3-(dimethylamino)propylurea

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January - 25 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-(dimethylamino)propylurea

EC Number:

401-950-2

EC Name:

3-(dimethylamino)propylurea

Cas Number:

31506-43-1

Molecular formula:

C6H15N3O

IUPAC Name:

[3-(dimethylamino)propyl]urea

Test material form:

liquid: viscous

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: Manufacturer, UA21412002
- Purity, including information on contaminants, isomers, etc.: Content: 75% 3-(N,N-Dimethyl)propyl-urea and 25% Polyethylenglycol

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store container tightly closed at a well-ventilated place. Store in the original container. Do not store with acids.

Method

Target gene:

His-, Trp -

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Metabolic Activation System: test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post mitochondrial S9 fraction.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Charles River Laboratories Hungary Kft. according to Ames et al. [1] and Maron and Ames [2]. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly. The composition of solution refers to 1000 mL.

Induction of Liver Enzymes: Male Wistar rats (502-719 g, animals were 26-29 weeks old) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg bw/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study were 28 June 2021.

Preparation of Rat Liver Homogenate S9 Fraction: On Day 4, the rats were euthanized, and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl.
The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1- 5 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fractions for this study was 01 July 2021 (Charles River Laboratories Hungary Kft. code: E13612, Expiry date: 01 July 2023).
The sterility of the preparation was confirmed in each case. The protein concentration of the
preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Charles River Laboratories Hungary Kft. The mean protein concentration of the
S9 fraction used were determined to be 25.1 g/L.
The biological activity in the Salmonella assay of S9 was characterized in each case using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batches of S9 used in this study functioned appropriately.

The S9 Mix (containing 10 % (v/v) of S9): Salt solution for S9 Mix:
NADP Na 7.66 g
D-glucose-6 phosphate Na 3.53 g
MgCl2 x 6 H2O 4.07 g
KCl 6.15 g
Distilled water q.s. ad 1000 mL

Sterilization was performed by filtration through a 0.22 μm membrane filter.
The complete S9 mix was freshly prepared containing components as follows:

Ice cold 0.2 M sodium phosphate buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix (see above) 400 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.

Test concentrations with justification for top dose:

Concentration of the test item: 0.3162 mg/mL, 1 mg/mL, 3.162 mg/mL, 10 mg/mL, 31.62 mg/mL, 31.62 mg/mL

Concentration (μg test item/plate): 15.81 μg test tem/plate; 50 μg test tem/plate; 158.1 μg test item/plate); 500 μg test tem/plate; 1581 μg test tem/plate; 5000 test tem/plate

Concentrations: Concentrations for the main tests were selected on the basis of the Preliminary Compatibility Test and Preliminary Range Finding Test. In the main tests same concentrations were used.

Test Item Concentrations in the Mutagenicity Tests (Assay 1 and Assay 2)
Based on the results of the preliminary test, a 100 mg/mL stock solution was prepared in distilled water, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in the main tests were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.

Vehicle / solvent:

In the study two vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive control chemicals. The following chemicals were used for vehicle (solvent) control groups:
1. Dimethyl sulfoxide (DMSO):
Supplier: VWR
Batch No.: 20B204006
Expiry date: 11 February 2025
Purity: 100%
2. Distilled water:
Manufacturer: MAGILAB Kft.
Batch No.: 2201-8186*/ 2109-8099
Expiry date: 05 July 2022 / 14 March 2022
*Note: This batch was used in the main tests.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO) and Distilled water:

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine (NPD); 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

Preparation of formulations: Distilled water was used as solvent to prepare the stock solution of the test material (based on the available information and the performed trial formulation). Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent.
As agreed with the Sponsor, correction factor of 1.333 for purity was used

Preliminary Compatibility Test: The solubility of the test item was examined using Distilled water, DMSO (Dimethyl sulfoxide) and N,N-Dimethylformamide (DMF). The test item was soluble at 100 mg/mL concentration using Distilled water, DMSO and DMF. Due to the better biocompatibility and agree with the Sponsor distilled water was selected as vehicle for the study. The obtained stock formulation (50 μL) with the solution of top agar and
phosphate buffer (section 9.3.4.) was examined in a test tube without test bacterium suspension.
The results of the Preliminary Compatibility Test are summarized in Table 5.

Preliminary Range Finding Test:
Based on the solubility test, a 100 mg/mL stock solution was prepared in distilled water. Six test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and 4 times approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation.
In the Preliminary Range Finding Test the plate incorporation method was used.

Rationale for test conditions:

Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli bacterial
strains were selected to be used in the study as these are the preferred bacterial strains for AMES
Test based on the OECD No. 471 guideline, and it was also used in the method validation study of the Test Facility.
Three replicates were used in the study which is the minimum requirement of the OECD 471.

