N,N'-diphenylguanidine monohydrochloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guideline for 28-day repeated dose toxicity in mammalian species (Chemical Substances Control Law of Japan)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

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:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River jpan Co. (Yokohama, Kanagawa Prefecture)
- Age at study initiation: 5 weeks of age
- Weight at study initiation: males=129-139g, females=103-119g
- Fasting period before study: no data
- Housing: individually in cages with aluminium faces
- Diet: NIH open formula rat and mouse ration manufactured by Oriental Yeast Co. Ltd, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 13+/-3
- Humidity (%): 55+/-20
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12 (lighted at 7 am)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days 4 weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, 1 and 5 hours after administration, 3 times a daily during the administration period, and 2 times a day during the recovery period (morning and afternoon).
DETAILED CLINICAL OBSERVATIONS: Yes, on all living of the animals scheduled for necropsy at the end of the administration period and at the end of the recovery period.
BODY WEIGHT: Yes, weekly from the beginning of administration to the end of the recovery period.
FOOD CONSUMPTION & FOOD EFFICIENCY: Yes, weekly
WATER CONSUMPTION : No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on all living of the animals scheduled for necropsy at the end of the administration period and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes with ether
- Animals fasted: Yes, 16 hours for blood sampling
- How many animals: 5/sex or all survivors
- Parameters examined : hematocrit, hemoglobin, red blood cells, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobine concentration, platelets, white blood cells, white blood cell percentage + blood coagulation examination

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on all living of the animals scheduled for necropsy at the end of the administration period and at the end of the recovery period.
- Animals fasted: Yes, 16 hours for blood sampling
- How many animals: 5/sex or all survivors
- Parameters examined : total protein, albumin, A/G, glucose, neutral fats, total cholesterol, blood urea nitrogen, creatinine, T.bilirubin, aspartate aminotransferase, alanine transaminase, alkaline phosphatase, calcium, inorganic phosphorus, sodium, potassium, chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: for 3 hr (10am to 1pm) and 24 hr (10 am to 10 am on the following day)
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- How many animals: 5/sex or all survivors
- Parameters examined (3 hr) : pH, accult blood, ketone bodies, glucose, protein, bilirubin, urobilinogen.
- Parameters examined (24 hr) : urine volume, color, urine specifique gravity, urine sediments.

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: Organ weights

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: brain, lungs, liver, kidney, adrenals, thymus, heart, spleen, testes, epididymis and ovaries
HISTOPATHOLOGY: Yes
Thymus, heart, lungs (including trachea), larynx, liver, spleen, kidney, adrenals, stomach, testes, epididymis, uterus, ovaries, bone marrow (femur), brain (including the brain stem), spinal cord, sciatic nerve and target organs

Other examinations:

no

Statistics:

The body weights, food consumption, food efficiency, hematological examination values, blood coagulation examination values, blood chemical examination values, urine examination values (urine volume and specific gravity), organ weights and organ weight/body weight ratio were subject to Bartlett’s distribution testing. Equal distribution was determined using the significant differences between the control groups and each of the administration groups according to Dunnett’s multiple comparison test. Inequal distribution was determined using the significant differences between the control groups and each of the administration groups according to Bartlett’s distribution test. For inequal distributions using Bartlett’s distribution test, the significant differences between the control groups and each of the administration groups were determined according to Steel’s test.
The significant standards for the quantitative values were conducted using 5 and 1% single assays.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: significantly high values for platelet counts in females as well as significantly low values for blood glucose in males in the 30 mg/kg group

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1 : Hematology of rats treated orally with 1,3-diphenylguanidine in the twenty-eight-day repeated dose toxicity test

	28 -day dosing groups (mg/kg)				14 -day recovery groups (mg/kg)
Item	0	10	30	90	0	90
MALE
No. ofanimals	5	5	5	5	5	4
HCT(%)	46.2± 2.6N	45.4±0.4	45.6±0.7	46.4±1.7	47.0±1.7	45.5±2.1
HGB(g/dL)	14.9±0.7	14.7±0.3	15.1±0.2	15.4±0.5	15.9±0.4	15.4±0.5
RBC(X10^6/mm3)	7.56 ±0.51	7.50±0.27	7.62±0.24	7.91±0.27	8.29±0.43	7.91±0,32
MCV (µm3)	61,1±1.1	60.6±2.1	59.9±1.4	58.7±1.7*	56.7±1.7	57.4±0.7
MCH (pg)	19,7±0.6	19.7±0.7	19.8±0.6	19.5±0.5	19.2±0.7	19.5±0.4
MCHC(%)	32.3±0.5	32.5±0.6	33.1±0.4*	33.2±0.5*	33.8±0.4	33.9±0.6
PLT(x10^3/mm^3)	1188±111	1118±78	1153±86	1118±76	1121±59	1217±109
WBC(x10^3/mm^3)	13.7±1.9	11.0±2.5	13.2±3.6	11.8±2.8	13.8±4.3	12.4±3.0
Differential leukocyte Counts (%)
NEUT	9±4	11±3	11±4	13±5	10±3	11±2
LYMPH	88±4	85±3	86±4	83±5	85±3	85±2
MONO	1±1	2±1	1±1	2±1	2±0	2±1
EOSN	1±1	1±0	1±1	1±0	1±0	1±1
BASO	0±0	0±0	0±0	0±0	0±0	0±1
LUC	1±0	1±0	1±0	1±0	1±0	1±0
FT (sec.)	14.2±0.5	14.2±0.6	14.7±0.8	14.5±0.5	14.8±0.8	14.1±0.5
APTT(sec.)	25.5±1.6	26.4±0.8	25.8±16	25.0±1.6	27.6±1.6	25.7±1.9
Fibrinogen (mg/dL)	212±12	222±7	214±8	209±24	229±16	222±17
FEMALE
No.ofanimals	5	5	5	1	5	2
HCT(%)	44.4±1.4	44.0±2.3	43.5±1.4	49.39	43.4±1.6	41.9±5.4
HGB(g/dL)	15.0±0.6	14.8±0.5	14.6±0.4	16.7	15.4±0.5	14.8±1.5
RBC(X10^6/mm3)	7.62±0.20	7.59±0.53	7.55±0.25	8.30	7.91±0.22	7.51±0.91
MCV (µm3)	58.3±2.1	58.1±1.4	57.7±2.0	59.4	54.8±0.9	558±0.4
MCH (pg)	19.7±0.8	19.6±0.8	19.4±0.5	20.2	19.4±0.5	19.7±0.4
MCHC(%)	33.8±0.5	33,6±0.7	33.6±0.6	33.9	35.5±0.6	35.2±1.0
PLT(x10^3/mm^3)	1213±83	1308±57	1436±117**	1412	1158±122	1114±192
WBC(x10^3/mm^3)	7.3±2.1	7.3±1.0	6.1±2.0	7.5	6.7±1.9	5.6±2.2
Differential leukocyte Counts (%)
NEUT	12±5	12±7	12±5	41	10±3	14±4
LYMPH	85±5	84±6	85±5	50	86±2	83±4
MONO	1±1	2±1	2±1	7	2±0	1±0
EOSN	1±0	2±1	1±1	0	1±0	1±0
BASO	0±0	0±0	0±0	0	0±0	0±0
LUC	1±1	1±0	1±1	3	1±0	1±0
FT (sec.)	14.8±0.6	15.0±0.34	15.0±0.3	13.8	14.9±0.4	15.1±1.3
APTT(sec.)	23.0±0.8	21.8±1.2	22.2±0.9	25.0	22.2±1.5	23.5±3.6
Fibrinogen (mg/dL)	194±10	191±20	183±11	285	176±15	179±18
NEUT : Neutrophil, LYMPH : Lymphocyte,MONO:Monocyte,EOSN: Eosinophil, BASO: Basophil,LUC:Large unstained cells. Valuesareexpressedas Mean±S.D. Significantdifferencefrom control group; *p<0.05, ** p<0.01 N:Nonparametric analysis

Table 2 : Blood chemistry of rats treated orally with 1,3-diphenylguanidine in the twenty-eight-day repeated dose toxicity test

		28 -day dosinggroups (mg/kg)			14-dayrecovery groups (mg/kg)
Item	0	10	30	93	0	90
MALES
No.ofanimals	5	5	5	5	5	4
Glucose (mg/dL)	157±7	156±12	136±19*	119±12**	152±16	135± 5*
T.cholesterol (mg/dL)	55±9	59 ± 15	55±11	66±20	63±8	55± 17
Triglycéride(mg/dL)	38.2±21.1	46.4±23.1	41.3±14.2	21.5± 6.8	50.4±6.2N	37.0± 28.3
BUN(mg/dL)	10.5±1.3	10.0±1.3	11.2±1.5	13.7±1.2**	13.5±1.1	13.5±1.3
Creatinine (mg/dL)	0.21±0.03	0.22±0.02	0.21±.0.03	0.21±0.02	0.22±0.02	0.23±0.06
T.bilirubin(mg/dL)	0.03±0.01	0.03±0.01	0.03±0.01	0.06±0,01**	0.04±0.01	0.04±0.01
T.protein(g/dL)	5.66±0.21	5.75±0.12	5.46±0.20	5.55±0.20	5.82±0.18	5.59±0.18*
Albumin(g/dL)	3.42±0.15	3.38±0.11	3.37±0.14	3.55±0.11	3.36±0.16	3.29±0.14
A/G	1.53±0.10	1.42±0.08	1.61±`0.11	1.80±0.21**	1.36±0.07	1.44^.±0.08
AST(U/L)	70±7	64±6	69±10	68±8	68±5	87±10**
ALT (UIL)	24±4	23±4	26±4	39± 6**	27±3	29±2
ALP(U/LI	784 ±116	781±176	929±208	1156±308*	608±97	617±140
FEMALES
No. ofanimals	5	5	5	1	5	2
Glucose(mg/dL)	126±15	126±9	116±11	107	132±2N	131±21
T.cholesteroi(mg/dL)	54±10	59±15	72 ±5*	73	65±9	59±4
Triglyceride(mg/dL)	13.1±6.9	21.4± 9.5	27.6±12.9*	15.8	30.2± 20.4N	11.4±0.7
BUN(mg/dL)	15.3±3.6	14.7±1.8	14.2±2.7	12.5	15.8±2.2	16.1±2.9
Créatinine (mg/dL)	028±0.06	0.27±0.02	0.26±0.03	0.21	0.31±0.03	0.31±0.03
T.bilirubin(mg/dL)	0.03±0.01	0.04±0.01	0.03±0.01	0.01	0.06±0.02	0.08±0.01
T.protein(g/dL)	5.87±0.12	5.67±0.12*	5.78± 0.15	6.70	5.85±0.18	5.81±0.08
Aibumin(g/dL)	3.62±0.16	3.48±0.16	3.58± 0.10	4.03	3.61±0.17N	3.60 ± 0.01
A/G	1.61±0.14	1.59±0.15	1.63±0.06	1.51	1.62 ±0.10	1.63±0.05
AST(WL)	70±10	70±8	80±7	80	72±10	88±8
ALT(LT/L)	19±3	21±3	24±3*	28	26±3 N	36±16
ALP(U/L)	466 ±40	488±98	424±37	521	328± 58	312±78
Values are expressed asMean ± S.E. Significantdifferencefrom controlgroup : *p<0.05 ; **p<0.01  N:Non parametric analysis

Applicant's summary and conclusion

Conclusions:

The NOEL for repeat dose toxicity is considered to be 10 mg/kg/day for both sexes.

Executive summary:

The repeated dose toxicity of DPG in corn oil was evaluated atdaily dose levels of 0, 10, 30 and 90 mg/kg in a 28-day oral repeated dose toxicity study in Crj:CD(SD) rats performed following the OECD guideline 407 and GLP. Five each of males and females were assigned to each group, and recovery groups were formed with five each of males and females, assigned to the control group and the maximum dose group. Clinical signs, body weight, food consumption, hematology, blood coagulation, blood chemistry, urinalysis, organ weights and histological examinations were conducted.

Dead or moribund animals were observed in both sexes in the 90 mg/kg group during the administration period.One male died during week 4, and 7 females died or were moribund during weeks 2~4, so the mortality rate was 10% for males and 70% for females. The results of the pathological examination did not exhibit any changes correlating to the cause of death. During observations on the general conditions, salivation was observed in both sexes in the 30 mg/kg Clinical symptoms including salivation, lying prone on the stomach, lying laterally, staggered gait, reduction in spontaneous motor activity and startle response were observed in both sexes in the 90 mg/kg group. Furthermore, in the dead or moribund females in the 90 mg/kg group, emaciation, fur loss on the rear limbs and dirty fur were observed. The symptoms observed during the administration period were not observed during the recovery period, and indicates reversibility. During the administration period, significantly low values or a tendency of low values was noted for body weight and food consumption in both sexes in the 90 mg/kg group. Also, significantly low values or a tendency of low values was noted for weight gain and total food consumption during the 4^thweek of administration. In males, significantly low values were observed for the mean food efficiency at the 4^thweek of administration. Reversibility from these changes was noted during the recovery period.The results of the hematological examination revealed no impact of the administration of the test substance on males.In females, significantly high values were noted for platelet counts in the 30 mg/kg group. There was one survivor in the 90 mg/kg group where a trend of high values was noted for hematocrit values, hemoglobin, red blood cell count, platelet count, neutrophil ratio, monocyte ratio and large unstained cell ratio, and a trend of low values was noted for lymphocyte ratio.At the end of the recovery period, neither sex exhibited any significant differences between the control group and the test substance group. The results of the blood coagulation examinations revealed one surviving case where a trend of high values were noted for fibrinogen by the end of administration in females in the 90 mg/kg group, but if this was excluded, differences were not noted between the control group and the test substance group for both sexes. Blood chemistry examination revealed significantly low values in blood glucose in males in the 30 and 90 mg/kg groups, and significantly high values in blood urea nitrogen, total bilirubin, A/G ratio, ALT and alkaline phosphatase in males in the 90 mg/kg group. A tendency for high values in total cholesterol, total protein, albumin, sodium, chloride, calcium, inorganic phosphorus and ALT was noted in one survivor by the end of administration in females in the 90 mg/kg group. At the end of the recovery period, slightly low values were noted in the males in the 90 mg/kg group. Urinalysis revealed an increase in urine volume and a decrease in specific gravity in both sexes in the 90 mg/kg group. Furthermore, there was an increase in ketone bodies and negative protein. At the end of the recovery period, significantly low values were noted in specific gravity in males in the 90 mg/kg group. Results of the organ weight measurements did not indicate differences that suggest a clear impact of administration of the test substance. Histopathological examination results and necropsy findings revealed emaciation in the moribund and dead animals. Additionally, while the toxicological significance is not clear, the liver turned brown in males in the 30 mg/kg group and in both sexes in the 90 mg/kg group, while the eardrums were observed to turn red in both sexes in the 90 mg/kg group. Pathological examinations revealed hydropic changes in renal collecting tubules in both sexes in the 90 mg/kg group. In the dead and moribund animals, there was a higher rate of incomplete uterus findings. However, there were only a few cases but atrophied or nuclear decay of the thymus, hydropic or hemorrhagic lung, liver congestion or necrosis, or dilation of the kidney was also noted. Furthermore, while the toxicological significance is not clear, a reduction in the fatty change in the liver was noted in males in the 30 and 90 mg/kg groups.

From the results given above, since salivation was noted in both sexes and there were significantly high values for platelet counts in females as well as significantly low values for blood glucose in males in the 30 mg/kg group, the NOEL is considered to be 10 mg/kg/day for both sexes.