N-cyclohexylcyclohexanamine 2-[1-[[[(1R)-1-[3-[(1E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanyl]methyl]cyclopropyl]acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: Guidelines for 28 day Repeat Dose Toxicity Test of Chemicals (Japan)

Deviations:

not specified

Principles of method if other than guideline:

Method: other: Guidelines for 28 day Repeat Dose Toxicity Test of Chemicals (Japan)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dicyclohexylamine

EC Number:

202-980-7

EC Name:

Dicyclohexylamine

Cas Number:

101-83-7

IUPAC Name:

N-cyclohexylcyclohexanamine

Test material form:

liquid

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, Atsugi Breeding Center production)
- Age at study initiation: 4 weeks old underwent preparatory handling for nine days
- Weight at study initiation: Males; 151.4 – 169.2 g Females; 129.9 – 149.6 g
- Fasting period before study:
- Housing: singly- metal cage wire mesh floor
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24.0 – 24.6°C
- Humidity (%): 46 – 63%
- Air changes (per hr): 15 times/hour
- Photoperiod (hrs dark / hrs light): illumination for 12 hours (7:00 a.m. to 7:00 p.m.)

IN-LIFE DATES: From: To: Animals received: November 20, 1996: Administration date: November 29, 1996 End of recovery 10th January 1997

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): test substance in the corn oil solution is stable for at least seven days in a range from 1.00 to 100 mg/ml
- Concentration in vehicle: The dicyclohexylamine was dissolved using corn oil to reach concentrations of 4.00, 14.0, and 40.0 mg/ml
- Amount of vehicle (if gavage): 5ml/kg
- Lot/batch no. (if required): not given
- Purity: not given

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

a content study was performed on the third formulation sample in this study, the test substance concentration in the administration sample was confirmed to be 102 – 113% of the prescribed concentration

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

70 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Group (concentration of administration solution) Number (animal numbers)
Male Female

Vehicle control group (Corn oil) 10 (1-10) 10 (31-40)
Dicyclohexylamine 20 mg/kg dosage group (4.00 mg/ml) 5 (11-15) 5 (41-45)
Dicyclohexylamine 70 mg/kg dosage group (14.0 mg/ml) 5 (16-20) 5 (46-50)
Dicyclohexylamine 200 mg/kg dosage group (40.0 mg/ml) 10 (21-30) 10 (51-60)

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: 14 days
- Dose selection rationale: The dosages were determined by referring to the results of the preliminary study for the dicyclohexylamine repeated dose 28-day oral toxicity study in rats that was performed prior to the start of this study. The results of document examination indicate that the 50% lethal dose for bolus administration of this test substance is 373 mg/kg, so a preliminary study was performed in which repeated doses of dicyclohexylamine were administered to male Sprague-Dawley rats of the same age as those used in this study once per day for seven days at dosages of 0, 50, 100, 250, and 500 mg/kg. As a result, all five rats to which 500 mg/kg was administered and four out of the five rats to which 250 mg/kg was administered died within the one week administration period, so these dosages were determined to exceed the maximum tolerated dose. However, no toxic changes were observed in the body weight, clinical signs, or autopsy findings of the rats to which 100 mg/kg was administered. We determined from these results that it was appropriate to set the maximum dosage in this study in the range between 100 and 250 mg/kg and that was confirmed by administering 150 and 200 mg/kg of this test substance to five rats each of the same age for a period of seven days. From those results, one of the five animals to which 200 mg/kg was administered died and indications such as convulsions and tremors were seen in clinical signs observations. From the above results, we determined that 200 mg/kg is appropriate for the maximum dosage in this study and then, dividing that by a common ratio of approximately three, the intermediate and lower dosages were set at 70 and 20 mg/kg.
- Rationale for animal assignment (if not random): Each animal was grouped through a random sampling method stratified by weight based on the weight of the animal the day prior to the start of administration
- Rationale for selecting satellite groups: For grouping, the 43 male and 43 females obtained were divided into groups of 36 and 7 starting from the lowest temporary animal number during preliminary handling. 30 animals were selected from this group of 36 to be the animals (ordinary test animals) investigated in accordance with the ‘Guidelines of the Act on the Evaluation of Chemical Substances and Regulation of Their Manufacture –Repeated Dose 28-day Oral Toxicity Study Using Mammals-’ and separately, six of the seven animals were selected to be the animals (neuropathological test animals) to undergo necropsy when the administration period was completed and have histopathological examinations of the nervous system performed. Of the ordinary male and female test animals that were alive when the administration period ended, five each from the vehicle control group and two each from the 200 mg/kg dosage group were used in the 14-day recovery study after the administration period ended.
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random):

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
-

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: In the ordinary test animals, weight was measured immediately before administration was started and at four days of administration, then after two weeks and during the recovery study period, the weight of all surviving cases was measured at a frequency of twice a week, and weight was also measured on the day that the administration period or the recovery study period ended as well as on the day of necropsy. In the neuropathological test animals, body weight measurement was performed at the same intervals as for the ordinary test animals in order to establish the dosage, but the data was not collected.


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data:
No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to the necropsies at the end of the administration period and the end of the recovery period on the ordinary test animals
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes -18-24 hours
- How many animals:
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to the necropsies at the end of the administration period and the end of the recovery period on the ordinary test animals
- Animals fasted: Yes 18-24 hours
- How many animals: 5/sex/dose
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: day 26
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: This behavior observation was conducted on all surviving cases of the 200 mg/kg dosage group at one, eight, 15, 22, and 28 days and at seven days during the recovery period. It was also conducted on all cases of the 70 mg/kg dosage group at eight, 15, 22, and 28 days, days on which abnormalities were observed in the 200 mg/kg dosage group, and it was conducted on all cases of the 20 mg/kg dosage group at 15, 22, and 28 days, days on which abnormalities were observed in the 70 mg/kg dosage group.
- Dose groups that were examined: 20, 70 and 200mg/kg
- Battery of functions tested: For the behavior observation, the animal’s condition in the cage, such as locomotor activity, posture, grooming and face washing, salivation, lacrimation, and the presence of convulsions and abnormal respiration, was observed and then, when the animal was removed from the cage, its reactivity to handling, vocalizations, and body temperature were observed. Furthermore, once the animal was moved to the work station, urination frequency, gait, tension of the limbs, and pupil diameter were observed.

OTHER:

Sacrifice and pathology:

When necessary in ordinary test animals, the axillary artery was severed and they were killed by exsanguination following the aforementioned blood collection and then gross examination of the organs and tissue was performed. In addition, the weight of each animal’s brain, liver, kidneys, adrenal glands, and testes or ovaries was measured and each organ weight was divided by the body weight on the necropsy date to calculate the relative weight. Additionally, the brain, spinal cord, pituitary, eyes, harderian gland, thyroid (including the parathyroid), submaxillary gland (including the sublingual gland), heart, lungs, liver, kidneys, spleen, adrenal glands, stomach, duodenum, jejunum, ileum, colon, rectum, testes or ovaries, bladder, prostate, seminal vesicle, bone marrow (femur), sciatic nerve, skeletal muscles (lower leg), and lesions were fixed in 10% formalin in 0.1 M phosphate buffer (pH 7.2). After paraffin embedding, hematoxylin-eosin stain samples of the brain, heart, liver, kidneys, spleen, adrenal glands, and sciatic nerve in all cases of the 200 mg/kg dosage group and the vehicle control group animals that were necropsied after the administration period were created in accordance with common procedures and they were examined histopathologically, as were the ovaries and the pituitary gland, which were suspected to have been affected by administration of the test substance from the results of the organ weight measurement in the necropsies at the end of the administration period. In addition, one case of extensive necrosis of the myocardium was observed in a male from the 200 mg/kg dosage group in the results of the histopathological examination of the cases that died during the study, so histopathological examination of the heart was performed on all of the males that died during the study as well as those that were necropsied when the administration period ended and when the recovery study period ended. Histopathological examinations of abnormal organs were also performed. At the same time, looking at the neuropathological test animals, perfusion fixation was performed at the end of the administration period on two males and three females of the vehicle control group that had survived at the end of the administration period and on one male and one female each of the 200 mg/kg dosage group and the perfusion fixation was performed from the aortic root under pentobarbital anesthesia with a fixing solution of 1.25% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer, the brain, spinal cord, and one section of the sciatic nerve were embedded in resin for electron microscopy, and they were examined histopathologically.

