N,N-bis[3-(dimethylamino)propyl]-N',N'-dimethylpropane-1,3-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is negative for mutagenicity (mutation, chomosomal abberation) in Ames, Micronucleus and Moue Lymphoma assays. Not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed in accordance with OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay). A GLP certificate is provided.

Justification for type of information:

see attached justifcation

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The stability of the test substance in the vehicle water has not been determined analytically .

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Target gene:

TA. 1535, TA 100, TA 1537, TA 98 and E . coli WP2 uvrA

Test concentrations with justification for top dose:

20 - 5,000 ug/plate

Details on test system and experimental conditions:

Standard plate test and preincubation test both with and without metabolic activation (Aroclor-induced rat liver S-9 mix)

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precitation of the test sub stance was found.

Occasionally, a slight decrease in the number of revertants was observed in the standard plate test with S-9 mix at 5,000 ug/ plate . In the preincubation assay bacteriotoxicity was found depending on the strain and test conditions_at doses > 2,500 ug / plate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

According to the result's of the present study, the substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental condition chosen.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed in accordance with OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay). A GLP certificate is provided.

Justification for type of information:

see attached justification

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The stability of the test substance in the vehicle water has not been determined analytically .

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Target gene:

TA. 1535, TA 100, TA 1537, TA 98 and E . coli WP2 uvrA

Test concentrations with justification for top dose:

20 - 5,000 ug/plate

Details on test system and experimental conditions:

Standard plate test and preincubation test both with and without metabolic activation (Aroclor-induced rat liver S-9 mix)

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precitation of the test sub stance was found.

Occasionally, a slight decrease in the number of revertants was observed in the standard plate test with S-9 mix at 5,000 ug/ plate . In the preincubation assay bacteriotoxicity was found depending on the strain and test conditions_at doses > 2,500 ug / plate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

According to the result's of the present study, the substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental condition chosen.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: in vitro micronucleus assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: OECG 487 - In Vitro Mammalian Cell Micronucleus Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The test substance was suspended in culture medium (RPMI 1640) and the highest test concentration of the test substance was 5000 g/mL. The starting solution (5000 g/mL) was diluted until formation of concentration series (5000, 2500, 1000, 500 and 250 g/mL). Fresh solutions of test substance were prepared before each experiment.

In the experiment with metabolic activation the concentration 5000 μg/mL was highly cytotoxic. Then the concentration of 2500 μg/mL was used as the highest one for the analysis of genotoxic effect with metabolic activation.

In experiments without metabolic activation the concentration 5000 μg/mL was used as the highest test concentration for analysis of genotoxicity without metabolic activation.

Vehicle / solvent:

20 l of the appropriate concentration of test substance solution in medium was added to lymphocyte culture (2.5 mL growth medium RPMI-M + ca 150-210 l human peripheral blood) in the presence and absence of a metabolic activation system (18.5 l S9 post mitochondrial fraction + 18.5 l cofactors). Duplicate cultures were used for each concentration and control.
The cultures were treated by test substance for 48 hours after mitogenic stimulation,

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Colchicine

Details on test system and experimental conditions:

Principle of test is the detection of binucleated cells with micronuclei, which are induced by the test substance in human peripheral blood lymphocytes. Lymphocytes are cultured in growth medium and test substance is added to them. Cell cycle is then stopped by cytochalasin B, cultures are sampled and microscopic preparations are prepared. Preparations are then analysed by microscope. Genotoxicity is indicated by increased incidence of binucleated cells with micronuclei.
Experiments with and without metabolic activation with short treatment (3 hours) are done at first. If both experiments of the short treatments are negative or equivocal, subsequently, extended exposure treatment without metabolic activation is performed.

The human peripheral blood lymphocytes used for testing were obtained from healthy non smoking donors.

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentration 5000 μg/mL was highly cytotoxic in experiment with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In the in vitro micronucleus test using human peripheral blood lymphocytes, the test substance POLYCAT 9 Catalyst was non mutagenic for the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: All the above strains have, mutation in the histidine operon, mutation (rfa-) - that leads to defective lipopolysaccharide coat, a deletion that covers genes involved in the synthesis of vitamin biotin.

