Perhydroimidazo[4,5-d]imidazole-2,5-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

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Perhydroimidazo[4,5-d]imidazole-2,5-dione

EC Number:

207-821-5

EC Name:

Perhydroimidazo[4,5-d]imidazole-2,5-dione

Cas Number:

496-46-8

Molecular formula:

C4H6N4O2

IUPAC Name:

perhydroimidazo[4,5-d]imidazole-2,5-dione

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Batch number: Zwischenprodukt aus Partie 692.
Date of manufacturing: 27 July 1998.
Degree of purity: 97.6-100.3% (determined via HPLC).

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S-9

Test concentrations with justification for top dose:

1st Experiment
Doses: 0, 20, 100, 500, 2500, 5000 ug/plate.

2nd experiment.
Doses: 0, 20, 100, 500, 2500, 5000 ug/plate.

Dose selection and evaluation as well as the number of plased used in repeat studies or further experiments are based on the findings of the 1st experiments.

Vehicle / solvent:

Due to the limited solubility of the test substance in water, acetone was selected as the vehicle. The vehicle choice was deemed appropriate in the bacterial reverse mutation tests. Additionally, historical control data were also available.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

The experimental procedure was split into the following;

>Mutagenicity tests
Standard plate test
Salmonella typhimurium plate incorporation method was based on the method of Ames et al., (1975). After mixing, the samples were poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. The plates were then incubated at 37C for 48 - 72 hours in the dark, the bacterial colonies (his* revertants) were counted.

Escherichia coli experimental procedure was based on the method of Ames et al., (1975). After mixing, the samples were poured onto minimal agar plates within approximately 30 seconds. The plates were then incubated at 37C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted.

>Preincubation test
The experimental procedure was based on the method as described by Yahagi et al., (1977) and Matsushima et al., (1980). After incubation at 37C for 48 - 72 hours in the dark, the bacterial colonies are counted.

>Titer determination
In both the standard plate and preincubation tests, 0.1mL of each culture was diluted to 10-6.

Standard plate test
The diluted culture was then added to; 2mL soft agar (containing 5mM tryptophan or 5mM histidine + 0.5mM biotin), 0.1mL vehicle (without and with test substance), 0.5mL S-9 mix.

Preincubation test
The diluted culture was then added to; 0.1mL vehicle (with and without test substance) and 0.5mL S-9 mix were incubated at 37C for 30 minutes using a shaker. Subsequently, 2mL of soft agar (containing 5mM tryptophan or 5mM histidine + 0.5mM biotin) is added.

After mixing, the samples were poured onto the agar plates within approximately 30 seconds. The plates were then incubated at 37C for 48 - 72 hours in the dark, the bacterial colonies were counted.

Control articles
Negative controls were performed for each experiment, in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).
Sterility control; plates treated with soft agar, S-9 mix, buffer, vehicle or the test substance but without the addition of tester strains.
Vehicle control; with and without S-9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.

Positive control
The following positive controls were utilised to check the mutability of the bacteria and the activity of the S-9 mix.
With S-9 mix; 2 -aminoanthracene (2 -AA).
Without S-9 mix; N-methyl-N-nitro-N-nitrosoguanidine (MNNG) , 4-nitro-o-phenylendiamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO).

Scope of tests and conditions are provided below;
1st Experiment
Strain: TA 1535, TA 100, TA 1537, TA 98, E. Coli WP2 uvrA.
Doses: 0, 20, 100, 500, 2500, 5000 ug/plate.
Vehicle: Acetone.
No. of plates: 3 per dose or per control.
Type of Test: Standard Plate Test - with and without S9 mix.

2nd experiment.
Strain: TA 1535, TA 100, TA 1537, TA 98, E. Coli WP2 uvrA.
Doses: 0, 20, 100, 500, 2500, 5000 ug/plate.
Vehicle: Acetone.
No. of plates: 3 per dose or per control.
Type of Test: Preincubation Test - with and without S9 mix.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study;

Mutagenicity was evaluated based on the individual plate counts, the mean number of revertant copies per plate and the standard deviations given for all the dose groups (including the positive and negative controls) in all experiments.

Toxicity was detected if the following observations were seen (this was tested and recorded for both with and without S9 in all experiments);
- decrease in the number of revertants,
- clearing of diminution of the background lawn (reducted his- or trp+ background growth),
- reduction in the titer.

The test substance is considered positive in the assay if the following criteria were met;
A dose-related and reproducible increase in the number of revertant colonies were observed (in at least one tester strain either with or without S-9 mix).

And the test substance is generally considered mutagenic if;
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

20 ug 27 28 4 0.9 34 36 2 1.0       24       37           32       37         100   ug   27   25   2   0.8   32   31   1   0.9     24       30           24       31         500   ug   24   24   1   0.8   24   27   3   0.8     24       26           25       30         2500   ug   17P   22   6   0.8   26P   23   2   0.7   30     28P       22P       32     22P       22P       21 5000 ug 19P 19   2 0.7   23P   24   3   0.7   26     18P       27P       26     21P       22P       26   NOPD   10   ug   731   792   65   26.7                 783                       861                 TONE 31 29 4 1.0 35 32 3 1.0 31 24       33       35 32       29       40 20   ug   25   29   5   1.0   27   30   3   0.9       34       31             29       32         100   ug   28   30   2   1.0   23   25   5   0.8       30       30             32       21         500 ug   35   32   3   1.1   32   30   4   0.9       31       25             29       33           2500   ug   30P   28   2   1.0   33P   31   7   0.9   31     28P       36P       32     27P       23P       35 5000   ug   24P   27   3   0.9   42P   36   6   1.1   32     30P       36P       29     28P       31P       30

Applicant's summary and conclusion

Conclusions:

Interpretation of results; negative under the conditions of this study. There was no evidence that the test substance has any mutagenic properties in the presence or absence of S-9 activation.

Executive summary:

The study was performed to the requirements of guideline OECD 471 (1997), and EEC Directive 92/69, B14 and B13 (December 1992), under GLP conditions, to evaluate the potential mutagenicity of glycouril in a bacterial reverse mutation assay using Salmonella typhimurium strains TA1535, TA100, TA1537, TA98 and Escherichia coli strain WP2 uvrA. All strains undertook a standard plate test, and a preincubation test, both with and without metabolic activation (Aroclor-induced rat liver S-9 mix).

All tests used a dosage range of 20ug-5,000 ug/plate. A phosphate buffer was used in the tests without metabolic activation, and due to the limited solubility of the test substance in water, acetone was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which, historical control data were available. Precipitation of the test substance was found from about 2,500 ug/plate onward, and a slight decrease in the number of revertants was occasionally observed in the preincubation test. An increase in the number of revertants was not observed in the standard plate test or in the preincubation test without S-9 mix, or after the addition of a metabolizing system.

According to the experimental conditions and test results, the test substance glycoluril is non mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay.