Thymidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Toxi-Coop ZRT

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: crystalline

Details on test material:

- Name of test material: Thymidine

Method

Target gene:

The purpose of this study was to evaluate the mutagenic potential of the test item by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of activated rat liver S9.
The bacterial reverse mutation test is a microbial assay which detects point mutations induced by chemicals causing base changes or frameshift mutations in the genome of amino-acid requiring strains of Salmonella typhimurium and Escherichia coli. The S. typhimurium histidine (his) reversion system measures his- to his+ reversions. The S. typhimurium strains are constructed to differentiate between base-pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The E. coli strain WP2 uvrA detects mutagens that cause base-pair substitutions (AT to GC) resulting in trp- to trp+ reversions.
The bacteria used in this assay do not possess the Phase I and Phase II metabolic capability to convert promutagens into mutagenic metabolites. In order to overcome this major drawback, an exogenous metabolic activation system is added in the form of a mammalian microsomal enzyme activation mixture (liver extract, S9 fraction). The activation system uses nicotinamide-adenine dinucleotide phosphate (NADP+)-cytochrome P450 dependent mixed function oxidase enzymes of the liver. The liver extract was obtained from rats, which were pre-treated with phenobarbital/β-naphthoflavone, two inducers of several drug-metabolizing enzymes.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media: Frozen 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 the assay. The cultures were incubated for approximately 10 hours in a 37 °C Benchtop Incubator Shaker.
- Properly maintained: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat Phenobarbital (PB) and β-naphthoflavone (BNF)-induced liver microsomal S9-fraction

Test concentrations with justification for top dose:

SOLUBILITY TEST
In the solubility test the test item was dissolved (and diluted accordingly) in ultrapure water. Clear solution was obtained at the concentration of 50 mg/mL. 100 μL of test item solution was examined in a test tube with the solution of top agar (2 mL) and phosphate buffer (0.5 mL) resulting in a clear solution of 5000 μg/tube.

CONCENTRATION RANGE FINDING TESTS (INFORMATORY TOXICITY TEST)
Based on the solubility test, 50 mg/mL stock solution was prepared in ultrapure water from the test item and diluted in 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) was determined at the concentrations of 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate of the test item.

Test Item Concentrations and Controls in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Based on the results of the preliminary tests, a concentrated stock solution with a concentration of 50 mg/mL was prepared from the test item with ultrapure water, that was diluted by serial dilutions each separated by approximately √10 factor to obtain six dosing solutions for lower doses. The maximum test concentration was 5000 μg test item/plate (± S9 Mix, limit concentration).
Concentrations tested: 5000, 1581, 500, 158, 50 and 15.8 μg/plate
Test solutions were freshly prepared at the beginning of the experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Initial mutation test: In agar (plate incorporation)
- Confirmatory mutation test: Preincubation

PROCEDURE FOR THE INITIAL MUTATION TEST
A standard plate incorporation procedure was performed, as an 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. Molten top agar was prepared and kept at 45 °C. 2 mL of top agar was aliquoted into individual test tubes. The equivalent number of minimal glucose agar plates was properly labeled. 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 reference controls 50 μL
- vehicle or solution of test item 100 μL
- over night 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 the properly labeled minimal agar plates (3 plates per control or concentration level). 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 and each of them with the addition of negative and positive controls. The plates were incubated at 37 °C for 48 hours.

PROCEDURE FOR THE CONFIRMATORY MUTATION TEST
As the Initial Mutation Test revealed unequivocal negative results a Confirmatory Mutation Test was carried out following a pre-incubation procedure. Before the overlaying of the test item the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes providing direct contact between bacteria and the test item (in its vehicle). Tubes were gently mixed and incubated for 20 min at 37 ºC 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 Confirmatory Mutation Test included test item exposure, negative and positive controls each without and with metabolic activation (S9 mix). After preparation the plates were incubated at 37 °C for ca. 48 hours.

NUMBER OF REPLICATIONS:
- 3 tubes per control or concentration level

DETERMINATION OF CYTOTOXICITY
- Method: The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) was determined at the concentrations of 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate of the test item.

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 reversion rate of the vehicle control
- in strain 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.