Metabolic activation:

with and without

Metabolic activation system:

s9

Test concentrations with justification for top dose:

All tests are run at a minimum of four concentrations. In the standard plate test, at least six dose levels of the test material, dissolved in a suitable solvent, are added to the test system. The standard test doses are 0.005, 0.01, 0.1, 1.0, 5.0 and 10.0 microliters per plate for liquids and 0.5, 1.0, 10.0, 100.0, 500.0 and 1000.0 vg per plate for solids.

Vehicle / solvent:

The bacterial strains were cultured in Oxoid Media #2 (nutrient Broth). The selective medium was Vogel Bonner Medium E with 2% glucose.

Untreated negative controls:

yes

Remarks:

Vehicle only

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: Activation: 2-anthramine

Evaluation criteria:

Plate test data consists of direct revertant colony counts obtained from a set of selective agar plates seeded with populations of mutant cells suspended in a semisolid overlay. Because the test material and the cells are incubated in the overlay for approximately 2 days and a few cell divisions occur during the incubation period, the test is semiquantitative in nature. Although these features of the assay reduce the quantitation of results, they provide certain advantages not contained in a quantitative suspension test:
- The small number of cell divisions permits potential mutagens to act on replication DNA, which is often more sensitive than nonrepl icating DNA.
- The combined incubation of the test article and the cells in the overlay permits constant exposure of the indicator cells for approximately 2 days.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

not applicable

Genotoxicity:

not determined

Remarks:

E.coli Strain is examinated in Study Marabottini 2020

Cytotoxicity / choice of top concentrations:

other: E.coli Strain is examinated in Study Marabottini 2020

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Not mutagenic under the conditions of the Ames test.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-01-20 to 2020-05-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

described in Council
Regulation (EC) No. 440/2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted 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:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Company ID no. 16753, Batch no. UW19417870
- Expiration date of the lot/batch: 16 April 2022
- Purity test date: 2019-12-12
- Purity: 97.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: +20°C (+14°C ÷ +25°C), under nitrogen atmosphere and protect from light
- Stability under storage conditions: No assay of test item stability, nor its concentration and homogeneity in solvent
-Solubity of the test item: the test item was found to be soluble in water (solvent) at 50.0 µL/mL.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Solutions were prepared on a volume/volume basis with correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. All test item solutions were used within 1 hours and 33 minutes from the initial preparation. All dose levels in this report are expressed to threesignificant figures.

Target gene:

Histidine and tryptophan locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: uvrA, uvrB Sensitivity to UV irradiation. rfa Sensitivity to Crystal Violet. pKM101 Resistance to Ampicillin.

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolism: S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

The maximum concentration of the test item to be used in the Main Assays was determined
taking into consideration solubility in the final treatment mixture and cytotoxicity
results obtained in the preliminary toxicity test. Based on these results, in Main Assay I and II,
using the plate incorporation method, the test item was tested at 5.00, 2.50, 1.25, 0.625 and 0.313 µL/plate with all tester strains. An additional experiment (Main
Assay III), was performed with TA1535 and TA100 tester strains both in the absence or
presence of S9 metabolism using the plate incorporation method and a narrowed dose
range: 5.00, 4.00, 3.20, 2.56 and 2.05 μL/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; aqueous solvents (sterile water).

- Justification for choice of solvent/vehicle: Sterile water was used was solvent for the test item treatment of the bacterial strain and as solvent for Sodium azide and Methylmethanesulfonate (MMS). This solvent was selected since it is compatible with the survival of the bacteria and the
S9 metabolic activity.
DMSO was used as solvent for the positive controls 9-Aminoacridine, 2-Nitrofluorene, 2-Aminoanthracene .

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (triplicate)
- Number of independent experiments: A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assays. Afterwards, three Main Assays were conducted.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation) during Main Assay I and III; Main Assay II was performed using pre-incubation method
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes
- Exposure duration/duration of treatment: The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates from the preliminary toxicity test and Main Assay I were held at 4°C for 24 hours while plates from the Main Assays II and III test were immediately scored.
- Harvest time after the end of treatment (sampling/recovery times): After incubation plates from the Main Assays II and III test were immediately scored by counting the number of revertant colonies on each plate. Plates from the preliminary toxicity test and Main Assay I were scored after being held at 4°C for 24 hours.

