Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 09 - June 29, 2016
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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: 2016JAN-15kg
Expiry Date: 01 January 2020
Appearance: Blue-white powder
Storage: Room temperature (15-25°C)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Specific details on test material used for the study:
Batch No.: 2016JAN-15kg
Expiry Date: 01 January 2020
Appearance: Blue-white powder
Storage: Room temperature (15-25°C)

Method

Target gene:
The S. typhimurium histidine (his) reversion system measures his- and his+ reversions. The S. typhimurium strains are constructed to differentiate between base-pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.
The E. coli strain WP2 uvrA detects mutagens that cause base-pair substitutions (AT to GC) resulting in trp- and trp+ reversions.
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 specified
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared from livers of phenobarbital/b-naphthoflavone-induced rats
Test concentrations with justification for top dose:
Based on the results of the preliminary tests, test item suspensions with a concentration of 50 mg/mL; 20 mg/mL, 8 mg/mL, 3.2 mg/mL, 1.0 mg/mL and 0.5 mg/mL were prepared from the test item with dimethyl sulfoxide (DMSO). The maximum test concentration was 5000 µg/plate (±S9 Mix). At the concentration choice the guideline criterion for non-toxic test compounds (where the recommended maximum test concentration is 5 mg/plate) and the results of the preliminary
Informatory Toxicity Test were taken into consideration. The Neodymium fluoride oxide, magnesium doped concentrations planned for the Initial and Confirmatory Mutation Tests: 5000, 2000, 800, 320, 100 and 50 µg/plate.
Vehicle / solvent:
Based on the preliminary information provided by the Sponsor, and the test item specific solubility properties, in the preliminary Range Finding Test
dimethyl sulfoxide (DMSO) was found as appropriate vehicle for preparing the test item suspension/solutions, and the behavior of the test item as a solute in top agar and in phosphate buffer was determined in the preliminary Solubility Test.
The chosen vehicle was compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary Solubility and Range Finding Tests. At the preparation of the test item suspensions, solutions, the appropriate amount of the test item was weighted and suspended in DMSO.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine, NPD
Details on test system and experimental conditions:
Origin of the Bacterial Strains:
The tester strains arrived to the test facility in a form of disc cultures. The origin of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA:
Supplier: Trinova Biochem GmbH (Rathenau Str. 2 (earlier: Kerkrader Str. 10));
D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.

Frozen stock cultures were prepared from the disc cultures.

Genotypes of the Strains Used for Mutagenicity Testing:
Strain Genotype Mutations Main DNA target Plasmid
trp./his mutation type of mutation cell wall DNA- repair
S.ty.mur.TA98 hisD3052 Frameshift rfa uvrB GC pKM101
S.ty.mur.TA100 hisG46 Base pair substitution rfa uvrB GC pKM101
S.ty.mur. TA1535 hisG46 Base pair substitution rfa uvrB GC No
S.ty.mur.TA1537 hisC3076 Frameshift rfa uvrB GC No
E.coli WP2uvrA trpE Base pair substitution + uvrA AT No
Abbreviations: S.ty.mur.: Salmonella typhimurium; E.coli: Escherichia coli

In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.

The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.

Spontaneous Reversion of Tester Strains

Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.

Procedure for Bacterial Cultures

The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 (5.4.2) for the overnight cultures in the assay. The cultures were incubated for approximately 11-13 hours at 37oC in a Benchtop Incubator Shaker.

Viability and the Cell Count of the Testing Bacterial Cultures

The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Media:
The Minimal Glucose Agar (MGA) Plates:
Ready-to-use minimal glucose agar (MGA) plates were used in the study. Heipha Dr. Müller GmbH was the manufacturer of the used MGA plates. The plates were provided by VWR. Certificate of Analysis was obtained from the supplier.

Typical composition (g/1000 mL) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g

Nutrient Broth No. 2:
Nutrient broth No. 2. 25.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121 ˚C in an autoclave.

Nutrient Agar:
Nutrient Agar 20.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121 ˚C in an autoclave.

Top Agar for Salmonella typhimurium Strains:
Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121 ˚C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 mL
Agar solution 900.0 mL

Top Agar for Escherichia coli Strain:
Tryptophan solution (2 mg/mL):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth 50.0 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution 947.5 mL

Metabolic Activation System:
The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Rat Liver S9 Fraction:
The S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2 (earlier: Kerkrader Str. 10); D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier . The copies of the corresponding Quality Control & Production Certificates of the S9 fractions used are attached as Appendix VI., the originals are stored in the Laboratory of TOXI-COOP ZRT.
Evaluation criteria:
The colony numbers of the untreated, vehicle control, positive control and test plates were determined; the mean values, standard deviations and the mutation rates were calculated.

