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

Endpoint summary

Description of key information

A Repeated Dose 28-day Oral Toxicity Study in Rodents equivalent to EU Method B.7, conducted on dicyclohexylamine was taken as the key study, resulting in NOAEL of 20 mg/kg body weight/day for males and females. When the point of departure was adjusted to account for the molecular weights of the molar equivalents in the registered substance, the adjusted NOAEL was 84.6 mg/kg/day for Montelukast.DCHA.

In addition, a 14 week oral toxicity Study in rodents, equivalent to EU Method B.26 was conducted on sodium Montelukast, which resulted in a NOAEL of 50 mg/kg/day for males and females. When the point of departure was adjusted to account for the molecular weights of the molar equivalents in the registered substance, the adjusted NOAEL was 65.5 mg/kg/day for Montelukast.DCHA.

Selection of the point of departure also took into account the appropriate adjustment factors to allow the derivation of the most conservative DNEL value.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

70 mg/kg bw/day (nominal)

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

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.

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.

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

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

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

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.

Reference 2

Endpoint:

repeated dose toxicity: oral

Remarks:

other: OECD 421

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

Qualifier:

equivalent or similar to guideline

Guideline:

other:

Deviations:

not specified

Principles of method if other than guideline:

Method: other: OECD TG421; preliminary reproduction toxicity screening,

GLP compliance:

yes

Limit test:

no

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
- Age at study initiation: 8 weeks
- Weight at study initiation: 306 – 335 g for males and 201 – 224 g for females
- Fasting period before study: N/A
- Housing: , the animals were kept separate in bracket-type wire mesh cages (W 410 × D 350 × H 170 mm: Lead Engineering Co., Ltd.) divided in two. A total of two animals, one male and one female, were housed together in the aforementioned cages during the mating period and the dam and pups were housed together in plastic econ cages (W 340 × D 400 × H 185 mm: Clea Japan) with a floor covering (white flakes: Charles River Japan) for the period from 17 days of gestation through four days of lactation.
- Diet (e.g. ad libitum): ad libitum solid food NMF (irradiation-sterilized and autoclave-sterilized)
- Water (e.g. ad libitum): ad libitum tap water (Gotenba Municipal Waterworks water: automated water supply equipment used).
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3°C
- Humidity (%): 50 ± 20%,
- Air changes (per hr): of 10 - 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours (7:00 a.m. to 7:00 p.m.).

IN-LIFE DATES: From: To:

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water):concentrations of the corn oil solution were stable after being stored for eight days refrigerated (approximately 4°C) away from the light followed by being stored at room temperature away from the light for 24 hours
- Concentration in vehicle:0, 4, 8 and 16 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no. (if required): chemical grade, Nacalai Tesque, lot number: V7R2020
- Purity: Chemical grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In formulating the test solution, the necessary amount of the test substance was measured for each concentration, it was dissolved in corn oil (chemical grade, Nacalai Tesque, lot number: V7R2020), and the specified amount was formulated. Test solution formulation was performed at a frequency of once per week as a general rule because it was confirmed at the Bozo Research Center that the test substance 4 and 40 mg/mL concentrations of the corn oil solution were stable after being stored for eight days refrigerated (approximately 4°C) away from the light followed by being stored at room temperature away from the light for 24 hours (Attached Data 2). After preparation, the daily amounts of the test solution were dispensed into brown glass bottles and stored refrigerated (approximately 4°C) away from light.
The percentage of the indicated value was always within the range of 97 – 101% and the concentrations were appropriate when each concentration of the test solution was confirmed at the Bozo Research Center using two administrations, one from before the start of administration to males and females and one in the final week of administration to males.

Duration of treatment / exposure:

Oral administration was selected as the administration route in accordance with the OECD Guideline for Testing of Chemicals, Preliminary Reproduction Toxicity Screening Test. The administration frequency was set at once per day and the administration period for males was set at the 14-day pre-mating period followed by the 49-day period until the day of necropsy after that; and for females, it was set at the 14-day pre-mating period, through the mating period, and the gestation period until the third day of lactation.

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 (twelve)

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: no
- Dose selection rationale: The dosages were determined using the results of the preliminary repeated dose 2-week oral administration study (Bozo Research Center, test number: U-1417, dosage: 0, 20, 40, 80, and 160 mg/kg) as a reference. Two of the five males and four of the five females in the 160 mg/kg dosage group died. There were no cases of death in the 80 mg/kg dosage group, but salivation was observed in both males and females in the clinical sign observations. No obvious changes in weight or food consumption were observed in the males of that group, but inhibited weight growth and a decrease in food consumption were observed in the females. However, no effects from administration of the test substance were observed on the female estrous cycle, necropsy findings, or organ weight. No obvious effects from the administration of the test substance were observed in the 20 or 40 mg/kg dosage groups other than the salivation that was observed in the 40 mg/kg dosage group. Consequently, the high dosage in this study was set at 80 mg/kg, at which effects on female body weight and food consumption was observed but at which there were no cases of death and the intermediate and low dosages were set at 40 and 20 mg/kg by dividing by a common ratio of two.
- Rationale for animal assignment (if not random): . Test animals’ were stratified by weight on the day of grouping (administration start day) and this was conducted through a combination of a block placement method and a random sampling method (the necessary groups were configured with the block placement method and the test groups and the individual numbers in the groups were allotted randomly) such that each group’s average weight was as equal as possible.
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random):

