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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:
9 January 2019 - 30 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st July 1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
DL-thyroxin
EC Number:
200-101-1
EC Name:
DL-thyroxin
Cas Number:
51-48-9
Molecular formula:
C15H11I4NO4
IUPAC Name:
O-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodotyrosine
Test material form:
solid: particulate/powder
Details on test material:
Name: LEVOTHYROXINE ACID
Batch No.: B486066
Expiry date: 27 August 2020
Storage condition: at room temperature, protected from light
Appearance: white slightly crème, powder

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
mammalian microsomal enzyme activation mixture (rat liver extract, S9 fraction).
Test concentrations with justification for top dose:
5; 16; 50; 160; 500; 1600 and 5000 μg/plate.
Vehicle / solvent:
In the study two vehicle control groups were used according to the solubility of the test item and the solubility of positive control reference items.
Dimethyl sulfoxide (DMSO) for Test Item, NPD, 9AA and 2AA;
Ultrapure water for SAZ and MMS.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO) for Test Item, NPD, 9AA and 2AA; Ultrapure water for SAZ and MMS.
True negative controls:
no
Positive controls:
no
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Remarks:
With metabolic activation [10 % (v/v) rat liver S9 mix
Positive control substance:
other: 2-aminoanthracene, 2AA
Remarks:
2 µg/plate for S. typhimurium strains;
50 µg/plate for E. coli WP2 uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
2 µL/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
50 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediammine, NPD; metyhl methanesulphonate, MMS
Remarks:
4 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
2 µg/plate
Details on test system and experimental conditions:
The exposure was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 1.38 x 10^9 cells per mL.
The plates were incubated at 37 °C for 48 hours and the growth of the bacterial background and the density of revertant colonies was determined.
The test substance was tested without as well as with an external metabolising system (S9-mix). The results were verified by a second, independent experiment. Three plates were used per concentration and per strain for the test substance; 6 for the (positive and negative) control.

Evaluation criteria:
The colony numbers on the controls (untreated, vehicle, positive) and the test item plates were determined (counted manually, evaluated by unaided eye), the mean values and appropriate standard deviations and 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 Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion 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.
According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a 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.

Results and discussion

Test resultsopen allclose all
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
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:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the performed experiments multiple test items were tested with reference values from the
common parallel 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 (ASTM Type I) 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.
In summary, the actual values of untreated, vehicle and positive controls were in line with the criteria for validity of the assay.
Remarks on result:
other: all strains/cell types tested

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 investigated strains: Salmonella typhimurium TA98, TA100,
TA1535 and TA1537 and of Escherichia coli WP2 uvrA.
In conclusion, the test item LEVOTHYROXINE ACID has no mutagenic activity on the
applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item LEVOTHYROXINE ACID 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/β-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 Confirmatory Mutation Tests (Pre-Incubation Tests).
As the evaluation of the Confirmatory Mutation Test in the case of Salmonella typhimurium strains was not possible (for an unforeseen technical reason), the Confirmatory Mutation Test (Pre-Incubation Test) was repeated, completed with these strains with the same conditions and concentration levels as the originally proposed procedure.
Based on the results of the Solubility Test and the Concentration Range Finding Test the test item was dissolved in dimethyl sulfoxide (DMSO).
At the concentration choice the solubility, the cytotoxicity and a possible positive effect of the test item (obtained in the Concentration Range Finding Test) were taken into consideration and based on the recommendations of OECD 471 guideline [6] the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:
-S9: 5000, 1600, 500, 160, 50 and 16 µg/plate;
+S9: 5000, 3200, 1600, 500, 160, 50 and 16 µg/plate.
Following the plate incorporation procedure test item precipitate was noticed in the
examined strains at the highest examined concentration of 5000 µg/plate (±S9); following the pre-incubation procedure precipitate was noticed in the Salmonella typhimurium strains at the concentration of 1600 and 500 µg/plate (-S9). The obtained precipitate did not influence, disturb the scoring in any case.
In the Initial Mutation Test slight, rather equivocal inhibitory effect of the test item on bacterial growth was observed in S. typhimurium strains at 5000 µg/plate, in the absence of exogenous metabolic activation (-S9). The slight cytotoxicity was indicated by affected background lawn development (slightly reduced background lawn).
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 all of 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.


Dose-related changed increased tendencies in revertant colony numbers were noticed in all
experimental phases of the study (non-GLP Concentration Range Finding Test, Initial Mutation Test and Confirmatory Mutation Test) in S. typhimurium TA98, in the presence of exogenous metabolic activation (+S9). The higher revertant colony numbers were above the historical control data range of vehicle control in the concentration range of 5000-1600 µg/plate following the plate incorporation procedures and at 5000 and 3200 µg/plate, following the pre-incubation procedure. The equivocal, borderline positive results, biological relevant revertant colony number increases obtained in the non-GLP Concentration Range
Finding Test (S. typhimurium TA98, +S9, 5000 µg/plate, precipitated concentration) were not repeated, the higher revertant colony numbers remained below the relevant genotoxicological threshold for being positive in the subsequent main experiments.
In summary unequivocal biologically relevant increases in revertant colony numbers were not observed in revertant colony numbers of any of the five test strains following treatment with LEVOTHYROXINE ACID 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 in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA. 
In conclusion, the test item LEVOTHYROXINE ACID has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.