Dicyclohexylcarbodiimide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 30, - December 10, 2010.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Cross-reference

Reason / purpose for cross-reference:

reference to other assay used for intermediate effect derivation

Reference

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

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

Specific details on test material used for the study:

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

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.

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)

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

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).

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.

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.

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

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

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

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

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN WITH JUSTIFICATIONS
- Premating exposure duration for parental (P0) animals: 2 weeks
- Basis for dose level selection: the dose of this study was set based on the results of the 28-day repeated dose toxicity test (See 'Cross-reference'): In this test, animals were dosed with 15, 100 or 500 mg/kg test item for 28 days. In the high dose group, 2 females died, and reduced locomotor activity, supression of body weight gain, increase in liver weight, thickening of the duodenal mucosa, etc. were observed in the remaining animals; no effects were observed at 100 mg/kg bw. In the range finding study, toxicity symptoms such as reduced locomotor activity were also observed in animals dosed with 125 and 250 mg/kg test item for 7 days. Based on the available information, the high dose of this study was set at 250 mg/kg bw, and the spacing factor was 2.5, so the medium dose was 100 mg/kg bw and the low dose 40 mg/kg bw.
- Route of administration: oral, gavage

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 OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Purity: 99.1%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated until use, storage under light shielding (measured temperature 3 to 7 ° C).
- Stability under test conditions: The stability of the test substance was confirmed at Hadano Laboratory before administration (August 30, 2010) and after the end of the administration period (December 10, 2010).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl: CD (SD), SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Atsugi Breeding Center (Japan).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 10 wks.
- Weight at study initiation: (P) Males: 354.0 to 436.0 g; Females: 215.5 to 280.2 g.
- Housing: individually housed, 2 animals per cage at the time of mating.
- Diet: ad libitum feed (CE-2, CLEA Japan).
- Water: ad libitum tap water.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25ºC
- Humidity (%): 40 - 75%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12h light/dark cycle.

IN-LIFE DATES: From: September 1, 2010 (62 males and 73 females, arrival date).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The medium was warmed at 40ºC or less, and the test item was weighed and added to the solution, at a concentration of 5% (w/v). Two further solutions were prepared at concentrations of 2 and 0.8 % (w/v). These solutions were stored in a refrigerated space (3 - 10ºC) and shielded from light, and used within 7 days after preparation (storage: from September 14th to October 30th, 2010). The doses were administered by gavage.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test item was found to be moisture sensitive; the stability of the test item in olive oil was confirmed by analysis.
- Concentration in vehicle: 5, 2 and 0.8% (w/v)
- Amount of vehicle (if gavage): 5 mL/kg
- Purity: according to Japanese Pharmacopoeia.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and sperm in vaginal smear, referred to as [day 0] of pregnancy.
- Further matings after two unsuccessful attempts: no.
- After successful mating each pregnant female was caged (how): females were individually housed in a plastic breeding cage for rats (350 w × 400 d × 180 h mm) from 18th day of pregnancy to 4th day of nursing, and a paper pulp chips (Pepper Clean, Japan SLC) were used as bedding. No anomalies in the breeding environment were observed during the period, the measured value of the temperature in the animal room was 23.5 to 24.0ºC, the measured value of humidity was 52.0 to 70.0%. In addition, it was confirmed that the analysis results of the feed, drinking water and bedding that were supplied were within the tolerance limits described in the standard operating procedure manual.
- Any other deviations from standard protocol: no.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item content was measured by a validated analytical method after preparation of the dosing solution; the average content was found to be 90 - 110%, and the variation between measurements was within the acceptable range (average value ± 10%). No further details on analytical method provided (Annex D-1, D-2 not included).

Duration of treatment / exposure:

Males were treated from 2 weeks before mating until the day before necropsy (42 administrations in total). Females were treated from 2 weeks before mating until delivery.

Frequency of treatment:

daily.

