box "Any other information on results incl. tables".

Table 1: Pre-Experiment for Toxicity, without metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]
C1	0	304000	742000	1310000	9.7	100.0
C2		314000	758000	1220000	9.2
1	50	256000	451000	1240000	5.6	59.0
2	150	229000	333000	939000	3.1	33.0
3	500	175000	200000	524000	1.6	16.6
4	1000	88900	81300	132000	0.4	4.2
5	1500	149000	154000	318000	1.0	10.1
6	2000	43600	37500	37500	0.1	1.2

C: Negative control

a: Suspension Growth, SG = [((value 24 h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h*x20))]

* : for value 24 h > 3x10⁵ then value 24 h = 3x10⁵

b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

Table 2: Pre-Experiment for Toxicity, with metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]
C1	0	248000	578000	1240000	7.2	100.0
C2		309000	688000	1230000	8.5
1	50	253000	504000	1250000	6.3	80.6
2	150	233000	406000	959000	3.9	49.8
3	500	212000	308000	1030000	3.2	40.6
4	1000	218000	280000	676000	2.0	26.0
5	1500	135000	154000	371000	1.1	14.2
6	2000	87500	77600	109000	0.3	4.2

C: Negative control

a: Suspension Growth, SG = [((value 24 h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h*x20))]

*: for value 24 h > 3x10⁵ then value 24 h = 3x10⁵

b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

Table 3: Main Experiment - Toxicity data, without metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]	RCE^c[%]	RTG^d[%]
C1	0	420000	989000	1510000	14.9	100.0	100.0	100.0
C2		407000	1010000	1400000	14.1
1	1	964000	992000	1370000	13.6	93.5	115.2	107.7
2	5	425000	993000	1430000	14.2	97.7	139.8	136.6
3	10	413000	1010000	1360000	13.7	94.5	123.2	116.4
4	50	367000	698000	1270000	8.9	61.0	95.5	58.2
5	175	259000	378000	954000	3.6	24.8	87.4	21.7
6	500	207000	267000	746000	2.2	15.4	98.4	15.2
7	1000	127000	140000	337000	1.0	7.0	97.0	6.7
EMS	300	295000	1070000	1220000	13.1	89.8	87.4	78.5
MMS	10	428000	1180000	949000	11.2	77.0	106.3	81.9

C: Negative control

a:  Suspension Growth, SG = [((value 24h x 30) / 1x10⁷) x ((value 48 h x 20) / (value 24 h*x20))]

*: for value 24 h > 3x10⁵ then value 24 h = 3x10⁵

b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

c:  Relative Cloning Efficiency, RCE = [(CE_{test group}/ CE_{of corresponding controls}) x 100]

Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)

d: Relative Total Growth, RTG = (RSG x RCE) / 100

EMS: Ethyl methanesulfonate

MMS: Methyl methanesulfonate

Table 4: Main Experiment - Mutagenicity data, without metabolic activation

		Cloning Efficiency (CE)			Mutagenicity Data
Test Group	Concen-tration [µg/mL]	Plate 1^e	Plate 2^e	CE^f[%]	Number of cultures / 96 wells					MF^g     [mutants / 10^6cells]	IMF^h      [mutants / 10^6cells]
					Plate 1^e	Plate 2^e	Plate 3^e	Plate 4^e	Mean
C1	0	71	73	86.6	20	14	9	14	14.3	93.4	/
C2		74	74	92.1	14	15	13	9	12.8	77.6	/
1	1	73	82	102.9	15	13	10	19	14.3	78.5	-7.0
2	5	82	84	125.0	11	6	16	14	11.8	52.6	-32.8
3	10	81	78	110.1	15	6	7	14	10.5	53.1	-32.4
4	50	74	69	85.4	8	11	14	9	10.5	68.1	-17.4
5	175	72	65	78.1	11	13	12	9	11.3	79.9	-5.6
6	500	72	73	88.0	16	18	14	16	16.0	103.7	18.2
7	1000	71	73	86.6	9	12	13	12	11.5	73.7	-11.8
EMS	300	62	75	78.1	70	70	74	70	71.0	862.6	777.1
MMS	10	76	74	95.0	49	53	58	50	52.5	418.4	333.0

C: Negative control

e: Number of cultures with cell growth.

