Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-25 to 2016-06-30
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 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:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PEGYZ-23
- Expiration date of the lot/batch: 31 March 2021
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70% relative humidity), protected from light.

OTHER SPECIFICS
Description: pink powder (the colour was determined by visual inspection upon arrival at test facility)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personal health and safety. Possibly irritative.
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- A 100 mg/mL stock solution was prepared in DMSO, which was diluted in 6 steps spaced by factors of 2, 2.5 and approximately √10.

Method

Target gene:
Histidine locus (S. typhimurium strains TA98, TA100, TA1535 and TA1537); tryptophan locus (E. coli strain WP2 uvrA)
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, 5000 µg/plate (TA98 only, plate incorporation);
Initial mutation test (5 strains)/complementary initial mutation test (S. typhimurium strains TA100, TA1535 and TA1537): 15.81, 50, 158.1, 500, 1581, 5000 µg/plate (plate incorporation);
Confirmatory mutation test: 5, 15.81, 50, 158.1, 500, 1581, 5000 µg/plate (all strains, pre-incubation).

The test item was found to form a slowly settling formulation in DMSO at 100 mg/mL (= 5000 µg/plate). Therefore, 5000 µg/plate was selected as top dose for the preliminary concentration 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 / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using distilled water, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetone and ethanol. The test item was insoluble (immediately settling formulation in each case) in distilled water and acetone at 100 mg/mL and 50 mg/mL concentration. The test item was insoluble at these concentrations using DMF (quickly settling formulation). The test item was insoluble at these concentrations using ethanol and DMSO (slowly settling formulation in each case). Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study. The obtained stock formulation (50 µL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility (preliminary compatibility test).

The formulations were stirred with magnetic stirrer during formulation and treatment.
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
Remarks:
without S9; 4 µg/plate (TA98)
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:
without S9; 2 µg/plate (TA100, TA1535)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9; 50 µg/plate (TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; 2 µL/plate (WP2uvrA)
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:
with S9; 2 µg/plate (TA98, TA100, TA1535, TA1537); 50 µg/plate (WP2uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Preliminary concentration range finding test/Initial mutation test/complementary 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. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C). 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 hours.
- Confirmatory mutation test: pre-incubation.
For the pre-incubation method, 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, 50 μL of test item formulations or its vehicle (or 50 μL of reference controls or their solvent), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. These 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 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 dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions was 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 was 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, a 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 item 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 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, other: TA98, TA100, TA1535, TA1537
Remarks:
Initial and Confirmatory Mutation test
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
Key result
Species / strain:
S. typhimurium, other: TA100, TA1535 and TA1537
Remarks:
Complementary Initial Mutation Test
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
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Initial Mutation Test/Confirmatory Mutation Test
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: insoluble (immediately settling formulation in each case)
- Precipitation:
Preliminary concentration range finding test: slight precipitate at 2500 and 5000 µg/plate with and without S9
Initial mutation test: (slight) precipitate at 5000 µg/plate with and without S9 in all strains
Complementary initial mutation test: (slight) precipitate at several concentrations with and without S9 in all strains
Confirmatory mutation test: (slight) precipitate at several concentrations with and without S9 in all strains

RANGE-FINDING/SCREENING STUDIES:
The observed number of revertant colonies was mostly in the normal range. Sporadically, minor differences compared to the solvent control numbers were detected. However, they did not have biological significance and were considered as biological variability of the test system.
Slight precipitate of the test item was detected in the Preliminary Range Finding Test in the examined bacterial strain with and without metabolic activation at 5000 and 2500 µg/plate concentrations.
No inhibitory, cytotoxic effects of the test item were observed 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.

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 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.
- 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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Inhibitory, cytotoxic effects of the test item were not detected in the Initial Mutation Test, in the Complementary Initial Mutation test* or in the Confirmatory Mutation Test.
*Note: Inhibitory, cytotoxic effects of the test item were detected in the Initial Mutation Test in case of the positive controls. However, in the performed Complementary Initial Mutation Test the study was fully valid.

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 bacterial strains used.
In conclusion, the test item erbium gadolinium yttrium zirconium oxide did not show any mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.