1,3-diphenylguanidine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

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:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: 6/7 weeks old at the beginning of study
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: five per cage
- Diet: NIH-07 Open Formula Diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum
- Water (e.g. ad libitum): ad libitum (not precised)
- Acclimation period: 11-15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 69° to 75°F
- Humidity (%): 35 to 65%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h of fluorescent light

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity analyses of a 250 ppm DPG feed mixture were conduted at MRI; analyses with HPLC indicated a maximum variation in concentration of 1.6% among three samping points. Analyses conduted by the study laboratory on feed mixtures of DPG with reverse-phase HPLC confirmed the homogeneity of the mixtures.
Because of the limited stability of the DPG feed mixtures, feeders were changed daily, 7 days per week and 13-weeks studies.
The stability of DPG feed mixtures was evaluated by HPLC. These analyses indicated that a 30 ppm DGP feed mixture was stable for 3 weeks when stored in the dark à 5 °C, and a feed mixture of 250 ppm dPG was stable for 3 weeks in the dark at -20°C. Analyses of 250 ppm feed miwtures after 7 and 14 days of storage in the dark at -20°C indicated minor (2.8% and 5.2% respectively) but statistically significant chemical losses at both time points. The small differences were attributed to analytical variation. Both the 30 and 250 ppm feed mixtures showed significant losses of DPG (6 and 11% respectively) after 14 day of storage under animal room conditions. Feeders were changed daily, 7 days per week in the 13-week studies.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

ad libitum

Doses / concentrations

Remarks:

Doses / Concentrations:
250, 500, 750, 1500 and 3000 ppm (17, 32, 50, 100, 181 mg/kg/d in males and 17, 32, 49, 95, 184 mg/kg/d in females)
Basis:
nominal in diet

No. of animals per sex per dose:

10 animals/ dose/sex
Additional rats (10 males and 10 females per exposure group) were used in a supplement clinical pathology study.

Control animals:

yes, concurrent no treatment

Details on study design:

DPG was mixed with NIH-07 Open Formula Diet in powder from.
A premixwith was prepared for each exposure concentration by mixing a weighed amount of DPG in a beaker with an equal weight of feed. The premix was then blended with a weighed amount of feed in a twin-shell blender for 15 minutes, with the intensifier bar on for the first 5 minutes.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Observed twice daily for mortality/morbidity and clinical signs of toxicity. Clinical observations were recorded weekly. Individual body weights were recorded at the start of the studies, weekly thereafter, and at the end of the studies, weekly thereafter, and at the end of the studies. feed consumption was recorded daily for 5 consecutive days per week for 13 weeks.

Sacrifice and pathology:

Necropsies were performed on all animals in the base studies. The heart, right kidney, liver, lungs, ovaries, prostate gland, seminal vesicles, spleen, right testis, and thymus were weighed at necropsy.
Histologic examinations were performed on all animals in the 1500 ppm groups and all animals that died early.

Other examinations:

Clinical pathology studies : hematology and chemical chemistry evaluations were performed on 10 supplemental-study rats per sex and exposure level at days 5 and 21 and on base-study rats at study terminaison (week 13).

Sperm motility and vaginal cytology evaluations : At the end of the 13-week studies, vaginal cytology and sperm motility evaluations were performed on all base-study rats in the 0, 500, 750, and 1500 ppm groups. begining 12 days prior to sacrifice, the vaginal vaults of 10 females from each exposure group were lavaged, and the aspirated lavage fluid and cells were stained with toluidine blue. Relative numbers of leukocytes, nucleated apithelial cells, and large squamous epithelial cells were determined and used as ascertain estrous cycle stage.