Evaluation criteria:

The colony numbers on the untreated / negative (solvent) / positive control and test item treated
plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The
mean number of revertants per plate, the standard deviation, and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft
ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

The study was considered valid if:
• the number of revertant colonies of the negative (vehicle/solvent) and positive controls
are in the relevant historical control range, generated at the test facility, in all tester strains
of the main tests (with or without S9-mix);
• at least five analysable concentrations are presented in all strains of the main tests.
• the highest tested concentration should be at the limit test concentration, or the highest
acceptable concentration based on cytotoxicity and/or precipitation.

Results and discussion

Test results

Test results 1

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 examined

Positive controls validity:

valid

Test results 2

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 examined

Positive controls validity:

valid

Test results 3

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 examined

Positive controls validity:

valid

Test results 4

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 examined

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Validity of the tests
Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates
were within the historical control range in all strains. At least five analysable concentrations, up to an acceptable maximal concentration, were presented in all strains with and without metabolic activation.
The reference mutagens showed a distinct increase of induced revertant colonies in each strain
with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Any other information on results incl. tables

Table 5: The Solubility of the Test Item in distilled water

Concentration of test item in distilled water (mg/mL)	Solubility in distilled water	Solubility in the top solution (test item formulation 50 µL + phosphate buffer 500 µL + top agar 2 mL)	Test item concentration in the test plate (mg/tube)
100	clear solution	clear solution	5000

Preliminary range finding test

In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate. No precipitate was detected on the plates in the preliminary experiment in both examined bacterial strains with and without metabolic activation. No inhibitory or toxic effects of the test item was observed in the preliminary experiment in both examined bacterial strains with and without metabolic activation.

Summary Tables of the Results

Table 7: Summary Table of the Range Finding Test

Concentrations (mg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains
		TA98		TA100
		-S9	+S9	-S9	+S9
Untreated control	Mean	21.3	20.7	104.3	93.3
	MF	1.05	0.97	1.00	0.93
DMSO control	Mean	21.0	22.0	--	109.0
	MF	1.03	1.03	--	1.09
Distilled water control	Mean	20.3	21.3	104.3	100.0
	MF	1.00	1.00	1.00	1.00
5000	Mean	22.7	20.7	96.0	90.0
	MF	1.11	0.97	0.92	0.90
2500	Mean	20.3	21.7	99.3	95.0
	MF	1.00	1.02	0.95	0.95
1000	Mean	18.3	21.0	90.7	95.7
	MF	0.90	0.98	0.87	0.96
316	Mean	21.0	23.0	97.7	98.7
	MF	1.03	1.08	0.94	0.99
100	Mean	19.7	22.3	97.3	95.7
	MF	0.97	1.05	0.93	0.96
31.6	Mean	18.0	21.0	94.3	99.7
	MF	0.89	0.98	0.90	1.00
10	Mean	17.3	20.0	97.3	97.7
	MF	0.85	0.94	0.93	0.98
NPD (4µg)	Mean	418.7	--	--	--
	MF	19.94	--	--	--
2AA (2µg)	Mean	--	2424.0	--	2457.3
	MF	--	110.18	--	22.54
SAZ (2µg)	Mean	--	--	1050.7	--
	MF	--	--	10.07	--

Mutagenicity Tests (Assay 1 and Assay 2)
In Assay 1, the plate incorporation method was used. In Assay 2 the pre-incubation method was used. The assays were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The assays were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate. Based on the results of the preliminary experiment, the examined test concentrations in the main tests were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.
No precipitate was detected on the plates in the main tests in the examined bacterial strains with
and without metabolic activation. In the Assay 1 no inhibitory, cytotoxic effect of the test item was observed in the main tests in all examined bacterial strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item (reduced background lawn development) was observed in the Assay 2 in all examined bacterial strains with and without metabolic activation at 5000 μg/plate concentration.
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA98 bacterial strain at 5000 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.38). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the
number of revertant colonies was within the historical control range. In Assay 2 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 1581 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.27). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases.
Furthermore, the observed mutations were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
In the main assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

