Registration Dossier
Registration Dossier
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EC number: 401-950-2 | CAS number: 31506-43-1 3-(DIMETHYLAMINO)PROPYL UREA; HST 2844
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test material was shown to be non-clastogenic to CHL cells in vitro.
3-(Dimethylamino)propvlurea was not mutagenic under the conditions used in this bacterial assay. (Ames test)
o alert has been found for genotoxicity in vitro non-specific and related to in vitro mammalian gene mutation with timidine Kinase gene
Link to relevant study records
- 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
- 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
- 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. - Target gene:
- His-, Trp -
- 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. - 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. - 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
- 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
- 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
- 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
- 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. - 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. - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- The molecular weight of the test material was 145.2 and therefore the maximum dose level of 1452 µg/ml was equivalent to the 10 mM maximum recommended concentration.
The dose selection for the main experiments was the 10 mM maximum concentration for all exposure groups. - Vehicle / solvent:
- DMSO
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- Cultures were established at least 16 to 72 hours prior to treatment, 5 x 10^4 to 2.5 x 10^5 cells were seeded per flask for the 6-hour and 24-hour cultures and 2 x 10^4 to 7.5 x 10^4 cells were seeded per flask for the 48-hour cultures. The cells were exposed to at least four doses of the test material, vehicle and positive controls, both with and without metabolic activation. All treatments were performed in duplicate (A + B). Cultures were maintained at 37OC in a humidified atmosphere of 5% CO2 in air.
Experiment 1 - Short Term Exposure Both With and Without Activation
i) 6 hours exposure to the test material without S9 mix followed by 18 hours culture in treatment-fiee media prior to cell harvest.
ii) 6 hours exposure to the test material with S9 mix followed by 18 hours culture in treatmentfree media prior to cell harvest.
Experiment 2 - Continuous Exposure Without Activation and Short Term With Activation
i) 24 hours continuous exposure to the test material prior to cell harvest.
ii) 48 hours continuous exposure to the test material prior to cell harvest.
iii) 6 hours exposure to the test material with S9 mix followed by 18 hours culture in treatment free media prior to cell harvest.
Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were trypsinised to detach them from the tissue culture flask and suspended in culture medium. A sample of the cell suspension from each harvest time was counted to estimate growth inhibition at each concentration. The cells were centrifuged, the culture medium drawn off and discarded, and the cells resuspended in 0.075M hypotonic KC1. After ten to fifteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed several times and the cells stored at 4 °C for sufficient time to ensure complete fixation.
Preparation of Metaphase Spreads, Staining and Coding
The cells were resuspended in fresh fixative before centrifugation and suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data. When the slides were dry they were stained in Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium. After checking that the slide preparations were of good
quality, the slides were coded using a computerised random number generator.
Mitotic Index
A total of 1000 cells were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 23 to 27 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.
Aberrations recorded by the slide scorer were checked by a senior cytogeneticist. Cells with 38 or more chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported. Endoreduplicated cells are recorded and are included in the polyploid cell total number.
If there was a dose-related increase in endoreduplicated cells then they are reported separately.
The percentage of cells showing structural chromosome aberrations (breaks and exchanges) was calculated and reported. The number of gap-type aberrations was recorded and reported. - Statistics:
- Statistical Analysis
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
If the study gives a positive response then a D20 value will be calculated, which is the presumed dose level of the test substance that is required to induce aberrations in 20% of metaphases. - Key result
- Species / strain:
- Chinese hamster lung (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- 10 mM
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test material was shown to be non-clastogenic to CHL cells in vitro.
- Executive summary:
This study was conducted according to a method which was designed to assess the potential chromosomal mutagenicity of a test material on the metaphase chromosomes of the Chnese Hamster Lung (CHL) cell line according to the requirements of the Japanese New Chemical Substance Law (METI), OECD 473 and the updated Annex V B10 Method.
Duplicate cultures of Chinese Hamster Lung (CHL) cells were treated with the test material at several dose levels, together with vehicle and positive controls.
Five exposure groups were used:
Experiment 1 included a 6(18)-hour exposure, both with and without the addition of an induced rat liver homogenate metabolising system;
Experiment 2 included a 24-hour continuous exposure, a 48-hour continuous exposure and a repeat of the 6(18)-hours exposure with metabolic activation.
The dose levels evaluated in the main experiments were selected from a range of dose levels based on the results of a preliminary toxicity test and were in the range of 18 1.5 to 1452 µg/ml for all of the exposure groups.
The vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line. All the positive control chemicals induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material did not induce any toxicological significant increases in the frequency of cells with aberrations in any of the exposure groups. There was a small but statistically significant increase in the number of cells with aberrations at the maximum dose level in the with-metabolic activation exposure group of Experiment 2. However, this was not observed at the same dose level in Experiment 1 and the increase was seen in one culture only and was therefore considered to have no toxicological significance. The test material was shown to be non-toxic to CHL cells in vitro and the maximum recommended dose level of 10 mM was used in all cases.
The test material was shown to be non-clastogenic to CHL cells in vitro.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1985
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver metabolic activation system, S9-mix
- Test concentrations with justification for top dose:
- Doses up to and including 500 µg per plate did not cause any bacteriotoxicffects. The total bacteria counts remained unchanged. No inhibition of growth was observed. In doses above this the substance had only a weak bacteriotoxic effect specific to strain, both with and without S-9 mix so that this range can nevertheless be used for evaluation purposes.
- Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: Endoxan, trypaflavine and 2-aminoanthracene
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 2500 µg/plate
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 2500 µg/plate
- Untreated negative controls validity:
- valid
- 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
- Remarks:
- above 2500 µg/plate
- Untreated negative controls validity:
- valid
- 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
- Remarks:
- above 2500 µg/plate
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- 3-(Dimethylamino)propvlurea was not mutagenic under the conditions used in this bacterial assay.
- Executive summary:
3-(Dimethylamino)propvlurea was not mutagenic under the conditions used in this bacterial assay.
Toxicitv was noted at doses of 2500 and 12500 µl/plate.
3-(Dimethylamino)propylurea was investigated in the Salmonella~microsome test for point - mutagenic effects in doses ranging from 20 to 12500 pg per plate on four Salmonella typhimurium LT2 mutants.
These were the histidine - auxotropic strains TA 1535, TA 100, TA 1537, and TA 98.
Doses up to and including 500 pg per plate did not cause any bacteriotoxic effects. The total bacteria counts remained unchanged. No inhibition of growth was observed. In doses above this the substance had only a weak bacteriotoxic effect specific to strain, both with and without a test item-induced rat liver metabolic activation system so that this range can nevertheless be used for evaluation purposes.
Evidence of mutagenic activity for 3-(Dimethylamino)propylurea was not found. Neither a dose - related doubling of the mutant count nor a biologically relevant increase in the same, in comparison with the
negative controls, was observed.
The positive controls Endoxan, trypaflavine and 2-aminoanthracene had a marked mutagenic effect, as can be seen from the biologically relevant increase of mutant colonies, compared with the corresponding
negative control.
Referenceopen allclose all
Table 5: The Solubility of the Test Item in distilled water
Concentration of test item | Solubility in distilled water | Solubility in the top solution | Test item concentration in the test plate |
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
| 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
| 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
| 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 | -- | ||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
After one-off oral applications of single doses of test material (5000 mg/kg b.w.) it leads to the conclusion that the test material fails to induce a significant response in the invivo clastogenicy study, similar to OECD 474.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1986
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- other: Bor: NMRI (SPF Han)
- Sex:
- male/female
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Duration of treatment / exposure:
- 1 off
- Frequency of treatment:
- 1 time
- Post exposure period:
- 24 h
48 h
72 h - Dose / conc.:
- 5 000 mg/kg bw (total dose)
- No. of animals per sex per dose:
- 5 males and 5 females
- Control animals:
- yes, concurrent no treatment
- yes, concurrent vehicle
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- After one-off oral applications of single doses of test material (5000 mg/kg b.w.) it leads to the conclusion that the test material fails to induce a significant response in the invivo clastogenicy study, similar to OECD 474.
- Executive summary:
Using the micronucleus test, 3-(Dimethylamino)propylurea was investigated in male and female mice for a possible clastogenic effect on the chromosomes or bone marrow erythroblasts. The known clastogen and cytostatic agent cyclophosphamide served as a positive control.
The animals received a single oral administration at a dose of 5000 mg/kg and 20 mg/kg of body weight for 3-(Dimethy1amino)propylurea and the positive control (cyclophosphamide), respectively. The femoral
marrow of the 3-(Dimethylarnino)propylurea - groups was prepared 24, 48, and 72 hours after the administration. Negative and positive controls were sacrificed after 24 hours only.
The animals treated with 3-(Dimethylamino)propylurea showed symptoms of toxicity lasting up to 24 hours after the administration. This effect was more pronounced in females than in males. 8 of 20 females but
none of the males died during the test.
Erythrocyte formation, as measured by the ratio of polychromatic to normochromatic erythrocytes, was clearly inhibited. Cyclophosphamide, the positive control, had a clear clastogenic effect, as can be seen from the biologically relevant increase in polychromatic erythrocytes with micronuclei. An inhibition of erythropoiesis was not found here.
After oral application of single doses of test material (5000 mg/kg b.w.) it leads to the conclusion that the test material fails to induce a significant response in the invivo clastogenicy study, similar to OECD 474.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
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