Dicyclohexylcarbodiimide

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:

October 2, 1991 - November, 1991.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dicyclohexylcarbodiimide

EC Number:

208-704-1

EC Name:

Dicyclohexylcarbodiimide

Cas Number:

538-75-0

Molecular formula:

C13H22N2

IUPAC Name:

N,N'-dicyclohexylcarbodiimide

Specific details on test material used for the study:

- Source: Tama Chemical Industry Co., Ltd.
- Lot: 10705
- Purity of 99.7 %

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 Japan Co., Ltd.
- Age at study initiation: 5 weeks
- Weight at study initiation: 170-197 (males), 137-158 (females)
- Housing: 2 per cage (same sex), in polycarbonate cages (265 W x 426 D x 200 H mm: Tokiwa Scientific Instruments Co., Ltd.) .) laid with animal bedding (Beta chip: Nippon Charles River Co., Ltd.). The cage, feeder and water bottle were sterilised once per week.
- Diet: MF: Oriental Yeast Co., Ltd. ad libitum.
- Water: tap water, irradiated with ultraviolet light and filtered with a 5 µm filter, ad libitum.
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25ºC
- Humidity (%): 40-70%
- Air changes (per hr): 12 times/per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark


IN-LIFE DATES: Obtained on October 2, 1991.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Remarks:

(Japanese Pharmacopoeia)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Olive oil (Japanese Pharmacopoeia) was added to the test substance, and the mixture was warmed at around 40 ºC and dissolved to a predetermined concentration;
the solution was prepared every 9 days, and it was kept in a cool dark place until immediately before administration.

VEHICLE
- Amount of vehicle (if gavage): the dosing volume was 5 ml/kg, based on the weight at the nearest measurement day.
- Purity: compliant with Japanese Pharmacopoeia standards.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stabilty of the solution (test item in olive oil) was analyzed, and it was confirmed that it was stable for 11 days after preparation, when stored in a cool dark place. No further details provided on the method. Accordingly, the test item solution was prepared every 9 days, and it was kept in a cool dark place until immediately before administration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily (once in the morning)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males / 6 females per group in 15 mg/kg and 100 mg/kg groups.
12 males / 12 females per group in control and 500 mg/kg group.

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were selected based on a preliminary oral toxicity study. Repeated oral administration of the test substance to SD rats at doses of 125, 250 and 500 mg/kg for 7 days resulted in toxic signs such as salivation, loss of locomotor activity, weight gain suppression, liver weight gain at 500 mg/kg; at 125 and 250 mg/kg, toxic signs such as spontaneous motor activity decrease were also observed. Based on this study, the high dose of this study was 500 mg/kg, the middle dose was 100 mg/kg, and the low dose was 15 mg/kg. In addition to this, a control group (only vehicle) was provided.
- Administration period: 28-days in the three doses-group and control group
- Post-exposure recovery period: 14 days in 6 males / 6 females for control and high dose groups (satellite groups).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: daily for morbidity, mortality, behaviour (4 weeks for all groups, up to 42 days for both recovery groups).

DETAILED CLINICAL OBSERVATIONS
- Time schedule: detalied observations including palpation once a week (4 weeks for all groups, up to 42 days for both recovery groups).

BODY WEIGHT:
- Time schedule for examinations: at the beginning of treatment and once a week thereafter (4 weeks for all groups, up to 42 days for both recovery groups).

FOOD CONSUMPTION: Food consumption was measured, although it is not a feeding study.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food was weighed at the beginning of the treatment and once a week thereafter, and the mean food intake per mouse per period was calculated (4 weeks for all groups, up to 40 days for both recovery groups).

FOOD EFFICIENCY: No.

WATER CONSUMPTION AND COMPOUND INTAKE: Water consumption was measured, although it is not a drinking water study.
- Time schedule for examinations: the tare was measured once a week and compared (4 weeks for all groups, up to 42 days for both recovery groups).

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY:
- Time schedule for collection of blood: suvirval animals at the time of each planned killing (28 or 42 days) were bled from posterior vena cava after anesthesia by pentobarbital sodium (Nembutal injection solution: Dynabot Corporation) intraperitoneally and examined on the following items: red blood and white blood cell count, platelet count, hemoglobin concentration, hematocrit value, leukocyte percentage, reticulocyte count, prothrombin time, activated partial thromboxane plastin time, mean red blood cell volume, mean red blood cell hemoglobin content and mean red blood cell hemoglobin concentration.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: in the remaining blood used for hematalogical examination the following parameters were examined: total protein, albumin, A/G ratio, glucose, triglyceride, total cholesterol, urea nitrogen, creatinine, calcium, inorganic phosphorus, GOT (AST), GPT (ALT), γ-GTP, ALP, sodium, potassium, chloride.