Positive control:

None

Observations and examinations performed and frequency:

1) Males (P)
(1) Clinical sign observation
Clinical sign observation was conducted on the outer body surface, nutrition status, and behavior three times a day, pre-administration, immediately post-administration, and two hours after administration. However, the observation on Saturdays and holidays occurred only pre-administration and immediately post-administration.
(2) Body weight measurement
Body weight was measured pre-administration on the first day of administration, then between 8:30 a.m. – 12:30 p.m.at four days, eight days, 11 days, 15 days, 22 days, 29 days, 36 days, and 43 days, and on the necropsy day (50 days after the start of administration).
(3) Food consumption measurement
For food consumption, the remaining food was measured between 8:30 a.m. – 12:30 p.m. on the same days that body weight was measured, excluding the mating period and one day’s food consumption was calculated by the difference from the previous day’s food consumption.

2) Females (P)
(1) Clinical sign observation
Clinical sign observation was conducted on the outer body surface, nutrition status, and behavior three times a day, pre-administration, immediately post-administration, and two hours after administration. However, the observation on Saturdays and holidays occurred only pre-administration and immediately post-administration.
(2) Body weight measurement
Weight was measured between 8:30 a.m. – 12:30 p.m. during the pre-mating period before administration at one day, then at four days, eight days, 11 days, and 15 days, followed by at zero days, seven days, 14 days, and 21 days of gestation during the gestation period, followed by at zero days and four days of lactation during the lactation period.
(3) Food consumption measurement
For food consumption, the remaining food was measured between 8:30 a.m. – 12:30 p.m. on the same days that weight was measured during the pre-mating period, then at one day, seven days, 14 days, and 21 days of gestation during the gestation period, followed by at one day and four days of lactation during the lactation period, and daily food consumption was calculated from the difference from the previous day’s food consumption.
(4) Vaginal smear examination
Vaginal smears were obtained from all females during the pre-mating administration period until copulation was established and were examined microscopically. The vaginal smear images during the pre-mating administration period were classified into proestrus, estrus, postestrus, and anestrous, and the frequency of estrus stage images and the number of days from one estrus stage until the next estrus stage (estrous cycle) were examined. The presence of sperm in the vaginal smear was checked during the mating period.
(5) Delivery and nursing observations
The dams delivered naturally and the presence of any delivery abnormalities was observed. Confirmation of delivery completion was conducted every day from 21 days through 23 days and if delivery had ended by 10:30 a.m., that day was considered ‘Day 0’ of lactation. If delivery occurred after 10:30 a.m., the following day was considered ‘Day 0’ of lactation. For dams for which a completed delivery had been confirmed, they were left to care for their pups until Day 4 of lactation and their lactation condition was observed using nest building, retrieving, and lactation as the indicators.

Sacrifice and pathology:

(4) Necropsy and organ weight measurement
After the weight of all cases was measured the day after the final administration, the subjects were exsanguinated through the abdominal aorta under ether anesthesia, necropsied, and the internal organs and tissue were observed macroscopically. In addition, the testes and epididymis were harvested and weighed, then fixed in Bouin solution. Additionally, the organs and tissue in which abnormalities were observed in the necropsy were fixed in 10% formalin solution prepared in a phosphate buffer solution (1/15 M, pH 7.1 – 7.4) (hereinafter abbreviated as ‘10% formalin solution in a phosphate buffer’) and preserved.
(5) Histopathological testing
The testes and epididymis of all cases were embedded in paraffin, segments were prepared for the control group and the high dosage group, hematoxylin-eosin staining and PAS dyeing was performed, and they were examined microscopically.
Female:
(6) Necropsy
After the weight of all cases was measured at four days of lactation, the subjects were exsanguinated through the abdominal aorta under ether anesthesia, necropsied, the internal organs and tissue were observed macroscopically, and the number of corpora lutea and the number of implantation sites were checked. In addition, the organs and tissue in which abnormalities were observed in the necropsy were fixed in a 10% formalin solution in a phosphate buffer and preserved. The two cases (No. 4102, 4110) from the 80 mg/kg dosage group that died during gestation and the six cases from the same group (No. 4101, 4106, 4108, 4109, 4111, 4112) in which all of the pups were stillborn or all of the pups died during the lactation period were necropsied in the same manner when they were discovered.
(7) Histopathological testing
The ovaries of all cases were embedded in paraffin, segments were prepared for the control group and the high dosage group, hematoxylin-eosin staining was performed, and they were examined microscopically.

Other examinations:

3) Parent animal (P) mating
After having administered the test substance for the 14-day pre-mating period, males and females of the same group were housed together overnight in one-on-one pairs. The cohabitation period lasted until copulation was confirmed, up to 14 days at most. The formation of a copulatory plug or the confirmation of sperm in the vaginal smear was regarded as confirmation of copulation and this day was considered ‘Day 0’ of gestation.

4) Pups (F1)
(1) Pup observation
At zero days of lactation, we counted the number of live born and the number of stillborn, and observed the existence of external malformations. Pups with external malformations were fixed in a 10% formalin solution in a phosphate buffer and preserved. The gender of the live born pups was determined and their weight was measured, then they were cared for by the dam. The stillborn pups were also fixed in a 10% formalin solution in a phosphate buffer and preserved.
(2) Nursling observation and measurement
Nurslings were observed once a day each day to determine whether they were alive or dead. