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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:

2021-12-03 to 2022-04-21

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,3-bis(3-(1H-imidazol-1-yl)propyl)urea

Cas Number:

1807618-04-7

Molecular formula:

C13H20N6O

IUPAC Name:

1,3-bis(3-(1H-imidazol-1-yl)propyl)urea

Test material form:

liquid: viscous

Remarks:

at room temperature crystallization is possible which does not lead to a quality loss according to the sponsor

Details on test material:

- Batch no.: RR1286
- Purity: 98%
- Appearance: yellow
- Expiry date: 2022-05-01

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: Atotech Deutschland GmbH, Batch no.: RR1286
- Purity, including information on contaminants, isomers, etc.: 98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 0 – 40°C; protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Prior to testing the test item was found to have crystallised. The sponsor was consulted and the crystallised test item was used for the purpose of this study. According to the sponsor, the crystallisation of the test item does not impede the quality of the substance. The test item was dissolved in A. dest. and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Method

Target gene:

histidine operon (S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537) and tryptophane operon (E.coli strain WP2 uvrA (pKM101))

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 liver microsomal fraction was prepared at Eurofins Munich and obtained from Trinova Biochem GmbH, Gießen, Germany. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route (Eurofins Munich) and male Sprague Dawley rats were induced with phenobarbital/β-naphthoflavone (Trinova). The S9 preparation obtained from Trinova Biochem GmbH was used in the pre-experiment and in main experiment I. The S9 preparation from Eurofins Munich was used in main experiment II.

- method of preparation of S9 mix: The S9 mix preparation was performed according to Ames et al. 100 mM of ice-cold sodium ortho-phosphate-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP. This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion: co-factor solution 9.5 parts; liver preparation 0.5 parts

- concentration or volume of S9 mix and S9 in the final culture medium: The S9 preparation mix consisted of 5% liver preparation. In the final culture medium, 500 µl S9 mix were used per 2700 µl total volume. The protein concentration in the S9 preparation of Eurofins Munich was 35.0 mg/mL (Lot: 191121), and in the S9 preparation of Trinova, 35.6 mg/mL (Lot: 4449). The protein concentration of Lot No.: 4449 was adjusted to 30 mg/mL.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
For Eurofins Munich-prepared S9 Homogenate, the following quality control determinations were performed by Eurofins Munich:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test

For Trinova Biochem GmbH-prepared S9 Homogenate, the following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of a pre-experiment for toxicity. As neither toxicity nor precipitation of the test item was observed in either tester strain used at the maximum recommended concentration of 5000 μg/plate (with and without metabolic activation), concentrations of 31.6, 100, 316, 1000, 2500 and 5000 μg/plate were selected for the main experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (for positive controls 4-nitro-o-phenylene-diamine and 2-aminoanthracene); Aqua dest. for test item and positive controls sodium azide/methylmethanesulfonate

- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide (CAS# 26628-22-8)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (4-NOPD; CAS# 99-56-9)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA; CAS# 613-13-8)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density: Samples of each tester strain were grown by culturing for 12 h at 37 °C in S. typhimurium medium (Nutrient Broth) and E. coli medium (Luria Bertani), respectively, to the late exponential or early stationary phase of growth (approx. 10E9 cells/mL).
- Test substance added in in agar (plate incorporation) in Experiment 1; preincubation in Experiment 2

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
For the pre-incubation method 100 μL of the test item-preparation is pre-incubated with the tester strains (100 μL) and sterile buffer or the metabolic activation system (500 μL) for 60 min at 37 °C prior to adding the overlay agar (2000 μL) and pouring onto the surface of a minimal agar plate.
- Exposure duration/duration of treatment:
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
- Any supplementary information relevant to cytotoxicity:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA1535 and TA1537 were counted manually. The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).

Evaluation criteria:

Acceptability criteria:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA (pKM101))
- the negative control plates (A. dest.) with and without S9 mix are within the mean values of the spontaneous reversion frequency of the historical control data range (January – December 2020 for all tester strains), see "Any other information on materials and methods incl. tables" for more detail):
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Evaluation of Mutagenicity:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

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

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
not specified
- Data on osmolality:
not specified
- Possibility of evaporation from medium:
not specified
- Water solubility:
The test item was dissolved in A. dest. and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.
- Precipitation and time of the determination:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
- Other confounding effects: The microbial contamination observed in one plate (experiment 1, TA1535, 31.6 μg/plate, with metabolic activation) did not affect the quality or integrity of the results as the microbial contamination could be clearly distinguished from the Salmonella typhimurium revertants and thus did not affect the evaluation.

RANGE-FINDING/SCREENING STUDIES (if applicable):
- The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. As neither toxicity nor precipitation of the test item was observed in either tester strain used at the maximum recommended concentration of 5000 μg/plate (with and without metabolic activation), concentrations of 31.6 to 5000 μg/plate were selected for the main experiments.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The negative control plates (A. dest.) with and without S9 mix were within the mean values of the spontaneous reversion frequency of the historical control data range. Corresponding background growth on both negative control and test plates was observed. The positive controls showed a distinct enhancement of revertant rates over the control plate

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
: No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
- Statistical analysis: not performed
- Any other criteria: not specified

Ames test:
- Signs of toxicity: No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
- Individual plate counts and mean number of revertant colonies per plate and standard deviation: Detailed information on individual plate counts and mean number of revertant colonies can be found in section "Overall remarks, attachments".

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data).
- Detailed information on historical control data for the negative and positive control can be found in section "Overall remarks, attachments".

Applicant's summary and conclusion

Conclusions:

Under the conditions of this bacterial reverse mutation assay, the test item 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea did not cause an increase in the frequency of revertants both with and without metabolic activation in any of the test strains under investigation (S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA (pKM101)).
Therefore, 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea is considered to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria (according to OECD 471), strains of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli (WP2 uvrA (pKM101)) were exposed to 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea (in Aqua dest.) at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 μg/plate in the presence and absence of mammalian metabolic activation (S9-mix). Experiment 1 was conducted according to the plate-incorporation test method, while in Experiment 2 the pre-incubation method was applied.

Due to an absence of cytotoxicity and precipitation, the test item was tested up to the recommended limit concentration of 5000 µg/plate.

The positive controls induced the appropriate responses in the corresponding strains. The response of the negative controls were within the historical control data range of the laboratory.

There was no evidence or a concentration-related positive response of induced mutant colonies over background for the test item.

Based on the results of this study, 1,3-bis(3-(1H-imidazol-1-yl)propyl)urea is considered to be non-mutagenic in the bacterial reverse mutation assay.

 

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