Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

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:

13 September 2017 to 05 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

EC Number:

945-733-5

Molecular formula:

See remarks

IUPAC Name:

Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Natural white powder
- Storage conditions: Room temperature (15-25 °C), protected from light and humidity

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: The appropriate vehicle (solvent) and the behaviour of the test material formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. DMSO was used as solvent to prepare the stock solution of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 4 hours after preparation. Analytical determination of the test material concentration, stability and homogeneity was not performed because of the character and the short period of study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The stock solution was placed in an ultrasonic water bath for 2 minutes before use.

Method

Target gene:

S. typhimurium: Histidine
E. coli: Tryptophan

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Methods for growing in culture: The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum was used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay. The cultures were incubated for 10 - 14 hours at 37°C in a Gyrotory water bath shaker.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
The Typical Composition (g/1 000 mL) of Minimal Glucose Agar
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g
Distilled water q.s. ad 1 000 mL
Minimal glucose agar plates (Batch number: 145659, Expiry date: 20 December 2017) were provided by Merck. Certificates of Analysis were obtained from the Supplier.

Nutrient Broth No.2:
Nutrient Broth No.2 25.0 g
Distilled water q.s. ad 1 000 mL
Sterilization was performed at 121 °C in an autoclave.

Nutrient Agar
Nutrient Agar 20.0 g
Distilled water q.s. ad 1 000 mL
Sterilization was performed at 121 °C in an autoclave.

Top Agar for Salmonella typhimurium Strains
Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Distilled water q.s. ad 1 000 mL
Sterilization was performed at 121 °C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin (F.W. 244.31) 122.2 mg
L-Histidine x·HCl x H2O (F.W. 209.63) 104.8 mg
Distilled water q.s. ad 1 000 mL
Sterilization was performed by filtration using a 0.22 μm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100 mL
Agar solution 900 mL

Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/mL):
L-Tryptophan (F.W. 204.23) 2 000 mg
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration using a 0.22 μm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth 50 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution 947.5 mL

- Properly maintained: Yes.
The strains are stored at -80 ± 10 ºC in the Culture Collection of the Microbiological Laboratory of Citoxlab Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures, to which DMSO was added as a cryoprotective agent.
- Periodically checked for phenotype stability: The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames. Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of Citoxlab Hungary Ltd.
- Periodically 'cleansed' against high spontaneous background: Yes. Each test strain reverts spontaneously at a frequency that is characteristic of the strain.
Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011-2016 were (as guide) as follows: Salmonella typhimurium TA98: 9-50, TA100: 54-210, TA1535: 1-46, TA1537: 1-24, Escherichia coli WP2 uvrA: 11-82.
- Viability: The viability of each testing culture was determined by plating 0.1 mL of the 10^5, 10^6, 10^7 and 10^8 dilutions prepared by sterile physiological saline on Nutrient Agar plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Examined concentrations in the Initial Mutation Test were 5 000, 1 581, 500, 158.1, 50 and 15.81 μg/plate.
Examined concentrations in the Confirmatory Mutation Test were 5 000, 1 581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the different concentrations were used.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: In a preliminary compatibility test the solubility of the test material was examined using Distilled water, N,N-Dimethylformamide (DMF), Dimethyl sulfoxide (DMSO) and Acetone. Partial dissolution was observed at the 100 mg/mL concentration using Distilled water and Acetone. At this concentration suspension with test material particles was observed using DMSO and DMF.
At 50 mg/mL concentration there was no change for Distilled water or Acetone, but a suspension of smaller test material particles was observed far DMSO and DMF. After 2 minutes ultrasonic water bath a homogeneous suspension was obtained for DMSO and DMF. Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. The obtained stock solution (100 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension.

Controls

Untreated negative controls:

yes

Remarks:

An untreated control was used to demonstrate that the chosen vehicle induced no deleterious or mutagenic effects. If the solvent of the positive control substance differed from the solvent of the test material, both solvents were run in the assay.

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO) and distilled water, depending on the solubility of the test material and the solubility of strain specific positive control chemicals.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine and 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION
Initial mutation test: plate incorporation method
Confirmatory mutation test: pre-incubation method

DURATION
- Pre-incubation period: 20 minutes
- Selection time:
Initial mutation test: 48 ± 1 h.
Confirmatory mutation test: 48 ± 1 h.

SELECTION AGENT
S. typhimurium: Histidine
E. coli: Tryptophan

NUMBER OF REPLICATIONS
Initial mutation test: Triplicate for each control or concentration level.
Confirmatory mutation test: Triplicate for each control or concentration level.

