6-hydroxy-1H-pyrimidin-4-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative (S. typhimurium TA98, TA100, TA1535, TA1537), OECD 471, Ebert 1995

Ames test: negative (S. typhimurium TA1535, TA100, TA97, TA98), NTP 1996

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1996 or before

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Published under the US National Toxicology Program, according to the quality rules of the NTP interagency program ("best science available"). Experimental details well documented, but no explicit reference to GLP. Only four S. typhimurium strains, but no E. coli tested.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

No explicit reference to this guideline

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

hisG46, hisD3052, hisC3076 (from choice of strains)

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rat liver S9, 10% and 30%; Aroclor 1254-induced male Syrian hamster liver S9, 10% and 30%

Test concentrations with justification for top dose:

100, 333, 1000, 3333, 10000 microgram/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation

Migrated to IUCLID6: for TA 98; sodium azide (TA100 + TA1535); 9-aminoacridine (TA97)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°
- Exposure duration: 2 days

NUMBER OF REPLICATIONS:
- not stated; sufficient to calculate mean and standard error of mean

NUMBER OF CELLS EVALUATED:
- no data

Evaluation criteria:

Significant increase of mutant colonies over spontaneous control rate
Pattern and strength of mutant response
Verification of response in repeat test

Statistics:

Mean and standard error of mean (SEM)

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

no toxicity up to 10000 microgram/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

no toxicity up to 10000 microgram/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

no toxicity up to 10000 microgram/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

no toxicity up to 10000 microgram/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The results are sufficiently detailed and unambiguous as to be reliable for risk assessment, classification and labeling.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1995-08-01 to 1995-09-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study according to OECD guideline. Only four strains of S. typhimurium were tested, but no strain of E. coli. One or more concentrations insoluble in the final treatment mixture (precipitating) were not tested; a saturated aqueous solution of the test material (2.5 mg/liter) served as a stock solution for the preparation of the final treatment mixtures, thus only amounts up to approximately 500 microgram/plate were investigated.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 strains of S. typhimurium, no E.coli strain tested. No precipitating concentration tested; maximum amount 500 microgram/plate.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Only 4 strains of S. typhimurium, no E.coli strain tested. No precipitating concentration tested; maximum amount 500 microgram/plate.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

hisG46, hisD3052, hisC3076

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (lot # 170795, Cytotest Cell Research , Rossdorf, Germany) induced with Aroclor 1254

Test concentrations with justification for top dose:

Test #1: Plate incorporation test: 5, 16, 50, 160, 501 microgram/plate
Test #2: Preincubation test: 5, 15, 48, 154, 482 microgram/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solubility of the test material in ten different solvents was tested; water turned out to be the most suitable, with a maximum solubility of 2.5 g/liter

Untreated negative controls:

yes

Remarks:

0.2 mL water

Negative solvent / vehicle controls:

yes

Remarks:

0.2 mL water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Migrated to IUCLID6: for TA98; sodium azide for TA100 and TA1535, 9-aminoacridine for TA1537

Untreated negative controls:

yes

Remarks:

0.2 mL water

Negative solvent / vehicle controls:

yes

Remarks:

0.2 mL water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Test #1: in agar (plate incorporation), Test #2: preincubation

DURATION
- Preincubation period: 30 min at 30°C
- Exposure duration (incubation time): 72 hours at 37°C

NUMBER OF REPLICATIONS: Triplicate, per strain and concentration of the test material

NUMBER OF CELLS EVALUATED: >=67'000'000 per plate

DETERMINATION OF CYTOTOXICITY
- other: examination of the bacterial background lawn

Evaluation criteria:

VALIDITY:
- Characteristic mean number of spontaneous revertants in solvent control
- Titers of overnight cultures > 100'000'000
- Significant increase in the number of revertants in the mean of each positive control, compared with the mean of the solvent control
- At least four non-toxic dose levels

EVALUATION:
- At least a doubling in the mean revertants per plate of at least one tester strain (in two independent experiments)
- This increase must be accompanied by a dose response to increasing concentrations of the test article
- Single increases in revertant frequencies (not dose-related, not reproducible in independent tests) are considered non-relevant
- If such increases occur in both independent tests, this will be taken as an indication of a mutagenic effect

Statistics:

Mean and standard deviation of replications
Test compound / control ratio: Mean no. of colonies/plate (test compound) / mean no. of colonies/plate (water)

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

up to 500 microgram/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 500 microgram/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not tested (pH of suspension: 3.9)
- Effects of osmolality: Not expected due to limited solubility
- Evaporation from medium: Not expected (solid, melting point >300°C
- Water solubility: 2.5 g/liter, this limited the maximum concentration introduced into the test system to approx. 500 microgram/plate. No concentrations were tested that were insoluble in the final treatment mixture.
- Precipitation: Not observed
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: None

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: A reduced growth of the bacterial background lawn, indicative of test compound induced cytotoxicity, was not detectable with any of the tester strains

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Only 4 strains of S. typhimurium, no E.coli strain tested. No precipitating concentration tested; maximum amount 500 microgram/plate.In both experiments (test #1: plate incorporation test; test #2: preincubation test) no indication of test compound induced mutagenicity was observed with either one of the tester strains TA 98, TA 100, TA 1535 and TA 1537, with or without metabolic activation by S9 mix.

Under the experimental conditions described, 4,6-dihydroxypyrimidine did not induce a mutageniceffect in four strains of S.typhimurium. It is therefore not considered to be a bacterial mutagen.

All criteria for a valid study were met according to the author: All tested bacterial strains exhibited a positive mutagenic response with the positive controls (with and without metabolic activation). Negative controls (water) were also tested with each strain, and the mean numbers of spontaneous revertants were considered acceptable. However, no concentrations insoluble in the final treatment mixture were tested, and the concentration for a limit test (5000 microgram/plate) was not reached.

Conclusions:

Interpretation of results (migrated information):
negative

The results are sufficiently detailed and unambiguous as to be reliable for risk assessment, classification and labeling.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test (bacterial reverse mutation in vitro): Negative

The study by Ebert (Huels,1995) is a completely documented company study report on Ames testing, under GLP and according to the OECD guideline 471 valid at the time of testing. Limitations are the low maximum concentrations applied (only 500 microgram/plate, non-precipitating), and the absence of E. coli tester strains, which are required by newer versions of the guideline OECD 471. Only S. typhimurium strains TA98, TA100, TA1535 and TA1537 were investigated.

The NTP study (1996) follows equally high scientific standards, but is documented in less detail. Again, only S. typhimurium strains were investigated (TA 1535, TA 100, TA97, and TA98). Doses up to 10000 microgram/plate were applied, without signs of precipitation or cytotoxicity.

Both studies show negative results for all strains tested, with and without metabolic activation.

It is known that the strains used in the two studies may not detect certain oxidizing mutagens, cross-linking agents and hydrazines, but since the test substance 4,6-dihydroxypyrimidine has neither of these properties, we believe that the missing E. coli strain does not disqualify the negative results obtained.

Based on a weight-of-evidence approach, according to Regulation (EC) No. 1907/2006, Annex XI, section 1.2., we conclude that 4,6-dihydroxypyrimidine is non-nutagenic in the Ames test.

Chromosome aberration: No data available

Justification for classification or non-classification

The Ebert (Huels, 1995) and the NTP (1996) studies are considered reliable with restrictions but adequate for the purposes of risk assessment, classification and labeling.

Based on the results of the Ebert (Huels, 1995) and the NTP (1996) studies, the substance does not meet the criteria for classification under Regulation (EC) No. 1272/2008 Annex I, Part 3, 3.5.2.