Details on study schedule:

- Animals arrival date: September 1, 2010
- Quarantine end date: September 14, 2010
- Dosing prior to mating: 2 weeks
- Mating: up to 2 weeks
- Age at mating of the mated animals in the study: [12] weeks
- End of administration period: December 10, 2010

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

13 male and 13 female per group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose of this study was set based on the results of the 28-day repeated dose toxicity test (See 'Cross-reference'): In this test, animals were dosed with 15, 100 or 500 mg/kg test item for 28 days. In the high dose group, 2 females died, and reduced locomotor activity, supression of body weight gain, increase in liver weight, thickening of the duodenal mucosa, etc. were observed in the remaining animals; no effects were observed at 100 mg/kg bw. In the range finding study, toxicity symptoms such as reduced locomotor activity were also observed in animals dosed with 125 and 250 mg/kg test item for 7 days. Based on the available information, the high dose of this study was set at 250 mg/kg bw, and the spacing factor was 2.5, so the medium dose was 100 mg/kg bw and the low dose 40 mg/kg bw.

Positive control:

Not required.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily before and after administration during administration and once daily during other rearing periods.

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Male animals were weighed on the day 1 (administration start date), 7, 14, 21, 28, 35, 42 days and on the autopsy day. Female animals were weighed on days 1, 7 and 14 of administration, pregnancy 0 (mating confirmation date), 7, 14, 20 days, day of delivery, 4 days after and on the autopsy day. If any animals died, they were weighed at the time of death.

FOOD CONSUMPTION AND COMPOUND INTAKE (not a feeding study):
- Male animals were measured for food intake on days 1 to 2, 7 to 8, 14 to 15, 29 to 30, 35 to 36, and 41 to 42.
- Female animals were measured for food intake on days 1 to 2, 7 to 8, 14 to 15 of administration; days 0 to 1, 7 to 8, 14 to 15, 20 to 21 of gestation and days 3 to 4 of rearing.

Oestrous cyclicity (parental animals):

For each group, vaginal smear specimens were prepared every day after the start of administration following the sexual cycle observation up to the grouping day, and the sex cycle was observed until the copulation was confirmed. In addition, the average number of days from estrous period to estrous period was calculated for each individual.

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not specified.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not specified.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litters were produced.
- Maternal animals: All surviving animals after the last litter of each generation was weaned (day 4).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- In males: liver, duodenum, prostate, seminal vesicle (including coagulated gland), mammary gland.
- In females: liver, duodenum, ovary, uterus, vagina, mammary gland. If the female or all babies died, also: heart, brain, kidney, lung. In females that had copulated but did not deliver or that died before delivery, liver and ovarian weights were measured.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed:
- In males, histopathological examination was performed on the liver, duodenum, testis, epididymis (0.1 M phosphate buffered 10% formalin solution).
- In females, histopathological examination was performed on the liver, duodenum and ovaries. In females that had copulated but did not deliver or that died before delivery, the number of corpus luteum was counted under a stereoscopic microscope and the implantation rate [(number of implantation / number of pregnant corpus luteum) X 100,%] was determined. In females that died or when all babies died, histopathological examinations also included: heart, brain, kidney, lung.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at [4] days of age.
- These animals were subjected to postmortem examinations as follows: all pups were subject to macroscopic examination, and any organ presenting abnormalities was fixed in 0.1 M phosphate buffered 10% formalin solution and stored.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

Fisher's exact test of significance (5%) was carried out on the frequency, the mating rate, the conception rate and the morphological abnormality frequency of the babies.From the histopathological findings of the treated groups, the graded pathological tissue findings were determined by Marm-Whitney's U test and the total value of positive groups was determined by Fisher's one-sided probability test. Significant difference test with the group was conducted (significance: 5%).
For the other data, a value obtained for each individual or an average value for each litter was taken. First, the uniformity of variance of each group was tested (significance: 5%) by Bartlett's method. When the variance was uniform, a one-way analysis of variance (significance level: 5%) was performed, and when significance was found between groups, multiple comparisons were performed by the Dmrnett method (significance Level: 5%). Finally, Kruskal-Wallis's rank test (significance level: 5%) was performed when the variance was 0 in any group and when the variance was not uniform, significance was observed between the groups In the case of multiple comparison by Dunnett type test method (significance level: 5%).

