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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998
Reference Type:
secondary source
Title:
SIDS Initial Assessment report for SIAM 14 - 1,3-diphenylguanidine
Author:
Anonymous
Year:
2002
Bibliographic source:
OECD - Paris, France, 26-28 March 2002

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.