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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro Ames test was performed according to the OECD 471 and in compliance with GLP in oder to investigate the mutagenic potential of the test substance based on the ability to induce back mutations in the S.thyphimurium strains TA 98, TA 100, TA 1535, and TA 1537 at concentrations ranging from 40 - 10,000 µg/plate (Standard plate test) and 250 - 3,000 µg/plate (preincubation test). No mutagenic effect in any strain was observed.

The in vitro gene mutation assay was performed according to the OECD 476 and in compliance with GLP in order to investigate the potential to induce gene mutations at the HPRT locus in Chinese hamster lung fibroblasts (V79) cells at concentrations ranging from 10 - 80 µg/ml. The test substance did not induce gene mutations.

The in vitro chromosome aberration assay was conducted according to the OECD 473 and in compliance with GLP in order to investigate the potential of the test substance to induce chromosome aberrations in Chinese hamster lung fibroblasts (V79) cells at concentrations ranging from 10 - 100 µg/ml. The test substance induced structural chromosome aberrations both with and without S9 mix at a concentration of 40 µg/ml.

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

HPRT

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

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.

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2001 - Feb 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 473

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF AG and Thor GmbH and 0767

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Purity: 49.5 g per 100 g (aqueous solution)
- Stability under test conditions: .The storage stability of the test substance covering the period of the study will be determined by re-characterisation after
completion of the experimental phase of the study. The results may be requested from the sponsors.
- Stability of the test substance in the solvent: The stability of the test substance in water were determined analytically by the sponsors. The results may be requested from the sponsors

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a liquid: On the day of the experiment (immediately before treatment), the test substance was dissolved in deionised water as requested from the sponsors.

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, LMP, Technical University Darmstadt, D-64287 Darmstadt
- Suitability of cells: used successfully for many years in in vitro experiments, high proliferation rate , reasonable plating efficiency and stable karyotype.
- Cell cycle length: 1.5 times of the normal cell cycle (18 h)
- Methods for maintenance in cell culture: subculture twice a week
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: 12 h

MEDIA USED
- Type and identity of media including CO2 concentration: MEM (Minimal Essentail Medium; SEROMED; D-12247) with 10%FCS, CO2 concentration 4.5% (95.5% air)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes. Before freezing, each batch was screened for mycoplasm contamination
- Periodically checked for karyotype stability: yes. Before freezing, each batch was checked for karyotype stability

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (Phenobarbital/b-Naphthoflavone induced rat liver S9)

Test concentrations with justification for top dose:

A pre-test on cell growth inhibition was performed in order to determine the toxicity of the test substance. Using reduced ceIt numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hrs treatment with 87.5 µg/mI and above in the absence and the presence of S9 mix. In addition, 24 hrs continuous treatment with 43.8 µg/mI and above in the absence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 100 µg/mI (with and without S9 mix) were chosen as top concentrations in the main experiment. Additional concentrations were 10, 20, 40, 60, 80 µg/ml although only 20, 40, and 60 µg/ml were the evaluated experimental points.

Vehicle / solvent:

- deionised water

Untreated negative controls:

yes

Remarks:

Culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

EMS as a positive control for tests without metabolic activation. CPA as a positive control for tests with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 1x10^4 - 6x10^4

DURATION
- Preincubation period: until exponentially growing stock cultures reached more than 50% confluent
- Exposure duration: 4 hours
- Fixation time (start of exposure up to fixation of cells): 18 hours

SPINDLE INHIBITOR (cytogenetic assays): colchincine (Colcemid) was added after 15.5 hours after the start of the treatment and incubated for 2.5 hours

STAIN (for cytogentic assay): Giemsa (E. Merck, D-64293 Darmstadt)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells on the slides were treated in the chambers with hypotonic solution (0.4% KCl) for 20 min at 37° C. C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts + 1 part). Per experiment both slides per group were prepared. After preparation the cells were stained

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100 weIll spread metaphase plates per culture were scored for cytogenetic damage on coded slides.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of our historical laboratory control data: 0.0 % - 4.0 %
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratories historical control data (see table 1 in "any other information")

