Tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001 - 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione

EC Number:

226-408-0

EC Name:

Tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione

Cas Number:

5395-50-6

Molecular formula:

C8H14N4O6

IUPAC Name:

1,3,4,6-tetrakis(hydroxymethyl)-octahydro-[1,3]diazolo[4,5-d]imidazole-2,5-dione

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG; THOR GmbH; Batch no. 0767
- Purity/Content: 49.5 g /100 g (aqueous solution)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The storage stability of the test substance covering the period of the study was determined by re-characterisation after completion of the experimental phase of the study.
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance in water was determined analytically.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: On the day of the experiment (immediately before treatment), the test substance was dissolved in deionised water.

FORM AS APPLIED IN THE TEST: dissolved in deionised water

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Technical University; D-64287 Darmstadt); Storage in liquid nitrogen in the cell bank of RCC Cytotest CelI Research GmbH
- Suitability of cells: Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability.
- Doubling time: 12 - 16 hours
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37° C in 80 cm^2 plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 10^5 cells per flask were seeded into 15 ml of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with. 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cälbe). The cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 4.5 % carbon dioxide (95.5 % air).
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: Doubling time: 12 hours

MEDIA USED
- Type and identity of media including CO2 concentration: MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with. 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cälbe); The cell cultures were incubated at 37° C in a humidified atmosphere with 4.5 % carbon dioxide (95.5 % air).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, before freezing
- Periodically checked for karyotype stability: yes, before freezing
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor-supplemented rat liver S9 (Phenobarbital/f3-Naphthoflavone induced); protein concentration: 30 mg/ml (pre-test), 31.3 mg/ml (main experiment)

Test concentrations with justification for top dose:

Experiment 1: ratio of S9/cofactor solution 1:2
without S9 mix: 10.0; 20.0; 40.0; 60.0; and 80.0 µg/ml
with S9 mix: 10.0; 20.0; 40.0; 60.0; and 80.0 µg/mI
Experiment 2: ratio of S9/cofactor solution 1:9
without S9 mix: 10.0; 20.0; 40.0; 60.0; and 80.0 µg/mI
with S9 mix: 10.0; 20.0; 40.0; 60.0; and 80.0 µg/mI

A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test substance was abserved and compared to the controls. Toxicity of the test substance is indicated by a reduction of the cloning efficiency (CE). 8 concentrations between 43.8 and 5600 µg/ml were applied for 4 hours with and without metabolic activation.
Toxic effects occurred down to the lowest concentration with and without metabolic activation. Already at 175.0 µg/ml with and without metabolic activation the cell growth was completely inhibited. Therefore, the maximum concentration of the test substance (80.0 µg/ml) in the first main experiment was based upon the toxicity of the test substance observed in the pre-experiment. The dose range of the second experiment was identical to the dose range of the first experiment. Detailed results of the pre-test in table 1
No precipitation occurred up to the highest concentration tested in any of the experiments.

Vehicle / solvent:

- Vehicle/solvent used: deionised water

Controls

Untreated negative controls:

yes

Remarks:

untreated cells (cells were cultivated without interruption)

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

7,12-dimethylbenzanthracene - with metabolic activation; ethylmethasulphonate - without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 1.5 x 10^6 (single culture); 5 x 10^2 cells (in dublicate; to determine viability)

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 hours
- Expression time (cells in growth medium): approx. 10 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation): approx. 18 days

SELECTION AGENT (mutation assays): 11 µg/ml thioguanine (6-TG)

STAIN (for cytogenetic assays): 10 % methylene blue

STAINING TECHNIQUE USED:
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment.
Three days after treatment 1 .5 x 10^6 cells per experimental point were sub-cultivated in 175 cm^2 flasks containing 30 ml medium. Following the expression time of approx. 7 days five 80 cm^2 cell culture flasks were seeded with about 3 - 5 x 10^5 cells each in medium containing 6-TG. Two additional 25 cm^2 flasks are seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures are incubated at 37°C in a humidified atmosphere with 4.5 % CO2 for about 8 days. The colonies were stained with 10 % methyhene blue in 0.01 % KOH solution.