f: Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)

g: Mutant frequency

MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

h:Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

EMS:   Ethyl methanesulfonate

MMS:  Methyl methanesulfonate

Table 5: Main Experiment - Colony sizing, without metabolic activation

Test Group*	Concentration [µg/mL]	Wells with at least 1 colony	Large colonies	Small colonies	% small colonies
C1	0	57	41	16	28.1
C2		51	37	14	27.5
EMS	300	284	211	73	25.7
MMS	10	210	75	135	64.3

C:        Negative control

EMS:   Ethylmethanesulfonate

MMS:  Methylmethanesulfonate

Table 6: Main Experiment - Toxicity data, with metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]	RCE^c[%]	RTG^d[%]
C1	0	308000	877000	1130000	9.9	100.0	100.0	100.0
C2		343000	903000	1110000	10.0
1	50	296000	724000	1130000	8.2	82.1	120.0	98.5
2	100	275000	602000	987000	5.9	59.6	0.0	0.0
3	250	256000	392000	1220000	4.8	48.0	108.7	52.2
4	500	263000	460000	931000	4.3	43.0	93.3	40.1
5	1000	217000	299000	868000	2.6	26.1	90.5	23.7
6	1200	215000	275000	715000	2.1	21.5	100.6	21.6
7	1500	166000	220000	497000	1.5	15.0	91.9	13.7
8	1600	116000	155000	406000	1.2	12.2	86.7	10.6
B[a]P	2.5	218000	484000	811000	3.9	39.4	79.5	31.3

C: Negative control

a: Suspension Growth, SG = [((value 24 h x 30) / 1 x 10⁷) x ((value 48 h x 20) / (value 24 h* x 20))]

 * : for value 24 h > 3 x 10⁵then value 24 h = 3 x 10⁵

b:  Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

c:  Relative Cloning Efficiency, RCE = [(CE_{test group}/ CE_{of corresponding controls}) x 100

Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)

d: Relative Total Growth, RTG = (RSG x RCE) / 100

B[a]P:  Benzo[a]pyrene

Table 7: Main Experiment - Mutagenicity data, with metabolic activation

		Cloning Efficiency (CE)			Mutagenicity Data
Test Group	Concen-tration [µg/mL]	Plate 1^e	Plate 2^e	CE^f[%]	Number of cultures / 96 wells					MF^g     [mutants / 10^6cells]	IMF^h      [mutants / 10^6cells]
					Plate 1^e	Plate 2^e	Plate 3^e	Plate 4^e	Mean
C1	0	71	72	85.4	9	8	8	12	9.3	59.5	/
C2		75	75	95.0	8	14	9	11	10.5	61.2	/
1	50	74	84	108.2	13	20	8	9	12.5	65.2	4.9
3	250	76	76	98.0	11	14	11	10	11.5	65.2	4.8
4	500	71	71	84.1	10	9	10	13	10.5	69.0	8.7
5	1000	75	65	81.6	20	15	22	18	18.8	133.4	73.1
6	1200	77	70	90.7	13	18	10	14	13.8	85.6	25.3
7	1500	73	68	82.9	20	15	17	23	18.8	131.6	71.3
8	1600	68	69	78.1	11	12	12	17	13.0	93.4	33.1
B[a]P	2.5	69	62	71.7	57	56	53	52	54.5	586.0	525.7

C: Negative control

e:  Number of cultures with cell growth.

f:   Cloning Efficiency, CE = ((-ln (((96 - (mean Plate 1, Plate 2)) / 96)) / 1.6) x 100)

g:  Mutant frequency,

MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

h: Induced mutant frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

B[a]P:  Benzo[a]pyrene

Table 8: Main Experiment - Colony sizing, with metabolic activation

Test Group	Concentration [µg/mL]	Wells with at least 1 colony	Large colonies	Small colonies	% small colonies
C1	0	37	31	6	16.2
C2		42	31	11	26.2
B[a]P	2.5	218	110	108	49.5

C: Negative control

B[a]P:  Benzo[a]pyrene

*: Based on the non-mutagenic effects of the test item, an assessment of clastogenicity was not feasible.