Evaluation criteria:

ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
-Mean plate counts for untreated and positive control plates should fall within two standard deviations of the current historical mean values.
- The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 Millions for each strain.
-No more than 5% of the plates should be lost through contamination or other unforeseen event.

CRITERIA FOR THE OUTCOME OF THE ASSAYS
The test item was considered mutagenic, when two-fold (or more) increases (depending of the strain) in the mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

linear regression and t-test

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:

valid

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:

valid

True negative controls validity:

not applicable

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

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:

valid

True negative controls validity:

not applicable

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (triplicate)
- Number of independent experiments: A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assays. Afterwards, three Main Assays were conducted.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation) during Main Assay I and III; Main Assay II was performed using pre-incubation method
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes
- Exposure duration/duration of treatment: The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates from the preliminary toxicity test and Main Assay I were held at 4°C for 24 hours while plates from the Main Assays II and III test were immediately scored.
- Harvest time after the end of treatment (sampling/recovery times): After incubation plates from the Main Assays II and III test were immediately scored by counting the number of revertant colonies on each plate. Plates from the preliminary toxicity test and Main Assay I were scored after being held at 4°C for 24 hours.

Acceptance criteria
The assay was considered valid if the following criteria were met:
-Mean plate counts for untreated and positive control plates should fall within two standard deviations of the current historical mean values.
- The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 Millions for each strain.
-No more than 5% of the plates should be lost through contamination or other unforeseen event.
Criteria for the outcome of the assays
The test item was considered mutagenic, when two-fold (or more) increases (depending of the strain tested) in the mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Evaluation
-The mean plate counts for untreated and positive control plates fell within
the normal range based on historical control data.

-The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million
for each strain. No plates were lost through contamination or cracking. The study was
accepted as valid.
N,N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine treatment yielded
reproducible biologically relevant, statistically significant (based on the “doubling rule”)
and dose related increases in the number of revertant colonies, both in the absence and
presence of S9 metabolism with TA1535 tester strain.


TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test item is soluble is water at 50.0 µL/mL
- Precipitation and time of the determination: no precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.

RANGE-FINDING/SCREENING STUDIES (if applicable):
The test item was assayed in the preliminary toxicity test at a maximum concentration of 5.00 µL/plate and at four lower concentrations spaced at approximately half-log intervals: 1.58, 0.500, 0.158 and 0.0500 μL/plate.
No precipitation of the test item was observed at the end of the incubation period at any concentration.
No toxicity was noticed at any concentration level, in the absence or presence of S9 metabolism, with any tester strain.
An increase in the revertant numbers (2.65, 2.26-fold) was observed at the highest does level with TA1535 tester strain, in absence and presence of metabolic activation, respectively. In addition, a slight increase in revertant numbers, which did not reach twice the concurrent negative control value, was also seen at the highest dose level with tester strains TA 98 and TA100 in the presence of S9 metabolism. No relevant increased was observed with the remaining tester strains with and without metabolic activation.

STUDY RESULTS
Ames test:
Three Main Assays were performed.
- Signs of toxicity: in the Main Assay I and III, conducted with plate incorporation method, no toxicity was observed in absence and presence of S9 metabolism, with any tester strain. In Main Assay II toxicity was noticed at the highest or at the two highest dose levels with all tester strains both in absence and presence of S9 metabolism.
- Individual plate counts: In main Assay I the test item induced an increase in revertant number both in absence and presence of S9 metabolism (2.05, 2.11-fold respectively) with TA1535 tester strain. This increase was statistically significant, based on the doubling rule, and exceeded the upper confidence limit of our historical negative control data. In the main Assay II (conducted with at the same concentration as Main Assay II and pre-incubation method), no increase was observed both in absence and presence of S9 metabolism in any tester strain. In Main Assay III (conducted in tester strain TA 100 and TA1535 at a narrowed dose range: 5.00, 4.00, 3.20, 2.56 and 2.05 µL/plate, an increase in revertant numbers was observed at higher dose levels with
TA1535 tester strain both in the absence and presence of S9 metabolism.
- Mean number of revertant colonies per plate and standard deviation : These increases were statistically significant, based on the "doubling rate" and biologically relevant as the number of revertant colonies fell outside the normal distribution of historical negative control data. In addition, a dose-effect relationship was indicated. Thus, the results obtained can be considered as clear evidence of mutation induction.
No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.