Statistics:

the mean and the standard deviation for all of the values obtained for body weight, food consumption, urinalysis (excluding semi-quantitative testing), hematological testing, blood biochemical testing, and organ weight. In addition, a variance uniformity assay (significance level: 5%) was performed using the Bartlett method when there were three groups, including the vehicle control group, with three or more animals per group, then when the variance was uniform, a one-way analysis of variance was performed, and when it was significant (significance level: 5), a multiple comparison was performed using the Dunnett or Scheffe method. However, if the variance was not uniform, a Kruskal-Wallis rank test was performed, and if it was significant (significance level: 5), a multiple comparison was performed using the Dunnett or Scheffe method. Moreover, for the histopathological findings, a significance test (significance level: 5%) was performed between the vehicle control group and each test substance administration group using the Mann-Whitney test for data that has been grade classified or performed using the one-sided Fisher’s exact test for positive total values. In the males and females of the 200 mg/kg dosage group, the results of the urinalysis excluding semi-quantitative testing, hematological testing, and blood biochemical testing, and the organ weight measurement values as well as the weight and food consumption amounts during the recovery period each had two cases, so only the mean value was obtained and assay was not performed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed in males and females of the dosage groups with dosages of 70 mg/kg or higher, convulsions were observed in the males at 70 mg/kg and higher and in females at 200 mg/kg, abnormal posture, decreased locomotor activity, abnormal vocalizations, respiration abnormalities, soiled fur, and mydriasis were observed in males and females at 200 mg/kg, straub tail was observed in males at 200 mg/kg, and tremors, piloerection, and reddish tears were observed in females at 200 mg/kg.

Mortality:

mortality observed, treatment-related

Description (incidence):

200 mg/kg dosage group: six of 10 of the males died by the end of the administration period, starting at 11 days, and six of 10 of the females died by the end of the administration period, starting at four days

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

200 mg/kg: significant decrease in body weight in both sexes compared to controls over the duraiton of the study. No effects at 70mg/kg or lower.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A decrease in food consumption was observed in males and females of the 200 mg/kg dosage group becomeing significnat at 22 dyays

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):

significant decrease in the segmented neutrophil percentage and a significant increase in the lymphocyte percentage were observed in females of the 70 mg/kg dosage group and an increase in white blood cells was observed in the females of the 200 mg/kg dosage group. In the testing at the end of the recovery study period, an increase in the white blood cell count was observed in females of the 200 mg/kg dosage group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significant changes in triglycerides levels (M, 70 mg/kg). Increased alkaline phosphatase (f, 200 mg/kg) Various significant changes to ion/counterion concentrations in both sexes at all doses (see below for detailed analysis)

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased adrenal gland (m/f, 200mg/kg) absolute and relative
decreased absolute liver weight (m/f 200mg/kg)
decreased absolute and relative weight in ovaries (70mg/kg and higher)
see below for further details

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the eight case of males and the eight cases of females from the 200 mg/kg dosage group that died, red spots on the thalamus, dark spots or dark areas on the lungs, paleness of the spleen, and soiled or wet fur or soiled or wet fur around the mouth and nose were observed in the necropsy results, but none of these were pronounced changes that could be a factor in the death and no pronounced changes were observed in other organs.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

salivation and convulsions in particular were seen in the dosage groups of 70 mg/kg and higher

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

Clinical signs:
Clinical study groups: In the 200 mg/kg dosage group, six of 10 of the males died by the end of the administration period, starting at 11 days, and six of 10 of the females died by the end of the administration period, starting at four days. One case of ulceration and crust formation was observed in the males of the vehicle control group, but it was a change that did not indicate dosage correlation. Salivation was observed in males and females of the dosage groups with dosages of 70 mg/kg or higher, convulsions were observed in the males at 70 mg/kg and higher and in females at 200 mg/kg, abnormal posture, decreased locomotor activity, abnormal vocalizations, respiration abnormalities, soiled fur, and mydriasis were observed in males and females at 200 mg/kg, straub tail was observed in males at 200 mg/kg, and tremors, piloerection, and reddish tears were observed in females at 200 mg/kg. The ulceration and crust formation observed in the male vehicle control group did not recover by the end of the recovery study period and the soiled fur observed in the females of the 200 mg/kg dosage group continued until the sixth day of recovery, but none of the other signs seen during the administration period were observed during the recovery study period.
In the examination of the animals’ behavior, salivation was observed in males and females of the 70 mg/kg dosage group starting on the observation at 15 days and in males and females of the 200 mg/kg dosage group starting on the observation at eight days and it was observed in all of the males and females at 70 mg/kg or higher at 22 days and 28 days. In addition, posture abnormalities such as crawling on its belly and crouching, as well as abnormal vocalizations were occasionally seen in the 200 mg/kg dosage group males and mydriasis was occasionally seen in the 200 mg/kg dosage group males and females. No abnormalities were observed in locomotor activity, grooming and face washing, lacrimation, abnormal respiration, reactivity to handling, body temperature, urination frequency, gait, or limb tension in any of the animals except for those with convulsions.

Neuropathological test animals: Of the three males in the 200 mg/kg dosage group, one died at 11 days and one died at 25 days. Two of the three females in the same group died at 28 days. In the males, the clinical signs of salivation, convulsions, posture abnormalities, decreased locomotor activity, soiled fur, and mydriasis were seen and in addition to the signs observed in the males, tremors, reddish tears, and piloerection were also observed in the females of the same dosage group.
In the tests related to animal behavior, salivation was observed at eight days in males of the 200 mg/kg dosage group and at 15 days for the females of that group, convulsions were observed at eight and 22 days in the males and at eight days, 15 days, and 28 days in the females. In addition, posture abnormalities and abnormal vocalizations were observed at 22 days in males. No abnormalities were observed in locomotor activity, grooming and face washing, lacrimation, abnormal respiration, response to touch, body temperature, urination frequency, tension in the limbs, or pupil condition, except in the animals that had convulsions.

2. Body weight
After administration was started, the mean body weight value of the males and females in the 200 mg/kg dosage group dropped below the mean body weight value of the vehicle control group and a significant difference was observed versus the vehicle control group continuously from eight days until the necropsy after the administration period ended for the males, and at four days, 25 days, and 28 days for the females. The mean body weight value continued to be low for both males and females even after the administration period ended. No change was observed in the body weight of the dosage groups of 70 mg/kg and lower.

3. Food consumption
A decrease in food consumption was observed in males and females of the 200 mg/kg dosage group. The decrease in food consumption was seen in males from eight days and it continued through the administration period, with a significant difference being observed at 22 days. A decrease was observed in the females only in the measurement at 22 days. A recovery in food consumption was observed in both males and females at eight days into the recovery study period. No change was observed in mean food consumption volume in the dosage groups of 70 mg/kg and lower.

4. Urinalysis
In all of the dosage groups, including the vehicle control group, there were cases of positive or false positive values for protein, ketone bodies, bilirubin, and urobilinogen in both males and females at the measurement periods in the week the administration period ended and the week the recovery test period ended and there were also cases in which epithelial cells or crystals were observed in the urine sediment. In the testing of females during the week that the administration period ended, a decrease in the potassium concentration in the urine was observed in the 20 and 200 mg/kg dosage groups and decreases in sodium and chlorine concentrations were observed in the 200 m/kg group, but these changes are due to the fact that the urine volume was slightly higher than in the vehicle control group and no difference was seen in the volume of these electrolytes excreted over 24 hours between these groups and the vehicle control group. No difference was observed between the vehicle control group and the test substance administration groups in the other test items.

