Registration Dossier

Administrative data

Description of key information

Ninety-day feeding experiments in rats and/or mice have been performed according to OECD guidelines and GLP in the frame of the US National Toxicology Program (1995).  A NOAEL (rat) of 17 mg/kg bw/d was determined in the rat for decreases in body weight gain and food consumption. No treatment related effects were seen on organs, haematological and clinical-chemistry parameters, or urine analysis.
In a 28-day gavage study in rats, mortalities were observed at the top dose level of 90 mg/kg/day, a dose level closed to the acute LD50 of 107 mg/kg in rats. at lower dose levels, minor hematological and biochemical changes were observed. The NOEL was considered to be 10 mg/kg/day for both sexes.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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
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
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
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.
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.
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
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
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
Critical effects observed:
not specified

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

Controls2 (%)

Average Feed

Consumption3

(g/day)

Average

Dose3

(mg/kg/day)

 

Survival1

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

4/104

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

0/105

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 ± 1.52

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 ± 2.33

1.3 ± 0.34

6.4 ± 1.93  

7.6 ± 3.93

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 ± 2.82

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.


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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
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
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals 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 (e.g. ad libitum): NIH open formula rat and mouse ration manufactured by Oriental Yeast Co. Ltd, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 13+/-3°C
- Humidity (%): 55+/-20%
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12/12 (lighted at 7 am)
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
No
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days (4 weeks)
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 10, 30, 90 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
No
Positive control:
No
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.
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
Details on results:
CLINICAL SIGNS AND MORTALITY
Dead animals in the 90 mg/kg group were observed during weeks 2 to 4 of the administration period, with 10 % of the males and 70 % of the females in the 90 mg/kg group dying within the administration period. Pathological examination revealed no obvious changes accounting for the deaths. Salivation was observed in both sexes in the 30 and 90 mg/kg groups, and adoption of a prone or lateral position, staggering gait, decrease in spontaneous motor activity and startle response were observed in both sexes in the 90 mg/kg group.
All clinical signs in surviving animals disappeared during the recovery period.

BODY WEIGHT AND WEIGHT GAIN & FOOD CONSUMPTION AND COMPOUND INTAKE
Body weight gain and food consumption were suppressed in both sexes in the 90 mg/kg group.

FOOD EFFICIENCY :
Among males, in the 90 mg/kg group, significantly low values were noted for the food efficiency during week 1 and week 3 of administration, with one animal having negative values during both week 2 and week 3 of administration. Additionally, significantly low values were noted for mean food efficiency during the administration period but significantly high values were noted for food efficiency during the recovery period.
Among females, in the 90 mg/kg group, a trend of low values was noted for the food efficiency during week 1 and week 3 of administration, with one animal having negative values during week 3 of administration. Additionally, significantly high values were noted during the beginning of the recovery period and there was a trend of low values during week 1 of recovery but there was a trend of high values during the second week.

HAEMATOLOGY (table 1)
Hematological examination revealed an increase in platelet counts in females in the 30 mg/kg group.

CLINICAL CHEMISTRY (table 2)
Blood chemistry examination revealed a decrease in blood glucose in males in the 30 and 90 mg/kg groups, and increases in blood urea nitrogen, total birilubin, A/G ratio, ALT and ALP values in males in the 90 mg/kg group.

URINALYSIS
Urinalysis revealed an increase in urine volume and a decrease in specific gravity in both sexes in the 90 mg/kg group.

ORGAN WEIGHTS (table 3)
Organ weight analysis revealed no changes attributable to the administration of the test substance.

GROSS PATHOLOGY
Macroscopic examination revealed an increased incidence of brown liver in males in the 30 mg/kg group and in both sexes in the 90 mg/kg group and reddish thympanic bulla in both sexes in the 90 mg/kg group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examination revealed increased incidences of hydropic changes in renal collecting tubules in both sexes in the 90 mg/kg group.
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
Critical effects observed:
not specified

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. of animals

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/mm3)

1188±111

1118±78

1153±86

1118±76

1121±59

1217±109

WBC(x10^3/mm3)

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/mm3)

1213±83

1308±57

1436±117**

1412

1158±122

1114±192

WBC(x10^3/mm3)

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. Values are expressedas Mean±S.D.