According to the criteria laid down in the OECD test guidance 471 (2020) there is no evidence supporting a specific requirement for a two- or threefold increase over background, this rule of thumb is the most common method used on the evaluation of data. Data sets, for tester strains TA 98, TA100, TA 102, TA 97, TA 97a, and E.coli WP2 urvA (pKM101) are judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean solvent control value. Data set for tester strains TA 1535, TA 1537, and WPs urvA are judged positive if the increase in the mean revertants at the peak of the dose response is equal to or greater than three times the mean solvent control value (Derelanko. MJ. 2014, Handbook of Toxicology, Third Edition, CRAC Press).
Thus, being the increase of the mean revertants “only” twice the mean revertant solvent control value, we can conclude that, the substance is not mutagenic in the Ames test conducted with five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA under the experimental condition chosen.

Conclusions:

It is concluded that the test item did not induce mutation in any of the tester strain , both in the absence and presence of S9 metabolism, under the reported experimental conditions.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 15, 2012 to March 18, 2013

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

The study was conducted in accordance with the Study Plan agreed upon. There were no major deviations from this Study Plan. However, the following minor deviations were noted:
-The following additional parameters were evaluated using the existing data following the recommendations of OECD guideline for testing of chemicals (No. 476, July 21, 1997 and the Mouse Lymphoma Workgroup, Aberdeen, 2003): suspension growth (SG), cloning efficiency (CE), relative suspension growth (RSG) and relative total growth (RTG).
-In the presence of TFT the plates were incubated for 11 to 14 days and wells containing clones were identified microscopically and counted and not 11 to 12 days as stated in the Study Plan caused by a typing error.
These minor deviations did not affect the validity of the scientific results obtained in this study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: the test item was provided from sponsor / registrant; Batch No. 1133437
- Purity, including information on contaminants, isomers, etc.: 98.2%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, do not store near acids. Store in steel containers. Keep containers tightly closed in a dry, cool and well-ventilated place.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: the test item was completely dissolved in aqua ad iniectabilia. The vehicle aqua ad iniectabilia served as the negative control

Target gene:

Thymidine kinase (TK) is a cellular enzyme that allows cells to salvage thymidine from the surrounding medium for use in DNA synthesis. If the thymidine analogue 5-trifluoro-thymidine (TFT) is included in the growth medium, the analogue will be phosphorylated via the TK pathway and will cause cell death by inhibiting DNA synthesis. Cells which are heterozy¬gous at the TK locus (TK+/-) may undergo a single-step forward mutation to the TK-/- genotype in which little or no TK activity remains. Such mutants are as viable as the heterozygotes in normal medium because DNA synthesis may still proceed by de novo synthetic pathways that do not involve thymidine as an intermediate. The basis for selection of the TK-/- mutants is the lack of any ability to utilize toxic analogues of thymidine, which enable only the TK-/- mutants to grow in the presen¬ce of TFT. Cells which grow to form colonies in the presence of TFT are therefore assumed to have mutated, either spontaneously or induced by the test item, to the TK-/- genotype.
Two types of mutated cells can be distinguished, large, normal-growing colonies and small, slow-growing colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions or deletions), whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations. Thus, in this system, gene mutations within the tk gene and chromosomal events involving the gene may be detected and distinguished.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

For cell lines:
- Absence of Mycoplasma contamination:
- Periodically checked for karyotype stability: [yes]
- Periodically ‘cleansed’ of spontaneous mutants: [yes]

Additional strain / cell type characteristics:

other: To reduce the background mutant frequency (spontaneous frequency) of TK-/- mutants to a level as low as possible, cell cultures were exposed to conditions that select against the TK-/- phenotype (exposure to aminopterin or methotrexate).

