N,N'-diphenylguanidine monohydrochloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is considered to be clastogenic based on the results of an in vitro chromosome aberration test in mammalian cells in the absence and presence of metabolic activation.

Data of a bacterial reverse mutation assay according to OECD 471 (Ames test) show that under the experimental conditions applied, the test item induced gene mutations by base pair changes and frameshifts in the genome of the Salmonella typhimurium TA98 and TA100 strains investigated. In conclusion, the test item N,N’-Diphenylguanidinmonohydrochlorid showed a mutagenic activity on Salmonella typhimurium TA98 and TA100 strains in the presence of an exogenous metabolic activation system.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

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

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.

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.

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-04-04 to 2018-05-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use, June 2012

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9

Test concentrations with justification for top dose:

Concentrations were based on prelim. non-GLP Test:

Plate incorporation (initial) Test (all strains):
-S9 mix: 5000, 1600, 500, 160, 50 and 16 μg/plate;
+S9 mix: 5000, 3600, 2400, 1600, 500, 160 and 50 μg/plate.

Repetition of plate incorporation (confirmatory) test with TA98 and TA100:
+S9 mix: 5000, 4800, 4500, 4000, 3600, 2400 and 1600 μg/plate.

Pre-incubation Test:
±S9 mix: 5000, 1600, 500, 160, 50 and 16 μg/plate. (E. coli and TA1535)

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water (test item and positive controls); DMSO (other positive controls)
- Justification for choice of solvent/vehicle: recommended by guidelines and well tolerated by bacteria.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine: without S9, TA98, 4µg/plate in DMSO; 2-aminoanthracene: with S9, all strains, 2 µg or 50 µg/plate with DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min at 37 °C (bacterial culture and the S9 Mix or phosphate buffer)
- Exposure duration: 48 hours in the dark at 37 °C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
A dose level is considered toxic if:
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs
- other: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.

Rationale for test conditions:

According to guidelines.

Evaluation criteria:

The colony numbers on the control, positive control and the test plates were determined, the mean values, standard deviations and the mutation rates were calculated.

Mutation Rate = Mean revertants at the test item (or control) treatments / Mean revertrants of vehicle control

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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: none
- Effects of osmolality: none
- Evaporation from medium: no
- Water solubility: well soluble
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: yes

HISTORICAL CONTROL DATA: Please refer to "Any other information on results incl. tables"

Table 1: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	14.3	0.98	18.0	1.10	83.0	1.09	83.3	1.05	9.0	1.04	13.7	1.05	7.3	1.22	7.3	1.38	28.0	1.06	36.3	0.99
DMSO Control	11.7	1.00	13.3	1.00	–	–	75.0	1.00	–	–	10.0	1.00	7.0	1.00	5.0	1.00	–	–	31.0	1.00
Ultrapure Water Control	14.7	1.00	16.3	1.00	76.0	1.00	79.7	1.00	8.7	1.00	13.0	1.00	6.0	1.00	5.3	1.00	26.3	1.00	36.7	1.00
5000	19.3	1.32	59.7	3.65	70.3	0.93	208.7	2.62	12.3	1.42	15.0	1.15	7.0	1.17	6.0	1.13	26.7	1.01	39.7	1.08
3600	–	–	28.7	1.76	–	–	109.7	1.38	–	–	13.3	1.03	–	–	6.3	1.19	–	–	43.0	1.17
2400	–	–	24.0	1.47	–	–	110.3	1.38	–	–	12.0	0.92	–	–	9.7	1.81	–	–	40.0	1.09
1600	15.3	1.05	22.3	1.37	78.7	1.04	107.0	1.34	7.3	0.85	10.7	0.82	7.3	1.22	7.3	1.38	34.7	1.32	35.0	0.95
500	13.0	0.89	23.7	1.45	86.0	1.13	98.7	1.24	11.0	1.27	13.3	1.03	7.3	1.22	7.0	1.31	34.3	1.30	39.7	1.08
160	19.7	1.34	23.3	1.43	75.3	0.99	107.0	1.34	6.3	0.73	11.3	0.87	7.3	1.22	6.0	1.13	34.7	1.32	39.3	1.07
50	19.3	1.32	26.7	1.63	90.0	1.18	89.3	1.12	10.7	1.23	10.3	0.79	5.3	0.89	4.7	0.88	29.7	1.13	32.0	0.87
16	19.3	1.32	–	–	81.0	1.07	–	–	8.3	0.96	–	–	6.7	1.11	–	–	38.3	1.46	–	–
NPD (4mg/plate)	370.0	31.71	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg/plate)	–	–	–	–	628.0	8.26	–	–	701.3	80.92	–	–	–	–	–	–	–	–	–	–
9AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	456.7	65.24	–	–	–	–	–	–
MMS (2mL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	850.7	32.30	–	–
2AA (2mg/plate)	–	–	1224.0	91.80	–	–	1234.7	16.46	–	–	148.3	14.83	–	–	141.3	28.27	–	–	–	–
2AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	190.0	6.13

 

Table 2Summary Table of the Results of the Confirmatory Mutation Test (Pre-Incubation Test)

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

–

–

18.3

0.95

–

–

90.7

1.04

9.7

0.78

12.0

1.13

30.0

1.05

37.0

1.23

DMSO Control

–

–

20.0

1.00

–

–

70.3

1.00

–

–

12.7

1.00

–

–

24.7

1.00

Ultrapure Water Control

–

–

19.3

1.00

–

–

87.3

1.00

12.3

1.00

10.7

1.00

28.7

1.00

30.0

1.00

5000

–

–

77.7

4.02

–

–

252.0

2.89

5.0

0.41

10.0

0.94

24.3

0.85

34.3

1.14

4800

–

–

55.3

2.86

–

–

219.3

2.51

–

–

–

–

–

–

–

–

4500

–

–

77.7

4.02

–

–

237.3

2.72

–

–

–

–

–

–

–

–

4000

–

–

80.0

4.14

–

–

218.3

2.50

–

–

–

–

–

–

–

–

3600

–

–

64.7

3.34

–

–

227.7

2.61

–

–

–

–

–

–

–

–

2400

–

–

51.3

2.66

–

–

248.7

2.85

–

–

–

–

–

–

–

–

1600

–

–

55.0

2.84

–

–

250.0

2.86

15.7

1.27

11.7

1.09

25.7

0.90

37.7

1.26

500

–

–

–

–

–

–

–

–

12.0

0.97

8.3

0.78

30.0

1.05

34.7

1.16

160

–

–

–

–

–

–

–

–

14.0

1.14

11.0

1.03

32.7

1.14

35.0

1.17

50

–

–

–

–

–

–

–

–

13.3

1.08

8.3

0.78

32.3

1.13

40.0

1.33

16

–

–

–

–

–

–

–

–

12.3

1.00

9.0

0.84

36.0

1.26

41.7

1.39

SAZ (2mg/plate)

–

–

–

–

–

–

–

–

746.0

60.49

–

–

–

–

–

–

MMS (2mL/plate)

–

–

–

–

–

–

–

–

–

–

–

–

853.3

29.77

–

–

2AA (2mg/plate)

–

–

1057.3

52.87

–

–

986.7

14.03

–

–

118.3

9.34

–

–

–

–

2AA (50mg/plate)

–

–

–

–

–

–

–

–

–

–

–

–

–

–

202.0

8.19

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks:Ultrapure water was applied as solvent of the test item and the positive control substance: SAZ and MMS; the DMSO was applied as solvent for positive control substance 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of 2AA is given referring to DMSO.

 

 

 

Table 3Summary Table of the Results of the Confirmatory Mutation Test (Repeated Plate Incorporation Test)

Confirmatory Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	–	–	17.0	1.04	–	–	84.3	1.10
DMSO Control	–	–	15.7	1.00	–	–	76.3	1.00
Ultrapure Water Control	–	–	16.3	1.00	–	–	76.7	1.00
5000	–	–	64.0	3.92	–	–	235.3	3.07
4800	–	–	64.3	3.94	–	–	262.7	3.43
4500	–	–	60.0	3.67	–	–	238.0	3.10
4000	–	–	67.0	4.10	–	–	188.7	2.46
3600	–	–	59.0	3.61	–	–	220.7	2.88
2400	–	–	28.7	1.76	–	–	149.7	1.95
1600	–	–	24.3	1.49	–	–	118.0	1.54
2AA (2mg/plate)	–	–	1197.3	76.43	–	–	898.0	11.76

 

Table 4 historical control data (2015-2017)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	19.7	94.6	10.9	8.9	25.2
		SD	1.7	2.4	0.4	1.3	5.2
		Minimum	8	67	4	3	11
		Maximum	40	133	21	20	52
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	24.5	112.9	11.0	9.2	31.7
		SD	1.4	6.1	0.5	1.2	6.4
		Minimum	11	74	3	3	13
		Maximum	43	159	20	20	60
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	18.1	87.0	10.8	8.5	24.6
		SD	1.1	3.6	0.6	1.3	3.3
		Minimum	9	58	4	3	10
		Maximum	36	131	23	20	54
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	23.0	102.4	11.0	9.2	31.4
		SD	0.8	7.7	0.4	1.3	5.8
		Minimum	11	69	3	3	12
		Maximum	42	148	23	21	59
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	19.5	92.8	11.3	9.1	26.6
		SD	1.4	5.0	0.5	1.8	6.6
		Minimum	12	62	4	3	10
		Maximum	30	139	22	18	52
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	24.4	109.9	11.2	9.5	34.0
		SD	1.2	6.7	0.8	1.9	6.1
		Minimum	13	83	5	4	16
		Maximum	37	149	18	18	63

 

TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2 uvrA, SD: Standard deviation; DMSO: Dimethyl sulfoxide; n: number of studies

Conclusions:

Data of this bacterial reverse mutation assay according to OECD 471 (Ames test) show that under the experimental conditions applied, the test item induced gene mutations by base pair changes and frameshifts in the genome of the Salmonella typhimurium TA98 and TA100 strains investigated. In conclusion, the test item N,N’-Diphenylguanidinmonohydrochlorid showed a mutagenic activity on Salmonella typhimurium TA98 and TA100 strains in the presence of an exogenous metabolic activation system, under the test conditions used in this study.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD 471 guideline (Ames test). The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. In the Confirmatory Mutation Test the Salmonella typhimurium TA1537 strain (based on the experience of the performed non-GLP Screening Test, Study code: 805-471-3509); furthermore the non-activation part of the TA98 and TA100 strains (-S9 mix) were not further investigated. The study included an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test in Salmonella typhimurium TA98, TA100, TA1535 and E.  coli WP2 uvrA and a repeated plate incorporation test in Salmonella typhimurium TA98 and TA100). A non-GLP solubility test and screening test was performed with three of the tester strains (Salmonella typhimurium TA98, TA100, TA1537). In this screening test the pre-incubation procedure was applied. The above strains were investigated in the absence and presence of exogenous metabolic activation (±S9 mix) with parallel running controls: untreated, solvent and positive reference controls.

Based on the results of the non-GLP solubility test and screening test, in the test phases of the present study the test item was dissolved in ultrapure water (ASTM Type I). At the preparation of the test item stock solution any correction factor (based on its purity, constituents) was not taken into consideration. Selection of the concentrations for the initial mutation test was based on the results of the non-GLP screening test and in accordance to the OECD guideline 471. The test item concentrations investigated in the initial mutation test were as follows:

-S9 mix: 5000, 1600, 500, 160, 50 and 16 µg/plate;

+S9 mix: 5000, 3600, 2400, 1600, 500, 160 and 50 µg/plate

The results of the non-GLP screening test allowed the applying of the recommended maximum test concentration of 5000 µg/plate.

In the initial mutation test (plate incorporation test) significant, colony number increases, revertant colony numbers above the solvent control data, above the historical control data ranges and above the thresholds for being positive were obtained in S. typhimurium TA98 and TA100 at 5000 µg/plate the presence of exogenous metabolic activation (+ S9 mix).

To confirm and to investigate the reproducibility of the positive results in the confirmatory mutation test the strains of S. typhimurium TA98 and TA100 were investigated in the presence of exogenous metabolic activation (+S9 mix). In the confirmatory mutation test at these two strains in parallel with the pre-incubation test, the plate incorporation procedure was repeated and the following concentration levels were investigated:

+S9 mix: 5000, 4800, 4500, 4000, 3600, 2400 and 1600 µg/plate.

In the confirmatory mutation test the strains Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA were further investigated with a pre-incubation procedure at following concentration levels:

±S9 mix: 5000, 1600, 500, 160, 50 and 16 µg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 mix) throughout the study. In this study a slight cytotoxic effect of the test item was observed following the pre-incubation procedure in Salmonella typhimurium TA1535 at 5000 µg/plate, in the absence of exogenous metabolic activation (-S9 mix). The cytotoxicity was indicated by decreased revertant colony counts (below the corresponding solvent data range) and slightly reduced background lawn development. The revertant colony numbers of solvent control ultrapure water (ASTM Type I) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges in different test phases. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. Unequivocal confirmed, repeated positive results were noticed following treatment with N,N’-Diphenylguanidinmonohydrochlorid in the investigated Salmonella typhimurium TA98 and TA100 strains at 5000 µg/plate, in the presence of exogenous metabolic activation (+S9 mix). In the performed experimental phases, following plate incorporation and pre-incubation procedures, the increased revertant colony numbers were above the corresponding historical control data ranges and above the thresholds for being positive in S. typhimurium TA98 and TA100 at the concentration of 5000 µg/plate (+S9 mix).  

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item induced gene mutations by base pair changes and frameshifts in the genome of the Salmonella typhimurium TA98 and TA100 strains investigated. In conclusion, the test item N,N’-Diphenylguanidinmonohydrochlorid showed a mutagenic activity on Salmonella typhimurium TA98 and TA100 strains in the presence of an exogenous metabolic activation system, under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Combined in vivo study according to OECD 474 and 489 with the structural analogue substance, i .e. the uncharged base (CAS 102 -06 -7), revealed negative results in the oral in vivo micronucleus test in rat and in the in vivo Comet assay on liver cells. Equivocal results were obtained in the in vivo Comet Assay on stomach cells.
Based on the weight of evidence with ex vivo results, the read-across substance, and consequently the test item itself, are considered to be not mutagenic in systemic organs.

Link to relevant study records

Reference

Endpoint:

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

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The test item (target substances, CAS 24245-27-0) is the salt of 1,3-diphenylguanidine and mono-hydrochloride. As read-across source substance the uncharged free base of 1,3-diphenylguanidine (CAS 102-06-7) is applied. This is considered appropriate since the target substance is expected to readily dissociate in contact with water such as surface water in the environment or the intestinal fluids or mucosal membranes of an organism. 1,3-diphenylguanidine is considered a weak base. Therefore, it is expected to be present as uncharged molecule at neutral and high pH solutions. However, acidic conditions will lead to protonation of the molecule and the charged ion is expected to be the main present form. Since transformation of the target to the source substance and vice versa is anticipated, kinetics and toxicological behaviour are well transferable from the source to the target substance. This applies for human health endpoints as well as for the aquatic environment.
Moreover, chloride ions are ubiquitously present in every-day nutrition and are part of numerous physiological processes. Therefore, this constituent does not require further assessment in regards to its toxicological behaviour. It is expected to be well regulated by endogenous physiological mechanisms of the organism.
Based on the considerations above, a read-across is considered appropriate and further tests, especially vertebrate studies, are not needed to adequately address this endpoint. Information Requirements are fulfilled according to REACH Annex XI Section 1.5.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

on liver cells

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

other: equivocal on stomach cells

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies with the test item

Ames test in vitro

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD 471 guideline (Ames test). The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. In the Confirmatory Mutation Test the Salmonella typhimurium TA1537 strain (based on the experience of the performed non-GLP Screening Test, Study code: 805-471-3509); furthermore the non-activation part of the TA98 and TA100 strains (-S9 mix) were not further investigated. The study included an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test in Salmonella typhimurium TA98, TA100, TA1535 and E.  coli WP2 uvrA and a repeated plate incorporation test in Salmonella typhimurium TA98 and TA100). A non-GLP solubility test and screening test was performed with three of the tester strains (Salmonella typhimurium TA98, TA100, TA1537). In this screening test the pre-incubation procedure was applied. The above strains were investigated in the absence and presence of exogenous metabolic activation (±S9 mix) with parallel running controls: untreated, solvent and positive reference controls.

Based on the results of the non-GLP solubility test and screening test, in the test phases of the present study the test item was dissolved in ultrapure water (ASTM Type I). At the preparation of the test item stock solution any correction factor (based on its purity, constituents) was not taken into consideration. Selection of the concentrations for the initial mutation test was based on the results of the non-GLP screening test and in accordance to the OECD guideline 471. The test item concentrations investigated in the initial mutation test were as follows:

-S9 mix: 5000, 1600, 500, 160, 50 and 16 µg/plate;

+S9 mix: 5000, 3600, 2400, 1600, 500, 160 and 50 µg/plate

The results of the non-GLP screening test allowed the applying of the recommended maximum test concentration of 5000 µg/plate.

In the initial mutation test (plate incorporation test) significant, colony number increases, revertant colony numbers above the solvent control data, above the historical control data ranges and above the thresholds for being positive were obtained in S. typhimurium TA98 and TA100 at 5000 µg/plate the presence of exogenous metabolic activation (+ S9 mix).

To confirm and to investigate the reproducibility of the positive results in the confirmatory mutation test the strains of S. typhimurium TA98 and TA100 were investigated in the presence of exogenous metabolic activation (+S9 mix). In the confirmatory mutation test at these two strains in parallel with the pre-incubation test, the plate incorporation procedure was repeated and the following concentration levels were investigated:

+S9 mix: 5000, 4800, 4500, 4000, 3600, 2400 and 1600 µg/plate.

In the confirmatory mutation test the strains Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA were further investigated with a pre-incubation procedure at following concentration levels:

±S9 mix: 5000, 1600, 500, 160, 50 and 16 µg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 mix) throughout the study. In this study a slight cytotoxic effect of the test item was observed following the pre-incubation procedure in Salmonella typhimurium TA1535 at 5000 µg/plate, in the absence of exogenous metabolic activation (-S9 mix). The cytotoxicity was indicated by decreased revertant colony counts (below the corresponding solvent data range) and slightly reduced background lawn development. The revertant colony numbers of solvent control ultrapure water (ASTM Type I) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges in different test phases. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. Unequivocal confirmed, repeated positive results were noticed following treatment with N,N’-Diphenylguanidinmonohydrochlorid in the investigated Salmonella typhimurium TA98 and TA100 strains at 5000 µg/plate, in the presence of exogenous metabolic activation (+S9 mix). In the performed experimental phases, following plate incorporation and pre-incubation procedures, the increased revertant colony numbers were above the corresponding historical control data ranges and above the thresholds for being positive in S. typhimurium TA98 and TA100 at the concentration of 5000 µg/plate (+S9 mix).  

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item induced gene mutations by base pair changes and frameshifts in the genome of the Salmonella typhimurium TA98 and TA100 strains investigated. In conclusion, the test item N,N’-Diphenylguanidinmonohydrochlorid showed a mutagenic activity on Salmonella typhimurium TA98 and TA100 strains in the presence of an exogenous metabolic activation system, under the test conditions used in this study.

In vitro chromosome aberration test in mammalian cells

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.

Read-across approach:

There are experimental data available on gene mutation in bacteria and chromosomal aberration in mammalian cells in vitro with the test item. However, no in vivo studies are available on genotoxicity with the test item, but with a structural analogue substance. These data are considered suitable and adequate to fulfill this endpoint. Moreover, in vitro studies with the structural analogue substance are provided in order to support the read-across approach. For detailed read-across justification please refer to the respective IUCLID Section.

In vitro CAS 102 -06 -7 (supporting studies)

In the first study (Simar 2014), DPG was assessed by means of the Ames's test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested in absence of metabolic activation and in presence of metabolic activiation using both rat and hamster liver S9 -mix, in two independant assays. The validity criteria for the assay were fulfilled. Under these experimental conditions, a clear mutagenic activity was revealed in both strains TA98 and TA100 exclusively in presence of hamster S9 -mix. This specific metabolic activation by hamster liver S9 -mix rich in N-acetyl transferases is in favour of the formation of the highly reactive nitrenium ions from the aromatic amine moiety of the test item DPG (Simar 2014).

In the second study (Simar 2015), the mutagenic activity of the read-across substance, DPG, was assessed by means of the Ames's test in the two Salmonella typhimurium strains TA98 and TA100 tested in presence of metabolic activation using human liver S9 -mix, in two independant assays. The validity criteria for the assay were fulfilled. Neither statistically nor biologically significant increases in the number of revertants were noted in both strains. Under these experimental conditions, it was considered that the test item, DPG, is not mutagenic in strains TA98 and TA100 in presence of human S9-mix.

All other in vitro investigations, chromosome aberrations (Nakagawa 2000) and gene mutations (Myhr 1979) tests were negative. In the study of Nakagawa (2000), DPG was tested on Chinese cells lung, the results (chromosome aberrations) were negative at the concentrations of 50-400 µg/plate, with and without activation rat-S9.

In the study of Myhr (1979) tested DPG, the results on L5178Y mouse lymphoma assay at the concentrations of 16.4 -525 µg/mL, with and without rat-S9 showed negative results.

Ex vivo mammalian alkaline comet assay: Study performed on rat and hamster hepatocytes (Simar 2016a):

The investigation of a genotoxic activity of the read-across substance 1,3-diphenylguanidine has been carried out compliance using the mammalian alkaline comet assay performed ex vivo on rat and hamster hepatocytes.

By comparing data in the rat and the hamster, this study aimed at assessing a systemic risk in more or less sensitive species in order to give a better relevant transposition to Human. In the assay performed in both rat and hamster hepatocytes, no biologically significant increases in the percentage of DNA in tail were observed at the 3 concentrations analysed ranging from 31.25 to 125 µg/mL when compared to the respective negative controls. Indeed, the values for percent DNA in tail were comprised in the range of historical data for negative control.

The structural analogue is thus considered as unable to induce primary DNA damage in the liver cells from either rat or hamster. Interestingly, beyond the lack of genotoxicity, there was no specific effect on hamster liver cells known to be more sensitive to aromatic amines.

In vivo CAS 102 -06 -7

The structural analogue substance, 1,3 -diphenylguanidine, was investigated for genotoxic potential by the means of the in vivo micronucleus test in bone marrow and the in vivo comet assay under alkaline conditions (SCGE) in the liver and stomach, in male OFA Sprague-Dawley rats, according to OECD Guidelines (Nos. 474 and 489, 2014). Animals were treated orally once a day for 3 consecutive days, 24 hours apart, at dose levels of 80, 40 and 20 mg/kg. The validity criteria for the results were fulfilled.

Under these experimental conditions, in male OFA Sprague-Dawley rats, 1,3-diphenylguanidine induced no genotoxic activity in bone marrow cells as assessed in the micronucleus assay.No statistically significant reduction in the PCE/NCE ratio was observed, but the systemic exposure was clearly demonstrated in the range-finding toxicity study. The absence of a significant reduction in the PCE/NCE ratio means that DPG induced no toxicity to the bone marrow at the maximal dose of 80 mg/kg/d.

Furthermore,1,3-diphenylguanidine does not induce DNA strand breaks and/or alkali-labile sites inducer activities toward the liver from OFA Sprague-Dawley male rats. Both these results tend to indicate a lack of systemic genotoxic in the rat.

In contrast, 1,3-diphenylguanidine induced a biologically increase, but not statistically significant, in DNA strand breaks at 80 mg/kg/day (x3) in stomach cells isolated from male rats after a 3-day treatment by oral route of administration. A concurrent statistically significant increase in the percentage of hedgehogs was noted.

To summarize,the increase in Tail DNA in stomach cells was statistically dose-related, the mean percent tail DNA in stomach cells in the 80 mg/kg/day, highest dose group was outside the distribution of the historical data for vehicle control, and, none of the treatment group exhibited a statistically significant increase in the mean of medians of percentage of DNA in tail in stomach cells compared with the concurrent negative control. Therefore, only 2 out of the 3 criteria for a positive response were met. Results are considered as equivocal. However, to assess the biological relevance, the follow-up histopathological and specific apoptosis quantification studies were performed. They failed at demonstrating any significant occurrence of cell death or necrosis in stomach cells. Therefore, the increase in the percentage of DNA in tail is not due to cytotoxicity and/or apoptosis interference but is probably due to a local genotoxic activity of the test item toward the stomach cells.

Overall conclusion

According the Guidance to Regulation (EC) No 2072/2008 on CLP of substances and mixtures (version 4.1, june 2015),"the hazard classification for germ cell mutagenicity primarily aims to identify substances causing heritable mutations or being suspected of causing heritable mutations. A secondary aim is that the hazard class germ cell mutagenicity offers supporting information with respect to the classification of carcinogenic substances". (page 358)

"It is also warranted that where there is evidence of only somatic cell genotoxicity, substances are classified as suspected germ cell mutagens. Classification as a suspected germ cell mutagen may also have implications for potential carcinogenicity classification. This holds true especially for those genotoxicants which are incapable of causing heritable mutations because they cannot reach the germ cells (e.g. genotoxicants only acting locally, 'site of contact' genotoxicants". This means that positive results in vitro are supported by at least one positive local in vivo, somatic cell test, such an effect should be considered as enough evidence to lead to classification in Category 2. If there is also negative or equivocal data, a weight of evidence approach using expert judgment has to be applied." (page 358)

Based on negative results obtained with the structural analogue in in vitro (OECD 473) and in vivo (OECD 474/475) tests, there are no concern of clastogenicity and aneugenicity with the structural analogue DPG or the test item itself.

The Ames test (OECD 471) demonstrated clear positive results when using hamster S9 -mix. In contrast, when performed with rat S9 -mix or human S9 -mix, the Ames test did not elicited biologically significant effects. This indicates that the hamster liver cells may metabolize the DPG in potentially genotoxic metabolites for the bacteria. To evaluate the relevance of this bacteria-specific effect in the Ames test, a comparative ex vivo comet assay in both rat and hamster hepatocytes was performed. Negative results were obtained with both rat and hamster liver cells, confirming that DPG or their metabolite did not induce genotoxicity on mammalian cells. This indicates that the hamster liver cells may metabolize the DPG in substances potentially genotoxic for the bacteria but not for the mammalian cells. This bacteria-specific effect is also supported by the negative results from the in vitro mammalian gene mutation assay (OECD 476).

In the in vivo comet assay in stomach and liver cells isolated from DPG-exposed male rat, no increase in primary damage was observed in the liver cells. An equivocal effect was observed in the stomach cells, in particular at the high dose. This result is questionable because DPG was not considered to be a direct mutagen (negative results obtained in all in vitro tests without S9), and the stomach cells are not known to have a high metabolism potential. Considering the potential routes of exposure of the workers and the general population, this result in stomach cells is considered of low relevance for human hazard assessment.

In conclusion, based on the weight of evidence, there is sufficient evidence to conclude that the structural analogue DPG, and consequently the test item itself, is not able to induce heritable mutations in somatic cells by systemic exposure. Moreover, DPG did not induce a direct toxicity to the reproductive organs in repeated dose toxicity studies, a genotoxic effect on the germ cells is also not expected. Therefore, the hazard classification for germ cell mutagenicity (category 2) according to the Regulation EC 1272/2008, is not relevant for the test item.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data with the test item and a structural analogue substance are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Available data on genotoxicity are evaluated in a weight of evidenve approach. Positive findings on mutagenicity or clastogenicity in vitro were not confirmed by in vivo examinations. Therefore, the test item does not require classification as germ cell mutagen or genotoxic according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.