Significant difference from control group; *p<0.05, ** p<0.01

N:Non parametric 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

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/kgin 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 4thweek of administration. In males, significantly low values were observed for the mean food efficiency at the 4thweek 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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
11 february 2010- 5 march 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
2-week range-finding toxicity study
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, l’Arbresle, France.
- Age at study initiation: 10 weeks old.
- Weight at study initiation: males = 363 g (range: 353 g to 371 g), females = 230 g (range: 210 g to 262 g).
- Fasting period before study:
- Housing: individually housed in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm).
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet,
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water
- Acclimation period: 6 days before the
beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
. temperature : 22 ± 2°C,
. relative humidity : 50 ± 20%,
. light/dark cycle : 12h/12h (7:00 - 19:00),
. ventilation : approximately 12 cycles/hour of filtered, non-recycled air.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose aqueous solution
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle in order to achieve the concentrations of 6, 12 and 15 mg/mL.
The dosage forms were prepared daily and were stored at room temperature and delivered to the study room in brown flasks.

VEHICLE
The vehicle was 0.5% methylcellulose aqueous solution prepared using:
. purified water, obtained by reverse osmosis using an ELIX 5 apparatus (Millipore SA, Saint-Quentin en Yvelines, France),
. methylcellulose, batch No. 107K0081, supplied by Sigma (Saint-Quentin-Fallavier, France).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
30, 60 and 75 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
3 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Allocation to groups: during the acclimation period, the required number of animals (12 males and 12 females) were selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a computerized stratification procedure so that the average
body weight of each group was similar.

Identification: each animal was identified by an individual ear tattoo. At the beginning of the study, each animal received a unique CIT identity number.

Rationale for dose-level selection
The dose-levels were selected on the basis of a 28-day rat study performed at dose-levels of 10, 30 and 90 mg/kg/day. Dead or moribund animals in the 90 mg/kg group were observed during weeks 2 to 4 of the administration period. Salivation was observed in both sexes in the 30 and 90 mg/kg dose groups, and many other clinical signs including startle response were observed in both sexes in the 90 mg/kg group. Body weight gain and food consumption were suppressed in both sexes in the 90 mg/kg group. Hematological examination revealed an increase in platelet counts in females in the 30 mg/kg group. Blood chemistry examination revealed a decrease in blood glucose in males in the 30 and 90 mg/kg groups, and increases in blood urea nitrogen, total bilirubin, A/G ratio, ALT and ALP values in 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. Histopathological examination revealed increased incidences of hydropic changes in renal collecting tubules in both sexes in the 90 mg/kg group. The NOEL for repeat dose toxicity is considered to be 10 mg/kg/day for both sexes.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, at least twice daily
DETAILED CLINICAL OBSERVATIONS: Yes, at least once daily
BODY WEIGHT: Yes, twice daily
FOOD CONSUMPTION : Yes, twice daily
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. A complete macroscopic post-mortem examination was performed on all animals. This included
examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the
brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the
neck with its associated organs and tissues.
HISTOPATHOLOGY: No. The heart, kidneys, liver, ovaries and testes were weighed and preserved although no microscopic examination was performed.
Other examinations:
no
Statistics:
Citox software (version D.05) was used to perform the statistical analyses of body weight and food consumption.
PathData software (version 6.2b5) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).
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:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
75 mg/kg/day : At this dose-level, one male out of six animals survived until scheduled sacrifice. Female U24511 was prematurely sacrificed on day 1, male U24490 was prematurely sacrificed on day 2, male U24492 was found dead on day 3, female U24510 was prematurely sacrificed on day 5 and female U24512 was prematurely sacrificed on day 13 because of body weight loss and clinical signs of locomotory difficulties and soiled urogenital and mouth regions. Lateral recumbency had been observed from day 6, with loss of balance, mydriasis, hypoactivity and half-closed eyes.
60 mg/kg/day: At this dose-level, two males and one female out of six animals survived until scheduled sacrifice. Male U24488 was prematurely sacrificed on day 5 and females U24507 and U24509 were prematurely sacrificed on day 6. All animals were prematurely sacrificed because of body weight loss and/or clinical signs.
0 and 30 mg/kg/day: There were no unscheduled deaths.

