Digadolinium trioxide

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:

from 2016-05-18 to 2016-09-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

Digadolinium trioxide

EC Number:

235-060-9

EC Name:

Digadolinium trioxide

Cas Number:

12064-62-9

Molecular formula:

Gd2O3

IUPAC Name:

Digadolinium trioxide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): digadolinium trioxide
- Physical state: solid
- Appearance: white powder

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: 100 mg/mL stock formulation was prepared in DMSO, which was diluted by serial dilutions in six steps to obtain seven dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate. The formulations were stirred with magnetic stirrer in the main tests.
- Dimethyl sulfoxide (DMSO) was used as solvent to prepare the stock solution of the test material. Test suspensions 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.
- As the theoretical correction factor (1.002) was nearly 1, in practice, there was no correction factor used in formulation preparation.

FORM AS APPLIED IN THE TEST (if different from that of starting material): formulation in dimethyl sulfoxide

Method

Target gene:

histidine and tryptophan

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial S9 fraction (rat liver)

Test concentrations with justification for top dose:

100 mg/mL (5000 µg/plate), 31.62 mg/mL (1581 µg/plate),10 mg/mL (500 µg/plate), 3.162 mg/mL (158.1 µg/plate), 1 mg/mL (50 µg/plate), 0.3162 mg/mL (15.81 µg/plate), 0.1 mg/mL (5 µg/plate).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The appropriate vehicle (solvent) and the behaviour of the test item 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 since better biocompatibility and homogeneity was observed in preliminary compatibility test.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylenediamine

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

INITIAL MUTATION TEST
- Method of application: Plate incorporation method
- Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item 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 item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes: top agar 2000 µL; vehicle or test item formulation (or reference controls) 50 µL; overnight culture of test strain 100 µL; phosphate buffer (pH 7.4) or S9 mix 500 µL
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.

CONFIRMATORY MUTATION TEST (pre-incubation method)
- A pre-incubation procedure was performed as a Confirmatory Mutation Test since no positive effect was observed in the Initial Mutation Test.
- Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item 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.

EVALUATION OF EXPERIMENTAL DATA
- 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.

In the main tests each sample (including the controls) was tested in triplicate.

Evaluation criteria:

A test item 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:
A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Preliminary Concentration Range Finding Test:
- In the Preliminary Concentration 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. In the test each sample (including the controls) was tested in triplicate.
- In the Range Finding Test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
- The observed numbers of revertant colonies were in the normal range. Minor differences compared to the solvent control numbers were observed in some sporadic cases. However, they had no biological significance and were within the historical control range in all cases; thus, they were considered as reflecting the variability of the test system.
- Precipitate was observed in both tester strains with and without metabolic activation at the concentration of 5000 µg/plate.
- Inhibitory or toxic effect of the test item was not detected in the Preliminary Concentration Range Finding Test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the Initial Mutation Test (using the plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 50 μg/plate concentration without metabolic activation (the observed mutation factor value was 2.00). Higher numbers of revertant colonies compared to the solvent control plates were observed at some other tested concentrations in this strain without metabolic activation. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.

In the Confirmatory Mutation Test (using pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA98 bacterial strain at 1581 μg/plate concentration with metabolic activation. The calculated mutation factor value at this dose level was 1.40. However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.

Precipitate was observed in the Initial Mutation Test and in the Confirmatory Mutation Test in all examined bacterial strains at 5000 µg/plate concentration with and without metabolic activation; furthermore precipitate/slight precipitate was observed in the Confirmatory Mutation Test in Salmonella typhimurium strains at 1581 µg/plate concentration with and without metabolic activation and in Salmonella typhimurium TA1537 strain at 500 µg/plate concentration without metabolic activation.

Note: In the Confirmatory Mutation Test strong precipitate was observed on the plates in all examined bacterial strains at 5000 µg/plate concentrations with and without metabolic activation.
The assessment of the background lawn development was difficult in this concentration, but counting of colonies was not affected.

There were no signs of inhibitory, cytotoxic effect of the test item in the Initial Mutation Test and the Confirmatory Mutation Test in the examined bacterial strains at any concentrations with or without metabolic activation.

Lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Test in some cases. However, the mean numbers of revertant colonies were within the historical control range, thus they were considered as biological variability of the test system.

Slight increases in the numbers of revertant colonies compared to the solvent control were detected in some other sporadic cases. However, no dose-dependence was observed and they were below the biologically relevant threshold value and were within the historical control range, they were considered as reflecting the biological variability of the test.

Any other information on results incl. tables

Validity of the tests:

Untreated, negative (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. At least five analysable concentrations were presented in all strains of the main tests. The selected dose range included a clearly toxic concentration or exhibited limited solubility as demonstrated by the preliminary toxicity range-finding test or extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event. The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Applicant's summary and conclusion

Conclusions:

The reported data of the 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. In conclusion, the test item digadolinium trioxide had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.