URINALYSIS:
- Time schedule for collection of urine: fresh urine of the survival animals was collected before the end of the administration period (3 weeks) and on the end of th recovery period. The following parameters were determined: pH, protein, glucose, ketones, bilirubin, urobilinogen, ocult blood, specific gravity, sediment, urine volume, sodium, potassium and chlorine.

NEUROBEHAVIOURAL EXAMINATION: No.
IMMUNOLOGY: No.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
Surviving animals, at the time of each planned killing, were exsanguined, sacrificied and necropsied. Dead animals were necropsied as soon as they were found. Brain, liver, kidney, adrenal gland, testis and ovary were weighed.

HISTOPATHOLOGY: Yes.
The following organs were collected, fixed and stored: Harder's glands, brain, pituitary, eyeball (including accessory gland), lung, stomach, thyroid (including parathyroid), heart, liver, spleen, kidney, adrenal gland, bladder, testis or ovary, bone marrow (femur). The testes suspected of being altered, heart, liver, kidney, spleen, adrenal glands and those duodena that had shown changes in the autopsy, were microscopical examined. All the organs that were found abnormal at necropsy were examined too. Examinations were made in all dosing groups.

Statistics:

The equidistance test by the Bartlett method was performed, one-way ANOVA was performed when the variance was uniform and Kruskal-Wallis test was performed when the variance was not uniform. Where the were significant differences between the groups, the method of Dunnett was applied. For uirne qualtitative test and urine sediments, Armitage's test was used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Reduced lococmotor activity and salivation were observed in the 500 mg/kg group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female of the 500 mg/kg group died on day 13 of the administration period and another died on day 4 of the recovery period. Animals showed crouching, prone position, ataxic gait, tiptoe gait, clonic convulsions and gasping before death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was reduced in the 500mg/kg males and females group. A recovery tendency was observed in both sexs during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption decreased in 500 mg/kg group (males and females) in the first week of administration, but no signifcant differences were observed after that.

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

effects observed, non-treatment-related

Description (incidence and severity):

Water intake was increased in the 500 mg/kg (males and females).

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Reduction of mean red blood cell hemoglobin level and reduction of prothrombin time was detected in males of the highest dose group. Shortening of activated partial thromboplastin time and increase of platelet count were observed in females (highest dose group).
No change was observed at the end of the recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A decrease in alkaline phospatase was observed in the 500 mg/kg males group and an increase in potassium in the 500 mg/kg males and females group.
At the end of the recovery period, an increase in alkaline phosphatase was observed in males (500 mg/kg). An increase in inorganic phosphorus in males was observed too, but it was within the physiological variation range.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urine volume tended to increase in both sexes although no significant differences was observed. Urinary pH was increased in the 500 mg/kg females group.

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males showed an increase in the liver/body weight ratio while females showed an increase in both actual weight and liver/body weight ratio in the 500 mg/kg group. Females in the 500 mg/kg recovery group showed an increase in liver weight but the difference with the control group was reduced. The weight of adrenal glands in females was decreased but there were no differences in the adrenal weight/ body weight ratios.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There was an elargement of the duodenum with thickening of the mucosa in both sexes in the 500 mg/kg group. This change was not observed in the recovery group.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the surviving animals, changes in the liver and duodenum of the 500 mg / kg group due to the administration of the test substance were observed in both sexes: enlarged hepatocytes around the Glissons capsules, eosinophilic hepatocyte cytoplasm and thickening of the duodenal mucosa.
In addition, fading foci in the kidney cortex, cyst formation, localized yellowing in the liver and brown / dark red spots in the lung were observed in a few cases, but it was considered to be a contingent lesion.

Details on results:

After the recovery period, histological changes of the liver and duodenum dissapeared and other charged parameters tended to show values almost within the normal range.

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

histopathology: non-neoplastic

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

duodenum

Treatment related:

yes

Dose response relationship:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Any other information on results incl. tables

Table 3. Body weight (group mean values)

	Days after commencement/cessation of treatment
Dose level (mg/kg)		0	7	14	21	28/0	35/7	42/14
MALE
0	Mean SD N	185 8.5 12	255 14.2 12	322 19.4 12	382 26.8 12	423 34.5 12	457 47.3 6	484 48.5 6
15	Mean SD N	187 6.7 6	255 11.3 6	319 19.1 6	377 27.0 6	415 37.8 6	-	-
100	Mean SD N	186 5.2 6	253 6.7 6	325 8.7 6	383 12.6 6	424 13.6 6	-	-
500	Mean SD N	187 8.4 12	239 15.9 12	300 22.5 12	351 23.9 12	368 24.3 12	427 23.5 6	468 28.1 6
FEMALES
0	Mean SD N	146 5.4 12	176 9.5 12	205 13.2 12	231 15.4 12	249 17.3 12	259 21.5 6	271 23.6 6
15	Mean SD N	145 6.6 6	181 8.7 6	207 10.8 6	233 16.0 6	245 13.5 6	-	-
100	Mean SD N	146 6.1 6	182 5.0 6	211 5.0 6	234 5.4 6	251 3.6 6	-	-
500	Mean SD N	147 4.9 12	170 8.5 12	196 10.3 11	216 13.2 11	225 26.6 11	250 21.5 4	268 15.1 4

Table 4. Organ weight and ratio organ weight/body weight (6 animals per group per dose, treatment groups)

Dose level (mg/kg)		Final body weight (g)	Brain	Brain/ body weight	Liver	Liver/ body weight	Kidneys	Kidneys/ body weight	Adrenals(x10^-3)	Adrenals / body weight	Testes /Ovaries (x10^-3)	Test-ovar/body weight
			(g)	%	(g)	%	(g)	%	(g)	%	(g)/ (mg)
MALE
0	Mean SD	430 31.3	2.00 31.3	0.47 0.029	18.06 2.234	4.19 0.249	3.06 0.297	0.71 0.054	58.3 0.276	13.7 1.58	3.03 0.276	0.71 0.055
15	Mean SD	415 37.6	1.97 0.089	0.48 0.051	17.61 2.925	4.22 0.316	3.01 0.210	0.73 0.021	61.3 7.57	14.8 1.69	3.24 0.464	0.79 0.178
100	Mean SD	424 13.8	2.04 0.037	0.48 0.024	18.20 1.611	4.29 0.272	3.08 0.225	0.73 0.055	57.4 7.75	13.5 1.49	3.10 0.092	0.73 0.044
500	Mean SD	4386 28.3	1.99 0.029	0.52 0.043	19.58 1.886	5.07 * 0.166	3.01 0.229	0.79 0.068	57.2 9.92	14.8 1.83	3.06 0.178	0.80 0.064
FEMALE
0	Mean SD	251 18.0	1.92 0.065	0.77 0.058	9.54 1.031	3.60 .0236	1.89 0.059	0.76 0.056	64.9 7.60	26.0 3.42	95.5 9.64	38.1 3.36
15	Mean SD	246 13.5	1.88 0.053	0.77 0.051	9.13 0.811	3.70 0.194	1.85 0.126	0.75 0.046	68.5 6.76	28.0 3.88	95.7 13.69	38.9 4.75
100	Mean SD	251 2.6	1.90 0.052	0.76 0.024	9.62 0.539	3.84 0.244	1.89 0.258	0.76 0.106	67.0 6.61	26.8 2.78	83.3 14.11	33.2 5.48
500	Mean SD	231 17.3	1.84 0.054	0.80 0.062	12.61* 0.796	5.43 * 0.328	1.87 0.109	0.81 0.039	63.9 7.25	27.9 4.95	86.0 13.53	37.2 4.67

* Significantly different from control value p < 0.01

Table 5. Organ weight and ratio organ weight/body weight (6 animals per group per dose, recovery groups)

Dose level (mg/kg)		Final body weight	Brain	Brain/ body weight	Liver	Liver/ body weight	Kidneys	Kidneys/ body weight	Adrenals(x10^-3)	Adrenals / body weight	Testes /Ovaries (x10^-3)	Test-ovar /body weight
		(g)	(g)	%	(g)	%	(g)	%	(g)	%	(g)/ (mg)
MALE
0	Mean SD	485 47.8	2.10 0.049	0.44 0.039	20.80 4.627	4.27 0.524	3.57 0.314	0.74 0.023	63.4 9.21	13.2 2.29	3.36 0.272	0.70 0.081
500	Mean SD	467 27.8	2.11 0.077	0.45 0.020	20.39 2.464	4.35 0.260	3.33 0.280	0.71 0.028	57.1 9.62	12.3 2.64	3.22 0.245	0.69 0.036
FEMALE
0	Mean SD	271 23.7	1.9 0.061	0.73 0.059	10.20 1.640	3.75 0.284	19.99 0.166	0.74 0.056	76.7 5.68	28.6 4.34	110.2 15.88	40.7 5.62
500	Mean SD	268 15.0	1.91 0.043	0.71 0.038	12.58 0.638*	4.69 * 0.080	1.97 0.137	0.74 0.045	66.4 * 6.44	24.9 3.65	113.1 11.99	42.2 3.15

* Significantly different from control value p < 0.01

Table 6. Total incidence macroscopic and microscopic findings

MACROSCOPIC FINDINGS
		28 DAYS								RECOVERY
	SEX	MALE				FEMALE				MALE		FEMALE
	DOSE LEVEL (MG/KG)	0	15	100	500	0	15	100	500	0	500	0	500
ORGAN FINDINGS	NUMBER OF ANIMALS	6	6	6	6	6	6	6	6	6	6	6	4
Duodenum enlargement		0	0	0	5	0	0	0	4	0	0	0	0
Kidneys Focal discoloration Cyst		0 0	1 0	0 0	0 0	0 0	0 0	0 1	0 0	0 0	0 0	0 0	0 0
Liver Focal yellowish change		0	0	0	0	0	0	0	0	1	0	0	0
Lungs Brownish/dark patch		0	0	0	0	0	0	1	0	0	1	0	0
MICROSCOPIC FINDINGS
		28 DAYS								RECOVERY
	SEX	MALE				FEMALE				MALE		FEMALE
	DOSE LEVEL (MG/KG)	0	15	100	500	0	15	100	500	0	500	0	500
ORGAN FINDINGS	NUMBER OF ANIMALS	6	6	6	6	6	6	6	6	6	6	6	4
Liver Enlarged/eosinophilic hepatocytes Neutrophilic infiltration Focal fatty change		0   0   0	0   0   0	0   0   0	5   0   0	0   0   0	0   0   0	0   0   0	6   1   0	0   0   1	0   0   0	0   0   0	0   0   0
Duodenum thickening of mucosa		0	0	0	5	0	0	0	4	0	0	1/5	0
Heart Focal myocardial degeneration		0	-	-	1	0	-	-	0	-	-	-	-
Kidneys Basophilic change of tubular epithelium Focal linphocytic in interstitium Hyaline droplets in tubular epithelium Cyst		3   1 0   0	0/1   0/1 1/1   0/1	-	2   0 0   0	1   1 0   0	-	0/1   0/1 0/1   1/1	2   2 0   0	-	-	-	-
Lungs Focal hemorrhage into alveoli		0	0	0	0	0	0	1	0	0	1	0	0

- not examined

Applicant's summary and conclusion

Conclusions:

The NOEL was determined to be 100 mg/kg bw/day in male and female rats.

Executive summary:

A 28-day repeated dose toxicity test was performed according toaccording to the Guidelines on Chemical Substitution Law (1986) (Guideline 28-Day Repeat Dose Toxicity Test of Chemical, Japan), similar to OECD 407 (GLP study). SD (Crj: CD) rats were treated with 0, 15, 100 and 500 mg/kg of the test substance by oral gavage for 28 days. 6 males and 6 females were selected for the groups of 15 and 100 mg/kg and 12 males/12 females for the control and 500 mg/kg groups. After the 28-days treatment, 6 males and 6 females for the control and 500 mg/kg groups were left to study their recovery over a period of 14 days. Animals of both sexes in the 500 mg/kg group showed reduced locomotor activity, salivation, inhibition of body weight gain, decreased food consumption and increased water intake. Two females in the 500 mg/kg dose group died, one during treatment and one during the recovery period. A decrease in alkaline phosphatase was also observed in males in the 500 mg / kg group. Pathological examination revealed increased relative liver weight, enlargement of the duodenum due to mucosal thickening and hepatocellular swelling in both sexes of the higher dose group. Histological changes in the liver and duodenum disappeared due to discontinuation of administration, and other changes also disappeared or showed a recovery tendency after the 14 days post-exposure. No effects were observed in the 100 and 15 mg/kg group in both males and females. Based on the above results, the NOEL under the test conditions was considered to be 100 mg/kg/day in both sexes.