CLINICAL SIGNS
Clinical signs were generally observed from day 2, although one female treated at 75 mg/kg/day (U24511) did have lateral recumbency and convulsions on day 1 and was prematurely sacrificed. Clinical signs were generally observed at approximately 30 minutes after dosing and lasted for up to 5 hours (up to 24 hours on occasion). At 30 mg/kg/day, the one female showing clinical signs (U24504) had staggering gait and loss of balance on day 2 only and the male (U24486) had loss of balance on day 9 only. At 60 mg/kg/day, the clinical signs were observed until mid- to late-week 2; one surviving male had no further clinical signs from day 12 while the other surviving male still had piloerection, staggering gait and salivation on day 14. The one surviving female had no important clinical signs from day 13. At 75 mg/kg/day, the one surviving male (U24491) still had several clinical signs on day 14 (lateral recumbency, hypersensitivity to touch and noise, locomotory difficulties and salivation).

BODY WEIGHT AND WEIGHT GAIN
By day 4 of dosing, the three remaining animals treated at 75 mg/kg/day had all lost weight (between -8 g and -25 g) as had 5/6 animals treated at 60 mg/kg/day (between -8 g and -34 g). the one remaining male lost weight until day 11, after which body weight gain was recorded but not sufficient to compensate for the 11 days of body weight loss (overall body weight change: -12 g).
At 60 mg/kg/day after day 4, the remaining animals had occasions where weight was lost but generally gained weight until the end of the study. Of these three surviving animals, two had an overall body weight gain (+46 g or +4 g) while the third had an overall body weight loss (-11 g).
At 30 mg/kg/day, the males showed no difference in mean body weight or body weight gain when compared with the controls. The females, however, gained little weight or lost weight throughout the study and finished with a small overall body weight gain (-74% when compared with the controls).

FOOD CONSUMPTION
The groups treated at 60 or 75 mg/kg/day had moderately to markedly reduced food consumption when compared with the controls from day 1 to day 11, after which the remaining animals consumed similar, or only slightly lower, amounts of food to the controls.
There were no effects of treatment with the test item on mean male food consumption at 30 mg/kg/day but mean female food consumption was lower than that of the controls throughout the study.

ORGAN WEIGHTS
Because of mortality, no statistics could be performed.
High absolute and body weight-related liver weights were observed in the surviving female (U24508) treated at 60 mg/kg/day. A relationship to treatment could not be excluded. When compared to controls, there was a trend to higher relative weights of the heart, kidneys, liver and testes in males treated at 60 or 75 mg/kg/day. These differences were minimal and most likely secondary to the decreased terminal body weights seen in these rats. These differences were therefore considered not to be treatment-related.

GROSS PATHOLOGY
The gross findings recorded in the liver, the thymus and the spleen in prematurely decedent rats at 60 or 75 mg/kg/day were possibly related to treatment with 1, 3-Diphenylguanidine.
Premature deaths:
- At 75 mg/kg/day: Males U24490 and U24492 treated at 75 mg/kg/day were respectively euthanized prematurely on ethical grounds or found dead on day 2 or 3. At the same dose-level, females U24510, U24511, U24512 were euthanized prematurely on days 5, 1 and 13 on ethical grounds.
Except for reduced sizes of the spleen and thymus in female U24512, no gross findings were recorded in these premature decedents.
- At 60 mg/kg/day: Male U24488 and females U24507 and U24509 were euthanized prematurely on days 5, 6 and 6, respectively. A few white areas (up to 0.4 cm in diameter) were noted in the liver of male U24488, on the dorsal face of the left lateral lobe. Though this finding was recorded in a single rat and in none of the surviving individuals, a relationship to treatment could not be excluded. A spleen reduced in size was observed in female U24509. The findings seen in the lymphoid tissues (spleen and thymus) were most likely secondary to stress, as a result of poor health condition observed in these individuals.
Terminal sacrifice: No gross findings were observed.
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: This dose was well tolerated by the males, the only effect being one male with loss of balance on day 9.
Dose descriptor:
LOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: The females did not tolerate the test item well, with very low body weight gains or body weight loss and reduced food consumption.
Critical effects observed:
not specified
Conclusions:
Under the experimental conditions of this study, it was considered that the high dose-level for an OECD 421 study should be below 30 mg/kg/day since this dose-level had an effect on the general condition of the female rats.
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item, 1,3-Diphenylguanidine, following daily oral administration (gavage) to rats for 2 weeks, in order to select dose-levels for an OECD No. 421 Study. Three groups of three male and three female Sprague-Dawley rats received the test item, 1,3-Diphenylguanidine, once daily for 2 weeks, by gavage. The dose-levels were 30, 60 and 75 mg/kg/day. A further group of three males and three females received the vehicle, 0.5% aqueous methylcellulose, alone, under the same experimental conditions and acted as a control group. A dose volume of 5 mL/kg/day was used. During the treatment period, the animals were checked at least twice daily for mortality and at least once daily for clinical signs. Body weight and food consumption were recorded twice weekly. Animals were sacrificed on completion of the treatment period (day 15) and a complete macroscopic post-mortem examination was performed. The heart, kidneys, liver, ovaries and testes were weighed and preserved although no microscopic examination was performed.

At 75 mg/kg/day, 4/6 animals (three females and one male) were prematurely sacrificed between days 1 and 13 because of loss of body weight and severe clinical signs (clonic convulsions, lateral recumbency, locomotory difficulties, staggering gait, loss of balance, mydriasis). Another male was found dead on day 3. At 60 mg/kg/day, 3/6 animals (one male and two females) were prematurely sacrificed on day 5 or day 6 because of loss of body weight and clinical signs (lateral recumbency, staggering gait, loss of balance, locomotory difficulties). The cause of the moribundity could not be determined. Thymus and/or spleen were reduced in size in two females (one at each dose-level) which was most likely related to stress associated with the poor health condition. A few white areas were noted in the liver of one male given 60 mg/kg/day. There were no unscheduled deaths at 30 mg/kg/day. Clinical signs were similar in incidence and duration between 60 and 75 mg/kg/day and included lateral recumbency, clonic convulsions, staggering gait, loss of balance, locomotory difficulties, hypoactivity, mydriasis, half-closed eyes and piloerection. In addition, one male treated at 75 mg/kg/day was hypersensitive to noise and touch. At 30 mg/kg/day, one male had loss of balance on day 9 and one female had staggering gait and loss of balance on day 2. At 75 mg/kg/day, both males and females had body weight losses until day 11, after which the one surviving male gained weight but not enough to compensate for the previous weight loss (overall body weight change of -12 g). At 60 mg/kg/day, initial body weight losses were followed by body weight gains but the overall body weight gains were lower than the controls (-70% for males and -85% for females). The males treated at 30 mg/kg/day had no effects of treatment with the test item on mean body weight gain but the females had a markedly lower mean body weight gain over the treatment period (-74%). Food consumption was moderately to markedly reduced for males and females treated at 60 or 75 mg/kg/day and also for females treated at 30 mg/kg/day. There were no effects on male food consumption at 30 mg/kg/day. The one surviving female at 60 mg/kg/day had increased absolute and body weight-related liver weights for which a relationship to treatment could not be excluded. No abnormalities were observed in scheduled sacrifice animals. In two prematurely sacrificed females (one treated at 60 mg/kg/day and one treated at 75 mg/kg/day) the spleen was reduced in size. One female treated at 75 mg/kg/day had a small thymus. One male treated at 60 mg/kg/day had a few white areas on the liver.

1,3-Diphenylguanidine, was administered once daily, for 2 weeks, by oral gavage, to Sprague-Dawley rats at dose-levels of 30, 60 or 75 mg/kg/day. The dose-levels of 60 and 75 mg/kg/day both exceeded the maximum tolerated dose with 50% and 17% survival rates, respectively. Prior to death or premature sacrifice, animals had general and neurological clinical signs which included lateral recumbency, clonic convulsions, staggering gait, loss of balance, locomotory difficulties, hypoactivity, mydriasis, half-closed eyes and piloerection. The dose-level of 30 mg/kg/day was well tolerated by the males, the only effect being one male with loss of balance on day 9. The females, however, did not tolerate the test item well, with very low body weight gains or body weight loss and reduced food consumption. All animals survived until scheduled sacrifice and had no macroscopic abnormalities and no effects on liver, kidney, heart, ovary or testis weight. Under the experimental conditions of this study, it was considered that the high dose-level for an OECD 421 study should be below 30 mg/kg/day since this dose-level had an effect on the general condition of the female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
17 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
It is a reliable study with a klimisch score of 1

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
No reliable study is available for this endpoint.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated toxicity / Oral route :

Sub-chronic feeding experiments in rats and/or mice have been performed according to OECD guidelines and GLP in the frame of the US National Toxicology Program (Irwin, 1995).

In F344/N rats and B6C3F1 mice which, in a dose-finding study, received 1,3-diphenylguanidine (98.9%) for 14 days in their feed in concentrations of 250, 500, 750, 1,500 and 3,000 ppm, only reduced feed intake and decreased body weights were observed as of 750 ppm (Irwin, 1995).

In the subsequent sub-chronic 13-week study, 1,3-diphenylguanidine (98.9%) was fed to the F344/N rat and B6C3F1 mouse in the same concentrations as in the sub-acute study (equivalent to daily dose of 17/17, 32/32, 50/49, 100/95 and 181/184 mg/kg bw/d for male/female rats and 38/46, 75/93, 114/141, 231/285 and 457/577 mg/kg bw/d in male/female mice at 250, 500, 750, 1500 and 3000 ppm, respectively).

In rats, due to the poor palatability of the 1,3-diphenylguanidine-treated feed, a decreased feed consumption and reduced body weights were observed as of 750 ppm compared to controls (final weight relative to controls: 750 ppm (m/f): 92%/93%; 1,500 ppm: 79%/86%; 3,000 ppm: 52%/-). Increased mortality was observed for both sexes after receiving 3,000 ppm in feed, whereby all females of this concentration group died. Primarily substance-induced effects on the organs were not observed. The diverse deviations from the controls, determined also for the haematological and clinical-chemical parameters particularly in both highest concentrations (1,500 and 3,000 ppm), are seen exclusively as the result of emaciation by reduced feed intake.

With the average feed intake comparable with the controls, body weight retardation was observed in the mice also as of 750 ppm. The reduced organ weights occurring as of 1,500 ppm were evaluated not as a specifically toxic response but rather were correlated to the clearly reduced body weights. Histopathological changes and significant deviations from the controls for the haematological and clinical-chemical parameters were not observed. Based on the secondary toxic effects, due to the poor palatability of the 1,3-diphenylguanidine-treated feed, the NOAEL lies at 250 ppm for rats (ca. 17 mg/kg b.w. and day) and 500 ppm for mice (ca. 75 mg/kg b.w. and day).

The repeated dose toxicity of DPG in corn oil was evaluated at daily dose levels of 0, 10, 30 and 90 mg/kgin a 28-day oral repeated dose toxicity study in Crj:CD(SD) rats performed following the OECD guideline 407 and GLP (Murata et al., 2000). 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 4th week of administration. In males, significantly low values were observed for the mean food efficiency at the 4th week 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.

Justification for classification or non-classification

Mandatory classification :

- Regulation (EC) No 1272/2008 Annex VI Table 3.1 : None

 

Self-classification

- Regulation (EC) No 1272/2008 : None