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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A study was performed to the requirements of OECD TG 471 (1997), EU Method B13/14 (December 1992) under GLP, in order to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA, in both the presence and absence of S-9 mix (standard plate test and preincubation test). The dose range was 20ug - 5000ug/plate. Further to this, tests were also conducted with a positive control (with S-9 mix; 2 -aminoanthracene (2 -AA) and 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)), and a negative controls (conducted to test for sterility control; plates treated with soft agar, S-9 mix, buffer, vehicle or the test substance but without the addition of tester strains and to determine the spontaneous mutation rate via the 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) were performed.

As the guidelines were met with no issues regarding the positive or negative controls, the conditions utilised for the study were considered to be suitable for test purposes. An increase in the number of his+ or trp+ revertants was not observed in both the standard and preincubation plates either with or without the presence of S-9 mix. It was therefore deemed that the test substance was not mutagenic in the S.typhimurium/E.coli reverse mutation assay and is not classified under Regulation 1272/2008.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

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

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).

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

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.

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.

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

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

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The study was performed to the requirements of OECD Guideline 474 (1997), Commission Directive 2000/32/EC (2000) to evaluate the potential mutagenicity of the test substance in a mouse micronucleus assay using an NMRI strain of mouse. The test substance was dissolved in 0.5% carboxymethylcellulose. The volume administered intraperitoneally was 10mL/kg bw, at 24 hours and 48 ours after a single administration of the test substance, the bone marrow cells were collected for micronuclei analysis. 5 males/test group were evaluated for the occurence of micronuclei. At least 2000 polychromatic erythrocytes/animal were scored for micronuclei.

The cytotoxic effects stemming from the treatment was described by the ratio between polychromatic and normochromatic erythrocytes in the same sample and reported as the number of normochromatic erythrocytes per 2000 polychromatic erythrocytes.

The dose range was 500, 1000 and 2000mg/kg bw for the 24 hour preparation interval, and the 48 hour preparation intercval was 2000mg/kg bw. The highest dose was estimated by a pre-experiment and it was deemed suitable. Further to this, tests were also conducted with a positive control (40mg/kg bw cyclophosphamide - administered intraperitoneally) and a negative control (vehicle only) to ensure the validity of the test results.

As the guidelines were met with no issues regarding the positive or negative controls the conditions was considered to be suitable for test purposes. The comparison of test results wtith the corresponding vehicle controls showed that there was not any enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used.

Based on the findings above, it was concluded that the test substance is considered to be non-clastogenic and non-aneugenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

Commision Directive 2000/32/EC, Annex 4C, dated May 19, 2000

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Micronucleus Assay

Specific details on test material used for the study:

Batch number: Fixapret 140 Nr. 766.
Date of manufacturing: March 24, 2001.
Purity: 96.3g/100g.

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal model for which suitable cytogenic investigations have been made, historic data from these investigations aid to elucidate interpretation of results from the micronucleus test. Additionally, the mouse is an experimental animal model that has been traditionally utilised for toxicological studies.

Sex:

male

Details on test animals or test system and environmental conditions:

Test Animals
Strain: NMRI.
Source: RCC Ltd. Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf.
Number: 42.
Initial age at the start of acclimatisation: 8-10 weeks.
Acclimatisation period: 5 days (minimum).
Body weight at beginning of study: mean value 40.9g (SD± 3.8g).

The animals were distributed into the test groups at random and identified by cage number.

Environmental Conditions
Housing: Single.
Bedding: Granulated soft wood bedding.
Feed: Pelleted standart diet, ad libitum (ALIROMIN 1324, D-32791 Lage/Lippe).
Water: Tap water, ad libitum (Gemeindewerke, D-64380 Roßdorf).
Temperature: 21C (SD± 4C).
Humidity: 30-70%.
Lighting: 12 hours Light / 12 Hours dark.

Route of administration:

intraperitoneal

Vehicle:

0.5% carboxymethyl cellulose (CMC).

Details on exposure:

Prior to treatment, all animals (including controls) were weighed and the individual volume to be administered was 10mL/kg body weight. The animals were then administered with the test substance, the vehicle or the positive control substance once intraperitoneally.

Duration of treatment / exposure:

The animals were sacrified by cervical dislocation 48 hours post test substance administration.

Frequency of treatment:

Once.

Post exposure period:

48 hours.

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6.

Control animals:

yes, concurrent vehicle

Positive control(s):

Historical positive control(s) were reported using a known clastenogestic and aneugenic substance; cyclophosphamide; 40 mg/kg b.w (oral administration).
Micronucleated polychromatic erythrocytes per thousand;
Range: 10.00 to 27.10.
Mean: 16.53*±4.09 (*= mean of 35 experiments (caiculated from 350 animals; 2000 polychromatic erythrocytes scored per animal).

Tissues and cell types examined:

Bone marrow.

Details of tissue and slide preparation:

The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald (MERCK, D-64293Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Evaluation criteria:

The test substance is classified as mutagenic if;
- it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes, that is statistically significant when compared with the negative control range or a relevant positive response for at least one of the test points.

The test substance is considered non-mutagenic if;
- neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a positive response at any of the test points.

Statistics:

Evaluation via statistical method analysis was conducted using a non-parametric Mann-Whitney test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Summary of Micronucleus Test Results

 

 

 

Test group	dose mg/kgb.w.	sampling time (h)	PCEs with micronuclei (%)	Range	PCE/ NCE ratio
vehicle	0	24	0.50	0-2	2000/1819
test substance	500	24	1.00	0-5	2000/1953
test substance	1000	24	0.30	0-2	2000/2597
test substance	2000	24	0.40	0-2	2000/2377
positive control	40	24	19.20	25-52	2000/1866
vehicle	0	48	0.60	0-2	2000/1699
test substance	2000	48	0.70	0-4	2000/2429

Conclusions:

Under the conditions of the study, it is concluded that the test substance did not cause any clastenogenic or aneugenic effects on the test animals.

Executive summary:

The study aimed to investigate the potential of the test substance glycoluril to introduce micronucleus abnormalities in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to the OECD Guideline No 475 (1997) and the Commission Directive 2000/32/EC, Annex 4C (2000). 

The test substance was dissolved in 0.5% CMC. 10mLkg body weight was administered intraperitoneally 24 hours, and 48 hours after a single administration of the test substance. The bone marrow cells were collected for micronuclei analysis. 5 male mice per test group were evaluated for the occurrence of micronuclei, and at least 2000 PCEs per animal were scored for the presence of micronuclei. The mice were treated with 1000 and 2000 mg/kg b.w. at the 24 hour preparation interval and 2000 mg/kg b.w. at the 48 hour preparation interval. 40 mg/kg b.w. cyclophosphamide administered intraperitoneally was used as a positive control. This showed a substantial increase of induced micronucleus frequency.

At the 48 hour preparation interval, there were slightly increased mean normochromatic erythrocytes (NCE) numbers, as compared to the mean values of NCEs of the corresponding vehicle control. This indicated that glycoluril had cytotoxic effectiveness in the bone marrow. However, when comparing with the corresponding vehicle controls, there was no enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used.

Therefore, it can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Thus, glycoluril is considered to be non-clastogenic and non-aneugenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity based on the results from the performed S.typhimurium/E.coli reverse mutation in vitro assay and genotoxicity from the mouse micronucleus in vivo assay.