- PRELIMINARY CONCENTRATION RANGE-FINDING TEST (INFORMATORY TOXIICTY TEST).
Based on the solubility test, a 50 mg/mL stock solution of the test material was prepared in DMSO. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and 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 5 000, 2 500, 1 000, 316, 100, 31.6 and 10 μg/plate of the test material, in the absence and presence of metabolic activation. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

PROCEDURE FOR EXPOSURE IN THE INITIAL MUTATION TEST
The Initial Mutation Test followed the standard plate incorporation procedure.
Bacteria (cultured in Nutrient Broth No.2) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test material and other components were prepared freshly and added to the overlay (45°C).

The content of the tubes:
Top agar 2 000 μL
Vehicle or test material formulation (or reference controls) 100 (50)* μL
Overnight culture of test strain 100 μL
Phosphate buffer (pH 7.4) or S9 mix 500 μL
*Treatment volume was 100 μL for test material formulations and its solvent; treatment volume was 50 μL for positive control substances and their solvents.

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

PROCEDURE FOR EXPOSURE IN THE CONFIRMATORY MUTATION TEST
The Confirmatory Mutation Test followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the Initial Mutation Test.
Bacteria (cultured in Nutrient Broth No.2) were exposed to the test material both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled.
Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

Rationale for test conditions:

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the different concentrations were used.

Evaluation criteria:

Criteria for Validity:
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;

Criteria for a Positive Response:
A test material was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response:
The test material was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

The colony numbers on the untreated / negative (solvent) / positive control and test material 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 material and for the controls using Microsoft Excel™ software.
* Mutation factor (MF): Mean number of revertants on the test material plate / mean number of revertants on the vehicle control plate.

Results and discussion

Test results

Key result

Species / strain:

other: Salmonella typhimurium TA 98, TA100, TA1535, TA1537; Escherichia coli WP2 uvrA

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate of the test material was detected in all examined bacterial strains with and without metabolic activation in the Initial Mutation Test on the plates at 5 000 μg/plate concentration. The same effect was observed in the Confirmatory Mutation Test in all Salmonella typhimurium bacterial strains with and without metabolic activation on the plates at 5 000 and 1 581 μg/plate concentrations and in Escherichia coli WP2 uvrA strain with and without metabolic activation at 5 000 μg/plate concentration. In these concentrations, the background lawn development could not be assessed due to the strong precipitate, but the colony counting was not affected.


RANGE-FINDING/SCREENING STUDIES
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.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
Precipitate/slight precipitate was detected on the plates in the Preliminary experiment in Salmonella typhimurium TA98 strain with and without metabolic activation at 5 000 and 2 500 μg/plate concentrations and in Salmonella typhimurium TA100 strain with and without metabolic activation at 5 000 μg/plate concentration.
Inhibitory or toxic effects of the test material were not detected in the preliminary experiment.
Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5 000 μg/plate


ADDITIONAL INFORMATION ON CYTOTOXICITY
Inhibitory, cytotoxic effect of the test material was not detected in the Initial Mutation Test and Confirmatory Mutation Test.

INITIAL AND CONFIRMATORY MUTATION TESTS
In the Initial Mutation Test and Confirmatory Mutation Test, 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.
In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 15.81 μg/plate concentration without 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 the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 5 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.31). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls 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 and they 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.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Any other information on results incl. tables

Validity of the Test

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 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.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test the test material was not mutagenic in any of the strains tested with and without metabolic activation.

Executive summary:

The test material 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 post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test material was dissolved in DMSO at a concentration of 50 mg/mL. Concentrations of 5 000; 2 500; 1 000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test material concentrations in the Initial Mutation Test were 5 000, 1 581, 500, 158.1, 50 and 15.81 μg/plate, and in the Confirmatory Mutation Test were 5 000, 1 581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Precipitate of the test material was detected in all examined bacterial strains with and without metabolic activation in the Initial Mutation Test on the plates at 5 000 μg/plate concentration. The same effect was observed in the Confirmatory Mutation Test in all Salmonella typhimurium bacterial strains with and without metabolic activation on the plates at 5 000 and 1 581 μg/plate concentrations and in Escherichia coli WP2 uvrA strain with and without metabolic activation at 5 000 μg/plate concentration.

Note: In these concentrations, the background lawn development could not be assessed due to the strong precipitate, but the colony counting was not affected. Inhibitory, cytotoxic effect of the test material was not detected in the Initial Mutation Test and Confirmatory Mutation Test.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test material has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.