Statistics:

Not applicable.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

VALIDITY OF THE PERFORMED EXPERIMENTS
Valid tests were performed since the tester strains (used in this study) demonstrated the specific phenotype characteristics, agreed with the corresponding historical control data ranges, and showed the adequate strain culture titer. Each batch of the S9 fraction used in this test had the appropriate biological activity (according to the provided Certificates) and were active in the applied system (2-aminoanthracene treatments).
The spontaneous revertant colony numbers of the ultrapure water vehicle control plates were slightly lower than the characteristic mean numbers agreed with the actual historical control data ranges in the Initial and Confirmatory Mutation Tests in absence of metabolic activation (-S9 Mix); however were evaluated as acceptable without any influence on the final conclusion of the study.
Each of the investigated reference mutagens showed the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in all experimental phases. Seven concentration levels were investigated in the Informatory Toxicity Test and six concentration levels in the main mutation experiments. In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain.

RANGE-FINDING TEST (INFORMATORY TOXICITY TEST)
In the preliminary Range Finding Test the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and TA100 tester strains under activation and non-activation conditions (in presence and absence of metabolic activation (S9 Mix)) with appropriate positive and negative controls.
In the test the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. Concentrations examined were: 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate.
In comparison with the revertant colony numbers of the vehicle control plates sporadic changes slightly lower or higher revertant colony counts were observed in both strains examined; however all of the obtained changes were considered as reflecting the biological variability of the applied test system.
The obtained revertant colony numbers were lower than the revertant colony numbers of the vehicle control and below the corresponding historical control data range in S. typhimurium TA100 at 5000 μg/plate, with addition of metabolic activation (+S9 Mix). The lower revertant colony counts remained in the historical control data range in S. typhimurium TA98 at 5 μg/plate (+S9 Mix).
The obtained revertant colony numbers remained in the vehicle control data range, however were below the corresponding historical control data range in S. typhimurium TA100 in the whole concentration range in absence (-S9 Mix) and at the concentrations of 1581, 500, 50, 15.8 and 5 μg/plate, in presence of metabolic activation (+S9 Mix).
Higher revertant colony numbers (above the historical control data range, however without any significance) were noticed in S. typhimurium TA98, at 5000 μg/plate, without metabolic activation (-S9 Mix), and the higher revertant colony numbers remained in the historical control data range in S. typhimurium TA98 at 158 and 5 μg/plate (-S9 Mix).
In the case of S. typhimurium TA100, at the untreated and ultrapure water controls the obtained revertant colony numbers were below the corresponding historical control data ranges, in absence of metabolic activation (-S9 Mix). The slightly lower colony counts did not have any effect on the results of the study.
All of the obtained (above detailed) changes were considered as reflecting the biological variability of the applied test system.