Table 9: Main Experiment - Biometry, without metabolic activation

Test Group	Concentration [µg/mL]	mean Mutant Frequency	mean induced Mutant Frequency	p-value	statistical Significance
C1	0	93.4	/	/	/
C2		77.6	/	/	/
1	1	78.5	-7.0	0.5434	-
2	5	52.6	-32.8	0.0465	+*
3	10	53.1	-32.4	0.0465	+*
4	50	68.1	-17.4	0.4020	-
5	175	79.9	-5.6	0.7758	-
6	500	103.7	18.2	0.1394	-
7	1000	73.7	-11.8	0.2242	-
EMS	300	862.6	777.1	0.0040	+
MMS	10	418.4	333.0	0.0040	+

C: Negative control

EMS:   Ethyl methanesulfonate

MMS:  Methyl methanesulfonate

+: significant

-: not significant

+*: significantly decreased compared to negative control, therefore not relevant for interpretation of results

Table 10: Main Experiment - Biometry, with metabolic activation

Test Group	Concentration [µg/mL]	mean Mutant Frequency	mean induced Mutant Frequency	p-value	statistical Significance
C1	0	59.5	/	/	/
C2		61.2	/	/	/
1	50	65.2	4.9	0.7758	-
3	250	65.2	4.8	0.4424	-
4	500	69.0	8.7	0.1414	-
5	1000	133.4	73.1	0.0040	+
6	1200	85.6	25.3	0.0444	+
7	1500	131.6	71.3	0.0040	+
8	1600	93.4	33.1	0.0121	+
B[a]P	2.5	586.0	525.7	0.0040	+

C: Negative control

B[a]P:  Benzo[a]pyrene

+: significant

-: not significant

Conclusions:

In a mammalian cell gene mutation assay conducted according to OECD 490, the test item N1,N3-diallylpropane-1,3-diamine dihydrochloride did not induce the mutation frequency at any tested concentrations and can be therefore considered as non-mutagenic.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD Guideline 490, mouse lymphoma L5178Y cells cultured in vitro were exposed 4 hours to N1,N3-diallylpropane-1,3-diamine dihydrochloride (100% purity) at concentrations of 1, 5, 10, 50, 175, 500 and 1000 µg/mL without S9 metabolic activation and at concentrations of 50, 250, 500, 1000, 1200, 1500 and 1600 µg/mL with S9 metabolic activation. The selection of the concentrations used in the main experiment were based on data from a pre-experiment. No precipitation of the test item was noted any experiment and growth inhibition was observed in the main experiment without and with metabolic activation. The relative total growth (RTG) was 15.2 and 6.7% (without metabolic activation) and 10.6 and 13.7% (with metabolic activation) for the highest concentrations evaluated. The positive controls showed distinct effects in mutation frequency, thus proving the ability of the test system to detect potential mutagenic effects. Furthermore, in the main experiment without and with metabolic activation, all validity criteria were met.

No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF) was not exceeded by the induced mutant frequency at any concentration.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 490 for in vitro mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

N1,N3-diallylpropane-1,3-diamine dihydrochloride was evaluated for genotoxicity/mutagenicity in an in vitro testing battery (OECD test guidelines 471, 487 and 490).

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, S. typhimurium strains TA98, TA100, TA1535 and TA1537 and tester strain E. coli WP2 uvrA (pKM 101) were exposed to N1,N3-diallylpropane-1,3-diamine dihydrochloride (100% purity) in purified water at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in all tester strains and both experiments (plate incorporation and pre-incubation). Based on the results, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

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.

In a mammalian cell gene mutation assay conducted according to OECD Guideline 490, mouse lymphoma L5178Y cells cultured in vitro were exposed 4 hours to N1,N3-diallylpropane-1,3-diamine dihydrochloride (100% purity) at concentrations of 1, 5, 10, 50, 175, 500 and 1000 µg/mL without S9 metabolic activation and at concentrations of 50, 250, 500, 1000, 1200, 1500 and 1600 µg/mL with S9 metabolic activation. The selection of the concentrations used in the main experiment were based on data from a pre-experiment. No precipitation of the test item was noted any experiment and growth inhibition was observed in the main experiment without and with metabolic activation. The relative total growth (RTG) was 15.2 and 6.7% (without metabolic activation) and 10.6 and 13.7% (with metabolic activation) for the highest concentrations evaluated. The positive controls showed distinct effects in mutation frequency, thus proving the ability of the test system to detect potential mutagenic effects. Furthermore, in the main experiment without and with metabolic activation, all validity criteria were met. No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF) was not exceeded by the induced mutant frequency at any concentration.

The negative results obtained from all three tests (OECD 471, OECD 487 and OECD 490, GLP), indicate that N1,N3-diallylpropane-1,3-diamine dihydrochloride is not genotoxic/mutagenic.

Justification for classification or non-classification

Based on the available results obtained from an in vitro test battery (OECD 471, 487 and 490), the substance N1,N3-diallylpropane-1,3-diamine dihydrochloride is not considered to be genotoxic and no classification is warranted in accordance with CLP Regulation (EC) 1272/2008.