N,N'-diphenylguanidine monohydrochloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-12-08 to 2011-04-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-diphenylguanidine monohydrochloride

EC Number:

246-107-8

EC Name:

N,N'-diphenylguanidine monohydrochloride

Cas Number:

24245-27-0

Molecular formula:

C13H13N3.ClH

IUPAC Name:

N,N'-diphenylguanidine hydrochloride

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany
- proliferation rate: 14 hours
- Modal number of chromosomes: 22 +/-1
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induce rat liver S9

Test concentrations with justification for top dose:

With and without S9: 9.7; 19.4; 38.8; 77.5; 155.0; 310.0; 620.0; 1240.0; 2480.0 µg/mL
Concentrations were selected based on prelim. dose-range finding assay. At the highest concentration of 2480 µg/mL precipitation was observed at the end of treatment.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding: 1 x 10^4 - 6 x 10^4 cells

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 14 h
- Fixation time (start of exposure up to fixation or harvest of cells): 15.5 h after start of treatment

SPINDLE INHIBITOR: Colcemid

STAIN: Giemsa

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: at least 100 well spread metaphases per culture
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

DETERMINATION OF CYTOTOXICITY
- Method: reduced cell number

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Rationale for test conditions:

According to respective guidelines

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory's historical control data.
and
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory's historical control data.
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory's historical control data.

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (7) (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 155 µg/mL and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: the substance is considered to be well soluble in water
- Precipitation: yes, at concentrations of 2480 µg/mL and above, with and without S9

RANGE-FINDING/SCREENING STUDIES: yes

Any other information on results incl. tables

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix. According to the OECD Guideline only one experiment was performed, since the test item was considered to be clastogenic after 4 hours treatment. The chromosomes were prepared 18 hours after start of treatment with the test item. The exposure period was 4 hours with and without metabolic activation. In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations. No relevant influence of the test item on pH value or osmolarity was observed (solvent control 398 mOsm, pH 7.43 versus 389 mOsm and pH 7.45 at 2480.0 µg/mL). Precipitation of the test item in culture medium was observed after 4 hours treatment with 2480.0 µg/mL and above in the presence and absence of S9 mix. Concentrations between 9.7 and 2480.0 ug/mL were applied. Clear cytotoxic effects were observed after 4 hours treatment with 155.0 µg/mL and above in the absence of S9 mix, where the cell numbers were reduced to about 60 % of solvent control cells. In the presence of S9 mix dose groups showing relevant cytotoxic effects were not evaluable for cytogenetic damage. In the absence of S9 mix a dose-dependent increase in the number of aberrant cells, excluding gaps was observed (5.5, 7.0 and 16.0 %, respectively) after treatment with 38.8, 77.5 and 155.0 µg/mL. All values exceeded the total range of the laboratory's historical solvent control (0.0 - 4.0 % aberrant cells, excluding gaps). In addition, the value obtained at 155.0 µg/mL (16.0 % aberrant cells, excluding gaps) was statistically significantly increased. In the presence of S9 mix a statistically significant and dose-dependent increase in the number of aberrant cells (5.5 %) was observed at the highest evaluable concentration (310.0 µg/mL). This statistically significant value also exceeded the total range of the laboratory's historical solvent control (0.0 - 4.0 % aberrant cells, excluding gaps). No relevant evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the controls. Therefore, biologically relevant increases of chromosomal aberrations were observed in the absence and presence of metabolic activation and the test item is thus considered clastogenic under the conditions of this study.

Table 1: Summary of results of the chromosome aberration study with the test item

 * Inclusive cells carrying exchanges

Preparation interval	Test item concentration in µg/mL		Polyploid cells in %		Cell numbers in% of control		Mitotic indices in % of control		Incl. gaps*		Aberrant cells in % excl. gaps*		with exchanges
Exposure period 4 hrs without S9 mix
18 hrs	Solvent control^1	2.7		100.0		100.0		4.5		3.5		1.0
	Positive control^2	1.7		n.t.		88.1		44.0		44.0^S		33.5
	38.8	2.1		92.0		106.0		6.0		5.5		3.0
	77.5	2.4		95.5		111.5		7.0		7.0		3.5
	155.0	2.5		60.4		84.5		16.5		16.0^S		5.0
Exposure period 4 hrs with S9 mix
18 hrs	Solvent control^1	2.0		100.0		100.0		3.0		2.0		0.5
	Positive control^3	2.1		n.t.		67.6		21.5		20.5^S		6.5
	77.5	1.8		106.5		107.3		2.5		2.5		1.5
	155.0	3.0		108.5		84.6		5.0		3.5		1.0
	310.0	2.9		100.0		95.8		6.0		5.5		1.5

n.t.     Not tested

^S                    Aberration frequency statistically significant higher than corresponding control values

¹                     DMSO 0.5 % (v/v)

²                     EMS 1000.0 µg/mL

³                     CPA 1.4 µg/mL

Applicant's summary and conclusion

Conclusions:

The test item is considered to be clastogenic in this chromosome aberration test in the absence and presence of metabolic activation.

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in one experiment. The following study design was performed:

	Without S9 mix	With S9 mix
Exposure period	4 h	4 h
Recovery	14 h	14 h
Preparation interval	18 h	18 h

  

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations. The highest applied concentration (2480.0 µg/mL; approx. 10.0 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data. In the absence and presence of S9 mix concentrations higher than the evaluated were not evaluable for cytogenetic damage due to exceedingly strong cytotoxic effects. The cell numbers were reduced to approximately 60 % in the highest evaluable dose group in the experimental part without S9 mix. In the absence of S9 mix a dose-dependent increase in the number of aberrant cells, excluding gaps was observed (5.5, 7.0 and 16.0 %%, respectively) after treatment with 38.8, 77.5 and 155.0 µg/mL All values exceeded the total range of the laboratory's historical solvent control (0.0 - 4.0 % aberrant cells, excluding gaps). In addition, the value obtained at 155.0 µg/mL (16.0 % aberrant cells, excluding gaps) was statistically significantly increased. In the presence of S9 mix a statistically significant and dose-dependent increase in the number of aberrant cells (5.5 %) was observed at the highest evaluable concentration (310.0 µg/mL). This value exceeded the total range of the laboratory's historical solvent control (0.0 - 4.0 % aberrant cells, excluding gaps) and is also statistically significant. No relevant evidence of an increase in polyploid metaphases was found after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.