Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 March 2017 - 30 May 2017
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): yttrium trifluoride
- Physical state: solid
- Appearance: white powder
- Further information on test material confidential.
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: Based on the results of the preliminary tests, 100 mg/mL stock formulation was prepared in distilled water, which was diluted by serial dilutions in seven steps to obtain eight dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
- Distilled water was used as solvent to prepare the stock formulation of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock formulation using the selected solvent and were used within 4 hours after preparation.
- No correction factor for purity of the test item was applied.

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

Method

Target gene:
histidine and tryptophan
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial S9 fraction (rat liver)
Test concentrations with justification for top dose:
- Range finding test: 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
- Initial mutation test: 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate.
- Confirmatory mutation test: 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate.

The selection of the doses for the main test was based on the results of the preliminary range finding study. The observed number of revertant colonies was in the normal range. Slight decreases of the revertant counts were observed compared to the solvent control sporadically. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system. No precipitate of the test item was 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.
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
- 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. The solubility of the test item was examined using distilled water, N,N-dimethylformamide (DMF), ethanol and dimethyl sulfoxide (DMSO). Partial dissolution was observed at 100 mg/mL using DMF, ethanol and DMSO. The test item was soluble at this concentration using distilled water. Therefore, distilled water was selected as vehicle (solvent).
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylenediamine
Remarks:
Salmonella TA98, non-activation, 4 µg
Negative controls:
yes
Solvent controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Salmonella TA100 and TA1535, non-activation, 2 µg
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA1537, non-activation, 50 µg
Negative controls:
yes
Solvent controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E. coli WP2 uvrA, non-activation, 2 µL
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All Salmonella strains, activation, 2 µg
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
E. coli WP2 uvrA, activation, 50 µg
Details on test system and conditions:
INITIAL MUTATION TEST
- Method of application: in agar (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 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 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 Excel TM 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 E. 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 E. 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 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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY 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.
- The following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
- The observed number of revertant colonies was in the normal range. Slight decreases of the revertant counts were observed compared to the solvent control sporadically. However, they had no biological relevance and were situated within the historical control range most probably reflecting the variability of the test system.
- No precipitate of the test item was detected in the Preliminary Range Finding Test.
- Inhibitory or toxic effects of the test item 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 5000 µg/plate.

INITIAL AND CONFIRMATORY MUTATION TESTS
- No precipitate of the test item was detected in the main tests.
- Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test 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 TA98 bacterial strain at 158.1 μg/plate without metabolic activation (the observed mutation factor value was 1.67). 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 TA98 bacterial strain at 50 μg/plate without metabolic activation (the observed mutation factor value was 1.35). 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 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 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 yttrium trifluoride had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.