3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

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

Study period:

2005-09-15 to 2005-09-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only three strains are tested. However, an expert statement is added in the field "any other remarks" to justify the fact that no further testing is necessary.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only three tester strains were used instead of five

Test guideline 2

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 instead of five

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

EC Number:

225-795-3

EC Name:

3-(2-chloroethyl)(1H,3H)quinazoline-2,4-dione

Cas Number:

5081-87-8

Molecular formula:

C10H9ClN2O2

IUPAC Name:

3-(2-CHLOROETHYL)QUINAZOLINE-2,4(1H,3H)-DIONE

Details on test material:

- Name of test material (as cited in study report): T 1201
- Substance type: white to pale beige solid
- Physical state: solid
- Purity test date: no data
- Lot/batch No.: batch 00464885 RT001201G1A251
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: room temperature, kept away from direct sunlight
- Other:
Solubility:
0.09 g/L in water
26.7 g/L in acetone
7.9 g/L in ethanol

Specific details on test material used for the study:

Description: white to pale beige solid
Batch number: 00464885 RT001201G1A251
Purity: not indicated by sponsor
Stability in solvent: not indicated by sponsor
Solubility in water: 0.09 g/L
Storage conditions: Room temperature, keep away from direct sunlight
Expiry date: not indicated by sponsor

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA98, TA100 and TA102

Additional strain / cell type characteristics:

other: see "Any other information on materials and methods"

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.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation (S9): for TA100 at 10 μg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Without metabolic activation (S9): for TA98 at 10 μg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation (S9): for TA102 at 4 μg/plate

Controls 4

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 metabolic activation (S9): for all strains, 2.5 μg/plate for strains TA98 and TA100 and 10 μg/plate for TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1- in agar (plate incorporation); Experiment 2-preincubation assay

Experiment I: in agar (plate incorporation)
- In the plate incorporation assay, 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 substit
ution buffer (for test without metabolic activation); 100 μL bacteria suspension (test system, pre-culture of the strains); and 2000 μL overlay agar.

Experiment II: preincubation
- In the pre-incubation assay, 50 μL test solution, solvent or 100 μL positive control, 500 μL S9 mix/S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated 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 minimal agar plates.
- After solidification, the plates from both assays were incubated upside down for at least 48 hours at 37°C in the dark.

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: at least 48 hours
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in the number of spontaneous revertants or a clearing of the bacterial back ground 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 (strains TA98, TA100 and TA102).
- 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 1 - with and without S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: 0.09 g/L
- Precipitation: No precipitation of the test substance in the overlay agar was observed.
- Other confounding effects: not applicable

RANGE-FINDING/SCREENING STUDIES:
- The pre-experiment was reported as main Experiment 1. No toxic effects, evident as a reduction in the number of revertants was observed in the pre-experiment, except in strain TA102 a reduction of the revertant colonies was observed with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
- No substantial increase in revertant colony numbers of any of the three tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
- The data in the negative control (Exp. 1 without S9 mix, Exp. 2 with and without S9 mix) and solvent control (Exp. 1 and 2 with and without S9 mix) of strain TA102 were slightly above the historical control range. Since this deviation was rather small, this effect was considered to be based upon biologically irrelevant fluctuations in the number of colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The plates incubated with the test substance showed normal background growth in all strains used with and without metabolic activation.

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

Under the conditions of the study, the test item was determined to be negative for mutagenic potential in all tester strains in the absence and presence of metabolic activation.