Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,3-Diphenylguanidine gave positive results in the Ames test with hamster's S9-mix only (negative results with and without rat's S9-mix, and with human's S9). Negative results were observed in the in vitro mammalian cell assays (MLA/TK, in vitro chromosomal aberration test).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Litton Bionetics Inc. standard protocol of Mouse Lymphoma Forward Mutation Assay
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 (male Fischer rats)
Test concentrations with justification for top dose:
16.4, 32.8, 65.6, 131, 188 µg/ml without S9,
32.8, 131, 188, 375, 525 µg/ml with S9
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethan sulfonate (without S9), dimethylnitrosamine (with S9)
Details on test system and experimental conditions:
The solubility of the test chemical in growth medium and/or DMSO is first determined. Then a wide range of chemical concentrations is tested for cytotoxicity, starting with a maximum applied dose of 10 mg/ml for test chemicals soluble in media or 1 mg/ml for solutions in organic solvents. After an exposure time of four hours, the cells are washed and a viable cell count is obtained the next day. Relative cytotoxicities expressed as the reduction in growth compared to the growth of untreated cells are used to select seven to ten doses that cover the range from 0 to 50-90% reduction in 24-hour growth. These selected doses are subsequently applied to cell cultures prepared for mutagenicity testing, but only four or five of the doses will be carried through the mutant selection process. This procedure compensates for daily variations in cellular cytotoxicity and ensures the choice of four or five doses spaced from 0 to 50-90% reduction in cell growth.
1. Nonactivation Assay:Cultures exposed to the test chemical for four hours at the preselected doses are washed and placed in growth medium for two or three days to allow recovery, growth and expression of the induced TK-/- phenotype. Cell counts are determined daily and appropriate dilutions are made to allow optimal growth rates.

At the end of the expression period, 3 x 10e6 cells for each selected dose are seeded in soft agar plates with selection medium and resistant (mutant) colonies are counted after 10 days incubation. To determine the actual number of cells capable of forming colonies, a portion of the cell suspension is also cloned in normal medium (nonselective). The ratio of resistant colonies to total viable cell number is the mutant frequency.
2. Activation Assay : The activation assay can be run concurrently with the nonactivation assay. The only difference is the addition of the S9 fraction of rat liver homogenate and necessary cofactors (CORE) during the four-hour treatment period. CORE consists of NADP (sodium sait) and isocitric acid. The final concentrations of the activation system components in the cell suspension are: 2.4 mg NADP/ml; 4.5 mg isocitric acid/ml; and 50 pl S9/ml.
Evaluation criteria:
A compound is considered mutagenic in this assay if:
-A dose-response relationship is observed over 3 of the 5 dose levels employed.
-The minimum increase at the low level of the dose-response curve is at least 2.5 times greater than the solvent and/or negative control values.
-The solvent and negative control data are within the normal range of the spontaneous background for the TK locus.

All evaluations of mutagenic activity are based on consideration of the concurrent solvent and negative contrat values run with the experiment in question. Positive control values are not used as reference points, but are included to ensure that the current ceil population responds to direct and promutagens under the appropriate treatment conditions.
Occasionally, a single point within a concentration range will show an increase 2.5 times greater than the spontaneous background. If the increase is at the high dose, is reproducible, and if an additional higher dose level is not feasible because of toxicity, the chemical can be considered mutagenic. If the increase is interna] within the dose range and is not reproducible, the increase will normally be considered aberrant. If the internat increase is reproducible, several doses clustered around the positive concentration will be examined to either confirm or reject the reliability of the effect.
Statistics:
No data
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: >= 65.6 µg/ml without S9, >=188 µg/ml with S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
With and without activation, the test compound did not induce a significant increase in mutant frequency at any of the tested doses. A 2.5-fold increase over the background frequency (average of the solvent and untreated negative control values) is considered necessary to demonstrate mutagenesis at any given dose level. Even at the relatively toxic doses of 188 ug/ml without activation (13.5% relative growth) and 375 µg/ml with activation (26.6% relative growth) the mutant frequencies observed were comparable to the negative controls.

The validity of the mutagenesis assay can be assessed by the results obtained for the positive and negative controls. The cloning efficiencies for the negative (solvent and untreated) controls varied from 100% without activation to 99% with activation, which demonstrates excellent culturing conditions for the assay. The negative control mutant frequencies were all within the normal range for nonactivation and activation tests, and the positive control compounds yielded frequencies in the normal range that were greatly in excess of the negative control values.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
DPG did not induce a significant increase in mutations at the TK locus in L5178Y mouse lymphoma cells at applied concentrations of 16.4 to 188.0 ug/ml without activation and at 32.8 to 525 ug/ml with microsomal activation. These concentration ranges included highly toxic treatments. Therefore, the test compound is considered to be inactive in the Mouse Lymphoma Forward Mutation Assay.
Executive summary:

This study evaluates DPG for its ability to induce forward mutation in the L5178Y TK+/- mouse lymphoma cell line, as assessed by colony growth in the presence of 5-bromo-2'-deoxyuridine (BrdU). DPG did not induce a significant increase in mutations at the TK locus in L5178Y mouse lymphoma cells at applied concentrations of 16.4 to 188.0 ug/ml without activation and at 32.8 to 525 ug/ml with microsomal activation. These concentration ranges included highly toxic treatments. Therefore, the test compound is considered to be inactive in the Mouse Lymphoma Forward Mutation Assay.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
The purpose of the ex vivo Comet assay following the alkaline version (pH > 13) developed by Singh et al. (1988) is to identify those agents which induce DNA damage such as single or double DNA strand breaks (SSB or DSB), alkali-labile sites, DNA-DNA / DNA-protein cross-linking and SSB associated with incomplete excision repair sites. The advantages of the Comet assay include its demonstrated sensitivity for detecting low levels of DNA damage.

Isolated cells are treated with the test item at different concentration levels. About 3 hours after the treatment, the cells are processed for the comet assay.
Single cells are embedded in agarose on microscope slides and the obtained microgels are successively submitted to lysis, unwinding and electrophoresis in alkaline conditions and under dimmed light to prevent any additional DNA damage. After neutralization, slides are dried and could therefore be stained with a fluorescent dye (e.g. propidium iodide) before analysis and scoring. The method used for quantifying DNA migration involves a computerized image analysis system in order to collect comet data; then, the dedicated software allows indeed the calculation of metrics for DNA migration.
GLP compliance:
no
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
not applicable
Species / strain / cell type:
hepatocytes: rat
Details on mammalian cell type (if applicable):
One young adult OFA Sprague-Dawley rat (Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE) weighing approximately 200 g and 5 to 10 weeks old.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
hepatocytes: hamster
Details on mammalian cell type (if applicable):
One young adult chinese hamster (Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE) weighing approximately 200 g and 5 to 10 weeks old.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
31.25, 62.5 and 125 µg/ml for rat and hamster
Vehicle / solvent:
The test item will be dissolved in dimethylsulfoxide (DMSO) in which it is soluble at least up to 100 mg/mL giving a possible highest final concentration of 1000 µg/mL when added at 1% in culture medium.
Half successive dilutions will be prepared also in DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
methylmethanesulfonate
Details on test system and experimental conditions:
TREATMENTS FOR THE DEFINITIVE GENOTOXICITY ASSAY
Three ml of cell suspension at 1.5 x 10^5 viable cells/mL will be transferred to each well of 6-wells plates (i.e. 4.5 x 10^5 viable cells/well). Three replicate wells per concentration and per control will be prepared for the comet assay. The plates will be incubated at 37°C in an atmosphere of 5% CO2 for approximately 90 to 120 minutes to attach. The cell cultures will then be treated with the different dilutions of the test item (3 wells per concentration).
Approximately 3 hours after the treatment, the culture medium will be discarded and the cells will be washed twice with PBS. The cells will then be gently scrapped (after an eventual tripsinization for 2 minutes at 37°C), transferred in a 15-mL tube containing 2 mL of culture medium (containing serum) and centrifuged for 6 minutes, 1000 rpm at ambient temperature. The supernatant will be then eliminated.
Isolated cells will be assessed for cytotoxicity and processed for the comet assay.

Determination of the cytotoxicity of the test item
Cytotoxicity will be determined through the enumeration of hedgehogs and also the use of the Trypan blue technique. A cell viability that is below 70 or 50% when compared to the control may be considered as excessive when measured by enumeration of hedgehogs or the Trypan blue technique, respectively.

-the median per slide of the percentage of DNA in tail for at least 50 cells,
-the mean of medians of the percentage of DNA in tail per replicate (i.e. 2 slides per well, i.e. 100 cells),
-the mean of medians per concentration (i.e. 3 replicates, 300 cells).

Evaluation criteria:
A study is accepted if the following criteria are fulfilled:
-Concurrent negative control should be within the control limits of the distribution of the laboratory’s historical negative control database.
-The concurrent positive control should induce responses that are compatible with those generated in the historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
-The appropriate number of concentration and cells must be analysed.
Moreover:
-In the negative control, an eventual increase in the frequency of hedgehogs, must not be >50%.
Statistics:
In order to quantify the test item effects on DNA, the following statistical analysis strategy will be applied, using the statistical software Stat view®, version 5.

As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test will be then performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups.
The non-parametric Mann-Whitney U-test will be applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each concentration versus the solvent control. This test will be also used to compare solvent control and positive control to determine acceptable criteria of a valid test.
Species / strain:
hepatocytes: rat
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
hepatocytes: hamster
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OBTAINED WITH 3 SLIDES ANALYSED PER CULTURE
Assay in Rat Hepatocytes
A statistically significant increase in percent DNA tail was noted at the intermediary concentration of 62.5 µg/mL with a value of 2.39% vs. 1.26% in the relative control. However, this value was within the intervals of confidence of historical data for negative control, and no dose-effect relationship was observed when analysing using the Kruskal-Wallis test.
This statistical increase is thus considered as not biologically relevant. After complementary reading, mean percentage of tail DNA from 3 independent cultures ranged from 1.41 to 2.73% vs. 1.26% in the corresponding negative control.
The test item is considered as not genotoxic toward Rat hepatocytes.

Assay Hamster Hepatocytes
No statistically significant increase in percent DNA tail were noted at the concentrations ranging from 31.25 to 125 µg/mL with values ranging from 3.39 to 4.89% vs. 3.26% in the relative negative control. Moreover, the values were within or very close to the intervals of confidence of historical data for negative control (i.e. 1.14 – 4.46). In contrast, a dose-effect relationship was observed when using the Kruskal-Wallis test. It is however noteworthy that one of the 9 slides analysed in the negative control was out of the intervals of historical data and was aberrant when comparing to the results obtained in the 8 other slides for which the values ranged from 0.60 to 3.64%, vs. 12.46% for the slide BC-16-129C. When analysing the data without taking into account this aberrant result, the percent DNA in tail in the control is of 2.09%, and no statistically significant effect is noted anymore.
After complementary reading, mean percentage of tail DNA from 3 independent cultures ranged from 3.39 to 4.89% vs. 2.09% in the corresponding negative control.
Under these conditions, the test item can be considered as not genotoxic toward Hamster hepatocytes.

From the overall results obtained in both rat and hamster hepatocytes, 1,3-diphenylguanidine was considered as not genotoxic in liver cells meaning that 1,3-diphenylguanidine does not induce primary DNA damage toward cells from a systemic organ cells. On the other hand, it is interesting to note that the profile of response is comparable between the 2 species. Interestingly, beyond the lack of genotoxicity, there was no specific effect on hamster liver cells known to be more sensitive to aromatic amines.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

 

Table 1 : Assay in RAT hepatocytes

 

Conc. In µg/mL

Total relative survival (1)(%)

% of DNA in tail

Relative ratio of hedgehogs (4)

Statistical significance

Mean of medians per culture (/3 cultures)

Non-parametric statistical assessment

P Kruskal-Wallis (2)

P mall Whitney (3)

Negative control

0

100

1.26

 

N.S.

-

-

-

DPG

125

107.0

1.41

N.S.

1.19

N.S.

62.5

25.2

2.39

<0.05

0.77

N.S.

31.25

57.0

2.73

N.S.

0.95

N.S.

2-AAF

200

35.3

4.66

-

<0.05

0.89

N.S.

MMS

10

53.3

13.58

-

<0.05

1.61

N.S.

NS: not statistically significant

(1): [(Adjusted number of cells x Relative survival) in treated x 100] / [(Adjusted number of cells x Relative survival) in control]

(2): Total groups without positive control

(3): Mean values obtained in treated group compared to Mean values obtained in negative control group

(4): Corresponds to the percentage of hedgehogs per treated group / percentage of hedgehogs in negative control group.

Table 2 : Assay in HAMSTER hepatocytes

 

Conc. In µg/mL

Total relative survival (1)(%)

% of DNA in tail

Relative ratio of hedgehogs (4)

Statistical significance

Mean of medians per culture (/3 cultures)

Non-parametric statistical assessment

P Kruskal-Wallis (2)

P mall Whitney (3)

Negative control

0

100

3.26

2.09 *

 

<0.05

N.S.*

-

-

-

DPG

125

77.6

3.59

N.S.

0.46

<0.05

62.5

87.5

3.39

N.S.

0.64

N.S.

31.25

135

4.89

N.S.

0.77

N.S.

2-AAF

200

52.9

5.84

-

N.S.

<0.05*

0.67

N.S.

MMS

10

66.0

31.06

-

<0.05

0.46

<0.01

*Results obtained without slide BC-16 129C

(1): [(Adjusted number of cells x Relative survival) in treated x 100] / [(Adjusted number of cells x Relative survival) in control]

(2): Total groups without positive control

(3): Mean values obtained in treated group compared to Mean values obtained in negative control group

(4): Corresponds to the percentage of hedgehogs per treated group / percentage of hedgehogs in negative control group.

 

 

Conclusions:
Based on theis assay, the test item 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.
Executive summary:

The investigation of a genotoxic activity of the test item 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 test item 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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster CHL/IU cells
Details on mammalian cell type (if applicable):
CHL/IU cell strains derived from female Chinese hamster lungs were used.
The cells were purchased from dainippon pharmaceuticals. DMSO was added until the final ratio to the cell suspension was 10%, which was then divided into 1 ml quantities and stored frozen in liquid nitrogen.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 (liver of SD rats, induced with phenobarbital and 5,6-benzofurabon)
Test concentrations with justification for top dose:
DPG : 50, 100, 200, 400 µg/plate (with and without S9).
Short term treatment : The dose for the positive control MMC and B(a)P was 0.1 and 20 µg/ml respectively, due the known chromosomal aberration inductivity.
Continuous treatment : MMC = 0.03 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 1% sodium carboxymethylcellulose solution (CMS-Na) for DPG
- Justification for choice of solvent/vehicle: Based on the result of the solvent investigation, 50mg/ml of DPG was insoluble in a solution of sodium chloride (saline solution) and 500 mg/ml was insoluble in acetone. 500 mg/ml was soluble in DMSO. And the other hand, 50 mg/ml was almost uniformly suspended in a 1% carboxymethyl cellulose sodium solution.

MMC (positive control) was diluted in saline at 1 µg/ml in short term treatment method.
B(a)P (positive control) was diluted in DMSO at 4 mg/ml.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
CMS-Na
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: S9 mix (-) : Mitomycin C ; S9 mix (+) : B(a)P
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (called MEM). 100 ml of inactivated fetal bovine serum was added to 9000 ml of MEM.
5 ml each of a cell suspension prepared with 4x10^3/ml were spread on a 6 cm plate and incubated for 3 days. After removing the incubation solution, a cells treated for 6 hours. After 6 hours, the cell surface was washed with MEM once and then it was incubated for an additional 18 hours in 5 ml of new incubation solution.
Short-term treatment : Test substance suspension = 0.3 ml (+0.5 ml S9). Incubation solution = 2.7 ml.
Continuous treatment : Test substance suspension = 0.5 ml. Incubation solution = 4.5 ml.

DURATION
- Preincubation period: 3 days
- Exposure duration: 6 + 18 hours (short-term treatment), 24 hours (continuous treatment)
- Expression time (cells in growth medium): no data
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SPINDLE INHIBITOR (cytogenetic assays): colcemid (colchicine)
NUMBER OF REPLICATIONS: 2 (plates/test : 4)
NUMBER OF CELLS EVALUATED: Two hundred cells were analysed in each group.
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: no data
- Determination of endoreplication: no data
Evaluation criteria:
Judgement was done on the basis of criteria of Ischidate et al. (1987).
Statistics:
no data
Species / strain:
other: Chinese hamster CHL/IU cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
IC50 = 232µg/ml (short-term), IC50 = 189µg/ml (continuous)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: Chinese hamster CHL/IU cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
IC50=192µg/ml (short-term)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
See tables
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1 : Chromosomal analysis of chinese hamster cells (CHL/IU) treated with DPG with and without S9 mix (Short-term treatment)

Group

Concen-tration (µg/ml)

S9 mix

Time of exposure (hr)

Cell growth index (%)

Number of cells analyzed

Number of cells with structural aberrations

ctb

cte

csb

cse

frg

total

Solventa

0

-

6-18

100

200

0

0

0

0

0

0

Test substance

50

-

6-18

117

200

0

0

0

0

0

0

100

-

6-18

95

200

0

0

0

0

0

0

200

-

6-18

85

200

0

0

0

0

0

0

400

-

6-18

18

200

0

1

0

0

0

1

MMC

0.1

-

6-18

104

200

61

61

0

0

0

122

Solvent

0

+

6-18

100

200

0

1

1

0

0

2

Test substance

50

+

6-18

75

200

1

0

1

0

0

2

100

+

6-18

62

200

1

0

0

0

0

1

200

+

6-18

55

200

2

0

0

0

0

2

400

+

6-18

45

200

0

0

0

0

0

0

BP

20

+

6-18

118

200

62

134

0

0

0

196

Group

Concentration (µg/ml)

S9 mix

Time of exposure (hr)

Numbers of cells with aberrations (without gap) (%)

Number of cells with gap

Polypoidb(%)

Judgementc

SA

NA

Solventa

0

-

6-18

0 (0.0)

1

0.0

-

-

Test substance

50

-

6-18

0 (0.0)

2

0.0

-

-

100

-

6-18

0 (0.0)

0

0.0

-

-

200

-

6-18

0 (0.0)

2

2.0

-

-

400

-

6-18

1 (0.5)

1

4.0

-

-

MMC

0.1

-

6-18

1 (0.5)

5

0.0

+

-

Solvent

0

+

6-18

2 (1.0)

1

0.0

-

-

Test substance

50

+

6-18

2 (1.0)

1

1.0

-

-

100

+

6-18

1 (0.5)

0

1.5

-

-

200

+

6-18

2 (1.0)

0

1.0

-

-

400

+

6-18

0 (0.0)

1

3.0

-

-

BP

20

+

6-18

143 (71.5)

3

0.0

+

-

Abbreviations : ctb = chromatid break ; cte = chromatid exchange ; csb = chromosome break ; cse = chromosome exchange (dicentric and ring), frg = fragment

SA = structural aberration, NA = numerical aberration, MMC = mitomycin C (positive control), BP = benzo(a)pyrene (positive control)

aTwo hundred cells were analysed in each group.

bJudgement was done on the basis of criteria of Ischidate et al. (1987).

c1% sodium carboxymethylcellulose solution was used as solvent.

Table 2 : Chromosomal analysis of chinese hamster cells (CHL/IU) treated with DPG without S9 mix (continuous treatment)

Group

Concen-tration (µg/ml)

S9 mix

Time of exposure (hr)

Cell growth index (%)

Number of cells analyzed

Number of cells with structural aberrations

ctb

cte

csb

cse

frg

total

Solventa

0

-

24

100

200

0

0

0

0

0

0

Test substance

50

-

24

107

200

0

0

0

1

0

1

100

-

24

79

200

0

0

0

1

0

1

200

-

24

51

200

1

1

0

0

0

2

400

-

24

12

200

0

0

0

0

0

0

MMC

0.03

-

24

125

200

19

34

0

0

0

53

Group

Concen-tration (µg/ml)

S9 mix

Time of exposure (hr)

Numbers of cells with aberrations (without gap) (%)

Number of cells with gap

Polypoidb(%)

Judgementc

SA

NA

Solventa

0

-

24

0 (0.0)

0

0.0

-

-

Test substance

50

-

24

1 (0.5)

0

0.0

-

-

100

-

24

1 (0.5)

0

0.5

-

-

200

-

24

1 (0.5)

0

0.0

-

-

400

-

24

0 (0.0)

0

0.0

-

-

MMC

0.0.3

-

24

50 (25.0)

1

0.5

+

-

Abbreviations : ctb = chromatid break ; cte = chromatid exchange ; csb = chromosome break ; cse = chromosome exchange (dicentric and ring), frg = fragment

SA = structural aberration, NA = numerical aberration, MMC = mitomycin C (positive control)

aTwo hundred cells were analysed in each group.

bJudgement was done on the basis of criteria of Ischidate et al. (1987).

c1% sodium carboxymethylcellulose solution was used as solvent.

Conclusions:
DPG did not include structural chromosomal aberrations or polyploidy under the conditions of this experiment.
Executive summary:

In vitro chromosomal aberration tests were conducted using DPG on CHL/IU cell strains derived from female Chinese hamster lungs.  

The results of the cell growth inhibition tests using the short term treatment method indicated that the 50% cell growth inhibition dose was 232 µg/ml without the S9 mix and 192 µg/ml with the S9 mix. Under all of the treatment conditions, the doses for all of the chromosomal aberration tests were set to ½, ¼ and 1/8 of the maximum dose of 400µg/ml. For all of the doses both with and without the S9 mix, the results indicated that the frequency of appearance of cells with chromosomal aberration or numerical aberration was less than 5%.

The results of the cell growth inhibition tests using the 24 hour continuous treatment method indicated that the 50% cell growth inhibition dose was 189µg/ml. The doses for the chromosomal aberration tests were set to ½, ¼ and 1/8 of the maximum dose of 400 µg/ml. The results indicated that for all of the doses, the frequency of appearance of cells with chromosomal aberration or numerical aberration was less than 5%.

From the aforementioned results, the authors have concluded that the chromosomal aberration induction for 1,3-diphenylguanidine on CHL/IU cells is negative under these test conditions.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September-December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat and Hamster S9-mix
Test concentrations with justification for top dose:
First assay:
-without S9: 5-15-50-150-500-1500 µg/plate (all the strains)
-with rat's S9-mix (without preincubation): 50-150-500-1500-5000 µg/plate (TA1535, TA1537), 50-150-500-1500-3000 µg/plate (TA98, TA100, TA102)
-with hamster's S9-mix (without preincubation): 15- 50-150-500-1500-3000 µg/plate (all the strains)

Second assay:
-without S9: 5-15-50-150-500-1500 µg/plate (all the strains)
-with rat's S9-mix (without preincubation): 5-15-50-150-500-1500-3000 µg/plate (TA98, TA100)
-with hamster's S9-mix (without preincubation): 15-50-150-500-1500-3000 µg/plate (TA98)
-with rat's S9-mix (with preincubation): 15-50-150-500-1500-5000 µg/plate (TA1535, TA1537), 15-50-150-500-1500-3000 µg/plate (TA98, TA100), 5-15-50-150-500-1500 µg/plate (TA102)
-with hamster's S9-mix (with preincubation): 15-50-150-500-1500-3000 µg/plate (all strains)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: As the test item was not freely soluble in aqueous solvent, trials for the search of the solubility of the test item were performed in dimethylsulfoxide (DMSO) in which it was soluble at 100 mg/mL, and in ethanol in which solubility could not be observed when tested at 200 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-antramine, benzidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate-incorporation and pre-incubation.
Without metabolic activation : For each strain, 0.1 mL of a bacterial suspension from a culture agitated overnight at 37°C and 0.1 mL of the test item at the relevant initial concentrations were successively added to 2 mL of top agar to which 10% of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C has been added. The contect of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar. The plates were then incubated at 37°C for approximately 48H. At the end of the expression time, colonies of revertants were counted for each plate.
With metabolic activation: The method was the same as the one described above except that immediately before spreading in the plates, 0.5 mL of the S9 mix metabolic activation system was added in soft agar.
On a technical point of view, the pre-incubation mehod is the same as the one using agar plate incorporation with, however, the following modifications: the following solutions were added in this order: the bacterial strain to be test (100µl), S9-mix (500 µl) and at the end the test item solution (50 µl as an organic was used). The mixture was preincubated with stirring at 37°C for 60 minutes prior to adding soft agar and spreading out in a Petri plate.

NUMBER OF REPLICATIONS: three plates per dose

DETERMINATION OF CYTOTOXICITY: bacterial growth
Evaluation criteria:
Interpretation criteria of the results:
Given that the procedures followed in order to evaluate mutagenic activity are semi-quantitative, the criteria used to determine a positive effect may have been to some extent subjective and based mainly on experience. However, under the expeirmental conditions, and when the validity criteria are reached, the following decision criteria may be used.
-Criteria based on biological significance: 1/Strains TA1535 & TA1537: a test item causing a positive response proportional to the dose for at least 3 doses with, the highest increase, a value greater than or equal to 3 times the value for the solvent control, is considered positive in the assay. 2/ Strains TA98 & TA100 & TA102: A test item causing a positive response proportional to the dose for at lease 3 doses with, for the highest increase, a value greater than or equal to 2 times the value for the solvent control, is considered positive in the assay.
-criteria based on statistical significance.
-reproducibility: if a test item causes a positive response during a single assay and that result cannot be reproduced in at least one independant assay, the initial positive result may be considered as not significant.
-comparison to historical control data: In some borderline cases, an additional criterion to be considered is the comparison between the number of revertants induced by the test item and the laboratory historical control data. Indeed, an increase in each individual value that is above the highest value of corresponding historical control data can help supporting a conclusion such as "equivocal" or "weak" mutagen.

All these criteria are not absolute, but they however, help in coming to a decision, which can be conclusive in the majority of the cases.

Statistics:
Data were analysd by means of Dunnett's method allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
with TA98 and TA100 strains
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the first assay in absence of metabolic activation except in train TA1535, in the highest dose of 1500 µg/plate induced a slight or moderate toxicity in all strains. A statistically significant in the number of revertants was noted in strain TA102 at 1500 µg/plate. Under these conditions, the highest dose to be tested in the second assay in absence of metabolic activation was kept at 1500 µg/plate in all strains. In the second assay, the highest dose of 1500 µg/plate induced either a slight or a string toxicity with no bacterial growth.

In the first assay in presence of rat metabolic activiation system, a slight to moderate toxicity was noted in all strains at the highest doses tested of either 3000 or 5000 µg/plate. Moreover, an important statistically significant decrease in the number of revertants was noted in strain TA102 at the highest dose of 3000 µg/plate. under these conditions, the highest dose to be tested in the second assay in presence of rat metabolic activation was set at 5000 µg§plate in strains TA1535 and TA1537, 3000 µg/plate in strains TA98 and TA100, and 1500 µg/plate in strain TA102.
In the second assay without pre-incubation, a string toxicity was noted in strain TA98 at the 2 highest doses tested of 3000 and 1500 µg/plate, respectively, without pre-incubation. In the second assay with pre-incubation, a slight toxicity was noted in strains TA1535 and TA1537 at the highest doses tested of 5000 µg/plate or a moderate toxicity in strain TA100 at 3000 µg/plate. No toxicity was observed in strains TA98 and TA102 with pre-incubation.

In the first assay in presence of hamster metabolic activation system, a slight to moderate toxicity was noted in all strains at the highest or 2 highest doses tested of 3000 and/or 1500 µg/plate. Moreover, an important statistically significant decrease in the number of revertants was noted in strain TA1537 at the highest dose of 3000 µg/plate.
Under these conditions, the highest dose to be tested in the second assay in presence of hamster metabolic activation was set at 3000 µg/plate in strains TA1535, TA98, TA100 and TA102, and 1500 µg/plate in strain TA1537.
In the second assay with pre-incubation, a strong toxicity was noted in strains TA1535, TA98 and TA102 at the highest dose tested of 3000 µg/plate, with no bacterial growth. The dose of 1500 µg/plate induced either a moderate or a slight toxicity in strains TA1535, TA1537, TA98 and TA102.
In the second assay without pre-incubation, a moderate toxicity was noted in strains TA98 and TA100 at the highest dose tested of 3000 µg/plate. The dose of 1500 µg/plate induced a slight toxicity in strain TA98.

