Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01.10.2010 – 03.12.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Council Regulation (EC) No.440/2008. Published in O.J. L 142, 2008
Deviations:
yes
Remarks:
(see Any other information ...)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Expiration: 03/2011
Storage condition of test material: at the room temperature in tightly closed container

Method

Target gene:
gene for histidine or tryptophan synthesis
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
supernatant of rat liver and a mixture of cofactors
Test concentrations with justification for top dose:
30, 100, 300, 1000, 3000 μg
Selection of doses/toxicity:
As the test substance is insoluble in water and relatively bad soluble in acceptable solvents (DMSO 1mg/ml). We tried make a suspension in water, but the test substance vas very little wettable. Behaviour of the test substance in dimethylformamide was similar as in dimethylsulfoxide (DMSO), what is one our favourite non-toxic solvents. So, toxicity test was performed with suspension of the test substance in DMSO in the maximum concentration recommended in guidelines (5000 µg/0.1 mL/ plate).

The test substance was then diluted until formation of concentration series (10-5000 µg per plate), which was tested for toxicity in strain TA 100 without metabolic activation. Particles of the test substance were observable from 500 µg per plate. In the maximum dose of 5000 µg per plate test substance in background made evaluation very difficult and some colonies could be omitted.
Therefore, in the first mutagenicity experiments the dose of 5000 µg per plate was omitted and experiments were done with the maximum dose of 3000 µg per plate. The starting dose was diluted according to the guidelines. The doses used were 30, 100, 300, 1000 and 3000 µg per plate.
The maximum dose was not too suitable for evaluation due to presence of the test substance in the background. This dose was omitted and maximum dose in the second mutagenicity experiments was 1000 µg per plate.
All concentrations of the test substance suspension were dosed in the volume of 0.1 mL per plate. Fresh suspensions of test substance were prepared before each experiment. The suspensions were shaken during dilution, before dosing to the top agar and before pouring onto the plates.
Vehicle / solvent:
Dimethylsulfoxide for analysis (purity>99.9%), Merck, Lot No. K40982552 019
- Justification for choice of solvent/vehicle: solubility of the substance


Controls
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent; negative controls contain 0.1 mL of DMSO for injection. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
(AS)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: N-methyl-N´-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine; 2-aminofluorene; 2-aminoanthracene; 9-aminoacridine hydrochloride monohydrate;
Remarks:
(other: MNNG; NPD; AF; AA; AAc)
Details on test system and experimental conditions:
The bacterial tester strains
histidine dependent Salmonella typhimurium TA 98 (CCM 3811), TA 1535 (CCM 3814), and tryptophan dependent strain Escherichia coli WP2 uvrA (CCM 4751) - were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno
and TA100 (CIP 103796, lot. No.1008) and TA 1537 (CIP 103799, lot No. 34508) were from Biological Resource Center of Institut Pasteur (CRBIP), Paris.
Strains TA 1537 and TA 98 detect frame shift mutations, strains TA 100 and TA 1535 serve to detect base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

NUMBER OF REPLICATIONS: two series

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). After this rule the result is positive, if a reproducible doseresponse effect occurs and/or a doubling of the ratio Rt/Rc is reached.
Statistics:
For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods:
Dunkel V. C.. Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation. Elsevier North-Holland Biomedical Press. 231 - 417
Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity. Mutat. Res. 189. 83 - 91

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
HISTORICAL CONTROL DATA:
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent; negative controls contain 0.1 mL of DMSO. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
As the test substance is insoluble in water and relatively bad soluble in acceptable solvents (DMSO 1mg/ml). We tried make a suspension in water, but the test substance vas very little wettable. Behaviour of the test substance in dimethylformamide was similar as in dimethylsulfoxide (DMSO), what is one our favourite non-toxic solvents. So, toxicity test was performed with suspension of the test substance in DMSO in the maximum concentration recommended in guidelines (5000 µg/0.1 mL/ plate).
The test substance was then diluted until formation of concentration series (10-5000 µg per plate), which was tested for toxicity in strain TA 100 without metabolic activation. Particles of the test substance were observable from 500 µg per plate. In the maximum dose of 5000 µg per plate test substance in background made evaluation very difficult and some colonies could be omitted.

Therefore, in the first mutagenicity experiments the dose of 5000 µg per plate was omitted and experiments were done with the maximum dose of 3000 µg per plate. The starting dose was diluted according to the guidelines. The doses used were 30, 100, 300, 1000 and 3000 µg per plate.
The maximum dose was not too suitable for evaluation due to presence of the test substance in the background. This dose was omitted and maximum dose in the second mutagenicity experiments was 1000 µg per plate.
All concentrations of the test substance suspension were dosed in the volume of 0.1 mL per plate. Fresh suspensions of test substance were prepared before each experiment. The suspensions were shaken during dilution, before dosing to the top agar and before pouring onto the plates.

Applicant's summary and conclusion

Conclusions:
Under the above-described experimental design, the test substance 2-(3',5'-Dichloro-2'-hydroxyphenyl)-4-quinazolinone was non-mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without and with metabolic activation.
Executive summary:

Test substance 2-(3',5'-Dichloro-2'-hydroxyphenyl)-4-quinazolinone was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four  indicator Salmonella typhimurium strains TA 98, TA 100,  TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was suspended in DMSO and assayed in doses of 10-3000 ug which were applied to plates in volumes of 0.1 mL.

Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance 2-(3',5'-Dichloro-2'-hydroxyphenyl)-4-quinazolinone was non-mutagenic for all the used bacterial strains with as well as without metabolic activation.