The test substance was classified as clastogenic if:
- the number of induced structural chromosome aberrations are not in the range of the historical control data (0.0 - 4.0 % aberrant cells exclusive gaps). and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

The substance was classified as aneugenic if:
- the numbe rof induced numerical aberrations were not in the range of the historical control data (0.0% - 8.5% polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fishers exact test (p < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

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
- At 25° C only 3.5 % of its potency is lost after 24 hours
- Water solubility: yes
- Precipitation: no
- Definition of acceptable cells for analysis: 100 well spread metaphase plates per culture

RANGE-FINDING/SCREENING STUDIES: clear toxic effects were observed after 4 hours treatment with 87.5 µg/ml in the absence and presence of S9 mix. In addition, 24 hrs continuous treatment with 43.8 pg/ml and above in the absence of S9 mix induced strong toxic effects.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 1
- Negative (solvent/vehicle) historical control data: see table 1

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The toxicity was examined using the determination of the cell number. Strong toxicity indicated by reduced cell numbers below 50 % of control were observed after 4 hrs treatment with 60 µg/mI in the absence and the presence of S9 mix (30 % and 38 % of control, respectively)

Table 1.1 - 1.3: Pre-test on toxicity; cytotoxicity of Protectol TD to cultures of chinesse hamster cell line V79

Without S9 mix, 4 hours exposure
Concentration [µg/ml]	Number of cells	% of solvent control
Solvent control	929	100
43.8	745	801
87.5	279	30
175.0	322	35
350.0	201	22
700.0	201	22
1400.0	430	46
2800.0	249	27
5600.0	269	29

Table 1.2

With S9 mix, 4 hours exposure
Concentration [µg/ml]	Number of cells	% of solvent control
Solvent control	623	100
43.8	567	91
87.5	276	44
175.0	150	24
350.0	103	17
700.0	171	27
1400.0	181	29
2800.0	160	26
5600.0	245	39

Table 1.3

Without S9 mix, 24 hours exposure
Concentration [µg/ml]	Number of cells	% of solvent control
Solvent control	762	100
43.8	151	20
87.5	129	17
175.0	188	25
350.0	73	10
700.0	204	27
1400.0	263	35
2800.0	239	31
5600.0	219	29

Table 2.1 - 2.2: Main experiment - determination of toxicity with number of cells in % of solvent control

Without S9 mix
Preparation interval	Concentration [µg/ml]	Cells in % of solvent control
18 hours	10.0	93
“	20.0	74
“	40.0	64
“	60.0	30
“	80.0	30
“	100.0	25

Table 2.2

With S9 mix
Preparation interval	Concentration [µg/ml]	Cells in % of solvent control
18 hours	10.0	95
“	20.0	78
“	40.0	68
“	60.0	38
“	80.0	26
“	100.0	22

Table 3: Summary of results of the chromosome aberration study

Experiment	Preparation Interval	Test substance concentration [µg/ml]	Polyploid cells [%]	Cell numbers in % of control	Mitotic indices in % of control	Incl. gaps	Aberrant cells in % excl. gaps	With exchanges
	Exposure period 4 hours without S9 mix
1	18 h	Negative control	1.9	n.t.	100	1.0	1.0	0.0
		Solvent control	1.6	100	100	1.5	1.5	0.5
		Positive control	2.7	n.t.	100	30.0	28.5	11.0
		20	2.7	74	91	1.0	0.5	0.0
		40	2.2	64	96	13.0	13.0	6.0
		60	2.4	30	95	31.5	28.5	21.0
	Exposure period 4 hours with S9 mix
1	18 h	Negative control	1.9	n.t.	100	2.0	1.0	0.5
		Solvent control	3.6	100	100	2.5	2.0	0.5
		Positive control	2.0	n.t.	99	16.0	14.0	5.0
		20	2.9	78	102	2.0	1.5	1.0
		40	2.8	68	79	8.5	8.5	4.5
		60	1.8	38	100	19.0	17.0	5.5

Conclusions:

In conclusion, it can be stated that under the experimental conditions selected, the test substance induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro, at a concentration that also induced cytotoxicity.
Therefore, the test substance is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

Executive summary:

The test substance, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 ceils of the Chinese hamster in vitro in one experiment. Following study design was performed:

	With and without S9 mix
Exposure period	4 hours
Recovery	14 hours
Preparation interval	18 hours

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structurai chromosome aberrations. 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 cytogenetic experiment was performed considering the toxicity data. The following concentrations were tested in the main experiment:

without S9 mix: 10; 20; 40; 60; 80; and 100 µg/ml

with S9 mix: 10; 20; 40; 60; 80; and 100 µg/ml

The evaluated experimental points and the results are summarised in Table 3 (any other information on results). Clear toxic effects indicated by reduced cell numbers were observed after treatment with 60 µg/ml in the absence and the presence of S9 mix (Table 5, page 28). In the absence and the presence of S9 mix, statistically significant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test substance at 40µg/ml. This concentration already reduced cell survival to 64% to 68%. At 1.5 times the concentration, survival was only 30%. Consequently, chromosomal aberrations were only induced at cytotoxic concentrations. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test substance as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jul 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

No Version specified

Deviations:

yes

Remarks:

does not conform with the current guideline (1997), because no E.Coli WP2 or S.thyphimurium TA 102 have been used

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF AG and batch no.: continuous production
- Date of manufacturing: Fourth quarter 1994
- Purity: 74.9 area %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Has not been determined analytically throughout the study. A test substance characterization has been performed in Jan/Feb 1995.
- Solubility and stability of the test substance in the solvent: The stability of the test substance in aqueous solution is guaranteed. Its consistency is a colorless liquid (about 50% aqueous solution)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
- Treatment of test material prior to testing: dissolved in purified water

Target gene:

His G 46, his C 3076, his D 3052

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix derived from aroclor induced rat liver

Test concentrations with justification for top dose:

1st experiment: 0; 40; 200; 1,000; 5,000 and 10,000 µg/plate (Standard plate test, SPT)
2nd and 3rd experiment: 0; 250; 500; 1,000; 2,000 and 3,000 µg/plate (Pre-incubation test, PIT)

Vehicle / solvent:

purified water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation), Vogel-Bonner E medium

DURATION FOR THE PREINCUBATION TEST.
- Preincubation period: 48 hours
- Exposure duration: 20 minutes

NUMBER OF REPLICATES: Three test plates per dose or per control

DETERMINATION OF CYTOTOXICITY:
- Method: observation of reduced his-background growth

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle 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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No increase in the number oh his+ revertants in any strain both with and without metabolic activation. A bacteriotoxic effect was observed from about 2,000 µg - 3,000 µg/plate onward depending on the strain and experiment. No precipitation of the test substance was found.

For detailed results see attached background material.

Conclusions:

The test substance is, according to the results of the present study, not mutagenic in the Ames test under the experimental conditions chosen here.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce back mutations in selected loci in several strains of Salmonella typhimurjum in the Ames test.

Strains: TA 1535, TA 100, TA 1537, TA 98

Dose range: 40 µg - 10,000 µg/plate (SPT)

250 µg - 3,000 µg/plate (PIT)

Test conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor induced rat liver S-9 mix).

Solubility: No precipitation of the test substance was found.

Toxicity: A bacteriotoxic effect was observed depending on the strain and test conditions from about 2,000 µg - 3,000 µg/plate onward.

Mutagenicity: An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Genetic toxicity in vivo

Description of key information

The in vivo micronucleus assay in bone marrow cells of the mouse was conducted according to the OECD 474 and in compliance with GLP in order to investigate the potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse at concentrations ranging from 375 - 1,750 mg/kg bw. Six mice (NMRI) were used per dose per sex. The test substance did not induce micronuclei.

The in vivo Unscheduled DNA synthesis assay was performed according to the OECD 486 and in compliance with GLP in order to investiagte the potential to induce DNA repair (UDS) in the hepatocytes of rats at concentrations of 1,000 and 2,000 mg/kg bw. Four male Wistar rats per dose were used. The test substance did not induce DNA damage leading to increased repair synthesis.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF, Batch No 0767
- Purity: 49.5 g in 100 g (aqueous solution)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance in water was determined analytically and may be requested from the sponsors.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment the test substance was formulated in deionised water.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Hanlbm: WIST (SPF)
The rat has been used for many years as suitable experimental animal in genotoxicity investigations. Thus, many data are available from such investigations which may be helpful in the interpretation of the results.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Switzerland
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation: mean value 191.2 g (SD +/- 22.6 g)
- Assigned to test groups randomly: yes
- Housing: single
- Fasting period: overnight
- Diet (e.g. ad libitum): pelleted standard diet
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: non toxicity for test animals
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight

Details on exposure:

administration: oral, gavage, once

Duration of treatment / exposure:

single exposure
hours after treatment until reading: 2 h and 16 h

Frequency of treatment:

once

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

maximum tolerated dose, corrected for purity

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

corrected for purity

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

- 2-acetylaminofluorene dissolved in dimethylsulfoxide/polyethylene glycol 400 (1+9) for 16 h preparation interval
- Route of administration: orally
- Doses / concentrations:100 mg/kg body weight

- N,N´-dimethylhydrazinedihydrochloride (sym.) dissolved in 0.9 % NaCl solution for 2 h preparation interval
- Route of administration: orally
- Doses / concentrations: 40 mg/kg body weight

Tissues and cell types examined:

hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The active ingredient content of the test substance was originally given by the sponsor as approximately 47 %. Therefore, the doses used in this study and reported presently are expressed in terms of this indicated content.
For genotoxicity investigations it is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The volume to be administered has to be compatible with physiological space available.
The highest dose recommended by the OECD guideline to be used in this test system (2000 mg/kg body weight) was estimated by the pre-experiment to be suitable. The low dose was half of the high dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were starved overnight (2 h treatment) or approximately 6 h (16 h treatment) before receiving the test substance, the positive or the vehicle control substance. Water was available ad libitum. Before the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the body weight of the animals. The animals received the test substance once. Four animals (males) were treated per dose group.

DETAILS OF SLIDE PREPARATION: After anaesthetizing the rats with Na-thiopental the liver was perfused through the vena portae with HBSS supplemented with collagenase adjusted to pH 7.4 and maintained at 37 °C.
The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh to yield a single cell suspension. The quality of the performed perfusion was determined by the trypan blue dye exclusion method for cell viability. In addition, the number of the cells was determined.
The washed hepatocytes were centrifuged and transferred into Williams medium E supplemented with Hepes (2.38 mg/mL), Penicillin (100 u/mL), Streptomycin (0.10 mg/mL), L-Glutamine (0.29 mg/mL), Insulin (0.50 µg/mL) and Fetal calf serum (100 µL/mL). This complete medium was adjusted to pH 7.6.
At least three cultures were established from each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (2.0 x 10^5 viable cells/mL) were added to 35 mm six-well dishes containing one 25 mm round plastic coverslip per well coated with gelatine.
After an attachment period of approximately 1.5 h in a 95 % air / 5 % CO2 humidified incubator at 37 °C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently, 3HTdR in 2.0 mL culture medium was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1 % (v/v) FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 20 minutes each, rinsed with 96 % (v/v) ethanol, and air-dried.
The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 photographic emulsion in the darf. The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 4 °C. The photographic emulsion was then developed with KODAK Dektol Developer at room temperature, fixed in TETENAL and stained with hematoxylin/eosin.

METHOD OF ANALYSIS: Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the ARTEK 880 or 982 counter. In addition, the number of grains of the most heavily labeled nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per animal and 50 cells per slide were evaluated. Heavily radiolabeled cells undergoing replicative DNA synthesis were excluded from counting. Three animals per group were evaluated as described above.

Evaluation criteria:

Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.
A test substance is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.
A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response less than 5 net grains.
Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test.
A test substance producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system.

Statistics:

A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test substance were in the range of the corresponding controls.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg body weight
- Clinical signs of toxicity in test animals: reduction of spontaneous activity, abdominal position, eyelid closure, apathy, ruffled fur

- viability of the hepatocytes was not affected

Table 1: Viability

Treatment	Period	Animal No.	Viability [%]
Deionised water	2 h	1	70
		2	73
		3	73
1000 mg/kg b.w.	2 h	5	71
		6	71
		7	74
2000 mg/kg b.w.	2 h	9	71
		10	77
		11	75
40 mg/kg b.w. DMH	2 h	13	79
		14	76
		15	70
Deionised water	16 h	17	83
		18	71
		20	73
1000 mg/kg b.w.	16 h	21	72
		22	77
		23	77
2000 mg/kg b.w.	16 h	26	72
		27	70
		28	71
100 mg/kg b.w. 2-AAF	16 h	29	74
		30	74
		31	73

Table 2: Mean Nucleus, Cytoplasmic Area, and Net Grains

Treatment	Period	Grains per nucleus		Grains per cytoplasmic area		Net grains per nucleus
		Mean	SD	Mean	SD	Mean	SD
Deionised water	2 h	11.46	± 4.74	16.20	± 6.18	-4.74	± 5.59
1000 mg/kg b.w.	2 h	15.16	± 6.75	20.71	± 8.84	-5.55	± 7.46
2000 mg/kg b.w.	2 h	15.62	± 7.28	22.80	± 9.61	-7.18	± 7.58
40 mg/kg b.w. DMH	2 h	33.38	± 17.69	15.88	± 8.08	17.50	± 14.21
Deionised water	16 h	8.77	± 4.31	13.64	± 5.98	-4.87	± 5.65
1000 mg/kg b.w.	16 h	12.89	± 6.12	18.96	± 8.09	-6.06	± 6.96
2000 mg/kg b.w.	16 h	8.87	± 4.27	12.46	± 5.78	-3.59	± 5.08
100 mg/kg b.w. 2-AAF	16 h	54.93	± 22.12	19.06	± 9.14	35.87	± 16.99

Conclusions:

In conclusion, it can be stated that under the experimental conditions selected, the test substance did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats. Therefore, the test substance is considered to be non-effective in this in vivo UDS test system.

Executive summary:

The test substance was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats.

The test substance was formulated in deionised water, which was also used as vehicle control. The volume administered orally was 10 mL/kg body weight. After a treatment period of 2 and 16 hours, respectively, the animals were anaesthetised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to ³HTdR (methyl-³H-thymidine) which is incorporated if UDS occurs.

The test substance is an aqueous solution. The active ingredient content of the test substance was originally given by the sponsor as approximately 47 %. Therefore, the doses used in this study are expressed in terms of this indicated content. The exact value of the test substance purity is, however, 49.5 g per 100 g as indicated later by the sponsor.

The test substance was tested at the following dose levels:

2 and 16 hours preparation intervals: 1000 and 2000 mg/kg b.w..

The highest dose was estimated by pre-experiments to be suitable.

For each experimental group including the controls, hepatocytes from three treated animals were assessed for the occurence of UDS.

The viability of the hepatocytes was not substantially affected due to the in vivo treatment with the test substance.

The test substance did not reveal at any dose level UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls.

Appropriate reference mutagens (DMH, 40 mg/kg b.w. and 2 -AAF, 100 mg/kg b.w.) were used as positive controls. Treatment with the positive control substances revealed distinct increases in the number of nuclear and net grain counts.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001 - 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Micronucleus assay in bone marrow cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF AG and THOR GmbH and batch no.: 0767

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability of the test substance in the solvent: The stability of the test substance in water was determined analytically and may be requested from the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment, the test substance was formulated in deionised water.
- Purity: 49.5 g / 100 g (aqueous solution)

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse has been used for many years as suitable experimental animal. Thus, many data are available which may be helpful in the interpretation of the results from the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllingsdorf
- Age at study initiation: 8 -10 weeks
- Weight at study initiation: mean weight males: 39.6 +/- 3.3 g and females: 31.7 +/- 2.0 g
- Assigned to test groups randomly: yes
- Housing: individually
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum five days
- number of animals: 84 (42 males and 42 females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 4
- Relative humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

deioised water
The vehicle was chosen to its relative non-toxicity for the animals.

Details on exposure:

- Single injection intraperitoneally
- Dose volume: 10 mg/kg bw

Duration of treatment / exposure:

- single expsoure
- 24 h and additionally 48 h for the vehicle control and highest dose

Dose / conc.:

375 mg/kg bw/day (nominal)

Remarks:

male animal

Dose / conc.:

437.5 mg/kg bw/day (nominal)

Remarks:

female animal

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

male animal

Dose / conc.:

875 mg/kg bw/day (nominal)

Remarks:

female animal

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Remarks:

male animal

Dose / conc.:

1 750 mg/kg bw/day (nominal)

Remarks:

female animal

No. of animals per sex per dose:

six males and six females were assigned to each test group and additionally six males and six females for control group and highest dose for the 48 h period. Animals were identified by their cage number as shown in table 1 (any other information).

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide disolved in deionised water
- Route of administration: intraperitoneally, once
- Doses: 40 mg/kg bw
- Dose volume: 10 ml/kg bw

Tissues and cell types examined:

bone marrow
polychromatic erythrocytes (PCE)
normochromatic erythrocytes (NCE)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The active ingredient content of the test substance was originally given by the sponsor as approximately 47%. Therefore, the doses used in this study and reported presently are expressed in terms of this indicated content.
lt is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test substances. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available. Three adequate spaced dose leveis spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animais (including the controls) were weighed and the individual volume to be administered was adjusted to the animais body weight. The animais received the test substance, the vehicle or the positive control substance once i.p.. Six males were treated per dose group and sampling time. The animais of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the test substance. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION:
The animais were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf Serum, using a syringe. The ceII Suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supenatant was discarded. A small drop of the resuspended celI pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the sildes was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To desoribe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides. Five males per test group were evaluated as described.

Evaluation criteria:

The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data (0.20 - 1.50 %; mean = 0.86 ± 0.32 % PCEs with micronuclei).
- the positive controls are in the range of our historical control data (10.0 - 27.1 %;mean = 16.78±4.79 % PCEs with micronuclei).
- at least 80 % of animals are evaluable

The test substance was classified as mutagenic if it induced either a dose-related increase in the number of micronucleated polychromatic erythrocytes (which clearly exceeds the negative control range) or a relevant positive response for at least one of the test points. Statistical methods can be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

A test substance producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) can be used as an aid in evaluating the results. Statistical significance at the five percent level (p < 0.05) was evaluated. However, the primary point of consideration is the biological relevance of the results.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

In animals treated with the highest dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1,000 - 2,000 mg/kg
- Clinical signs of toxicity in test animals: clear signs of toxicity in the highest test groups. Reduction of spontaneous activity, abdominal position, eyelid closure, apathy, ruffled fur

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): affected by treatment (see tables 1 and 2 in any other information on results)
- Statistical evaluation: Mann-Whitney Test
- Appropriateness of dose: yes

In comparison to the corresponding vehicle control there was no substantial enhancement in the frequency of the detected micronuclei at the 24 h preparation interval. The value obtained for the males treated with 1500 mg/kg at the 48 h preparation interval (1 .6 %) was statistically significant. However, this increase is within the historical range for the negative control males (0.0 - 2.1%) and significance was obtained due to the low vehicle control value. Therefore, it is considered as biologically irrelevant.

Historical controls (1999 - 2000)

	Micronucleated PCE’s per thousand (‰)
	Negative controls		Positive controls
	Males	Females	Males	Females
Mean*	0.76	0.62	18.70	14.54
SD**	0.42	0.34	5.79	5.32
range	0.0 – 2.1	0.0 – 1.4	9.30 – 34.10	7.00 – 35.50

*= mean of 62 experiments

Table 1: Summary of micronucleus test results for males

Test group	Dose [mg/kg bw]	Sampling time [h]	PCEs with micronuclei [‰]	Range	PCE/NCE	significance	P
Vehicle	0	24	0.3	0 – 1	2000/1587
Test substance	375	24	0.2	0 – 1	2000/1778	n.t.	-
“	750	24	1.4	0 – 6	2000/1612	-	0.0873
“	1500	24	1.2	0 – 4	2000/2176	-	0.0873
Positive control	40	24	15.5	12 – 46	2000/1419	+	0.0040
vehicle	0	48	0.0	0 – 0	2000/1706	-	-
Test substance	1500	48	1.6	2 – 6	2000/2564	+	0.0040

-     = not significant

+ = significant (p < 0.05)

n.t. = not tested, as the mean micronucleus frequency was not above the vehicle control value

Table 2: Summary of micronucleus test results for females

Test group	Dose [mg/kg bw]	Sampling time [h]	PCEs with micronuclei [‰]	Range	PCE/NCE	significance	P
Vehicle	0.0	24	0.8	0 – 3	2000/1288
Test substance	437.5	24	0.4	0 – 2	2000/1615	n.t.	-
“	875.0	24	0.5	0 – 3	2000/1854	n.t.	-
“	1750.0	24	0.9	1 – 2	2000/2335	-	0.5000
Positive control	40.0	24	16.5	27 – 39	2000/1894	+	0.0040
vehicle	0.0	48	0.4	0 – 2	2000/1572	-	-
Test substance	1750.0	48	0.6	1 – 2	2000/2191	-	0.3571

-   = not significant

+ = significant (p < 0.05)

n.t. = not tested, as the mean micronucleus frequency was not above the vehicle control value

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow celIs of the mouse. Therefore, the test substance is considered to be non-clastogenic and non-aneugenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test substance was formulated in deionised water, which was also used as vehicle control. The volume administered intraperitoneally (i.p.) was 10 mI/kg b.w.. 24 h and 48 h after a single i.p. administration of the test substance the bone marrow cells were collected for micronuclei analysis. Five males or five females per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The test substance is an aqueous solution. The active ingredient content of the test substance was originally given by the sponsor as approximately 47%. Therefore, the doses used in

this study are expressed in terms of this indicated content. The exact value of the test substance purity is, however, 49.5 g / 100 g as indicated later by the sponsor.

The following dose levels of the test substance were investigated:

males:

24 h preparation interval: 375, 750, and 1500 mg/kg b.w..

48 h preparation interval: 1500 mg/kg b.w..

females:

24 h preparation interval: 437.5, 875, and 1750 mg/kg b.w..

48 h preparation interval: 1750 mg/kg b.w..

The highest dose (1500 mg/kg b.w. for males and 1750 m g/kg b.w. for females) was estimated by pre-experiments to be suitable.

After treatment with the highest dose of the test substance the number of NCEs was increased as compared to the mean value of NCEs of the vehiole controls thus indicating that the test substance had cytotoxic effectiveness in the bone marrow.

In comparison to the corresponding vehicle controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered intraperitoneally (i.p.) was used as positive control which showed a substantial increase of induced micronucleus frequency.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test substance was investigated for its mutagenic potential based on the ability to induce back mutations in selected loci in several strains of Salmonella typhimurjum in the Ames test. Strains used were TA 98, TA 100, TA 1535 and TA 1537. For the standard plate test concentrations of 0; 40; 200; 1,000; 5,000 and 10,000 µg/plate , and for the preincubation test concentrations of 0; 250; 500; 1,000; 2,000 and 3,000 µg/plate both with and without S9 mix derived from aroclor induced rat liver have been tested. Appropriate negative and positive controls were included. A bacteriotoxic effect was observed depending on the strain and test conditions from about 2,000 µg - 3,000 µg/plate onward. An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or with S9 mix. The test substance is, according to the results of the present study, not mutagenic in the Ames test under the experimental conditions chosen here.

The test substance was investigated for its mutagenic potential at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, with and without microsomal activation derived from phenobarbital/b-naphthoflavone induced rat liver and a treatment period of 4 hours. Test concentrations for both experiments were 10, 20, 40, 60, and 80 µg/ml with and without S9 mix. Appropriate negative and positive controls were included. Strong toxic effects occurred in both experiments at 60 and 80 µg/ml in the absence of S9 mix and also in the first experiment in the presence of S9 mix. 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. 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.

The in vitro chromosome aberration test in Chinese hamster V79 cells was conducted in order to investigate the potential mutagenic effect of the test substance. Concentrations tested were 10, 20, 40, 60, 80, and 100 µg/ml with and without S9 mix obtained by phenobarbital/b-naphthoflavone induced rat liver although only 20, 40, and 60 µg/ml were the evaluated experimental points. A solvent, a vehicle and positive controls in the presence and absence of S9 mix were performed. Clear toxic effects indicated by reduced cell numbers were observed after treatment with 60 µg/ml in the absence and the presence of S9 mix. In the absence and the presence of S9 mix at 40 µg/ml, statistically significant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test substance. This concentration already reduced cell survival to 64% to 68%. At 1.5 times the concentration, survival was only 30%. Consequently, chromosomal aberrations were only induced at cytotoxic concentrations. Appropriate mutagens were used as positive controls inducing statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions selected, the test substance induced structural chromosome aberrations in vitro. Therefore, the test substance is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

The test substance was investigated for its potential to induce in polychromatic erythrocytes (PCE) in the bone marrow of the mouse in vivo. The volume administered intraperitoneally (i.p.) was 10 mI/kg b.w.. Appropriate negative and positive controls were included. 24 h and 48 h after a single i.p. administration of the test substance the bone marrow cells were collected for micronuclei analysis. Five males or five females per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample. The following dose levels corrected for the purity of the test substance were investigated (i.e., they refer to the amount of tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione, and not to the 50% dilution in water as registered):

males:

24 h preparation interval: 375, 750, and 1500 mg/kg b.w..

48 h preparation interval: 1500 mg/kg b.w..

females:

24 h preparation interval: 437.5, 875, and 1750 mg/kg b.w..

48 h preparation interval: 1750 mg/kg b.w..

After treatment with the highest dose of the test substance the number of NCEs was increased as compared to the mean value of NCEs of the vehicle controls thus indicating that the test substance had cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test substance did not induce micronuclei. Therefore, the test substance is considered to be non-clastogenic and non-aneugenic in this micronucleus assay.

The test substance was investigated for its potential to induce DNA repair (UDS) in the hepatocytes of rats in the in vivo UDS assay. The volume administered orally was 10 mL/kg bw. After a treatment period of 2 and 16 hours, respectively, the animals were anaesthetised and sacrificed. Primary hepatocyte cultures were established and exposed for 4 hours to ³HTdR (methyl-³H-thymidine) which is incorporated if UDS occurs. The test substance was tested at the following dose levels

corrected for the purity of the test substance (i.e., they refer to the amount oftetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione, and not to the 50% dilution in water as registered):

2 and 16 hours preparation intervals : 1000 and 2000 mg/kg b.w.

For each experimental group including the controls, hepatocytes from three treated animals were assessed for the occurence of UDS. The viability of the hepatocytes was not substantially affected due to the in vivo treatment with the test substance. The test substance did not reveal at any dose level UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. In conclusion, it can be stated that under the experimental conditions selected, the test substance did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats. Therefore, the test substance is considered to be non-effective in this in vivo UDS test system.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The substance did not induce gene mutations in bacteria or mammalian cells, but induced chromosomal aberrations in vitro at cytotoxic concentrations. As this positive result was not confirmed in an in vivo MNT (exposure of the bone marrow was confirmed by an altered NCE/PCE ratio) and an in vivo UDS assay, the substance does not need to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.