Purine-2(3H),6(1H)-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Purine-2(3H),6(1H)-dione

EC Number:

200-718-6

EC Name:

Purine-2(3H),6(1H)-dione

Cas Number:

69-89-6

Molecular formula:

C5H4N4O2

IUPAC Name:

xanthine

Specific details on test material used for the study:

Purity: > 99%

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Molecular Toxicology, Inc.; sourced from male Sprague-Dawley rats induced with phenobarbital and 5,6-benzoflavone.
- method of preparation of S9 mix: freshly prepared on day of use; maintained on ice prior to and during use.
- concentration or volume of S9 mix and S9 in the final culture medium: contained 5% v/v S9 fraction. Sodium phosphate buffer was used as the substitution buffer for plates treated in the absence of S9.
- quality controls of S9: The prepared S9 mix contained the following sterile cofactors: 8 mM MgCl2, 33 mM KCl, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP.

Test concentrations with justification for top dose:

1.58, 5.0, 15.8, 50, 150, 500, 1580, and 5000 µg/plate. The top dose is the limit recommended by guideline.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water (vehicle control); DMSO
- Justification for choice of solvent/vehicle: test substance was found to be soluble in sterile water; dilutions in DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

vehicle

Controls 2

Positive control substance:

other:

Remarks:

for all tester strains

Controls 3

Positive control substance:

methylmethanesulfonate

Remarks:

for tester strain E. coli WP2 uvrA

Controls 4

Positive control substance:

other:

Remarks:

for tester strain S. typhimurium TA98

Controls 5

Positive control substance:

other:

Remarks:

for tester strain S. typhimurium TA1537

Controls 6

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for tester strain S. typhimurium TA100, TA1535

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2 (Main and Confirmatory)

METHOD OF TREATMENT/ EXPOSURE (MAIN):
- Following materials mixed and added over surface of minimal agar plate: 100 µL of the prepared test substance solutions, negative (vehicle) control, or prepared positive control substance; 500 µL S9 mix or substitution buffer; 100 µL bacteria suspension (ST or EC); and 2000 µL overlay agar maintained at approximately 45°C.
- Plates placed on level surface until agar gelled then inverted and incubated at approximately 37⁰C until growth was adequate for enumeration. Appropriate sterility control check plates (treated with critical components in absence of bacteria) included as a standard procedural check.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: approximately 65 hours
- Harvest after the end of treatment (sampling/recovery times): After incubation, number of colonies per plate counted manually and/or with the aid of a plate counter.

CONFIRMATORY TEST
Employed pre-incubation modification of plate incorporation test. Test or control substances, bacteria suspension, and S9/substitution buffer were incubated under agitation for approximately 30 minutes at approximately 37°C prior to mixing with overlay agar and pouring onto minimal agar plates before proceeding to main test. The study design for the confirmatory test, including strains, dose levels etc. was as described for main test.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Evaluated by spectrophotometric optical density measurement.

Evaluation criteria:

Criteria for Validity: Background lawn for vehicle control plates should appear normal. Mean revertant colony counts for each strain treated with vehicle should lie close to or within expected range taking into account the laboratory historical control range and/or published values. Positive controls (with S9 where required) should produce substantial increases in revertant colony numbers with appropriate bacterial strain. In case where part of study is invalid based on these criteria, detailed results for that part of study will not be reported and affected part of study would be subjected to an automatic repeat at the discretion of the Study Director.

Evaluation of Toxicity: Toxic effects of test substance are indicated by partial or complete absence of background lawn of non-revertant bacteria or a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle control taking into account laboratory historical control range. Where precipitation obscures observations on condition of background lawn, lawn can be considered normal and intact if revertant colony counts are within expected range based on results for lower dose levels and historical control counts for that strain.

Evaluation of Mutagenicity: For each experimental point, the Mutation Factor (MF) calculated by dividing mean revertant colony count by mean revertant colony count for corresponding concurrent vehicle control. Results considered positive if substantial increase in revertant colony counts. Otherwise, results were considered negative. This increase must be dose related and/or reproducible. Test substance that produces neither a concentration related increase in number of revertant colonies nor a reproducible substantial increase in revertant colonies is considered to be non-mutagenic in test system.

Statistics:

Calculated means and standard deviations for all quantitative data collected.

Results and discussion

Test results

Test results 1

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No signs of toxicity were seen in any of the strains at all dose levels. For all strains, the plates containing 1580 µg and 5000 µg of test substance had precipitate. Contamination was observed in few plates throughout the assay; however, it did not obscure the count with the exception of one replicate in TA98 for the pre-incubation method. Although the one plate was unreadable there were still two replicate plates that were able to be evaluated. For all strains, at least six non-toxic dose levels without precipitate were evaluated, therefore bacterial mutagenicity was adequately assessed. For negative control validity, at least six non-toxic dose levels without precipitate were evaluated, therefore bacterial mutagenicity adequately assessed. For positive control validity, expected substantial increases in revertant colony counts in both absence and presence of S9 in each phase of test confirmed sensitivity of test and activity of S9 mix.

Applicant's summary and conclusion

Conclusions:

The test substance was negative for mutagenicity in a Bacterial Reverse Mutation Test (Ames Test).

Executive summary:

The purpose of the study was to evaluate the potential for the test material to induce gene mutations in bacteria using the Ames assay. Point mutations which involve substitution, addition or deletion of one or a few DNA base pairs are detected in amino acid-requiring strains of Salmonella typhimurium (S. typhimurium, ST) and Escherichia coli (E. coli, EC) by their ability to functionally reverse mutations. These reverse mutations result in revertant colonies of bacteria with restored capability to synthesize the essential amino acid. This Ames test was conducted under test guidelines U.S. EPA Health Effects Test Guidelines, OPPTS 870.5100 “Bacterial Reverse Mutation Test” (1998) and OECD Guidelines for Testing of Chemicals, Section 4 (Test No. 471): “Bacterial Reverse Mutation Test" (1997). The test was run with test substance levels of 1.58, 5.0, 15.8, 50, 158, 500, 1580, and 5000 µg/plate, with the high level being the standard limit for the test. The main test was conducted using the plate incorporation method in both the absence and presence of metabolic activation (chemically-induced rat liver S9 mix). The results of the test were confirmed using a similar study design but employing the pre-incubation modification of the Ames test.
The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values. The positive control substances caused the expected substantial increases in revertant colony counts in both the absence and presence of S9 in each phase of the test confirming the sensitivity of the test and the activity of the S9 mix. Therefore, each phase of the test is considered valid. No signs of toxicity were seen in any of the strains at all dose levels. For all strains, the plates containing 1580 µg and 5000 µg of test substance had precipitate. Contamination was observed in few plates throughout the assay; however, it did not obscure the count with the exception of one replicate in TA98 for the pre-incubation method. Although the one plate was unreadable there were still two replicate plates that were able to be evaluated. For all strains, at least six non-toxic dose levels without precipitate were evaluated, therefore bacterial mutagenicity was adequately assessed.There was no concentration-related or substantial test substance related increases in the number of revertant colonies observed with strains TA98, TA100, TA1535, TA1537, or E.coli WP2 uvrA in both the absence and presence of S9 using either the plate incorporation or the pre-incubation method. In conclusion, based on these findings and on the evaluation system used, the test substance did not elicit evidence of bacterial mutagenicity in the Ames assay.