Metabolic activation:

with and without

Metabolic activation system:

Description of the metabolic activation system

The in vitro metabolic activation system was comprised of rat liver enzymes (S9 fraction) and an energy producing system comprised of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt) and glucose-6-phosphate. The enzymes were contained in a 9000 x g supernatant from liver homogenate prepared from male rats treated with 500 mg/kg of Aroclor 1254 five days prior to sacrifice. The treatment with Aroclor 1254 was used to induce mixed function oxidase enzymes capable of transforming chemicals to more active forms.
The preparation of the Aroclor 1254-induced rat S9 fraction was carried out according to MARON & AMES (1983). The S9 fraction was stored in liquid nitrogen at below -80°C until used. The S9 fraction was tested for protein content (according to LOWRY), and relative P-448/P-450 activity. The protein content of the S9 fraction was 33.1 mg/mL S9, cytochrome P-450: 0.40 nmol/mg protein.
The S9 fraction was thawed immediately before use and was combined to form the activation system described below:

Amount Component Final concentration
8.0 mL sterile aqua ad iniectabilia
720.0 mg glucose-6-phosphate 138 mM
100.0 mg NADP 6.7 mM
4.0 mL 150 mM KCl salt solution
(sterile stock solution)30 mM
8.0.mL rat liver S9 (Aroclor 1254-induced) 5.3 µM P-450

The S9 mix was sterilized through a 0.45 µm filter and kept on ice at all times. The S9 mix was always prepared freshly for each experiment.

Test concentrations with justification for top dose:

The appropriate concentration ranges was established by performing a preliminary cytotoxicity (reduction in the number of colonies by more than 50% compared with the vehicle control) experiment, with and without metabolic activation.
A wide range of test item concentration of 25, 100, 250, 1000, 2500, and 5000 µg/mL was tested for cytotoxicity. The preliminary cytotoxicity information was then used to select concentration levels for the mutation assay. Concentrations were selected using the following criteria:
At least four analyzable concentrations were used. Where there is cytotoxicity, these concentrations cover a range from the maximum to little or no toxicity and are separated by no more than a factor between 2 and √10. If the maximum concentration is based on cytotoxicity then it should result in approximately 10 - 20% (but not less than 10%) relative survival (relative cloning efficiency) or relative total growth.
Although more concentrations may be used to initiate a mutation experiment, the objective is to carry at least 5 concentrations through the entire experiment. This procedure compensates for normal variations in cellular toxicity and helps to ensure the choice of at least 4 concentrations appropriately spaced in the relative growth range of approximately 10% to 100%.
Based on the results of the preliminary study five concentrations of 15.63, 31.3, 62.5, 125 and 250 µg POLYCAT 9 Catalyst/mL medium were employed in the mutagenicity tests.
The lowest separation factor of 2 as recommended by the guidelines was used. No increase in the mutant frequency was observed. Hence, it was considered acceptable not to add any further lower concentrations, as these additional lower concentrations would provide no further information.

Vehicle / solvent:

Justification for choice of solvent/vehicle: the test item was completely dissolved in aqua ad iniectabilia. The vehicle aqua ad iniectabilia served as the negative control
- Justification for percentage of solvent in the final culture medium: A correction factor of 1.02 was used as the purity of test item was 98.5% only. As recommended by the guidelines at least 106 cells (4.0 x 106) were suspended in treatment medium and diluted to 5 x 105 cells/mL.
Fresh preparations of the test and reference item solutions were prepared on each day of biological testing.

Concurrent negative controls were performed for each portion of the assay by exposing the cells to the vehicle in culture medium.
In the activation portion of the assays, the negative controls were also exposed to the S9 metabolic activation mix. A single negative control culture was used in the cytotoxicity assay and two negative control cultures were used in the mutation assays, each.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Remarks:

Methylmethanesulfonate (MMS) is mutagenic via alkylation of cellular DNA and is highly mutagenic to mouse lymphoma cells without S9 metabolic activation. MMS was used at 10 µL/mL and 15 µL/mL medium as a positive control for the mutation studies without

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- Number of independent experiments: 2

