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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2016-11-09 to 2017-02-15
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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
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

Constituent 1
Reference substance name:
trichloropraseodymium; heptahydrate
Cas Number:
10025-90-8
Molecular formula:
PrCl3.7H2O
IUPAC Name:
trichloropraseodymium; heptahydrate
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Praseodymium trichloride heptahydrate
- Physical state: Solid
- Appearance: Green crystalline powder
- Further information on test material 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 distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10.
- A correction factor of 1.53 was used at dose formulation preparation considering the composition. All concentrations and dose levels are expressed as praseodymium trichloride content of the test item (i.e., the correction was done to correct for the amount of hydration water present in the test item).

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).
Initial Mutation Test and Confirmatory Mutation Test: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate (all strains).
Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98, TA100 and TA1535 strains: 0.001581, 0.005, 0.01581, 0.05, 0.1581, 0.5, 1.581, 5 and15.81 µg/plate.

The selection of the doses was based on the results of the range finding study. Inhibitory, cytotoxic effects of the test item (slightly reduced background lawn development) were observed in the range finding study in Salmonella typhimurium TA100 strain at 5000 and 2500 µg/plate concentrations with and without metabolic activation. Precipitate/slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000, 2500 and 1000 µg/plate. The highest dose tested in the initial mutation test and confirmatory mutation test was 5000 µg/plate, i.e. the test item was tested up to both precipitating and cytotoxic concentrations.

Because excessive cytotoxicity was observed in the confirmatory mutation test in Salmonella typhimurium TA98, TA100 and TA1535 strains without metabolic activation at several concentrations, the number of analysable doses did not meet the recommendation of the test guidelines. Therefore, a complementary confirmatory mutation test was performed using a lower concentration series.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: In order to obtain a suitable solvent, the solubility of the test item was examined in distilled water, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). The formulations at 100 mg/mL concentration using distilled water, DMSO or DMF were solutions. Due to the better biocompatibility, distilled water was selected as vehicle.
Controlsopen allclose all
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 (NDP)
Remarks:
Salmonella TA98, non-activation, 4 µg
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:
Salmonella TA100 and TA1535, non-activation, 2 µg
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA1537, non-activation, 50 µg
Untreated negative controls:
yes
Negative solvent / vehicle 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
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:
All salmonella strains, activation, 2 µg
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:
E. coli WP2 uvrA, activation, 50 µg
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 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 TESTS (pre-incubation method)
- A pre-incubation procedure was followed in the confirmatory mutation test and the complementary 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 37°C 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 and complementary 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 resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
Inhibitory, cytotoxic effects of the test item (slightly reduced background lawn development) were observed in the Preliminary Range Finding Test in Salmonella typhimurium TA100 strain at 5000 and 2500 µg/plate concentrations with and without metabolic activation.
Precipitate / slight precipitate was observed in both tester strains with and without metabolic activation at the concentrations of 5000, 2500 and 1000 µg/plate.

INITIAL AND CONFIRMATORY MUTATION TESTS
Precipitate / slight precipitate was observed in all tester strains with and without metabolic activation in the Initial Mutation Test at 5000, 1581 and 500 µg/plate concentrations and in the Confirmatory Mutation Test at 5000, 1581, 500 and 158.1 µg/plate concentrations.

Slight decreases of the revertant counts were observed compared to the solvent control at some non-cytotoxic concentrations in the study. However, the mean number of revertant colonies was within the historical control range, thus they were considered as biological variability of the test system.

Inhibitory, cytotoxic effects of the test item (slightly reduced background lawn development) were observed in the Initial Mutation Test at 5000 µg/plate concentration in Salmonella typhimurium TA100 strain with and without metabolic activation and in Salmonella typhimurium TA1535 strain without metabolic activation. Similar but stronger inhibitory, cytotoxic effects of the test item (slightly reduced background lawn development) were observed in the Confirmatory Mutation Tests at 5000 and 1581 µg/plate concentrations in all Salmonella typhimurium strains with metabolic activation; in Salmonella typhimurium TA1537 strain without metabolic activation; at 15.81, 5, 1.581 and 0.5 µg/plate concentrations in Salmonella typhimurium TA98, TA100 and TA1535 strains without metabolic activation and at 5000 µg/plate concentration in Escherichia coli WP2 uvrA strain with and without metabolic activation.

In the Initial Mutation Test, Confirmatory Mutation Test and Complementary 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 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 Tests (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 158.1 μg/plate concentration without metabolic activation. The calculated mutation factor value at this dose level was 1.56. 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.

Increases in the numbers of revertant colonies were detected compared to the solvent control during the study in several 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.

Any other information on results incl. tables

VALIDITY OF THE TESTS

- 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 has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.