N,N'-diphenylguanidine monohydrochloride

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

1,3-diphenylguanidine

EC Number:

203-002-1

EC Name:

1,3-diphenylguanidine

Cas Number:

102-06-7

Molecular formula:

C13H13N3

IUPAC Name:

1,3-diphenylguanidine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system 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: A04C-10 from SAFE (batch 14169).
- Water: softened by reverse osmosis and filtered on 0.22 µm membrane, was provided ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 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.)

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

80 mg/kg bw/day (actual dose received)

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 bw and once 2 to 6 hours before sacrifice at a dose of 70 mg/kg bw.

Examinations

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.

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

on liver cells

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Sex:

male

Genotoxicity:

other: equivocal on stomach cells

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

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.

Any other information on results incl. tables

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

 

 

 

 

Applicant's summary and conclusion

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.