Cell cultures were maintained in cleansing medium for one day, placed in recovery medium for one day and then returned to normal growth medium for three to eight days before use.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): As recommended by the guidelines at least 106 cells (4.0 x 106) were suspended in treatment medium and diluted to 5 x 105 cells/mL.
- Test substance was added in medium
- Media: The cells used during the experimental studies were maintained in RPMI 1640 medium supplemented with 0.05% Pluronic® F68 , 2 mM L-glutamine , 220 µg/mL sodium pyruvate, 100 µg/mL gentamycin , 2.5 µg/mL fungizone and fetal bovine serum (10% by volume), hereinafter referred to as growth medium. Treatment medium was growth medium without sodium pyruvate, gentamycin and fungizone. Cleansing medium used for reducing the spontaneous frequency of TK-/- mutants prior to experimental studies consists of growth medium supplemented with approximately 4.0 x 10-5 M thymidine, 1.2 x 10-4 M hypoxanthine, 3.3 x 10-5 M glycine and 7.2 x 10 7 M methotrexate. Recovery medium is similar to cleansing medium, except that the methotrexate component is removed. Selection medium is growth medium that contains 3 µg/mL of TFT.

TREATMENT AND HARVEST SCHEDULE:
- The cells for the first and second experiments were obtained from logarithmically growing laboratory stock cultures of 2.75 x 106 cells/mL and were seeded into a series of tubes, diluted to 5 x 105 cells/mL per tube. The cells were pelleted by centrifugation, the culture medium was removed, and the cells were resuspended in treatment medium that contained 5% heat inactivated fetal bovine serum and the corresponding concentration of test substance. The dosed tubes were closed, vortexed and placed on a roller drum at approx. 37°C at 10 - 15 rpm for an exposure period of 3 hours.
Thereafter the cells were washed and resuspended in growth medium.:
- Exposure duration/duration of treatment: The test was carried out employing two exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; the experiment with S9 mix was carried out in two independent assays. The experiments were conducted with the same concentrations as in the first experiment.


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Cell densities were adjusted to 2 x 105/mL and the cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture were placed in two 96 well microtiter plates (^ 192 wells, averaging 1.6 cells/well) and incubated for 1 week whereas the rest of the cells was incubated for 2 days for the expression period (plating efficiency step 1). The cells for the plating of survival were counted after 1 week and the number of viable clones was recorded. The cells incubated for the expression period were maintained below 106 cells per mL and a minimum of 4 concentration levels plus positive and negative control was selected for 5-trifluoro-thymidine (TFT) resistance. At the end of the second expression period the selected cultures were diluted to 1 x 104 cells/mL and plated for survival (plating efficiency step 2) and TFT resistance in parallel (plating efficiency for TFT resistance).
- Selection time (if incubation with a selective agent): The plates were incubated for 11 to 14 days and wells containing clones were identified microscopically and counted.
- Fixation time (start of exposure up to fixation or harvest of cells): 17 days
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 5-trifluoro-thymidine (TFT) Batch no. BCB41284V; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany
- Criteria for small (slow growing) and large (fast growing) colonies: The number of large and small colonies was recorded with an automated colony counter that can detect colony diameters equal to or greater than 0.2 to 0.3 mm. Large colonies are defined as ≥ 1/4 and small colonies < 1/4 of the well diameter of 6 mm

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative Total Growth (RTG) is the measure of cytotoxicity relative to the control, that takes into account all cell growth, and cell loss during the treatment period and the 2 day expression period (RSG), and the cells' ability to clone 2 days after treatment (viability). RTG is calculated as follows: RTG=RSG x Individual viability value/Mean control viability value.
Relative suspension growth (RSG) is a measure of the growth in suspension during treatment and the expression period relative to the mean control and was calculated as follows:
RSG (%) = (Individual SG Value/Mean control SG value) x 100.
Viability is the measure of the cells' ability to clone i.e. Cloning efficiency (CE)

Rationale for test conditions:

In the preliminary experiment without and with metabolic activation (24-hour or 3-h exposure) cytotoxicity (decreased survival) was noted starting at a concentration of 250 µg/mL . Hence, in the main study the concentration-range of 15.63 to 250 µg/mL was used in the experiments without and with metabolic activation

Evaluation criteria:

