Didysprosium 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 2016-07-05 to 2016-08-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

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:

Didysprosium trioxide

EC Number:

215-164-0

EC Name:

Didysprosium trioxide

Cas Number:

1308-87-8

Molecular formula:

Dy2O3

IUPAC Name:

dysprosium(3+); oxygen(2-)

Test material form:

solid: particulate/powder

Details on test material:

Name of the test material (as cited in the report): didysprosium trioxide
Physical state: solid
Appearance: (pale) yellow powder
Further information on test item confidential.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Based on the results of the solubility test, a 100 mg/mL stock solution was prepared in dimethyl sulfoxide (DMSO). Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10.
- No correction for purity of the test item was applied.

Method

Target gene:

Histidine locus (S. typhimurium strains TA98, TA100, TA1535 and TA1537); tryptophan locus (E. coli strain WP2uvrA)

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital-/β-naphthoflavone-induced rat liver post-mitochondrial S9 fraction

Test concentrations with justification for top dose:

Preliminary concentration range finding test: 10, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate (TA98 and TA100, plate incorporation);
Initial mutation test: 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate (all strains, plate incorporation);
Confirmatory mutation test: 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate (all strains, pre-incubation).

The selection of the doses was based on the results of the range finding study. Slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000 and 2500 μg/plate. Inhibitory or toxic effects of the test item were not detected. Based on these findings, the maximum final concentration to be tested in the main experiments was decided to be 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: In order to obtain a suitable solvent, a solubility test was performed using distilled water, dimethyl sulfoxide (DMSO), ethanol, acetone and N,N-dimethylformamide (DMF). Each formulation was stirred in order to limit observed sedimentation, which rate depends on the solvent used. The following observations were made on the formulations at 100 mg/mL concentration:
- distilled water: fast sedimentation,
- DMSO: slower sedimentation,
- ethanol: fast sedimentation,
- acetone: fast sedimentation,
- DMF: slower sedimentation.
Due to the speed of the sedimentation and the better biocompatibility, DMSO was selected as vehicle for the study.

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

Remarks:

non-activation, 4 μg, Salmonella TA98

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

Remarks:

non-activation, 2 μg, Salmonella TA100 and TA1535

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

Remarks:

non-activation, 50 μg, Salmonella TA1537

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

Remarks:

non-activation, 2 μL, E. coli WP2 uvrA

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

Remarks:

activation, 2 μg, all Salmonella strains; 50 μg, E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION
- Preliminary concentration range finding test / Initial mutation test: in agar (plate incorporation)
Bacteria (cultured in Nutrient Broth) 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
Bacteria (cultured in Nutrient Broth) 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.

DURATION
- Pre-incubation period: 48 h (confirmatory mutation test)
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): 48 h (simultaneously with exposure duration)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains); tryptophan (E. coli strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background inhibition; decrease in the number of revertant colonies

Evaluation criteria:

Criteria for a Positive Response:
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.

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.

Statistics:

According to the guidelines, statistical methods 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.

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 Excel TM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: fast sedimentation in formulation at 100 mg/mL
- Precipitation:
Preliminary range finding test: slight precipitate at 2500 and 5000 µg/plate with and without metabolic activation (TA98, TA100)
Initial mutation test: slight precipitate at 5000 µg/plate with and without metabolic activation in all tester strains
Confirmatory mutation test: slight precipitate at 5000 µg/plate with and without metabolic activation in all tester strains

RANGE-FINDING/SCREENING STUDIES
The observed number of revertant colonies was in the normal range. Sporadically, minor differences compared to the solvent control numbers were observed. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
Slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000 and 2500 μg/plate.
Inhibitory or toxic effects of the test item were not detected in the Preliminary Range Finding Test.
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 5000 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%))
- Positive historical control data: 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.
- Untreated, negative (solvent/vehicle) and positive control plates were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY
- No signs of inhibitory, cytotoxic effect of the test item (such as reduced background lawn development and/or reduced number of revertant colonies) were observed in the Initial Mutation Test and the Confirmatory Mutation Test in the examined bacterial strains at any concentrations with or without metabolic activation.

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 exhibited limited solubility as demonstrated by the preliminary range finding test and extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event. The tests were considered to be valid.

Initial Mutation Test/Confirmatory Mutation Test

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

In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain with metabolic activation at the concentration of 50 μg/plate. The mutation factor value was 1.74. 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 TA100 bacterial strain at 158.1 and 5 μg/plate concentrations with metabolic activation. The calculated mutation factor value at these dose levels was 1.22. 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.

Slight increases in the numbers of revertant colonies were detected compared to the solvent control in the Initial Mutation Test in some sporadic cases. However, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and were within the historical control range. They were considered as reflecting the biological variability of the test.

Applicant's summary and conclusion

Conclusions:

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.
In conclusion, the test item didysprosium trioxide has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.