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY
- cloning efficiency

OTHER EXAMINATIONS:
- spontaneous mutation rate

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
a) the numbers of mutant colonies per 10^6 cells found in the negative and/or solvent controls fall within the laboratory historical control data range of 1996 - 2000 (see table 2)
b) the positive control substances must produce a significant increase in mutant colony frequencies within the laboratory historical control data range of 1996 - 2000 (see table 2)
c) the cloning efficiency (absolute value) of the negative and/or solvent controls must exceed 50 %

A test substance is classified as positive if it induces a concentration-related increase of the mutant frequency.
A test substance producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test substance is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency (4.0 mutant colonies (Experiment 1) and 11.2 (Experiment 2) mutant colonies per 10^6 cells) at least at one of the concentrations in the experiment and if this increase exceeds the range of our historical negative or solvent control data.

The test substance is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 - 33.1 mutants per 10^6 cells) a con centration-related increase of the mutations within this range has to be discussed.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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
- Effects of osmolality: no
- Precipitation: no

HISTORICAL CONTROL DATA:
- Positive historical control data: see table 2
- Negative (solvent/vehicle) historical control data: see table 2

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In both main experiments strong toxic effects in both parallel cultures occurred at 60.0 and 80.0 µg/ml in the absence of metabolic activation. In the presence of metabolic activation strong toxic effects were observed at 60 and 80 µg/ml in the first and at 80 µg/ml in the second experiment. Moderate toxic effects occurred already at 40 µg/ml in some of the cultures with and without metabolic activation. In the first main experiment the relative cloning efficiency at 80 µg/ml with metabolic activation fell short of the range of 10 - 20 % recommended by the guidelines.

Any other information on results incl. tables

Table 3: Mutagenicity data (Mutationrates), Experiment 1, Culture 1

 

	conc.	S9	number of mutant colonies per flask						standard	mutant	induction
	µg/ml	mix	found after plating in TG medium						deviation	colonies	factor
		#	1	II	III	IV	V	mean		per 10^6cells
column	1	2	3	4	5	6	7	8	9	10	11
Negative control		-	3	4	3	0	1	2.2	1.6	7.1	1.0
Solvent control with water		-	1	2	3	1	5	2.4	1.7	8.0	1.0
Positive control with EMS	150.0	-	40	40	45	32	41	39.6	4.7	140.4	19.7
Test substance	5.0	-	culture was not continued##
Test substance	10.0	-	1	2	2	2	4	2.2	1.1	7.0	0.9
Test substance	20.0	-	10	3	5	1	0	3.8	4.0	14.3	1.8
Test substance	40.0	-	2	2	1	2	3	2.0	0.7	8.5	1.1
Test substance	60.0	-	5	4	2	3	6	4.0	1.6	12.9	1.6
Test substance	80.0	-	3	3	4	4	5	3.8	0.8	17.4	2.2
Negative control		+	2	2	1	5	5	3.0	1.9	8.8	1.0
Solvent control with water		+	3	2	2	1	4	2.4	1.1	7.4	1.0
Solvent control with DMSO		+	2	1	4	1	5	2.6	1.8	8.5	1.0
Positive control with DMBA	2.7	+	123	132	134	125	127	128.2	4.7	389.3	45.9
Test substance	5.0	+	culture was not continued##
Test substance	10.0	+	5	1	1	3	4	2.8	1.8	8.3	1.1
Test substance	20.0	+	1	2	1	2	3	1.8	0.8	6.5	0.9
Test substance	40.0	+	2	5	2	4	2	3.0	1.4	11.6	1.6
Test substance	60.0	+	3	1	4	2	1	2.2	1.3	9.9	1.3
Test substance ###	80.0	+	4	1	5	3	6	3.8	1.9	16.4	2.2