The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment is a mutant frequency that is >= two times the concurrent background mutant frequency.
The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations is not sufficient evidence to evaluate a test item as a mutagen. For further details see "Any other information on materials and methods incl. tables"

Statistics:

Mean and Standard deviation determination.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 250 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mutation frequencies of the cultures treated with POLYCAT 9 Catalyst ranged from 59.06 to 106.72 per 106 clonable cells (3 hours exposure) and from 64.82 to 124.38 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and from 51.57 to 107.58 per 106 clonable cells (3 hours exposure, first assay) and from 50.87 to 107.94 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 106 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

In the preliminary experiment without and with metabolic activation (24-hour or 3-h exposure) cytotoxicity (decreased survival) was noted starting at a concentration of 250 µg/mL.

Thus, in the main study the concentration-range of 15.63 to 250 µg/mL was used in the experiments without and with metabolic activation.

Methylmethanesulfonate (at 10 or 15 µL/mL) was employed as a positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene (at 2.5 or 4.0 µg/mL) in the presence of exogenous metabolic activation.

In the main study, cytotoxicity (decreased survival) was noted in the absence and presence of metabolic activation at the top concentration of 250 µg/mL.

The values of mutation frequencies of the negative controls ranged from 67.13 to 99.73 per 106 clonable cells in the experiments without metabolic activation and from 72.70 to 87.01 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range.

The mutation frequencies of the cultures treated with the test item ranged from 59.06 to 106.72 per 10⁶ clonable cells (3 hours exposure) and from 64.82 to 124.38 per 10⁶ clonable cells (24 hours exposure) in the experiments without metabolic activation and from 51.57 to 107.58 per 10⁶ clonable cells (3 hours exposure, first assay) and from 50.87 to 107.94 per 10⁶ clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and the normal range of 50 to 170 mutants per 10⁶ viable cells and, hence, no mutagenicity was observed according to the criteria for assay eIn addition, no change was observed in the ratio of small to large mutant colonies, ranging from 0.41 to 1.33 for POLYCAT 9 Catalyst treated cells and from 0.58 to 1.08 for the negative controls.

The positive controls Methylmethanesulfonate (MMS) and 3-Methylcholanthrene (3-MC) caused pronounced increases in the mutation frequency ranging from 1706.96 to 1914.27 per 10⁶ clonable cells in the case of MMS and ranging from 1208.85 to 1797.76 per 10⁶ clonable cells in the case of 3-MC.

In addition, the colony size ratio was moderately shifted towards an increase in small colonies, ranging from 1.82 to 2.69 in the case of MMS valuation.

For detailed information please see the Results in Tabular form In the attached background material. 

 

Conclusions:

Interpretation of results
negative

Under the present test conditions, the test substance, tested up to a cytotoxic concentration of 250 µg/mL, in the absence and presence of metabolic activation in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, the test substance also did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that the test substance, tested up to a cytotoxic concentration of 250 µg/mL in the absence and presence of metabolic activation, did neither induce mutations nor have any chromosomal aberration potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

Two Ames tests conducted with the substance N,N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine, CAS  33329-35-0  are available.

In a first Ames test the substance of N,N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine, CAS  33329-35-0  was examined for mutagenic activity in a series of in vitro microbial assays employing Salmonella thyphimurium strains (TA 1535, TA 1537, TA 1538, TA 98, TA 100) in a study conducted with a method similar to OECD Guideline 471 in 1987. The compound was tested, directly and in the presence and absence of liver microsomal enzyme preparations from Aroclor -induced rats, which served as metabolic activation, at the following range concentrations 0.005 -10 µL per plate. The test substance exhibited toxicity with the strain TA-1535 in the activation assay at 10.0 µL per plate dose level.

The results of the Ames test conducted with the substance N,N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine, CAS  33329-35-0 in absence of the metabolic activation system were all negative. The test was repeated with TA 100 because of the contamination the initial assay. The repeated was also negative.

The results of the test conducted on the compound in the presence of metabolic activation system were all negative.

In the second Ames test The test item N,N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used.

Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

On the basis of the results obtained in the preliminary toxicity test, in Main Assay I, using the plate incorporation method, the test item was assayed at 5.00, 2.50, 1.25, 0.625, 0.313 µL/plate with all tester strains.