Statistics:

For the 13-week studies, two approaches were employed to assess the significance of pairwise comparisons between exposed and control groups in the analysis of continuous variables. Organ and body weight data, which are approximately normally distributed, were analyzed using the parametric multiple comparisons procedures of Williams or Dunnett. Clinical chemitry, hematology which typically have skewel distributions, were analyzed using the nonparametric multiple comparisons methods of Shirley or Dunn. Jonckheere's test was used to assess the significance of dose-reponse trends and to determine whether a trend-sensitive test was more appropriate for pairwise comparisons than a test capable of detecting departures from monotonic dose response. If a P-value from Jonckheere's test was greater than or equal to 0.10, Dunn's or Dunnett's test was used rather than Shirley's or William's test.
The outlier test of Dixon and Massey was employed to detect extreme values. No value selected by the outlier test was eliminated unless it was at least twice the next largest value or at most half of the next smallest value. The extreme values chosen by the statistical test were subject to approval by NTP personnel. In addition, values indicated by the laboratory report as being inadequate due to technical problems were eliminated from the analysis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of toxicity were noted primarily in rats in the 1,500 and 3,000 ppm groups beginning at Week 2. The majority of rats in these groups appeared thin and had ruilled fur, with discolorations of the tail, ears, and scrotum or vaginal area. Salivation, hypoactivity, and convulsions and seizures were also observed in some male and female rats in these groups, and abnormal posture (staggering) was noted in most males and females. Other clinical signs observed in these groups included hyperactivity, hunched posture, ptosis, ataxia, dyspnea, and bristly hair.

Mortality:

mortality observed, treatment-related

Description (incidence):

Six males and all females in the 3,000 ppm groups died or were killed moribund before the end of the 13-week study; all rats in the lower exposure groups survived to the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of male and female rats that were exposed to 1,500 or 3,000 ppm were markedly lower than those of controls throughout the 13-week study. Mean body weights of the 3,000 ppm groups decreased during the first week of the study for males and the first 2 weeks of the study for females before starting to increase. No final mean body weight or body weight gain was determined for female rats administered 3,000 ppm 1,3-diphenylguanidine due to 100% mortality in this exposure group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Average feed consumption decreased as exposure concentrations increased above 500 ppm with feed consumption 34% to 40% less than the controls during the 13-week study period in males and females that received 3,000 ppm. During the first week of the study feed consumption by groups receiving 3,000 ppm were 57% and 63% lower than control for males and females respectively, indicating poor palatability at this exposure concentration.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In general, changes in hematology parameters were limited to rats receiving 1500 and 3000 ppm. A mild polycythemia occurred at day 5 in the 3000 ppm male and female rats, and to a lesser extent in the 1500 ppm females. It was indicated by greater erythrocyte counts hematocrit values, and hemoglobin concentrations than controls and would be consistent with a relative polycythemia related to dehydratation and hemoconcentration. There were slightly lower reticulocyte counts at day 5 in 3000 ppm male and female rats and 1500 ppm females. other changes in hematology parameters were minor, sporadic, and did not suggest a treatment effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in clinical chemistry parameters occurred primarily in the 1,500 and 3,000 ppm groups, although some minor changes were observed in other groups. Greater alkaline phosphatase activity and bile acid concentration than controls occurred in an exposure-related manner in male and female rats. Males exhibited greater increases in activity and at earlier time periods. By Week 13, alkaline phosphatase activity and bile acid concentration were greater than the controls in all groups of exposed rats; these changes are consistent with cholestasis. The lack of an increase of alkaline phosphatase activity in groups that received 3,000 ppm was probably related to inanition and a decreased contribution of the intestinal fraction of alkaline phosphatase to the total serum activity. Total protein, creatinine, cholesterol, and triglyceride concentrations in the 1,500 and 3,000 ppm groups were lower than the controls and these differences are consistent with inanition.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights for groups receiving 750 ppm or greater were significantly lower than those of the controls and were the result of low body weights and low feed consumption by these groups rather than a specific toxic response to 1,3-diphenylguanidine.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross necropsy observations related to 1,3-diphenylguanidine treatment were limited to thinness of the carcass in higher exposure rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic changes associated with chemical administration were observed in the bone marrow, thymus, uterus, testes, prostate gland/seminal vesicle, and salivary glands. All of the gross and microscopic changes occurred in the two highest exposure groups and were attributed to the lower feed intake, reduced weight gains, and poor body condition of these animals.

In the thymus, lymphoid depletion and necrosis were present in several 3,000 ppm females which were found dead or were killed in moribund condition. Depletion of hematopoietic cells in the femoral bone marrow was also variably present in the 3,000 ppm females which died early. Both of these lesions are common in moribund animals and are not considered to be direct toxic effects of chemical administration.
An exposure-related effect in the uterus of females was characterized by an overall reduction in size and was diagnosed as hypoplasia. This finding occurred with greater incidence and severity in the three highest exposure groups. In general, this change was attributed to poor body condition and delayed development due to lower feed consumption; the younger age of those females which died or were killed during the study may have been a reason for the smaller size of the uterus.
Several lesions were noted sporadically in the reproductive organs of 3,000 ppm males. In two of ten 3,000 ppm males, lower numbers of mature spermatozoa were present in the seminiferous tubules than in the controls; lower numbers of spermatozoa were also noted in the epididymal tubules than in the controls. Secretory depletion of the prostate gland and seminal vesicles was observed in several 3,000 ppm males; this difference was characterized by alveolar size smaller than controls and smaller amounts of secretory material within the lumen. Decreased spermatogenesis and secretory depletion of the accessory sex glands were considered secondary to poor body condition. In the salivary glands of several 3,000 ppm males and females, a change diagnosed as cytologic alteration was observed, characterized by smaller size and increased basophilia of the secretory acini. This change was interpreted to be a reflection of physiological atrophy due to reduced feed intake. No specific cause of death could be determined for the early death animals from the 3,000 ppm groups.
Evaluation of male reproductive tissues in groups that received 500, 750, or 1,500 ppm revealed a significant reduction in sperm motility in 1,500 ppm males. Among 750 and 1,500 ppm group females the length of the estrous cycle was greater than the controls.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

17 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

2

Dose descriptor:

LOAEL

Effect level:

32 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Decrease of food consumption, increase of alkaline phosphatase and bile acids

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

TABLE 1 : Survival, Body Weight, Feed Consumption, and Compound Consumption Data forF344/NRats in the 13-Week Feed Study of 1,3-Diphenylguanidine

Dose (ppm)	Mean Body Weight (grams)								Final Weight Relative to Controls^2 (%)		Average Feed Consumption^3 (g/day)		Average Dose^3 (mg/kg/day)
	Survival^1		Initial		Final		Change
MALE
0		10/10		147		368		221		-		17.3	-
250		10/10		142		351		210		96		17.0	17
500		10/10		150		350		200		95		16.7	32
750		10/10		141		340		198		92		16.4	50
1,500		10/10		145		290		145		79		14.8	100
3,000		4/10^4		142		192		49		52		10.3	181
FEMALE
0		10/10		118		202		84		-		11.4	-
250		10/10		120		195		75		96		11.2	17
500		10/10		120		191		71		95		10.6	32
750		10/10		116		187		71		93		10.4	49
1,500		10/10		116		174		58		86		9.5	95
3,000		0/10^5		119		–		–		–		7.5	184

¹Number surviving at 13 weeks/number of animals per group. For groups with no survivors, no final mean body weights or body weight changes are given.

²(Exposure group mean/control group mean) x 100.

³For all groups except 3,000 ppm females, consumption values are averaged for Weeks 1-13. For 3,000 ppm females, consumption values are averaged for Weeks 1-12.

⁴Week of death: 9, 9, 9, 10, 10, 12.

⁵Week of death: 4, 4, 5, 7, 7, 8, 9, 12, 12, 12.

TABLE 2 (male) :Selected Clinical Chemistry Data forF344/NRats in the 13-Week Feed Study of1,3-Diphenylguanidine¹

		Concentration (ppm)
		0	250	500	750	1500	3000
MALE
n	Day 5	10	10	10	10	10	10
	Day 21	10	10	10	10	10	10
	Week 13	10	10	10	10	10	4
Creatinine (mg/dL)	Day 5	0.68 ± 0.02	0.62 ± 0.03	0.68 ± 0.01	0.63 ± 0.03	0.65 ± 0.02	0.58 ± 0.01**
	Day 21	0.80 ± 0.03	0.75 ± 0.02	0.78 ± 0.03	0.76 ± 0.02	0.75 ± 0.02	0.65 ± 0.03**
	Week 13	0.69 ± 0.02	0.68 ± 0.02	0.65 ± 0.02	0.64 ± 0.02	0.62 ± 0.03*	0.60 ± 0.04*
Total protein (g/dL)	Day 5	6.1 ± 0.1	6.0 ± 0.1	6.0 ± 0.1	6.1 ± 0.1	6.1 ± 0.1	5.9 ± 0.1
	Day 21	6.4 ± 0.1	6.2 ± 0.1	6.2 ± 0.1	6.2 ± 0.1	6.0 ± 0.1**	5.6 ± 0.1**
	Week 13	6.8 ± 0.1	6.8 ± 0.1	6.8 ± 0.1	6.7 ± 0.1	6.5 ± 0.1*	5.8 ± 0.2**
Cholesterol (mg/dL)	Day 5	100 ± 4	100 ± 2	100 ± 3	91 ± 2*	91 ± 2	92 ± 3
	Day 21	93 ± 2	90 ± 2	87 ± 2	88 ± 2	88 ± 2	94 ± 5
	Week 13	88 ± 2	87 ± 3	91 ± 2	88 ± 2	78 ± 1**	70 ± 2**
Triglycerides (mg/dL)	Day 5	197 ± 11	227 ± 10	197 ± 7	210 ± 9	201 ± 12	105 ± 13**
	Day 21	316 ± 21	308 ± 19	294 ± 20	301 ± 17	239 ± 26*	133 ± 25**
	Week 13	237 ± 17	239 ± 17	241 ± 21	262 ± 24	210 ± 18	72 ± 9*
Alkaline phosphatase (IU/L)	Day 5	535 ± 17	640 ± 10	710 ± 11**	693 ± 12**	729 ± 11**	517 ± 19
	Day 21	391 ± 7	452 ± 6	506 ± 12**^2	528 ± 24**	537 ± 21**^2	376 ± 18
	Week 13	199 ± 7	258 ± 8**	282 ± 7**	291 ± 9**	359 ± 11**	349 ± 31**
Bile acids (µmol/L)	Day 5	4.9 ± 1.5^2	5.0 ± 1.1	6.5 ± 1.2	6.6 ± 1.4	23.6 ± 2.8**^2	37.7 ± 4.0**
	Day 21	5.4 ± 2.3^3	1.3 ± 0.3^4	6.4 ± 1.9^3	7.6 ± 3.9^3	17.8 ± 4.5*^5	75.2 ± 15.4**^2
	Week 13	5.1 ± 0.8	12.0 ± 1.9**^5	13.8 ± 2.4**	11.1 ± 2.5*^6	22.8 ± 3.0**	36.0 ± 1.7**^4

TABLE 2 (female) :Selected Clinical Chemistry Data forF344/NRats in the 13-Week Feed Study of1,3-Diphenylguanidine¹

		Concentration (ppm)
		0	250	500	750	1500	3000
MALE
n	Day 5	10	10	10	10	10	10
	Day 21	10	10	10	10	10	10
	Week 13	10	10	10	10	10	0
Creatinine (mg/dL)	Day 5	0.63 ± 0.02	0.61 ± 0.01	0.58 ± 0.03	0.59 ± 0.03	0.57 ± 0.02	0.58± 0.03
	Day 21	0.62 ± 0.02	0.62 ± 0.02	0.64 ± 0.02	0.61 ± 0.02	0.57 ± 0.02	0.43± 0.03**
	Week 13	0.63 ± 0.03	0.63 ± 0.02	0.62 ± 0.01	0.66 ± 0.02	0.56 ± 0.03	-
Total protein (g/dL)	Day 5	5.7 ± 0.1	5.7 ± 0.1	5.6 ± 0.1	5.5 ± 0.1	5.4 ± 0.1*	5.3± 0.1**
	Day 21	6.1 ± 0.1	6.0 ± 0.1	5.9 ± 0.1	5.9 ± 0.1	5.6 ± 0.1**	5.2± 0.1**
	Week 13	6.7 ± 0.1	6.7 ± 0.1	6.3 ± 0.1**	5.9 ± 0.1**	5.5 ± 0.0**	-
Cholesterol (mg/dL)	Day 5	107 ± 2	104 ± 3	104 ± 3	98 ± 3*	92 ± 2**	100± 2**
	Day 21	112 ± 3	106 ± 2	103 ± 2*	110 ± 3	107 ± 4	85± 4**
	Week 13	110 ± 4	109 ± 2	100 ± 3	88 ± 2**	86 ± 3**	-
Triglycerides (mg/dL)	Day 5	136 ± 7	159 ± 12	135 ± 10	138 ± 22	70 ± 6**	70± 10**
	Day 21	168 ± 12	136 ± 12*	124 ± 13*	145 ± 18	103 ± 11**	69± 7**
	Week 13	104 ± 11	113 ± 10	106 ± 11	100 ± 18	74 ± 7	-
Alkaline phosphatase (IU/L)	Day 5	373 ± 5	477 ± 13	469 ± 7	469 ± 13	383 ± 13	308± 10
	Day 21	305 ± 6	357 ± 12	356 ± 5*	385 ± 11**	406 ± 16**	313± 18
	Week 13	164 ± 6	185 ± 4**	249 ± 5**	251 ± 11**	254 ± 11**	-
Bile acids (µmol/L)	Day 5	14.0 ± 3.1	9.7 ± 2.4	12.5 ± 1.2	19.4 ± 3.4	38.5 ± 3.6**	31.7± 3.5**
	Day 21	11.3 ± 2.9	9.3 ± 2.8^2	25.4 ± 2.3**	13.6 ± 3.0	50.6 ± 3.8**	214.4± 27.0**
	Week 13	14.6 ± 2.1	12.3 ± 2.0	18.3 ± 2.8	17.8 ± 3.0	51.9 ± 4.1**	-

¹Data are given as mean ± standard error. Statistical tests were performed on unrounded data.

²n=9. ³n=5. 4n=3. ⁵n=8. 6n=7.

* Significantly different (P 0.05) from the control group by Dunn's or Shirley's test.

** Significantly different (P 0.01) from the control group by Dunn's or Shirley's test.

Applicant's summary and conclusion

Conclusions:

Consumption of feed containing DPG for 13 weeks was not associated with any histologic response which could be attributed to chemical exposure. Instead the observed changes were indicative of reduced nutrient intake and are consistent with similar changes observed in other studies of feed restricted rats. No organ specific toxicity attribuable to DPG exposure was apparent.

Executive summary:

To evaluate the toxicity associated with systemic exposure, 13 -week toxicology study was conducted by administering DPG in feed to groups of male and female F344/N rats. During 13 -week study, rats received feed containing 0, 250, 500, 750, 1500or 3000 ppm DPG. Six male and all female rats that received feed containing 3000 ppm died or were killed moribund before the end of the 13 -week study. Final mean body weights and feed consumption of male and female rats that received 1500 or 3000 ppm were lower than controls throughout the study. The values of several hematologic parameters were significantly different from the controls in groups of rats that received 1500 or 3000 ppm; however, these differences were attribuable to reduced nutrient intake as a result od reduced feed consumption. Lower total serum protein, cholesterol, triglyceride, and creatinine concentrations were also considered too be the consequence of reduced nutrient intake. Alkaline phosphatase activity and bile acid concentrations were greater than the controls in most groups exposed to DPG and were considered to be an indication of cholestasis.