Mutagenicity test:
In two independent assays performed without metabolic activation, no statistically or biologically significant increase in the number of revertants was noted in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested, in the présence of DPG. Moreover, no statistically or biologically significant increase in the mean number of revertants was noted in the 3 S.typhimurium strains TA1535, TA1537 and TA102 tested, in the presence of either rat or hamster liver S9 -mix.
In the first assay in strain TA98 in presence of rat metabolic activation, a statistically significant increase was observed at the lowest dose tested of 50 µg/plate with an induction ratio of 2.4, i.e. above the threshold ratio for a biologically significant response (set at 2 in the strain). The value observed for the mean number of revertants was higher than the maximum value already noted in historical data for negative control (i.e. 62.1 vs. 46). however, this effect was not reproductible as in the second assay performed in the same experimental condition, neither statistically nor biologically significant increases were observed at the 6 doses tested ranging from 5 to 3000 µg/plate. Moreover, following the pre-incubation method, no biologically significant increase was observed. This effect was thus considered as devoid of mutagenic hazard.
In the first assay in strain TA98 in presence of hamster metabolic activation, neither statistically nor biologically significant increases n the number of revertants were observed at the all the doses tested ranging from 15 to 3000 µg/plate. Nevertheless, although the preliminary toxicity assays is not aimed at assessing the mutagenic potential of a test item, it demonstrated clear increases in the number of TA98 revertants. Therefore, both the standard plate-incorporation technique and the pre-incubation method were used in the second assay. In the condition with-preincubation statistically and/or biologically significant increases in the number of revertants were observed at the doses tested of 15, 50 and 500 µg/plate with induction ratios of 1.7, 1.8 and 2, i.e. equal to the threshold ratio for a biologically significant response at the dose of 500 µg/plate. In the standard condition without-preincubation statistically and/or biologically significant increases in the number of revertants were observed at all the doses tested ranging from 15 to 3000 µg/plate with induction ratios ranging from 1.9 to 5.1, i.e. above the threshold ratio for a biologically significant response. The values observed for the mean numbers of revertants were higher than the highest value already noted in historical data for negative control (i.e. 39 to 107 vs.50). This effect was thus considered as mutagenic.
In the first assay in strain TA100 in presence of rat metabolic activation, a statistically significant increase was observed at the lowest dose tested of 50 µg/plate with an induction ratio of 1.7, i.e. below the threshold ratio for a biologically significant response (set at 2 in this strain). The mean number of revertants was higher than the maximum value already noted in historical data for negative control (i.e. 178.7 vs. 143). However, this effect was not reproductible as in the second assay performed under the same experimental conditions or following the pre-incubation method, no biologically significant increases were observed at the 6 or 7 doses tested ranging from 5 or 15 to 3000 µg/plate S9+ with pre-incubation. Statistically significant increases were observed at the intermediary doses tested of 500 and 1500 µg/plate with induction ratios of 1.4, i.e. clearly below the threshold ratio for a biologically significant response, and the value observed for the neam number of revertants was within the intervals of historical data for negative control (i.e. 117 and 117.3 vs. 59-143). This effect was thus considered as not mutagenic.
In the first assay in strain TA100 in presence of hamster metabolic activation, statistically and/or biologically significant increases in the number of revertants were observed at the all doses tested ranging from 15 to 3000 µg/plate with induction ratios of 1.7 to 2.5, i.e. above the threshold ratio for a biologically significant response at the doses of 50, 150 and 500 µg/plate. The values observed for the mean numbers of revertants were higher than the highest value already noted in historical data for negative control (i.e. 226 to 256 vs.126). Moreover, this effect was reproducible as in the second assay performed in the same experimental condition, statistically and biologically significant increases were observed at the 5 doses tested ranging from 50 to 3000 µg/plate. This effect was thus considered as mutagenic.






Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results:

Assay without and with Rat S9-mix

 

 

TA1535

TA1537

TA98

TA100

TA102

ASSAY1

DOSESin

µg/plate

Induction

Ratio

(a)

DOSESin

µg/plate

Induction

Ratio

(a)

DOSESin

µg/plate

Induction

Ratio

(a)

DOSESin

µg/plate

Induction

Ratio

(a)

DOSESin

µg/plate

Induction

Ratio

(a)

 

TESTITEMWITHOUT

S9-mix

0

5

15

50

150

500

1500

-

1.0

0.9

1.0

0.9

1.2

0.8

0

5

15

50

150

500

1500*

-

1.5

1.8

0.7

0.9

1.5

0.7

0

5

15

50

150

500

1500*

-

1.1

1.1

1.0

1.3

0.9

0.5

0

5

15

50

150

500

1500

-

0.9

0.9

1.0

1.1

1.2

0.7

0

5

15

50

150

500

1500*

-

1.2

1.2

1.3

1.0

0.9

0.4

 

TESTITEM

 

WITHRATS9-mixWITHOUT

PRE-INCUBATION

0

50

150

500

1500

5000

-

0.8

0.9

0.7

0.6

0.9

0

50

150

500

1500

5000*

-

2.5

1.1

1.6

1.4

0.9

0

50

150

500

1500

3000

-

2.4

1.0

1.0

1.1

1.0

0

50

150

500

1500

3000

-

1.7

1.0

1.2

1.0

0.8

0

50

150

500

1500

3000

-

1.2

1.2

1.1

0.9

0.3

 

 

TA1535

TA1537

TA98

TA100

TA102

ASSAY2

DOSESin

µg/plate

Induction

Ratio

(a)

DOSESin

µg/plate

Induction

Ratio

(a)

DOSESin

µg/plate

Induction

Ratio

(a)

DOSESin

µg/plate

Induction

Ratio

(a)

DOSESin

µg/plate

Induction

Ratio

(a)

 

TESTITEMWITHOUT

S9-mix

0

5

15

50

150

500

1500

-

1.6

1.3

1.2

1.2

1.0

0.7

0

5

15

50

150

500

1500*

-

0.8

0.6

0.7

0.9

1.0

-

0

5

15

50

150

500

1500*

-

1.6

1.2

1.2

1.2

1.0

-

0

5

15

50

150

500

1500

-

1.1

1.1

1.2

1.0

1.0

0.8

0

5

15

50

150

500

1500

-

0.9

0.9

0.9

1.0

0.7

0.3

 

 

TESTITEM

 

WITHRATS9-mixWITH

PRE-INCUBATION

0

15

50

150

500

1500

5000

-

1.2

1.7

1.1

0.9

1.2

1.5

0

15

50

150

500

1500

5000

-

1.0

1.2

0.9

1.2

1.3

1.5

0

15

50

150

500

1500

3000

-

1.1

0.8

1.0

1.2

1.2

1.6

0

15

50

150

500

1500

3000

-

1.2

1.2

1.2

1.4

1.4

1.1

0

5

15

50

150

500

1500

-

1.3

1.2

1.3

1.4

1.6

1.0

(a)Induction Ratio=number of revertants in the treated / number of revertants in the control

-:No calculation could be performed

*Presence of microcolonies of toxicity

 

 

Conclusions:
Under these experimental conditions, DPG showed a clear mutagenic activity in both strains TA98 and TA100 exclusively in presence of hamster S9 -mix.
Executive summary:

The mutagenic activity of the test item, 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. The study was thus considered as valid. 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 ioins from the aromatic amine moiety of the test item DPG.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2014 - January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
test performed on two strains and with humanliver metabolic activation only.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His operon
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with
Metabolic activation system:
human S9-mix (Commercial human liver S9 fraction with 5 different donors)
Test concentrations with justification for top dose:
First assay (TA98, TA100): 15-50-150-500-1500-3000-5000 µg/plate
Second assay (TA98, TA100): 15-50-150-500-1500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: As the test item was not freely soluble in aqueous solvent, trials for the search of the solubility of the test item were performed in dimethylsulfoxide (DMSO) in which it was soluble at 100 mg/mL, and in ethanol in which solubility could not be observed when tested at 200 mg/mL.
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate incorporation method (first assay), pre-incubation method (second assay)
For each strain, 0.1 mL of a bacterial suspension from a culture agitated overnight at 37°C and 0.1 mL of the test item at the relevant initial concentrations were successively added to 2 mL of top agar to which 10% of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C has been added. 0.5 mL of the human S9 mix metabolic activation system was added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar. The plates were then incubated at 37°C for approximately 48h. At the end of the expression time, colonies of revertants were counted for each plate.
On a technical point of view, the pre-incubation mehod is the same as the one using agar plate incorporation with, however, the following modifications: the following solutions were added in this order: the bacterial strain to be test (100µl), human S9-mix (500 µl) and at the end the test item solution (50 µl as an organic solvent was used). The mixture was preincubated with stirring at 37°C for 60 minutes prior to adding soft agar and spreading out in a Petri plate.

NUMBER OF REPLICATIONS: three plates per dose

DETERMINATION OF CYTOTOXICITY: bacterial growth
Evaluation criteria:
Interpretation criteria of the results:
Given that the procedures followed in order to evaluate mutagenic activity are semi-quantitative, the criteria used to determine a positive effect may have been to some extent subjective and based mainly on experience. However, under the expeirmental conditions, and when the validity criteria are reached, the following decision criteria may be used.
-Criteria based on biological significance: a test item causing a positive response proportional to the dose for at least 3 doses with, the highest increase, a value greater than or equal to 2 times the value for the solvent control, is considered positive in the assay. 2
-criteria based on statistical significance.
-reproducibility: if a test item causes a positive response during a single assay and that result cannot be reproduced in at least one independant assay, the initial positive result may be considered as not significant.


Statistics:
Data were analysd by means of Dunnett's method allowing the comparison of the mean value for each dose to the mean value for the corresponding solvent control.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Toxicity in mutagenicity assays:
IN the first assay in presence of human metabolic activation system, a slight to strong toxicity was noted in both strains at the 3 or 4 highest doses tested of 500 or 1500 to 5000 µg/plate. Moreover, in both strains, no bacterial growth was obtained at the 2 highest doses of 3000 and 5000 µg/plate; instead, microcolonies of toxicity were noted. Furthermore, a statistically significant decrease in the number of revertants was noted in strain TA100 at the dose of 1500 µg/plate. Under these conditions, the highest dose to be tested in the second assay was set at 1500 µg/plate.

Mutagenicity assays:
In two independent assays performed with metabolic activation using Human liver fraction, no statistically or biologically significant increase in the mean number of the revertants was noted in the 2 Salmonella typhimurium strains TA98 and TA100 tested, in the presence of DPG. The test item was thus considered as not mutagenic under these specific conditions.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: (with human S9-mix)
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation (human S9-mix)

Under these experimental conditions, DPG is not mutagenic in strains TA98 and TA100 in presence of human S9-mix.
Executive summary:

The mutagenic activity of the test item, DPG, was assessed by means of the Ames's test in the five 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. The study was thus considered as valid. 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo, negative results were obtained 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. The ex vivo comet assay performed on hepatocytes showed negative results in rat and hamster. In conclusion, based on the weight of evidence, DPG is considered to be not mutagenic in systemic organs.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
one dose of DPG used
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Portage, Portage, MI
- Age at study initiation: 8/9 wks old
- Weight at study initiation: no data
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: no data
- Housing: one or two per cage prior dosing, and one per cage after dosing ; in suspended, stainless steel cages with stainless steel mesh bottoms
- Diet (e.g. ad libitum): Certified Rodent Diet #5002 (PMI, St Louis, MO), ad libitum
- Water (e.g. ad libitum): supplied by the public water system of St Louis, MO, ad libitum
- Acclimation period: 10 days (minimum)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79°F
- Humidity (%): 40-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
The potential for 1,3-diphenylguanidine to induce chromosomal aberrations in the bone marrow cells of Sprague-Dawley rats was tested. 
In the range-finding experiment, 2 male and 2 female rats were treated  with 1,3-diphenylguanidine at 50, 100, 200, 400, 600, 800, 1000 or 5000  mg/kg body weight. 1,3-Diphenylguanidine was found to be toxic to male rats at 400 mg/kg and higher, and toxic to female rats at 200 mg/kg and at 600 mg/kg and higher as indicated by clinical signs of toxicity and death. The combined male and female LD50 was determined to be 427.3 mg/kg by the Probit method.
Based on results from the toxicity range-finding experiments,  DPG was administered via oral gavage to 20 male and 20  female rats at a target dose of 300 mg/kg body weight (approximately 70%  of the combined LD50). Control groups received 10 ml/kg of body weight of vehicle control (corn  oil) (15 males and 15 females) or a 40 mg/kg of body weight dose of  positive control (cyclophosphamide)(5 males and 5 females). Bone marrow  was sampled at 6, 24 and 48 hours after dosing with the vehicle or  1,3-diphenylguanidine. A single sampling time of 24 hours after dosing  was used for the cyclophosphamide control group. Slides were scored for increases in the proportion of aberrant metaphases and in the frequency  of aberrations/cell.

Solutions or suspensions of the test substance were prepared on the day of use using corn oil as the vehicle. Animals were treated by a single oral gavage dose of corn oil (vehicle control, 10 ml/kg body weight). 1.3-diphenylguanidine in corn oil (10 ml of solution/kg body weight) or cyclophosphamide in 0.9% saline (positive control, 10 ml of solution/kg body weight) The positive control used was commercial grade cyclophosphamide monohydrate.
Duration of treatment / exposure:
single administration
Frequency of treatment:
single administration
Post exposure period:
72 hours
Remarks:
Doses / Concentrations:
300 mg/kg (maximum tolerated dose)
Basis:
actual ingested
No. of animals per sex per dose:
Groups treated with DPG : 20 animals/sex/dose
Control group (vehicle) : 15 animals/sex
Positive group : 5 animals/sex
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
Tissues and cell types examined:
Bone Marrow cells (femur)
Details of tissue and slide preparation:
Extraction of bone marrow cells and slide preparation:
Two to three hours prior to harvest animals were injected intraperitoneally with colchicine (4 mg/kg) to arrest dividing cells in metaphase. Animals were sacrified by CO2 inhalation. Both femurs were removed and the muscle tissue cleaned away. The bone marrow cells were aspirated from each femur into a centrifuge tube containing approximately 10 ml of PBS at 37°C. The bone marrow cells were centrifuged at 1000 rpm for 5 minutes. The PBS was decanted and approximately 10 ml of hypotonic KCl (0.075 M), prewarmed at 37°C, was added to each tube. The cells were incubated at 37°C for 12/15 minutes to swell the cells. The bone marrow cells were centrifuged at 1000 rpm for 5 minutes and the supernatant decanted. The bone marrow cells were resuspended by gently tapping the bottom of the tube. While agitating the tube, 0.5 ml of fresh Carnoy's fixative (methanol/acetic acid, 3:1, v/v) was slowly added. The cells were resuspended with a Pasteur pipette to disperse clumps. Fixative was added to each sample ti achieve a final volume of 10 ml. The cells were centrifuged, the supernatant decanted, and 5 ml of fresh fixative was added to each tube 2-3 times more times. Cells were resuspended in an appropriate volume of fixative and dropped nto clean, wet slides. Slides were dried on a slide warmer or over a flame. Slides were stained with 2% Giemsa stain.

Scoring of slides
The scoring for mitotic index and chromosomal aberrations was performed by Pharmakon USA, Waverly, PA.
To eliminate bias, all slides were coded prior to scoring. Whenever possible, 50 metaphases/animal (500 metaphases/treatment group) were scored for the presence of chromosome aberration. Both chromatid- and chromosome-type aberrations were scored. A total of 5000 cells/treatment group were evaluated for mitotic frequency. Mitotic frequency is expressed as mitotic index, which is the fraction of mitotic cells in the cell population scored.
Statistics:
Each individual test animal was the unit used for analysis of mean body weight change. A Dunnett's t-test (one sided) was used for comparison of treatment groups and positive control values with vehicle control values. Chi-square analysis was performed to compare the proportion of aberrant cells in the cells from animals treated with the test substance to those from animals treated with vehicle only. Student's t-test was used to compare structural aberrations per cell in the treatment groups with vehicle controls Results were considered statistically significant at the probability level of p<= 5.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 300 mg/kg
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the range finding study, 1,3-diphenylguanidine was found to be toxic to male rats at 400 mg/kg and higher, and to female rats at 200 and  600 mg/kg and higher.The combined LD50 was determined to be 427.3 mg/kg. based on these result, a target dose of 300 kg/kg bw (approximately 70% of the combined LD50 value) was selected as the maximum dose for male and female rats that would insure a reasonable probability of observing signs of toxicity but allow survival of the treated animals through to the 48 hour time point.

In the main cytogenetic experiment, 1,3-diphenylguanidine was toxic to male and female rats dosed at 300 mg/kg as evidenced by clinical signs of toxicity (hypoactive and non responsive) and death. Five male rats and six female rats were found dead within 24 hours of dosing. No deaths or clinical signs were observed in the control groups (vehicle and positive control). Statistically significant decreases in mean body weight were observed in the DPG treated male and female rats at 6 and 24 hours alter treatment and in the positive control treated male rats 24 hours alter treatment.
No statistically significant increases in the proportion of aberrant cells or aberrations cell were observed at the 300 mg/kg treatment level at the 6, 24 and 48 hour time points. Significant induction of toxicity, measured as mitotic index depression was observed at the 300 mg/kg treatment level at the 6 hour (35%) and 24 hour (31%) lime points. No depression in mitotic index was observed at the 48 hour time point.
The positive control group (cyclophosphamide) yielded expected positive responses indicating the adequacy of our experimental conditions for the detection of clastogens.
Conclusions:
The observations and findings of this study indicate that 1,3-diphenylguanidine was nonclastogenic. It did not induce increases in the proportion of aberrant cells or aberrations/cell under the experimental conditions utilized in this study.
Executive summary:

The potential for 1,3-diphenylguanidine to induce chromosomal aberrations in the bone marrow cells of Sprague-Dawley rats was tested. Based on results from the toxicity rangefinding experiments, 1,3-Diphenylguanidine (DPG) was administered via oral gavage to male and female rats at a target dose of 300 mg/kg body weight (approximately 70% of the combined LD50). Control groups received 10 ml/kg of body weight of vehicle control (corn oil) or a 40 mg/kg of body weight dose of positive control (cyclophosphamide). Bone marrow was sampled at 6, 24 and 48 hours alter dosing with the vehicle or DPG. A single sampling time of 24 hours alter dosing was used for the cyclophosphamide control group. Slides were scored for increases in the proportion of aberrant metaphases and in the frequency of aberrations/cell.

In the main cytogenetic experiment, DPG was toxic to male and female rats as evidenced by clinical signs of toxicity (hypoactive and nonresponsive) and death. Five male rats and six female rats were found dead within 24 hours of dosing. Statistically significant decreases in mean body weight were observed in the DPG treated male and female rats at 6 and 24 hours alter treatment and in the positive control treated male rats 24 hours afier treatment.

No statistically significant increases in the proportion of aberrant cells or aberrations/cell were observed at the 6, 24 and 48 hour time points. Significant induction of toxicity, measured as mitotic index depression. was observed at the 6 hour (35%) and 24 hour (31%) time points. No depression in mitotic index was observed at the 48-hour time point.

The positive control group (cyclophosphamide) yielded expected positive responses indicating the adequacy of our experimental conditions for the detection of clastogens.

The observations and findings of this study indicate that 1,3-diphenylguanidine was non clastogenic. It did not induce increases in the proportion of aberrant cells or aberrations/cell under the experimental conditions utilized in this study.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2015- January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
The potential clastogenic activity of 1,3-diphenylguanidine was tested using both the in vivo micronucleus test in bone marrow and the comet assay in the liver and stomach in the rat. The actual treatment was carried out by oral route, using 1 daily treatment for 3 days.
The purpose of the in vivo micronucleus test in bone marrow is to detect any clastogenic or spindle poison activity of the test item by detecting micronucleated polychromatic erythrocytes in the bone marrow of treated rats, in compliance with the OECD guideline 474 (2014).
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE
- Age at study initiation: 8 weeks
- Weight at study initiation: 171 g to 210 g
- Assigned to test groups randomly: yes, based on weight
-The animals were not fasted at the treatment time.
- Housing: The bedding consists of dust-free, irradiated softwood pellets. The animals were dispatched in polypropylene cages by random-distribution.
- Diet (e.g. ad libitum): A04C-10 from SAFE (batch 14169).
- Water (e.g. ad libitum): softened by reverse osmosis and filtered on 0.22 µm membrane, was provided ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 15 %,
- Air changes (per hr): no details
- Photoperiod (hrs dark / hrs light): lighting 12 hours a day (8 a.m. - 8 p.m.)

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: carboxymethyl cellulose (CMC; Sigma, batch SLBH9727V) at 0.5% in distilled water (Fresenius, batch 13HAP132)
- Form: suspension
Details on exposure:
In the main genotoxicity assay, three suspensions of the test item at the concentrations of 8, 4 and 2 mg/mL were prepared, giving final doses of 80, 40 and 20 mg/kg, respectively when administered at 10 mL/kg.

For the preparation of the suspensions, the powder was firstly wetted with a little volume of vehicle, agitated with the help of a vortex. The required volume of vehicle was thus added and the suspensions were mixed with a vortex for 30 seconds, then with a turrax for either 30 or 20 seconds for the toxicity and the main assay, respectively. Samplings were done under magnetic agitation.
Duration of treatment / exposure:
3 daily treatments at 24-hour intervals
Frequency of treatment:
3 daily treatments at 24-hour intervals
Post exposure period:
no
Remarks:
Doses / Concentrations:
20, 40 and 80 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males for both micronucleus and comet assays (7 animals for the highest dose, but 5 animals were retained)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (Baxter, batch 3F752C) in NaCl at 0.9% in distilled water (Aguettant-PROAMP, Batch 140070), 25 mg/kg, intraperitoneal administration.
Tissues and cell types examined:
Bone marrow
Number of polychromatic erythrocytes observed for each animal : 4000
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on the preliminary and confirmatory studies

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Number of sampling times : one
-for the negative control, the comet positive control group (methylmethane sulfonate), and the 3 treated groups: 2-6 hours after the third treatment
-for the positive control group (cyclophosphamide): 24 hours after the single treatment (micronucleus test)

DETAILS OF SLIDE PREPARATION:
At the sampling time, the animals of the CPA positive control group were sacrificed by CO2 asphyxia; the femurs were removed, and the bone marrow was extracted with foetal calf serum (1 mL per animal). The animals also used for the comet assay were anaesthetized with isoflurane (see §11.1) and
exsanguined.
Bone marrows from treated and negative control animals were sampled just before the sampling of liver and stomach for the comet assay.
The cell suspensions were centrifuged for 5 minutes at 1000 rpm. The supernatants were removed. After homogenisation, the centrifugate was spread on slides. The smears were stained using a technique, derived from the May Grunwald Giemsa technique (Schmid, 1975), which makes it possible to distinguish between polychromatic (PCE) and normochromatic erythrocytes (NCE): PCE are purple whereas NCE are red.
After blind coding of slides by a person not involved in the study, two slides per animal were scored by two independent operators; for each animal, the number of polychromatic erythrocytes having one or more Howell-Jolly bodies (micronuclei) was determined from the microscopic examination of 4000 polychromatic erythrocytes (2000 PCE/slides).
The proportion of immature among total (immature and mature) erythrocytes was determined for each animal. The polychromatic/normochromatic erythrocyte ratio was determined from the microscopic examination of 1000 erythrocytes per animal (500/slides). When analysing slides, the proportion of immature erythrocytes among total erythrocytes should not be less than 20% of the control value.

METHOD OF ANALYSIS:
The results are reported in the form of tables giving the number of micronucleated cells per 2000 polychromatic erythrocytes for each animal, together with the mean on 1000 PCE and the statistical analysis.
The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE), calculated from 1000 polychromatic erythrocytes is also established for both treated and control animals.

Evaluation criteria:
A study is accepted if the following criteria are fulfilled:
- Concurrent negative controls should be within the control limits of the distribution of the laboratory’s historical negative control database.
- The concurrent positive controls or scoring controls should induce responses that are comparable to the historical positive control data and produce a statistically significant increase compared with the concurrent negative control.
- The appropriate number of doses and cells has been analyzed.
- When toxicity is observed, the highest dose may produce toxicity in the bone marrow (e.g. a reduction in the proportion of immature erythrocytes among total erythrocytes in the bone marrow of more than 50%, but to not less than 20% of the control value).
- If death(s) is(are) observed at the tested doses, the mortality rate must be less than 20 % per group.

Interpretations of the results:
For a test item to be considered positive in the micronucleus test, it must be observed:
- At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- This increase is dose-related when evaluated with an appropriate trend test, and
- Any of these results are outside the distribution of the historical negative control data.
Positive results in the micronucleus test indicate that a substance induces micronuclei that are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. Negative results indicate that, under the test conditions, the test substance does not produce micronuclei in the immature erythrocytes of the test species.
A test substance for which the results do not meet the above criteria is considered non-mutagenic in this test.
Statistics:
The statistical comparison for the polychromatic/normochromatic erythrocyte ratio was performed using Student's t test.
Statistical analysis was performed for micronucleus number using a non-parametric test, the Mann Whitney U rank test, recommended by UKEMS (Lovell et al., 1989). Statistical analysis for micronucleus number was conducted.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
high dose limited by systemic toxicity
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PCE / NCE ratio
The ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was established at each dose level. No statistically significant decrease in the ratio PCE to NCE was noted in the 3 1,3-diphenylguanidine treatment groups when compared to the negative control group.
In consequence, no toxicity toward medullar cells was evidenced and thus, no proof of systemic exposure was brought.

Frequency of micronucleated PCE
Regarding the frequency of micronucleated polychromatic erythrocytes, no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with 1,3-diphenylguanidine at any dose, when compared with the control group.
Therefore, the test item was considered as not genotoxic under these experimental conditions.

Sampling time (3-6 H after last treatment)*

Test item

Doses in mg/kg/day (x3)

sex

PCE/NCE RATIO

Micronuclei for 1000 PCE

Kruskall Wallis

mean

Student’s t Test (p)

Mean

Mann Whitney (p)

Negative control group

Vehicle 10 mL/kg

M

1.07

 

0.60

 

-

Positive control group

Cyclophosphamide (10mL/kg – IP route) 25 mg/kg/day (x1)

M

0.74

NS

13.35

P<0.01

-

1,3-diphenylguanidine treated groups

80

M

1.48

NS

1.00

NS

NS

40

M

1.00

NS

0.70

NS

20

M

1.17

NS

0.45

NS

* 24 H after last treatment for positive control

Conclusions:
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.
Executive summary:

The potential clastogenic activity of 1,3-diphenylguanidine was tested using the in vivo micronucleus test in male rat, in compliance with

OECD Guideline No. 474, by oral route (gavage), using 3 successive daily treatments at maximum dose compatible with the toxicity of the test item, i.e. 80 mg/kg/day (x3), followed by one sampling time 2 to 6 hours after the last treatment. The two lower doses of 40 and 20 mg/kg/day (x3) were also analysed.

The ratio of polychromatic (PCE) to normochromatic erythrocytes (NCE) was established at each dose level. No statistically significant decrease in the ratio PCE to NCE was noted in the 3 1,3 -diphenylguanidine treatment groups when compared to the negative control group. In consequence, no toxicity toward medullar cells was evidenced and thus, no proof of systemic exposure was brought.

Regarding the frequency of micronucleated polychromatic erythrocytes, no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes was found in the animals treated with 1,3- diphenylguanidine at any dose, when compared with the control group.

Therefore, the test item was considered as not genotoxic under these experimental conditions.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2015- January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD 489: In Vivo Mammalian Alkaline Comet Assay
Deviations:
no
Principles of method if other than guideline:
The potential clastogenic activity of 1,3-diphenylguanidine was tested using both the in vivo micronucleus test in bone marrow and the comet assay in the liver and stomach in the rat. The actual treatment was carried out by oral route, using 1 daily treatment for 3 days.
The purpose of the in vivo Comet assay following the alkaline version (pH > 13) developed by Singh et al. (1988), is to identify those agents which induce DNA damage such as single or double DNA strand breaks (SSB or DSB), alkali-labile sites, DNA-DNA / DNA-protein cross-linking and SSB associated with incomplete excision repair sites in compliance with the OECD guideline 489 (2014). The advantages of the Comet assay include its demonstrated sensitivity for detecting low levels of DNA damage.
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE
- Age at study initiation: 8 weeks
- Weight at study initiation: 171 g to 210 g
- Assigned to test groups randomly: yes, based on weight
-The animals were not fasted at the treatment time.
- Housing: The bedding consists of dust-free, irradiated softwood pellets. The animals were dispatched in polypropylene cages by random-distribution.
- Diet (e.g. ad libitum): A04C-10 from SAFE (batch 14169).
- Water (e.g. ad libitum): softened by reverse osmosis and filtered on 0.22 µm membrane, was provided ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 15 %,
- Air changes (per hr): no details
- Photoperiod (hrs dark / hrs light): lighting 12 hours a day (8 a.m. - 8 p.m.)

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: carboxymethyl cellulose (CMC; Sigma, batch SLBH9727V) at 0.5% in distilled water (Fresenius, batch 13HAP132)
- Form: suspension
Details on exposure:
In the main genotoxicity assay, three suspensions of the test item at the concentrations of 8, 4 and 2 mg/mL were prepared, giving final doses of 80, 40 and 20 mg/kg, respectively when administered at 10 mL/kg.

For the preparation of the suspensions, the powder was firstly wetted with a little volume of vehicle, agitated with the help of a vortex. The required volume of vehicle was thus added and the suspensions were mixed with a vortex for 30 seconds, then with a turrax for either 30 or 20 seconds for the toxicity and the main assay, respectively. Samplings were done under magnetic agitation.
Duration of treatment / exposure:
3 daily treatments at 24-hour intervals
Frequency of treatment:
3 daily treatments at 24-hour intervals
Post exposure period:
no
Remarks:
Doses / Concentrations:
20, 40 and 80 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males for both micronucleus and comet assays (7 animals for the highest dose, but 5 animals were retained)
Control animals:
yes, concurrent vehicle
Positive control(s):
For the comet assay, 5 male rats studied were treated orally twice with Methylmethane sulfonate (MMS; Sigma, batch MKBR6050V) at 24-hour intervals at a dose of 100 mg/kg and once 2 to 6 hours before sacrifice at a dose of 70 mg/kg.
Tissues and cell types examined:
liver and stomach
Details of tissue and slide preparation:
Cell isolation(s)
The 5 males1 of each group were assigned for cell isolation and assessed for DNA fragmentation. Single cell preparation was done within one hour after animal sacrifice. A 'V' shaped incision was made from the centre of the lower abdomen to the rib cage. The skin and muscles was removed to reveal the abdominal cavity. A portion of the liver and the stomach was removed and washed in the cold mincing buffer until as much blood as possible has been removed. The portion was minced with a pair of fine scissors to release the cells. The cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole
cell suspension was collected. Cells were enumerated on a haemocytometer, and sufficient cells to obtain 30 x 103 and 50 x 103 hepatocytes and stomach cells, respectively, per slide were harvested from each cell suspension for proceeding to slides preparation.

Preparation of specific reagents for comet assay
Agarose
- 0.8 and 1.5% (w/v) normal agarose (NA) gel for the bottom layer. Regular melting agarose was dissolved at either 0.8 or 1.5% (w/v) in phosphate buffer (Ca++, Mg++ free and phenol free) by heating in a microwave.
- 0.5 % (w/v) low-melting agarose (LMA) gel for the cell-containing layer was dissolved at 0.5% (w/v) in phosphate buffer (Ca++, Mg++ free and phenol free) by heating in a microwave. During the study this solution was kept at 37-45°C and discarded afterward.
Mincing buffer
The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++free, and phenol red free if available), pH 7.5 (DMSO was added immediately before use). This solution was refrigerated at <10°C until use.
Lysis stock solution
The lysing solution will consist of 100 mM EDTA (disodium), 2.5 M sodium chloride, and 10 mM tris hydroxymethyl aminomethane in purified water, with the pH adjusted to 10.0 with 1 M sodium hydroxide and/or hydrochloric acid. This solution may be refrigerated at <10°C until use. On the same day of use, 1 % (v/v) of triton-X100 and 10 % (v/v) DMSO was added to this solution and the complete lysing solution was refrigerated at <10°C for at least 30 minutes prior to use.
Electrophoresis buffer
The alkaline solution consists of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water, pH >13. This solution was refrigerated at <10°C until use. The pH of the solution was measured just prior to use.
Neutralization buffer
The neutralization solution consists of 0.4 M tris hydroxymethyl aminomethane in purified water, pH 7.5. This solution was either refrigerated at <10°C or stored in accordance with manufacturer’s specifications until use.
Staining stock solution
The staining stock solution will contain 1 mg/mL of propidium iodide in distilled water. Just prior to use, the staining stock solution was diluted 1/50th in distilled water to obtain the final staining solution (final concentration: 20 µg/mL).
Dried slides preparation (pre-layering)
Conventional slides were dipped in hot 1.5 % normal melting point agarose in PBS. After gentle removal, the underside of the slides was wiped in order to remove excess agarose. The slides were then laid in a tray on a flat surface to dry.
Slide preparation
Before use, a volume of 85 µL of 0.8% of Normal Agarose (NA) was added on the microscope slide prelayered with 1.5% of NA and then covered with a glass coverslip. Slides were placed at +2.8°C until the agarose layer hardens. The cells of the different doses tested were mixed with 0.5% of Low Melting Point Agarose (LMPA) (75 µL/slide) kept at ca. 37 °C and added on the microscope slide after gently sliding off the coverslip. The slides were then covered with a new glass coverslip, and were placed once again at +2.8°C.
Four slides per animal were prepared for the Comet assay.

Determination of the cytotoxicity of the test item
In the main assay conducted in the liver, the frequency of hedgehogs in the control group was inferior to 50%.
Otherwise, a statistically significant increase in the frequencies of hedgehogs was noted at 80 mg/kg/day (x3) with a value of 17.29% vs. 10.35% in the respective control. As this increase was not concurrent to an increase in percent tail DNA, it was probably due to a slight toxic effect of the test item and had no incidence in terms of genotoxicity.
In the main assay conducted in the stomach, the frequency of hedgehogs in the control group was inferior to 50%.
Otherwise, statistically significant increases in the frequencies of hedgehogs were noted at 40 and 80 mg/kg/day (x3) with values of 27.54 and 32.82% vs. 18.45% in the respective control. These increases were dose-related.

Protocol for the Comet assay
Lysis:
After the top layer of agarose has solidified, the glass coverslips were removed and the slides were immersed for at least 1 hour at ca. + 4 °C in the dark in a lysing solution consisting of 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, to which 1% Triton X-100 and 10% DMSO was freshly added (pH adjusted to 10 with NaOH).
Unwinding, electrophoresis and staining:
After this incubation period, the slides were then removed and placed on a horizontal gel electrophoresis unit and the unit filled with freshly prepared alkaline buffer (1 mM EDTA and 300 mM NaOH, pH > 13) to around 0.25 cm above the slides. In order to avoid excessive variation across the groups during each electrophoretic run, only one of the replicate slides were processed in each run for each animal (DNA –unwinding and electrophoresis). The cells were exposed to the alkali for 20 minutes to allow the DNA unwinding, and expression of single-strand breaks and alkali-labile sites. Next, electrophoresis was conducted for 20 minutes at <10°C by applying an electric current of 0.7 V / cm (25 V / 300 mA). All these steps were conducted protected from daylight to prevent the occurrence of additional DNA damage. After electrophoresis at pH >13, the slides were neutralized twice for 5 minutes with 0.4 M Tris (pH 7.5) and the DNA was exposed for 5 minutes to absolute ethanol in order to preserve all the Comet assay slides. Subsequently, the slides were airdried and then stored at room temperature until they were scored for DNA migration.
Image analysis:
Just prior to scoring, the DNA was stained using propidium iodide (final concentration of 20 µg/mL distilled water; 25 µL/slide).
Coded slides respectively were examined with a 200 x magnification, using a fluorescent microscope (Leica Microsystems SAS – DM 2000, Heerbrugg,
Switzerland), equipped with an excitation filter of 515-560 nm and a barrier filter of 590 nm, connected through a gated monochrome CCD IEEE1394 FireWire video camera (Allied Vision Technologies), to the Comet Assay IV Image Analysis System, version 4.11 with Windows XP Pro Software (Perceptive Instruments Ltd, Suffolk, UK). For all groups three slides were analysed with 50 nuclei per slide randomly scored. Five animals were retained per group, i.e. at least 15 slides per group, at least 750 analysed nuclei per group.
Tail parameters:
Recent publications focused on the interpretation of the results through the analysis of the median of the percentage of DNA in tail, with the animal as statistical unit (D. Lovell and T. Omori, 2008). In fact, this parameter appears to be the most linearly related to dose (B. Burlinson et al., 2007).

Expression of the results
The results obtained in the different treatments are presented in tabular form giving for the liver and for the stomach:
- the median per slide of the percentage of DNA in tail for at least 50 cells,
- the mean of medians of the percentage of DNA in tail per animal (i.e. 3 slides, 150 cells),
- the mean of median per concentration (i.e. 5 animals, 750 cells).
In addition, each slide was also examined for presence of hedgehogs (possible indicator of toxicity and/or apoptosis). Hedgehogs were excluded from image analysis data collection. However, determining their frequency might be useful for data interpretation. Therefore, the percentage of hedgehogs was recorded for
each slide per animal, and per organ. The hedgehogs, also known as clouds or ghost cells, are morphological indicative of highly damaged cells often associated with severe genotoxicity, necrosis and/or apoptosis. A hedgehog results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum.
Evaluation criteria:
A study is accepted if the following criteria are fulfilled:
- Concurrent negative controls should be within the control limits of the distribution of the laboratory’s historical negative control database. In the current study, the mean value for the negative control in the stomach (i.e. 11.87) was out of the limits of the distribution of the laboratory’s historical negative control database. However, it is to be underlined that this value is within the intervals of extreme values already noted in the laboratory historical data (i.e. 0.74
– 19.03).
- The concurrent positive controls or scoring controls should induce responses that are comparable to the historical positive control data and produce a statistically significant increase compared with the concurrent negative control.
- The appropriate number of doses and cells must be analysed. Moreover:
- In the vehicle group, an eventual increase in the frequency of hedgehogs, must not be >50%.
- If death(s) is(are) observed at the tested doses, the mortality rate must be less than 20 % per group. The dead animals from the high dose treated group are replaced by those in parallel treatment.


Statistics:
In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5. As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups. The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control. This test was also used to compare vehicle control and positive control to determine acceptable criteria of a valid test.
Sex:
male
Genotoxicity:
negative
Remarks:
on liver cells
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Sex:
male
Genotoxicity:
other: equivocal on stomach cells
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Liver cells
No statistically significant increases in the median percentage of DNA in tail at the three analysed doses of 80, 40 and 20 mg/kg/day (x3), vs. the negative control. Indeed, the values for the mean of medians of percentage of DNA in tail were of 0.36, 0.26 and 0.49% at 80, 40 and 20 mg/kg/day (x3), respectively vs. 0.40% in the concurrent negative control.
The test item 1,3-diphenylguanidine was thus considered as not genotoxic in liver from rats. Indeed, no primary DNA damage was detected in this organ.

Stomach cells:
No statistically significant increase in the mean of medians of percentage of DNA in tail was observed at the tested doses of 20, 40 and 80 mg/kg/day (x3) of 1,3-diphenylguanidine in stomach from OFA Sprague- Dawley male rats with values of 4.8, 11.09 and 27.22%, respectively vs. 11.87% in the relative control.
A statistically significant dose-related increase in the mean of medians of percentage of DNA in tail was noted (Kruskall-Wallis p <0.05). Moreover, at the highest dose tested of 80 mg/kg/day (x3) of 1,3-diphenylguanidine, the mean percentage of DNA in Tail was clearly higher than the maximum value already observed in historical data for negative control (27.22% vs. 19.03%). Therefore, although this effect could be considered as biologically significant, it was not statistically significant in the pair-wise statistical assessment. This may be explained by the heterogeneity noted in the distribution of the data. Indeed, in the vehicle control, the values of 2 slides from 1 animal (out of 5) were clearly over the range observed in this group (see animal No. 4652 with values of 29.98 and 48.25 % for slides Nos. 30B and 30C, respectively vs. 2.13 for the slide No. 30A and means ranging from 5.61 to 11.56 for the 4 other animals). Moreover, in the high dose group, only 2 animals out of 5 presented a value for the percentage of DNA in tail over the highest value noted in the concurrent negative control, i.e. 43.16 and 46.57% for animals Nos. 4667 and 4665 vs. 26.79% for the control animal No. 4652.
Finally, it is noteworthy that concurrent statistically significant increases in the incidence of hedgehogs were noted with ratios of 1.05, 1.49 and 1.78 at 20, 40 and 80 mg/kg/day (x3), respectively.
Therefore in order to allow discrimination between necrosis or apoptosis and an actual genotoxic potential of the test item, a histopathological study was performed. For this, a histopathological analysis on the pieces of stomach sampled on animals used in the main assay was performed.
Otherwise, a statistically significant decrease in the mean of medians of percentage of DNA in tail per slide was noted in the group treated with 20 mg/kg/day (x3) of 1,3-diphenylguanidine. However, this not dose-related increase is devoid of genotoxic hazard.
As (i) the increase in Tail DNA was statistically dose-related, and (ii) the mean percent tail DNA in the 80 mg/kg/day (x2) highest dose group was outside the distribution of the historical data for vehicle control, but (iii) none of the treatment group exhibited a statistically significant increase in the mean of medians of percentage of DNA in tail compared with the concurrent negative control, only 2 out of the 3 criteria for a positive response were met. Results regarding the genotoxic potential of the test item toward the stomach are thus considered as equivocal.

Histopathological study in the stomach
A provision for histopathological assessment was done to assess the biological relevance of the equivocal results.
The histopathology study failed at demonstrating any significant occurrence of necrosis and/or apoptosis.
This indicates that the increase in the percentage of DNA in tail is not due to cytotoxicity interference.

Quantification of apoptosis in the stomach
The results for the quantification of apoptosis in sections of stomach demonstrated no significant incidence of apoptosis, as detected by immunohistochemistry. Indeed, 0.58, 0.24 and 0.25% of apoptotic cells were seen at 80, 40 and 20 mg/kg/day (x3) of 1,3-diphenylguanidine vs. 0.17% in the concurrent control. The results were not statistically significant. When expressed as density of labelled cells per mm², the occurrence of apoptosis was of 1.95, 1.13 and 1.25 x 10-² cells/mm² at 80, 40 and 20 mg/kg/day (x3) vs. 0.75 in the vehicle control group.
Therefore, the quantification of apoptotic cells through a specific method failed at demonstrating any significant occurrence of apoptosis.

Comet assay in the liver

Number of cells observed: 150

Number of cells observed per dose: 750

Group

Test item

Doses in mg/kf/day (x3)

% of DNA in tail. Mean of medians per animal (/5 animals)

Non parametric statistical assessment

Hedgehogs

P Kruskall-Wallis

P Mann Whitney

Relative ratio of heghogs

p

Negative control

CMC at 0.5% in distilled water

0

0.40

NS

-

-

-

Treated

1,3-diphenylguanidine

80

0.36

NS

1.67

<0.001

40

0.26

NS

1.04

NS

20

0.49

NS

1.09

NS

Positive control

Methylmethane sulfonate

100 mg/kg/day (x2), 70 mg/kg/day (x1)

59.92

-

<0.01

1.86

<0.001

 

Comet assay in the Stomach

Number of cells observed: 150

Number of cells observed per dose: 750

Group

Test item

Doses in mg/kf/day (x3)

% of DNA in tail. Mean of medians per animal (/5 animals)

Non parametric statistical assessment

Hedgehogs

P Kruskall-Wallis

P Mann Whitney

Relative ratio of heghogs

p

Negative control

CMC at 0.5% in distilled water

0

11.87

<0.05

-

-

-

Treated

1,3-diphenylguanidine

80

27.22

NS

1.78

<0.001

40

11.09

NS

1.49

<0.001

20

4.80

<0.05

1.05

NS

Positive control

Methylmethane sulfonate

100 mg/kg/day (x2), 70 mg/kg/day (x1)

75.06

-

<0.01

1.40

<0.001

 

 

 

 

Conclusions:
Taking into account the overall results, it is concluded that the genotoxic potential of 1,3-diphenylguanidine is equivocal toward stomach in OFA Sprague-Dawley male rats as investigated by the in vivo Comet assay.
Executive summary:

The genotoxic potential of 1,3-diphenylguanidine was investigated in the in vivo comet assay performed under alkaline conditions, i.e. pH > 13 (Alkaline Single Cell Gel Electrophoresis) in the liver and the stomach of male rats in compliance with OECD Guideline 489 (2014) using 3 successive daily treatments at the maximum dose compatible with the toxicity of the test item, i.e. 80 mg/kg/day (x3), followed by one sampling time 2 to 6 hours after the last treatment. The two lower doses of 40 and 20 mg/kg/day (x3) were also analysed.

1,3-diphenylguanidine does not present DNA strand breaks and/or alkali-labile sites inducer activities toward the liver from OFA Sprague-Dawley male rats.

In return, 1,3-diphenylguanidine induced a significant increase 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 was statistically dose-related,

- the mean percent tail DNA in the 80 mg/kg/day (x2) highest dose group was outside the distribution of the historical data for vehicle control,

- none of the treatment group exhibited a statistically significant increase in the mean of medians of percentage of DNA in tail 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. Therefore, the increase in the percentage of DNA in tail is not

due to cytotoxicity and/or apoptosis interference but is probably due to an intrinsic genotoxic activity of the test item toward the stomach.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro assays

Ames tests :

In the first key 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 key study (Simar 2015), the mutagenic activity of the test item, 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.

Several supporting Ames studies are available and shows the same results: negative results with and without rat S9 (Enamoto 2000, JETOC 1996, Mortelmans et al., 1986; Irwin 1995), and questionable results with hamster S9 (Mortelmans et al. 1986)

In vitro mammalian cells tests :

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 test item 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 test item 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 assays

Key study : Combined in vivo micronucleus/comet assay in rat (Simar 2016b/c)

The test item 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. Moreover, toxicokinetics data included in the section 7.1 of the IUCLID show clearly that DPG is rapidly and completely absorded after oral administration in rats and widely distributred through the various tissues. 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 present 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.

Supporting in vivo studies

The potential for 1,3-diphenylguanidine to induce chromosomal aberrations in the bone marrow cells of Sprague-Dawley rats was tested (Kier 1998). Based on results from the toxicity range-finding experiments, 1,3-Diphenylguanidine (DPG) was administered via oral gavage to male and female rats at a target dose of 300 mg/kg body weight (approximately 70% of the combined LD50). Control groups received 10 ml/kg of body weight of vehicle control (corn oil) or a 40 mg/kg of body weight dose of positive control (cyclophosphamide). Bone marrow was sampled at 6, 24 and 48 hours alter dosing with the vehicle or DPG. A single sampling time of 24 hours alter dosing was used for the cyclophosphamide control group. Slides were scored for increases in the proportion of aberrant metaphases and in the frequency of aberrations/cell.

In the main cytogenetic experiment, DPG was toxic to male and female rats as evidenced by clinical signs of toxicity (hypoactive and nonresponsive) and death. Five male rats and six female rats were found dead within 24 hours of dosing. Statistically significant decreases in mean body weight were observed in the DPG treated male and female rats at 6 and 24 hours alter treatment and in the positive control treated male rats 24 hours afier treatment.

No statistically significant increases in the proportion of aberrant cells or aberrations/cell were observed at the 6, 24 and 48 hour time points. Significant induction of toxicity, measured as mitotic index depression was observed at the 6 hour (35%) and 24 hour (31%) time points. No depression in mitotic index was observed at the 48 hour time point. The positive control group (cyclophosphamide) yielded expected positive responses indicating the adequacy of our experimental conditions for the detection of clastogens.

The observations and findings of this study indicate that 1,3-diphenylguanidine was non clastogenic. It did not induce increases in the proportion of aberrant cells or aberrations/cell under the experimental conditions utilized in this study.

The micronucleus test on erythrocytes of the peripheral blood of mice, that obtained feed with a DPG- content of 250, 500, 750, 1500 and 3000 ppm in the subchronic test, led to a negative result in the male animals. The test result of the females was evaluated as "questionable" because of the statistically significant increase of the micronuclei-carrying normochromatic erythrocytes in the female animals of the middle concentration group (750 ppm) (Irwin 1995).

Justification for classification or non-classification

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 is 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 orequivocal data,a weight of evidence approach using expert judgment has to be applied. " (page 358)

Based on negative results obtained inin vitro(OECD 473) and in vivo (OECD 474/475) tests, there are no concern of clastogenicity and aneugenicity with DPG.

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 metabolized 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 metabolites didn’t induce genotoxicity on mammalian cells. This indicates that the hamster liver cells may metabolized 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 are sufficient evidence to conclude that DPG is not able to induce heritable mutations in somatic cells by systemic exposure. Moreover, DPG didn’t induce a direct toxicity to the reproductive organs in repeated dose toxicity studies, a genotoxic effect on the germ cells is also not expected.

So, in conclusion, the hazard classification for germ cell mutagenicity (category 2) according to the Regulation EC 1272/2008, is not relevant for DPG.