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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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:
From 2006-04-12 to 2006-04-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Non-GLP study conducted according to the OECD Guideline 471 with an acceptable restriction (only three tester strains were used). When considered with the other Ames study, five of the strains mentioned in the OECD guideline are investigated and enough to cover the endpoint.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: only three tester strains were used
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4D (dated May 19, 2000)
Deviations:
yes
Remarks:
: only three tester strains were used
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-hydroxyethyl)-1H,3H-quinazoline-2,4-dione
EC Number:
214-897-3
EC Name:
3-(2-hydroxyethyl)-1H,3H-quinazoline-2,4-dione
Cas Number:
1207-75-6
Molecular formula:
C10H10N2O3
IUPAC Name:
3-(2-hydroxyethyl)-1,2,3,4-tetrahydroquinazoline-2,4-dione
Test material form:
solid
Specific details on test material used for the study:
- Name of test material (as cited in study report): 3-(2-hydroxyethyl)-2,4(1H,3H)-quinazolinedione (T001200)
- Substance type: no data
- Physical state: white solid
- Analytical purity: 100 %
- Lot/batch No.: batch 00495665 RT001200G1A291
- Expiration date of the lot/batch: 2006-06-30
- Stability under test conditions: no data
- Storage condition of test material: room temperature

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98, TA100 and TA102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
- Pre-experiment/Experiment 1: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
- Experiment 2: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; for TA100 at 10 µg/plate
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without S9; for TA98 at 10 µg/plate
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; for TA102 at 4.0 µL/plate
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9; for TA98 and TA100 at 2.5 µg/plate and TA102 at 10 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: plate incorporation assay
Experiment II: pre-incubation assay

Experimental performance: The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL: Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 μL: S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL: Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL: Overlay agar

In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.

DURATION:
- Pre-incubation period: 60 minutes
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: Each concentration, including controls, was tested in triplicate.

DETERMINATION OF CYTOTOXICITY- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
- The test substance was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control was observed.
- A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.
Statistics:
A statistical analysis of the data was not required.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
: TA102 only (experiment 2)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Precipitation of the test item was observed in the test tubes in the overlay agar at 5000 µg/palte in both experiments. Precipitation of the test item on the plates in the overlay agar was observed at 5000 µg/plate in both experiments with and withotu S9-mix. The undissolved particles of the test item had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. The pre-experiment is reported as experiment I since no toxic effects were observed. Six concentrations were tested in experiment II and 5000 µg/plate was chosen as maximum concentration.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects, evident as a reduction in the number of revertants, occured in the test groups with and without metabolic activation with the exception in experiment II in strain TA102, where a reduction in the number of revertants was observed at 5000 µg/plate with and without S9-mix.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The test substance was evaluated for mutagenic potential in the bacterial reverse mutation assay in S. typhimurium strains TA98, TA100 and TA102 in the presence and absence of metabolic activation. Under the conditions of the study, the test substance was determined to be negative for mutagenic potential in all tester strains in the absence and presence of metabolic activation. Based on CLP regulation, the test item is not considered to be classified.