5. Hematological testing
An increase in mean corpuscular hemoglobin concentration and mean corpuscular hemoglobin content in males in the testing at the end of the administration period was observed in the 70 mg/kg dosage group, but no changes were seen in the red blood cell count or the hematocrit value, so we determined it to be an incidental change. However, a significant decrease in the segmented neutrophil percentage and a significant increase in the lymphocyte percentage were observed in females of the 70 mg/kg dosage group and an increase in white blood cells was observed in the females of the 200 mg/kg dosage group. In the testing at the end of the recovery study period, an increase in the white blood cell count was observed in females of the 200 mg/kg dosage group.

6. Blood biochemical testing
In the testing at the end of the administration period, a significant increase in the blood triglyceride level was observed in males of the 70 mg/kg dosage group, an increase in the inorganic phosphorous concentration was observed in males of the 200 mg/kg dosage group and females of the groups with dosages of 70 mg/kg or higher, an increase in the calcium concentration was observed in females of the groups with a dosage of 70 mg/kg or higher, an increase in the sodium concentration was observed in males of the groups with a dosage of 70 mg/kg or higher, an increase in the potassium concentration was observed in females of the 200 mg/kg dosage group, an increase in the chlorine concentration was observed in males of the 20 mg/kg dosage group, and an increase in the alkaline phosphatase activity was observed in females of the 200 mg/kg dosage group.
In the testing at the end of the recovery study period, a decrease in the triglyceride level and an increase in the inorganic phosphorous concentration were observed in females of the 200 mg/kg dosage group. No difference was observed between the vehicle control group and the test substance administration groups for the other test items.

7. Pathological testing
1) Organ weight
In the necropsies performed at the end of the administration period, increases in both absolute weight and relative weight of the adrenal gland were observed in males and females of the 200 mg/kg dosage group, a decrease in absolute weight of the liver was observed in males and females of the 200 mg/kg dosage group and a decrease in the relative weight of the liver was observed in males of the 200 mg/kg dosage group while an increase in the absolute weight of the liver was observed in females of the 200 mg/kg dosage group, and a decrease in the absolute weight and the relative weight of the ovaries was observed in the groups with dosages of 70 mg/kg and higher. Inhibited weight gain was observed in both males and females of the 200 mg/kg dosage group in the necropsies, so the relative weight of the testes as well as male and female brains and kidneys increased, but no change was seen in the absolute weight.
In the necropsies performed at the end of the recovery study period, an increase in the absolute weight and relative weight of the adrenal gland was seen in females of the 200 mg/kg dosage group, a decrease in absolute weight of the liver was observed in males and females, and a decrease in absolute weight of the ovaries was observed in females. In addition, the inhibited weight gain continued into the recovery study period, so an increase in the relative weight of the brain and kidneys was observed in males and females and increases in the relative weight of the testes and adrenal glands were observed in males, but no increase in absolute weight was observed.

2) Necropsy findings

(1) Necropsy findings of cases that died during the study
In the eight case of males and the eight cases of females from the 200 mg/kg dosage group that died, red spots on the thalamus, dark spots or dark areas on the lungs, paleness of the spleen, and soiled or wet fur or soiled or wet fur around the mouth and nose were observed in the necropsy results, but none of these were pronounced changes that could be a factor in the death and no pronounced changes were observed in other organs.

(2) Necropsy findings at the end of the administration period
In the necropsies performed at the end of the administration period, there were occasional cases of black spots or areas on the lungs, pale areas on the lungs, dilatation of the renal pelvis, pale areas on the liver, diaphragmatic nodules on the liver, and dark spots on the ovaries and soiled fur was observed in one male and one female case of the 200 mg/kg dosage group and crust formation was observed in one male of the control group, but dosage dependence was not observed in any of the findings except for the soiled fur, so we determined that they were not clear changes that could be considered to be due to administration of the test substance.

(3) Necropsy findings at the end of the recovery study period
In the necropsies performed at the end of the recovery period, enlargement of the mandibular lymph nodes, dark spots or areas on the lungs, pale areas on the liver, mild adhesion of the abdominal cavity and skin crust formation were observed, but all of these are findings that are occasionally observed in rats of the same age, so we determined that they are not clear findings that can be considered to be due to the test substance administration.

3) Histopathological findings

(1) Histopathological findings of cases that died during the study
In the histopathological results of the cases in the 200 mg/kg dosage group that died during administration, one case of extensive myocardial degeneration (Photo 1) was observed. Mild myocardial degeneration was observed in one other male case, but it was at a level that is typically seen and it was an altered distribution, so we found it to be a naturally-occurring change. No other heart changes were observed in the other cases that died during administration. In addition, periportal fatty changes were observed in the liver, extramedullary hematopoiesis and brown pigment deposits were observed in the spleen, and basophilic tubules of the cortex and casts were observed in the kidneys, but these are changes that are observed in rats from the same line at a relatively high rate and they are not thought to be an effect of the test substance administration.
As macroscopic lesion sites, we saw hemorrhaging in the lungs that underwent histopathological observation for all three males and two of the females, and, of those, there was foam cell accumulation in two of the male cases and edema was observed in one of the male cases. In addition, one case of thymus gland bleeding and one case of skin ulceration were seen in the males. No histopathological abnormalities were observed in the brain, spinal cord, or sciatic nerve. No abnormalities were observed in the adrenal glands or in the female pituitary gland or ovaries.

(2) Histopathological findings at the end of the administration period
Comparing the necropsies of the vehicle control group and the 200 mg/kg dosage group at the end of the administration period, periportal fatty changes were observed in the livers of all of the males and females in both groups and extramedullary hematopoiesis was observed in the spleen of all of the male and female cases in both groups, and brown pigment deposits were observed in all of the females of both groups. In addition, basophilic tubules in the cortex of the kidneys were observed in all five male cases of the vehicle control group, one of the two males in the 200 mg/kg dosage group, three of the five females of the vehicle control group, and one of the two females in the 200 mg/kg dosage group and eosinophilic bodies were observed in three of the five males of the vehicle control group and casts were observed in one of the five females of the vehicle control group, but no significant difference was observed between the vehicle control group and the 200 mg/kg dosage group in the occurrence frequency in any of these histopathological findings.
No histopathological abnormalities were observed in the brain, spinal cord, or sciatic nerve in either the ordinary test animals or the neuropathological test animals.
Only one case of cortex hyperplasia in the adrenal gland was observed in a female of the vehicle control group. No abnormalities were observed in the adrenal glands or in the female pituitary gland or ovaries.
All of the hearts of males were examined histopathologically because one case of myocardial degeneration was observed in a male that died during administration, but mild myocardial degeneration that was considered naturally-occurring was observed in only one case of the vehicle control group and only three cases were seen in the 70 mg/kg dosage group.
The dark spots or areas in the lungs observed in the macroscopic findings were hemorrhages, and in the case in which dilatation of the renal pelvis was observed in the macroscopic findings, only basophilic tubules in the cortex were observed in the histopathological finding results other than the confirmed dilatation of the renal pelvis. Fatty changes were observed in the histopathological test results in the case in which pale areas were observed on the liver. In addition, hemorrhages and periportal fatty changes were observed in the case of diaphragmatic nodules in the liver and corpus luteum hemorrhages were observed in the case with dark spots on the ovaries. Moreover, crust formation was observed in one male case in the vehicle control group, but we confirmed that it was ulceration in the histopathological test results.

(3) Histopathological findings at the end of the recovery study period
All of the hearts of males were examined histopathologically when the recovery study ended because one case of myocardial degeneration was observed in a male that died during administration, but no abnormalities were found. Only fatty changes were observed in the case in which pale areas were seen macroscopically in the liver. In addition, both the dark spots and dark areas on the lungs were lung hemorrhages and the mandibular lymph node enlargement was plasma cell hyperplasia [translator’s note: rest of sentence untranslatable due to missing words], and one case of skin ulceration was seen in the male vehicle control group.
Each of these findings occurs frequently in rats of the same age, so we determined that they were not changes due to the test substance administration.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

70 mg/kg bw/day (nominal)

System:

autonomic nervous system

Organ:

other: Sympathetic Nervous System

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

RS-Freetext:
CLINICAL OBSERVATIONS:
-Clinical signs: Salivation, convulsion observed in male and
female rats from 70 mg/kg bw/day onwards which disappeared
during recovery period.

200 mg-groups, male and females:
--Mortality:  8/13; reason of death unknown, 1/18 with
myocardial degeneration
--Body weight gain (as graphic): significantly decreased,
continued (but not significant) until the end of the
recovery period:
FINAL BODY WEIGHTS, m/f: control versus high dose: 
end of treatment time: 355g/232g versus 285g/184g  
end of recovery period: 399.9g/266g versus 333g/236g
--Food consumption(graphic): significantly reduced but had
recovered by the end of the test

ALL DOSE GROUPS:
--HAEMATOLOGY (significant changes):
Male, 70 mg-group versus control:
-----MCH(pg): 21.9 versus 20.7; MCHC (%): 34.4 versus 33.2
Female, 70 mg-group versus control:
-----Seg. Neutrophils (% of WBC): 4 versus 13; 
-----Lymphocytes (% of WBC): 94 versus 84
Female, 200 mg-group (2 rats) versus control
    (at the end of the treatment period but not at the end
of the recovery period)
-----WBC (x 10[exp2]/mm³): 113 versus 40 

--CLINICAL CHEMISTRY (significant changes):
male, 20mg-group versus control:
-----Chloride (mEq/L): 107.2 versus 104.9
male, 70 mg-group versus control:
-----Triglyceride (mg/dL): 85 versus 45; 
-----Sodium (mEq/L): 143.4 versus 141.9
male, 200 mg-group (2 rats) versus control:
     (at the end of the treatment period but not at the end
of the recovery period) 
-----Inorg. phosphate (mg/dL): 9.2 versus 9.0;
-----Calcium (mg/dL): 9.4 versus 9.0 
female, 70 mg-gr., 200 mg-gr.(2 rats) versus control:
     (at the end of the treatment period but not at the end
of the recovery period) 
-----Inorg. Phosphate (mg/dL): 7.2, 10.5 versus 6.2
-----Calcium (mg/dL): 9.3, 10.7 versus 8.8

URINALYSIS (significant changes):
--female, potassium (mEq/day): 
20 mg-gr., 200 mg-gr. versus control: 152, 100 versus 247   
                           (70 mg-gr.: 187 not sign.);      
  (no changes after recovery period)
--female, Sodium (mEq/L): 
200 mg-gr versus control: 46.2 versus 85.5 (no changes after
the recovery period)
--female, Chloride (mEq/L):
200 mg-gr. versus control: 60.9 versus 111 (no changes after
the recovery period)


GROSS AND HISTOPATHOLOGY
--ORGAN WEIGHTS (significant changes):
-----adrenal gland
absolute//relative (mg//mg/g), high dose versus control
---male: 62.5//0.220 versus 47.3//0.134
---female: 88.2//0.478 versus 61.0//0.263
-----ovaries
absolute//relative(mg//mg/g), mid, high dose versus control
---female: 79.6//0.357, 59,9//0.325 versus 98.2//0.425
--HISTOPATHOLOGY
no relevant changes in comparison to the concurrent controls
reported

Applicant's summary and conclusion

Conclusions:

From the above results, salivation and convulsions were observed in clinical sign observations with a 70 mg/kg dosage of dicyclohexylamine and toxicological effects on autonomic nerves were suspected, so the maximum no observed effect level in male and female rats was determined to be 20 mg/kg.

Executive summary:

The dicyclohexylamine repeated dose 28-day oral toxicity study (14-day recovery) was conducted using male and female Sprague-Dawley (Crj:CD) rats. The dosages were 0 (vehicle control group), 20, 70, and 200 mg/kg for both males and females. The study was started with 13 males and 13 females in both the vehicle control group and the 200 mg/kg dosage group and 5 males and 5 females in both the 20 and 70 mg/kg dosage groups. One subset of the animals that survived at the end of the administration period underwent systemic perfusion fixation and was used for pathological examination of the central nervous system and 5 males and 5 females of the vehicle control group and 2 males and 2 females of the 200 mg/kg dosage group underwent the 14-day recovery test. The remaining animals that survived at the end of the administration period were necropsied at that time. The results are summarized below.

 

1. In the 200 mg/kg dosage group, death occurred starting on the 11th day of administration for males and on the 4th day of administration for females and was observed intermittently until the end of the administration period. In that dosage group, 8 of each of the 13 male/female rats died.

 

2. Looking at clinical signs, neurological signs such as salivation, convulsions, abnormal posture, mydriasis, abnormal respiration, and abnormal vocalizations were observed in both the male and female test substance administration groups and salivation and convulsions in particular were seen in the dosage groups of 70 mg/kg and higher. A noradrenaline antiresorptive effect in the sympathetic nerve terminals has been reported for dicyclohexylamine and the cause of changes in clinical signs and death seen in this study is conjectured to be excessive excitation of the sympathetic nerve. However, these nerve signs resolved when the administration period ended and no changes were observed in the brain, spine, or sciatic nerve in the histopathological examination results.

 

3. Extensive myocardial degeneration was observed in the pathological examination of one of the males that died midway through the study, but no obvious changes that were thought to be due to the test substance administration were observed in the hearts of the other animals that died. No changes that were thought to have been the cause of death were observed in the other pathological findings, hematological test results, or blood biochemical test results.

 

4. The mean body weight value in the males and females of the 200 mg/kg dosage group fell below the mean body weight value of those in the vehicle control group and a decrease in food consumption was observed. No effect was observed on the body weight or food consumption of the groups with administration of 70 mg/kg or lower. The average value of

the body weight in the 200 mg/kg dosage group continued to be low even after the administration period was completed, but a recovery was observed in food consumption when the drug was discontinued.

 

5. An increase in white blood cell count was observed in females of the 200 mg/kg dosage group when the administration period ended and when the recovery test period ended, but such an increase was not observed in the males and no change was seen in the differential leukocyte count of the females.

 

6. In the cases necropsied after the administration period, an increase in adrenal weight was observed in males and females of the 200 mg/kg dosage group and a decrease in ovary weight was observed in females of the 70 mg/kg dosage group and higher. A recovery from the effects related to ovary and adrenal weight was observed when the drug was discontinued.

 

7. A decrease in liver weight was observed in males and females of the 200 mg/kg dosage group and it did not recover even after the recovery test period had passed. Changes that could be the origin of the liver weight decrease were not observed in either the pathological findings or in the blood biochemical test results, so it is unclear what mechanism is responsible for the effect on the liver due to administration of the test substance.

 

8. In the blood biochemical testing at the end of the administration period, an increase was observed in the inorganic phosphorus concentration in males of the 200 mg/kg dosage group and in females of the 70 mg/kg dosage group and higher, an increase was seen in the calcium concentration of the females of the 70 mg/kg dosage group and higher, and an increase in alkaline phosphatase activity was observed in females of the 200 mg/kg dosage group. The changes in the inorganic phosphorus of males and females and in the calcium concentration of females of the 200 mg/kg dosage group were thought to be changes that deviated from the normal range, so an effect on phosphorus/calcium metabolism caused by test substance administration was suspected.

 

9. From the above results, we determined that the maximum no-observed-adverse-effect level of male and female rats in the repeated dose 28-day oral toxicity study of dicyclohexylamine was 20 mg/kg.