#     ratio of S9/cofactor solution was 1:2

##   concerning concentration range five concentrations were selected to be evaluated at the end of the experiment

### value ignored due to excessive cytotoxicity (suvival < 10%)

Table 4: Mutagenicity data (Mutation rates), Experiment 1, Culture 2

	conc.	S9	number of mutant colonies per flask						standard	mutant	induction
	µg/ml	mix	found after plating in TG medium						deviation	colonies	factor
		#	1	II	111	IV	V	mean		per 10^6cells
column	1	2	3	4	5	6	7	8	9	10	11
Negative control		-	4	3	1	0	3	2.2	1.6	8.9	1.0
Solvent control with water		-	3	1	1	2	0	1.4	1.1	3.8	1.0
Positive control with EMS	150.0	-	44	35	36	40	41	39.2	3.7	135.9	15.4
Test substance	5.0	-	culture was not continued##
Test substance	10.0	-	0	3	4	0	2	1.8	1.8	4.3	1.1
Test substance	20.0	-	1	2	2	1	1	1.4	0.5	4.5	1.2
Test substance	40.0	-	3	2	2	2	2	2.2	0.4	9.7	2.6
Test substance	60.0	-	9	7	4	6	6	6.4	1.8	17.4	4.6
Test substance ###	80.0	-	4	1	5	6	8	4.8	2.6	22.1	5.8
Negative control		+	4	3	4	5	5	4.2	0.8	15.3	1.0
Solvent control with water		+	1	3	2	3	3	2.4	0.9	9.8	1.0
Solvent control with DMSO		+	2	4	3	1	1	2.2	1.3	8.1	1.0
Positive control with DMBA	2.7	+	143	165	153	138	123	144.4	15.8	591.6	73.2
Test substance	5.0	+	culture was not continued##
Test substance	10.0	+	4	3	3	1	2	2.6	1.1	10.2	1.0
Test substance	20.0	+	2	2	2	3	2	2.2	0.4	7.7	0.8
Test substance	40.0	+	7	0	4	4	3	3.6	2.5	9.8	1.0
Test substance	60.0	+	0	1	3	0	2	1.2	1.3	3.6	0.4
Test substance ###	80.0	+	5	5	4	4	2	4.0	1.2	12.7	1.3

        #     ratio of S9/cofactor solution was 1:2

## concerning concentration range five concentrations were selected to be evaluated at the end of the experiment

### value ignored due to excessive cytotoxicity (suvival <10%)

Table 5:   Mutagenicity data (Mutationrates), Experiment 2, Culture 1

 

	conc.	S9	number of mutant colonies per flask						standard	mutant	induction
	µg/ml	mix	found after plating in TG medium						deviation	colonies	factor
		#	1	II	111	IV	V	mean		per 10^6cells
column	1	2	3	4	5	6	7	8	9	10	11
Negative control		-	6	7	3	6	2	4.8	2.2	18.1	1.0
Solvent control with water		-	3	1	5	2	7	3.6	2.4	14.1	1.0
Positive control with EMS	150.0	-	40	31	32	42	35	36.0	4.8	156.2	8.6
Test substance	5.0	-	culture was not continued##
Test substance	10.0	-	2	6	4	6	0	3.6	2.6	13.1	0.9
Test substance	20.0	-	3	3	4	6	5	4.2	1.3	14.2	1.0
Test substance	40.0	-	8	7	4	3	7	5.8	2.2	18.3	1.3
Test substance	60.0	-	8	5	11	5	12	8.2	3.3	43.9	3.1
Test substance	80.0	-	3	9	12	5	8	7.4	3.5	32.2	2.3
Negative control		+	2	3	4	1	0	2.0	1.6	7.3	1.0
Solvent control with water		+	4	4	1	3	8	4.0	2.5	13.0	1.0
Solvent control with DMSO		+	4	1	2	5	7	3.8	2.4	13.8	1.0
Positive control with DMBA	2.7	+	63	61	84	83	89	76.0	13.0	318.7	23.0
Test substance	5.0	+	culture was not continued##
Test substance	10.0	+	2	1	1	0	2	1.2	0.8	4.3	0.3
Test substance	20.0	+	7	4	5	5	3	4.8	1.5	15.5	1.2
Test substance	40.0	+	1	5	2	3	6	3.4	2.1	13.3	1.0
Test substance	60.0	+	1	4	1	2	2	2.0	1.2	7.9	0.6
Test substance	80.0	+	9	7	3	7	1	5.4	3.3	20.2	1.5