A test item is considered mutagenic if:
- a dose–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 is considered biologically relevant if:
- in strain TA98, TA100 and Escherichia coli WP2 uvrA the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA1535, TA1537 the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results will be the criterion for the interpretation of results; statistical evaluation of the results is not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic 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 colony numbers of the untreated, vehicle control, positive control and test plates were determined; the mean values, standard deviations and the mutation rates were calculated by EXCEL software.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no 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:
no 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test) an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test). In the preliminary Range Finding Test as well as in the Initial Mutation Test the plate incorporation method was used.

The pre-experiments on solubility of the test item and the Range-Finding Test was not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study code of present study.

Informatory Toxicity Test

Based on the solubility test, test item suspensions with concentrations of 50, 16, 5 and 1.6 mg/mL were prepared and further diluted (by factor of approximately √10) with dimethyl sulfoxide (DMSO) to obtain seven dosing suspensions/solutions.

The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimuriumTA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item. The revertant colony numbers of vehicle control plates with and without S9 Mix were within the historical control data ranges in both examinedSalmonella typhimuriumstrains. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case; all of the obtained slight revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. The obtained revertant colony numbers were slightly lower than the revertant colony numbers of the vehicle control (without any biological significance) at 5000 µg/plate inS. typhimuriumTA98, and at 500 and 160 µg/plate in TA100, in the presence of exogenous metabolic activation (+S9 Mix). Based on the manual evaluation, by naked eye no precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9 Mix).

Initial Mutation Test and Confirmatory Mutation Tests

Based on the results of the preliminary tests, test item suspensions with a concentration of 50 mg/mL; 20 mg/mL, 8 mg/mL, 3.2 mg/mL, 1.0 mg/mL and 0.5 mg/mL were prepared from the test item with dimethyl sulfoxide (DMSO). The maximum test concentration was5000 µg/plate (±S9 Mix).At the concentration choice the guideline criterion for non-toxic test compounds (where the recommended maximum test concentration is 5 mg/plate) and the results of the preliminary Informatory Toxicity Test were taken into consideration. The Neodymium fluoride oxide, magnesium dopedconcentrations planned for the Initial and Confirmatory Mutation Tests: 5000, 2000, 800, 320, 100 and 50 µg/plate.

Confirmatory Mutation Test (Pre-Incubation Test)

A pre-incubation procedure [1], [2], [5] and [6] was performed, as a Confirmatory Mutation Test. Before the overlaying of the test item, 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). These tubes were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes, the content mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method. The entire test consisted of non-activation and activation test conditions, each of them inclusive negative and positive controls. After preparation the plates were incubated at 37°C for about 48 hours.

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 Neodymium fluoride oxide, magnesium doped has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of phenobarbital/b-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

In the Range Finding Test as well as in the Initial Mutation Test the plate incorporation method was used. Based on the results of the Solubility Test and the Range Finding Test the test item was suspended at different nominal concentrations in dimethyl sulfoxide (DMSO). This vehicle was compatible with the survival of the bacteria and the S9 activity; furthermore appropriate historical control database is available in the testing laboratory.

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests: 5000; 2000; 800; 320; 100 and 50 µg/plate.

In the Initial and Confirmatory Mutation Tests the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.

No biological relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Neodymium fluoride oxide, magnesium dopedat any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. The sporadic increases in revertant colony numbers compared to the vehicle control values observed in both independently performed main experiments were noticed as reflecting the biological variability of the applied test system. There was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the Initial and Confirmatory Mutation Tests inhibitory, cytotoxic effect of the test item was not noticed. The colony and background lawn development was not affected in any case.Based on the manual evaluation, by naked eye no precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9 Mix). Because of the specific solubility properties of the test item the treated plates were examined additionally with microscope at 40 X magnification. At this magnification test item particles were seen on the plates in whole examined concentration range. The amount of micro particles changed in dose-related manner, and their presence did not disturb the evaluation of the background lawn development in any case.

The revertant colony numbers of vehicle control (dimethyl sulfoxide, DMSO) plates with and without S9 Mix were within the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

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 Neodymium fluoride oxide, magnesium doped has no mutagenic activity on the applied bacterium tester strainsunder the test conditions used in this study.