Table 9: Summary Table of the Assay 2

Concentrations (mg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	19.0	22.0	102.0	92.0	10.7	10.7	8.3	7.3	33.7	38.7
	MF	1.02	0.96	1.00	0.96	0.91	0.97	1.14	1.05	0.92	0.86
DMSO control	Mean	19.3	21.0	--	96.3	--	12.3	7.0	7.3	--	42.3
	MF	1.04	0.91	--	1.00	--	1.12	0.95	1.05	--	0.94
Distilled water control	Mean	18.7	23.0	101.7	96.3	11.7	11.0	7.3	7.0	36.7	45.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	10.3	14.7	49.3	73.3	4.3	6.3	6.7	2.3	17.7	18.7
	MF	0.55	0.64	0.49	0.76	0.37	0.58	0.91	0.33	0.48	0.41
1581	Mean	19.3	21.03	91.0	106.7	9.3	7.0	9.3	7.0	40.0	39.3
	MF	1.04	0.93	0.90	1.11	0.80	0.64	1.27	1.00	1.09	0.87
500	Mean	18.7	23.0	99.0	102.3	8.0	8.7	8.0	7.3	38.7	40.0
	MF	1.00	1.00	0.97	1.06	0.69	0.79	1.09	1.05	1.05	0.89
158.1	Mean	18.0	26.7	94.3	99.7	9.7	7.3	7.0	8.3	38.3	38.7
	MF	0.96	1.16	0.93	1.03	0.83	0.67	0.95	1.19	1.05	0.86
50	Mean	19.3	21.7	96.7	99.3	9.3	8.3	7.0	7.0	36.7	38.7
	MF	1.04	0.94	0.95	1.03	0.80	0.76	0.95	1.00	1.00	0.86
15.81	Mean	19.0	21.7	90.0	106.0	11.3	9.0	8.0	7.0	39.7	41.3
	MF	1.02	0.94	0.89	1.10	0.97	0.82	1.09	1.00	1.08	0.92
NPD (4µg)	Mean	404.0	--	--	--	--	--	--	--	--	--
	MF	20.90	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2473.3	--	2454.7	--	204.3	--	208.7	--	--
	MF	--	117.78	--	25.48	--	16.57	--	28.45	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	255.3
	MF	--	--	--	--	--	--	--	--	--	6.03
SAZ (2µg)	Mean	--	--	1050.7	--	1042.7	--	--	--	--	--
	MF	--	--	10.33	--	89.37	--	--	--	--	--
9AA (50 µg)	Mean	--	--	--	--	--	--	412.0	--	--	--
	MF	--	--	--	--	--	--	58.86	--	--	--
MMS (2mL)	Mean	--	--	--	--	--	--	--	--	1053.3	--
	MF	--	--	--	--	--	--	--	--	28.73	--

Summary Tables of the Results

Table 8: Summary Table of the Assay 1

Concentrations (mg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	21.3	20.0	103.0	110.3	12.3	12.0	10.3	10.0	45.7	47.0
	MF	0.91	0.92	0.88	0.96	1.03	0.97	1.19	1.07	1.06	0.99
DMSO control	Mean	18.7	31.0	--	108.0	--	12.7	10.3	11.7	--	46.3
	MF	0.80	1.43	--	0.94	--	1.03	1.19	1.25	--	0.98
Distilled water control	Mean	23.3	21.7	116.7	115.0	12.0	12.3	8.7	9.3	43.0	47.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	21.3	30.0	96.0	96.7	12.3	11.0	9.7	10.0	52.7	55.3
	MF	0.91	1.38	0.82	0.84	1.03	0.89	1.12	1.07	1.22	1.17
1581	Mean	22.3	23.7	91.3	110.3	12.3	11.7	11.7	11.7	53.3	54.3
	MF	0.96	1.09	0.78	0.96	1.03	0.95	1.35	1.25	1.24	1.15
500	Mean	22.0	27.3	93.3	101.0	13.3	13.0	10.7	11.7	55.0	51.7
	MF	0.94	1.26	0.80	0.88	1.11	1.05	1.23	1.25	1.28	1.09
158.1	Mean	21.0	20.0	101.0	104.0	11.0	13.3	8.7	11.0	54.3	54.3
	MF	0.90	0.92	0.87	0.90	0.92	1.08	1.00	1.18	1.26	1.15
50	Mean	22.3	20.3	105.0	108.7	12.7	13.7	9.3	11.0	55.3	54.7
	MF	0.96	0.94	0.90	0.94	1.06	1.11	1.08	1.18	1.29	1.15
15.81	Mean	20.7	26.3	103.7	98.0	13.0	13.7	9.3	9.3	53.0	54.0
	MF	0.89	1.22	0.89	0.85	1.08	1.11	1.08	1.00	1.23	1.14
NPD (4µg)	Mean	408.0	--	--	--	--	--	--	--	--	--
	MF	21.86	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2477.3	--	2477.3	--	208.0	--	200.7	--	--
	MF	--	79.91	--	22.94	--	16.42	--	17.20	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	261.0
	MF	--	--	--	--	--	--	--	--	--	5.63
SAZ (2µg)	Mean	--	--	1069.3	--	1041.3	--	--	--	--	--
	MF	--	--	9.17	--	86.78	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	422.7	--	--	--
	MF	--	--	--	--	--	--	40.90	--	--	--
MMS (2mL)	Mean	--	--	--	--	--	--	--	--	1102.7	--
	MF	--	--	--	--	--	--	--	--	25.64	--

Applicant's summary and conclusion

Conclusions:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation
Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the
tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented
post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β naphthoflavoneinduced rats.
The reported data of this mutagenicity assay show that under the experimental conditions
applied the test item did not induce gene mutations by base pair changes or frameshifts in
the genome of the strains used.