#     ratio of S9/cofactor solution was 1:9

## concerning concentration range five concentrations were selected to be evaluated at the end of the experiment

Table 6:   Mutagenicity data (Mutationrates), Experiment 2, Culture 2

conc. S9 number of mutant colonies per flask standard mutant induction   µg/ml mix found after plating in TG medium deviation colonies factor     # 1 II III IV V mean   per 10^6cells   column 1 2 3 4 5 6 7 8 9 10 11 Negative control   - 3 6 2 6 5 4.4 1.8 21.0 1.0 Solvent control with water   - 1 3 3 0 6 2.6 2.3 10.1 1.0 Positive control with EMS 150.0 - 34 29 42 38 45 37.6 6.3 164.4 7.8 Test substance 5.0 - culture was not continued##           Test substance 10.0 - 3 5 9 5 7 .5.8 2.3 28.6 2.8 Test substance 20.0 - 5 3 1 5 6 4.0 2.0 13.0 1.3 Test substance 40.0 - 7 4 3 1 7 4.4 2.6 24.0 2.4 Test substance 60.0 - 6 6 5 1 5 4.6 2.1 22.8 2.3 Test substance 80.0 - 5 9 2 9 7 6.4 3.0 32.2 3.2                         Negative control   + 4 4 2 0 4 2.8 1.8 10.7 1.0 Solvent control with water   + 0 5 1 6 4 3.2 2.6 11.3 1.0 Solvent control with DMSO   + 7 4 2 1 6 4.0 2.5 11.0 1.0 Positive control with DMBA 2.7 + 100 121 100 113 109 108.6 9.0 385.9 35.1 Test substance 5.0 + culture was not continued##           Test substance 10.0 + 0 2 5 1 3 2.2 1.9 6.9 0.6 Test substance 20.0 + 2 2 8 4 3 3.8 2.5 11.5 1.0 Test substance 40.0 + 5 3 6 4 2 4.0 1.6 11.9 1.1 Test substance 60.0 + 1 2 7 8 4 4.4 3.0 13.8 1.2 Test substance 80.0 + 8 5 9 7 11 8.0 2.2 21.3 1.9

#    ratio of S9/cofactor solution was 1:9

## concerning concentration range five concentrations were selected to be evaluated at the end of the experiment

Applicant's summary and conclusion

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, with and without liver microsomal activation and a treatment period of 4 hours. Since experiment 1 showed no indication of a possible mutagenic potential of the test substance, a second confirmatory experiment was performed with and without metabolic activation. The ratio of S9/cofactor solution was 1:2.0 in experiment I, and 1:9.0 in experiment II with metabolic activation.

The test substance is an aqueous solution (49.5 g per 100 g). The concentrations reported refer to the aqueous test substance solution and not to the content of active ingredient.

Dose selection of the main experiments was performed considering the toxicity data. The cultures were evaluated at the following concentrations:

Experiment 1:

without S9 mix: 10.0; 20.0; 40.0; 60.0; and 80.0 µg/ml

with S9 mix: 10.0; 20.0; 40.0; 60.0; and 80.0 µg/ml

Experiment II:

without S9 mix: 10.0; 20.0; 40.0; 60.0; and 80.0 µg/ml

with S9 mix: 10.0; 20.0; 40.0; 60.0; and 80.0 µg/ml

No precipitation of the test substance was observed up to the maximum concentration in all experiments.

In both main experiments strong toxic effects in both parallel cultures occurred at 60.0 and 80.0 µg/ml in the absence of metabolic activation. In the presence of metabolic activation strong toxic effects were observed at 60 and 80 µg/ml in the first and at

80 µg/ml in the second experiment. Moderate toxic effects occurred already at 40 µg/ml in some of the cultures with and without metabolic activation. In the first main experiment the relative cloning efficiency at 80 µg/ml with metabolic activation fell short of the range

of 10 - 20 % recommended by the guidelines. The mutation frequency however, generally remained within the historical range of negative and solvent controls even at such severely toxic concentrations.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

No relevant and reproducible increase of the mutation frequency was observed up to the highest investigated concentration in both main experiments carried out independently of each other.