Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May - 08 June 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)
Version / remarks:
adopted 21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Ph armaceuticals Intended for Human Use
Version / remarks:
June 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial forward mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: crystalline
Remarks:
crystalline powder
Details on test material:
Batch No: 11679700
Storage: at room temperature, protected from light

Method

Target gene:
histidine locus (Salmonella typhimurium strains) and tryptophan locus (E. coli strain)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
10 % (v/v) rat liver S9 mix
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
10 % (v/v) rat liver S9 mix
Test concentrations with justification for top dose:
Initial Mutation Test and Confirmatory Mutation Test: 16, 50, 160, 500, 1600, and 5000 µg/plate

Concentration Range Finding Test (Informatory Toxicity Tests): The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5 000, 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 in line with the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains. In the performed 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 or increases (compared to the revertant colony numbers of the vehicle co ntrol) remained within the biological variability range of the applied test system.
No precipitation of the test item was observed on the plates at any examined concentration level (±S9 Mix).
Vehicle:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: In the solubility test the test item behaviour was inve stigated in the applied test system. The test item was dissolved and further diluted in DMSO, accor dingly. The obtained solutions with the solution of top agar and phosphate buffer were examined in a test tube without test bacterium suspension. No precipitation was observed.
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:
Positive Control concentration: 4 µg/plate for TA98
Negative controls:
yes
Solvent controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control concentration 2 µg/plate for TA100 and TA1535
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
positive control concentration: 50 µg/plate for TA1537
Negative controls:
yes
Solvent controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
positive control concetration: 2µL/plate for WP2 uvrA
Details on test system and conditions:
METHOD OF APPLICATION: initial mutation test: in agar (plate incorporation); confirmatory test: in agar (plate incorporation) with preincubation
- 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 for the overnight cultures in theassay. The cultures were incubated for approximately 11-14 hours in a 37°C Benchtop IncubatorShaker.

- Molten top agar was prepared and kept at 45°C. Two mL of top agar was aliquoted into individualtest tubes (3 tubes per control or concentration level). The equivalent number of minimal glucoseagar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45°C).
The typical content of the tubes:
top agar 2000 µL
vehicle or solution of test item or positive controls 100 µL
overnight culture of test strain 100 µL (containing aprox 10^9 CFU/ml)
phosphate buffer (pH: 7.4) or S9 mix 500 µL
This solution was mixed and poured on the surface of the properly labeled minimal agar plates. For incubations with metabolic activation, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube.

- Exposure duration: incubated at 37°C for about 48 hours

- Preincubation period (in confirmatory test): Before the overlaying of the test item, the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to allow 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, two mL of molten top agar was added to the tubes, and the content was mixed and poured onto minimal glucose agar plates.

NUMBER OF REPLICATIONS: Triplicates

METABOLIC ACTIVATION SYSTEM: Rat Liver S9 Fraction
The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided
by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX I
NC., P.O. BOX 1189; BOONE, NC 28607 USA).

VALIDITY CRITERIA
The tests (Initial and Confirmatory Mutation Tests) are considered valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers are in the 109 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three nontoxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.

- For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate or 5 µL/plate. For test compounds that are not soluble at 5 mg/plate or 5 µL/plate and that are not toxic at levels lower than an insoluble level, the highest doses tested is at least one insoluble concentration in the final treatment mixture under the actual conditions of the test at the start of the experiment. Insolubility is assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.

- The test has to be included five analyzable concentrations (where the precipitate does not interfere
with the scoring) and a minimum of three non-precipitated dose levels
Evaluation criteria:
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 Salmonella typhimurium TA100 the number of reversions is at least twice as high as the rev ersion rate of the vehicle control, - in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Statistics:
Based on the evaluation criteria no statistical analysis was required.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
- Validity criteria: All criteria for the validity of the performed experiments according to the OECD guideline were met.

- Controls: In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates with and without S9 Mix were in line with 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 revertant colony numbers of the untreated and ultrapure water control plates in different experimental phases were slightly higher or lower than the dimethyl sulfoxide (DMSO) control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.

-No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Adenosine-5’-monophosphate (AMP) at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.
In the performed experiments, sporadically increased revertant colony numbers were noticed. These increases did not show a clear dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.

- The highest revertant colony number increase was observed in the Initial Mutation Test (Plate Incorporation Test) in Salmonella typhimurium TA1537 strain at 5000 µg/plate, in the presence of metabolic activation (+S9). This value however remained in the range of the corresponding vehicle historical control data and additional concentration related increase in revertant colony counts was not noticed. The mutation rate was 1.70, which was far below the genotoxicological threshold for being positive.

- In the Initial and Confirmatory Mutation Tests, inhibitory effect of the test item on bacterial growth was not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.

- In the performed experiments no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix).

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 Adenosine-5’-monophosphate (AMP) (CAS Nr. 61-19-8) has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item Adenosine-5’-monophosphate (AMP) (CAS Nr. 61-19-8) was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella 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/ß-naphthoflavone-induced rats.  The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test). Based on the results of the Solubility Test and the Concentration Range Finding Test the test item was dissolved in dimethyl sulfoxide (DMSO).  Based on the results of the preliminary Range Finding Tests the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:  5000; 1600; 500; 160; 50 and 16 µg/plate. The maximum test concentration was in all strains 5000 µg/plate (±S9 Mix). At the concentration choice the guideline criterion regarding the soluble, non-cytotoxic test items was taken into consideration. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study. The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained 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 revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.  The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.  No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with Adenosine-5’-monophosphate (AMP) at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. 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 Adenosine-5’-monophosphate (AMP) (CAS Nr. 61-19-8) has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study