Reproductive indices:

- Copulation index = [Number of copulated pairs / number of mated pairs (%)]
- Fertility index = [number of fertile males / copulation index x 100 (%)]
- Implantation index = [Number of implantation scars / number of corpora lutea x 100 (%)]

Offspring viability indices:

- Gestation index = [Number of dams with live offspring / number of pregnant dams x 100 (%)]
- Sex ratio = [Number of male offspring / (number of male + female offspring)]
- Delivery index = [Number of offspring at birth / number of implantation scars x 100 (%)]
- Birth index = [Number of live offspring at birth / number of implantation scars x 100 (%)]
- Live birth index = [Number of live offspring at birth / number of offspring at birth x 100 (%)]
- Viability index = [Number of live offspring 21 days after birth / number of live offspring after culling x 100 (%)]

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

Females: one animal in the 100 mg/kg group died at perinatal stage, five in the 250 mg/kg group (table 2).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 250 mg/kg administration group, suppression of increase in body weight was observed in both males and females. No significant differences with the control were observed at any other dose.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the 250 mg/kg administration group, female food intake decreased. No effects were observed at 100 mg/kg or less.

Food efficiency:

not specified

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):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination of the liver revealed bile duct expansion and diffuse Kupffer cell proliferation around the Gleason sheath in males and females at 250 mg / kg administration group. On histopathological examination of the duodenum, thickening of the mucosa was observed in males and females at the dose of 250 mg / kg. Degeneration / necrosis of the proximal renal tubular epithelium in the kidney cortex was observed in females whose whole litter died by nursing day 4.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The female sex cycle, mating rate and conception rate, number of corpus luteums of pregnant animals, implantation number, implantation rate and pregnancy period were not affected by administration of test substance.
The estrous regression days in the 250 mg/kg group increased, but it had been observed before administration that the same group's estrus regression days tended to be extended.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

testis, epididymis, prostate and seminal vesicle weights were not affected by the administration of the test substance.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Failure of labor condition was observed in the group administered 100 and 250 mg / kg, and the birth rate decreased: the number of births and the number of babies born decreased, the live birth rate and the birth rate decreased. No effects on delivery rate, neonatal survival rate or sex ratio.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There was no effect of administration of the test substance on the sex ratio, the form and weight of the newborn infant.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The birth rate, fertility rate and neonatal survival rate decreased in the 250 mg/kg administration group.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of administration of the test substance on the sex ratio, the form and weight of the newborn infant.

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Any other information on results incl. tables

Table 2. Mortality in parental animals.

Animal number	Dose (mg/kg)	Death period	Internal uterine findings at necropsy
F03002	100	During labor	8 fetus remains
F04001	250	Nursing 3rd	No abnormality
F04004	250	Pregnancy 23rd	15 fetus remains
F04006	250	During labor	2 fetus remains
F04008	250	During labor	10 fetus remains
F04009	250	During labor	1 fetus remains

Table 3. Body weights of male rats.

Group		Olive oil (vehicle)			DCC	40mg/kg		DCC	100mg/kg		DCC	250mg/kg
Number of males		13			13			13			13
Days of administration	1	397.4	±	15.0	402.5	±	14.7	397.0	±	10.8	400.3	±	14.7
	7	429.3	±	19.5	427.2	±	22.0	421.2	±	14.8	420.7	±	16.6
	14	459.0	±	24.9	455.4	±	24.8	449.9	±	20.0	445.0	±	20.3
	21	481.7	±	24.1	479.8	±	26.1	470.2	±	22.0	460.9	±	23.6
	28	510.7	±	26.4	502.4	±	26.5	494.1	±	24.1	485.5	±	29.5
	35	533.7	±	30.6	526.6	±	26.3	516.2	±	28.0	506.9	±	32.2
	42	554.0	±	31.8	547.2	±	30.3	535.6	±	31.0	525.5	±	35.5

Each value shows mean (g)±S.D.

Significantly different from the control group (*: P<0.05, **: P<0.01).

Table 4. Body weights of female rats.  

Group		Olive oil (vehicle)	DCC 40 mg/kg	DCC 100 mg/kg	DCC 250 mg/kg
Number of females		13	13	13	13
Days of administration
	1	247.3 ± 11.9	248.1 ± 9.4	246.1 ± 12.2	250.2 ± 11.6
	7	258.6 ± 12.3	258.9 ± 10.3	259.7 ± 12.0	257.5 ± 13.9
	14	270.9 ± 15.9	270.9 ± 15.9	266.1 ± 16.7	260.7 ± 16.2
Number of dams		13	12	12	12
Days of pregnancy	0	280.9 ± 12.6	279.7 ± 14.4	276.9 ± 19.4	267.5 ± 15.3
	7	314.9 ± 17.1	309.7 ± 17.0	308.1 ± 21.4	293.9 ± 17.5 *
	14	351.6 ± 17.8	342.1 ± 18.4	347.7 ± 25.8	326.4 ± 18.5 **
	20	432.6 ± 25.8	415.0 ± 20.8	423.5 ± 30.0	392.3 ± 22.9 **
Number of dams		13	12	11	10
Days of lactation	0	323.8 ± 24.3	315.8 ± 34.0	307.7 ± 34.2	291.5 ± 36.1
	4	339.6 ± 26.7 (12)	335.3 ± 20.8 (10)	331.2 ± 26.9 (10)	325.2 ± 13.4 (4)

Each value shows mean (g) ± S.D. Significantly different from the control group (*: P<0.05, **: P<0.01). Figures in parentheses indicate number of dams.

 

Table 5. Organ weights of male rats

Group		Olive oil (vehicle)	DCC 40 mg/kg	DCC 100 mg/kg	DCC 250 mg/kg
Number of males		13	13	13	13
Body weight	(g)	528.4 ± 33.5	521.6 ± 28.4	507.6 ± 28.9	491.7 ± 33.8
Liver	(mg)	14917.5 ± 1803.5	14677.0 ± 1145.0	15266.9 ± 2121.6	15699.2 ± 1366.2
	(mg/g)	28.160 ± 2.004	28.149 ± 1.780	29.991 ± 2.914	31.930 ± 1.807**
Testes	(mg)	3257.5 ± 563.1	3441.9 ± 243.3	3293.5 ± 419.8	3387.7 ± 532.2
	(mg/g)	6.209 ± 1.230	6.618 ± 0.603	6.512 ± 0.912	6.922 ± 1.228
Epididymides	(mg)	1192.3 ± 194.7	1301.6 ± 102.0	1198.8 ± 88.7	1266.9 ± 158.8
	(mg/g)	2.268 ± 0.406	2.501 ± 0.223	2.368 ± 0.207	2.588 ± 0.377
Prostate, ventral	(mg)	626.5 ± 101.8	637.6 ± 162.5	638.5 ± 130.1	650.3 ± 104.6
	(mg/g)	1.194 ± 0.229	1.226 ± 0.313	1.256 ± 0.246	1.331 ± 0.245
Seminal vesicles	(mg)	1907.7 ± 430.4	1669.7 ± 338.9	1837.3 ± 216.5	1752.7 ± 126.9
	(mg/g)	3.614 ± 0.781	3.205 ± 0.638	3.617 ± 0.336	3.570 ± 0.214

Each value shows mean±S.D. Significantly different from the control group (*: P<0.05, **: P<0.01).

Table 6. Organ weights of female rats

Group		Olive oil (vehicle)	DCC 40 mg/kg	DCC 100 mg/kg	DCC 250 mg/kg
Number of females		12	10	10	4
Body weight	(g)	307.7±25.5	306.0±20.4	297.5±23.7	289.6±20.6
Liver	(mg)	10146.4±740.0	10223.8±555.2	15266.9±2121.6	15699.2±1366.2
	(mg/g)	33.120±3.014	33.501±2.342	34.760±2.501	35.956±4.321
Ovaries	(mg)	105.5±9.7	102.9±11.6	109.0±12.9	97.6±9.8
	(mg/g)	0.344±0.039	0.336±0. 032	0.367±0.044	0.338±0.033

Table 7. Reproductive performance.

Group	Olive oil (vehicle)	DCC 40 mg/kg	DCC 100 mg/kg	DCC 250 mg/kg
Number of pairs	13	13	13	13
Number of copulated pairs	13	13	13	13
Copulation index	100	100	100	100
Number of fertile males	13	12	12	12
Fertility index	100	92.3	92.3	92.3
Pairing days until copulation	2.6±1.3 (13)	3.2±1.0 (13)	2.8±1.2 (13)	2.6± 1.0(13)

Table 8. Reproduction/developmental toxicity.

Group	Olive oil (vehicle)	DCC 40 m/kg	DCC100mg/kg		DCC250mg/kg
Number of dams	13	12	11		8
Gestation length (days) Mean ± S.D. per dam Number of corpora lutea	22.5 ± 0.5	22.3 ± 0.5	22.5 ± 0.5		22.5 ± 0.5
Total	216	187	202		193
Mean ± S.D. per dam Number of implantation scars	16.6 ± 2,3	15.6 ± 1.4	16.8 ± 1.8	(12)	16.1 ± 1.9	(12)
Total	198	173	190		175
Mean ± S.D. per dam	15.2 ± 2.0	14.4 ± 1.4	15.8 ± 1.9	(12)	14.6 ± 5.3	(12)
Implantation index (%)	92.2 ± 8.7	92.7 ± 7.1	94.3 ± 7.6	(12)	90.7 ± 19.4	(12)
Gestation index (%)	100.0	100.0	91.7		66.7
Number of offspring at birth
Total	184	164	161		101
Mean ± S.D. per dam Number of live offspring at birth	14.2 ± 2.8	13.7 ± 1.4	14.6 ± 1.4		12.6 ± 4.3
Male	87	69	78		39
Female	92	90	73		39
Total	179	159	151		78
Mean ± S.D. per dam Sex ratio	13.8 ± 2.6	13.3 ± 1.4	13.7 ± 2.1		9.8 ± 5.7
Mean±S.D. per dam Number of dead offspring	0.49 ± 0.06	0.43 ± 0.10	0.51 ± 0.12		0.40 ± 0.28
Total	5	5	10		23
Mean ±S.D. per dam Delivery index	0.4 ± 0.9	0.4 ± 0.7	1.9 ± 3.9		2.9 ± 4.5
Mean%±S.D. per dam Birth index	92.1 ± 8.9	94.8 ± 5.5	95.3 ± 6.6		90.6 ± 12.4
Mean% ±S.D. per dam Live birth index	89.8 ± 9.5	910 ± 5.6	81.9 ± 28.3		72.2 ± 35.0
Mean% ±S.D. per dam	97.6 ± 5.4	97.1 ± 4.6	93.9 ± 12.1		77.6 ± 33.2
Number of offspring on day 4
Male	78	58	72		28
Female Sex ratio	83	74	63		17
Mean ±S.D. per dam Viability index	0.49 ± 0.06 (12)	0.44 ± 0.09 (10)	0.53 ± 0.11	(10)	0.62 ± 0.09	(4)
Mean% ±S.D. per dam	90.9 ± 27.4	83.3 ± 38.9	86.2 ± 31.7		57.1 ± 53.5	(7)
Number of external abnormalities 0	0	0	0		0
Mean% ± S.D. per dam	0	0	0		0

Applicant's summary and conclusion

Conclusions:

The NOAEL (based on toxicity) for both parent animals and first generation male/female Sprague Dawley rats was found to be 100 mg/kg bw; the NOAEL (based on reproductive ability) for parent male/female Sprague Dawley rats was found to be 40 mg/kg bw.

Executive summary:

A Reproduction / Developmental Toxicity Screening Test was conducted on the test item, according to OECD Guideline 421 (GLP study). 13 male and 13 female Sprague Dawley rats per group were exposed to 0 (control), 40, 100 or 250 mg/kg bw of test item by oral gavage, based on the results of a 28 -day repeated dose toxicity test performed previously. Males were dosed up to the day of conception and females were dosed throughout the study. Observations included clinical observations, body weight measurements, food intake, ostreus cycles, mating procedures, behavioural studies, evaluation of reproduction, weight of organs, and histopathological examinations. Toxicity signs were observed at the highest dose (lower body weight, enlarged liver, thickening of duodenum walls), and effects on reproduction were observed at the mid and high doses (lower birth rate, maternal deaths). Based on the available information, the NOAEL for general toxicity was set at 100 mg/kg bw, both for P0 and F1 generations, and the NOAEL for reproductive toxicity was set at 40 mg/kg bw.