INITIAL AND CONFIRMATORY MUTATION TESTS (PLATE INCORPORATION AND PRE-INCUBATION TESTS)
In the Initial Mutation Test, the plate incorporation method, in the Confirmatory Mutation Test, the pre-incubation test was used. The tests were performed using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA test strains under activation and non-activation conditions (in the presence and absence of metabolic activation (S9 Mix)) with appropriate positive and negative controls. In these tests, the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
The examined concentrations were: 5000; 1581; 500; 158; 50 and 15.8 μg/plate.
In the performed experiments the observed revertant colony number increases were of minor intensity, far below the biologically relevant thresholds for being positive. The obtained increases remained in the historical control data ranges and were considered as to reflect the biological variability of the test system.
In the Initial Mutation Test the revertant colony numbers were slightly higher than the revertant colony numbers of the vehicle control plates at 5000 μg/plate in S. typhimurium TA1535 (-S9 Mix) and in TA98 (±S9 Mix), at 500 μg/plate in TA98 (-S9 Mix); at 158 μg/plate in TA1535 (-S9 Mix), in E. coli WP2 uvrA (+S9 Mix); at 50 μg/plate in TA1537 (-S9 Mix) and at 15.8 μg/plate in TA1535 (+S9 Mix).
In the Confirmatory Mutation Test the revertant colony numbers were higher than the revertant colony numbers of the vehicle control plates at 5000 μg/plate in TA1537 (-S9 Mix), in TA98 (±S9 Mix); at 1581 μg/plate in TA98 and TA1537 (-S9 Mix); at 500 μg/plate in TA98 and in E. coli WP2 uvrA (-S9 Mix); at 158 μg/plate in TA98, TA1535 and TA1537 (-S9 Mix); at 50 μg/plate in TA98 and TA1537 (-S9 Mix) and at 15.8 μg/plate in TA1535 (-S9 Mix).
The revertant colony counts remained in the vehicle control data range, showed no inhibition; however were below the historical control data range in the Initial and Confirmatory Mutation Test in S. typhimurium TA100, in absence of metabolic activation (-S9 Mix).
In the Initial Mutation Test the revertant colony numbers were below the actual historical control data range without any biological significance at 5000, 1581, 500, 50 and 15.8 μg/plate; in the Confirmatory Mutation Test at the concentration range of 1581-15.8 μg/plate.
Inhibitory effect of the test item was not observed throughout the study. The obtained lower revertant colony numbers remained mostly in the corresponding historical control data ranges and the background lawn development was not affected in any case.
In the Initial Mutation Test the revertant colony numbers were lower than the revertant colony numbers of the vehicle control plates at 5000 μg/plate in S. typhimurium TA1535 (+S9 Mix); at 1581 μg/plate in TA1535 and TA1537 (-S9 Mix); at 500 μg/plate in E. coli WP2 uvrA (-S9 Mix), at TA1535 and TA1537 (+S9 Mix); at 158 μg/plate in TA1537 (-S9 Mix) and at 50 μg/plate in TA1535 (+S9 Mix).
In the Confirmatory Mutation Test the revertant colony numbers were lower than the revertant colony numbers of the vehicle control plates at 5000 μg/plate in E. coli WP2 uvrA (+S9 Mix); at 1581 μg/plate in S. typhimurium TA1537 and E. coli WP2 uvrA (+S9 Mix), at 500 μg/plate in TA1535 and E. coli WP2 uvrA (+S9 Mix); at 158 μg/pate in S. typhimurium TA1537 and E. coli WP2 uvrA (+S9 Mix); at 50 μg/plate in E. coli WP2 uvrA (+S9 Mix) and at 15.8 μg/plate in TA1537 (±S9 Mix).
The highest revertant colony number increase over the spontaneous rate of the vehicle control plates was observed in Salmonella typhimurium TA1537 at 50 μg/plate in the Confirmatory Mutation Test (Pre-Incubation Test), without metabolic activation (-S9 Mix). The mutation rate was: 2.43. This value was unique, and far below the genotoxicological threshold for being positive.
The lower revertant colony counts (compared with the revertant colony numbers of the vehicle control) were below the corresponding historical control data range in the Initial Mutation Test in E. coli WP2 uvrA, at 15.8 μg/plate (+S9 Mix) and in the Confirmatory Mutation Test in S. typhimurium TA100, at 5000 μg/plate, without (-S9 Mix). These changes were considered as reflecting the biological variability of the applied test system.

CONTROLS
The revertant colony numbers of vehicle control (ultrapure water) plates with and without S9 Mix were in line within the corresponding historical controls data ranges with the small differences obtained in the case of S. typhimurium TA100 in absence of metabolic activation in the range finding test .
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 DMSO control plates in the different experimental phases were slightly higher or lower than the ultrapure control plates. These higher or lower revertant counts of these controls remained mostly in the historical control data ranges (lower counts were obtained in the Confirmatory Mutation Test in S. typhimurium TA100 at the untreated control (-S9 Mix)).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, the test item revealed no mutagenic activity on the bacterial strains tested under the conditions used in this study.

Executive summary:

A study was carried out according to EU Method B.13/14, OECD Guideline 471 (Bacterial Reverse Mutation Assay) and OPPTS 870.5100. 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 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 preliminary Range Finding Test the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests: 5000; 1581; 500; 158; 50 and 15.8 μg/plate. The revertant colony numbers of vehicle control (ultrapure water control) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants and were in line with the corresponding historical control data. The obtained slightly lower values in Salmonella typhimurium TA100 were considered acceptable without any effect on the final conclusion of the study. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item 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 revealed no mutagenic activity on the bacterial strains tested under the conditions used in this study.