No toxicity was observed at any dose level, in absence or presence of S9 metabolism, with any tester strain.

Test item treatment at the highest dose level induced once again an increase in revertant numbers both in the absence and presence of S9 metabolism (2.05, 2.11-fold respectively) with TA1535 tester strain. Since no relevant increase was observed with TA100 tester strain, which contains the same base-pair substitution mutation (hisG46), the results obtained with TA1535 was considered equivocal, thus a confirmatory experiment (Main Assay II), was performed with all tester strains, using the same concentrations as Main Assay, and pre-incubation method.

Toxicity was noticed at the highest or at the two highest dose levels with all test strains both in absence and presence of S9 metabolism. No increase in revertant number was observed with any tester/ strain/activation condition combinations.

In order to clearly exclude or confirm a genotoxic effect and additional experiment (Main Assay II) was performed with TA1535 and TA 100 tester strains both in the absence or presence of S9 metabolism using the plate incorporation method and a narrowed dose range: 5.00, 4.00, 3.20, 2.56 and 2.05 µL/plate.

 

No toxicity was seen at any dose level, in the absence or presence of S9 metabolism, with any tester strain.

Once again, an increase in revertant numbers was observed at higher dose levels with 1535 tester strain both in the absence and presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.

According to the criteria laid down in the OECD test guidance 471 (2020) there is no evidence supporting a specific requirement for a two- or threefold increase over background, this rule of thumb is the most common method used on the evaluation of data. Data sets, for tester strains TA 98, TA100, TA 102, TA 97, TA 97a, and E.coli WP2 urvA (pKM101) are judged positive if the increase in mean revertants  at the peak of the dose response is equal to or greater than two times the mean solvent control value. Data set for tester strains TA 1535, TA 1537, and WPs urvA are judged positive if the increase in the mean revertants at the peak of the dose response is equal to or greater than three times the mean solvent control value.

Thus, being the increase of the mean revertants “only” twice the mean revertant solvent control value, we can conclude that, the substance is not mutagenic in the Ames test conducted with  five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA under the experimental condition chosen (Derelanko. MJ. 2014, Handbook of Toxicology, Third Edition, CRAC Press).

QSAR conducted with Vega, Derek, and Danish QSAR Database report the substance of interest to be not mutagenic for Ames test.

Furthermore, the analogue substance N-[3-(dimethylamino)propyl]-N,N',N'-trimethylpropane-1,3-diamine, CAS 3855-32-1, does not result to be mutagenic in an Ames test (OECD 471 and GLP condiction) in Salmonella Typhimurium strains and E.coli strains

In an OECD Guideline 487 study (In vitro Mammalian Cell Micronucleus Test N,N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine, CAS  33329-35-0tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times without S9 mix and one exposure time with S9 mix revealed no indications of chromosomal damage in the in vitro micronucleus test.

 In a OECD Guideline 490 study (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene) the substance N,N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine, CAS  33329-35-0 tested up to cytotoxic concentrations in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test.

After 28 and 90 day repeated oral exposure some treatment-related effects (including mortality) were observed at high dose levels. However, these effects were ascribed to a strong local effect of the test item due to its chemical nature (strong base) to the gastrointestinal and respiratory tracts. Thus, 28 and 90-day repeated dose studies conducted with N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine, CAS  33329-35-0 did not show any adverse effects on the reproductive organ of both genders.

Based on a toxicokinetic assessment of information from existing animal studies is it observed that toxic effects are primarily manifested on local level (irritation of skin and mucous membranes). The results of sensitisation studies (no sensitisation, potential skin corrosion) support this conclusion.

Overall, the substance N,N-bis[3-(dimethylamino)propyl]-N’,N’-dimethylpropane-1,3-diamine, CAS  33329-35-0 did not demonstrated genetic activity in any of the assay conducted and was considered not mutagenic.

Justification for selection of genetic toxicity endpoint

Current test performed to OECD standard and under GLP using the test substance.

Justification for classification or non-classification

Based on the available data on germ cell mutagenicity, the substance does not need to be classified as do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.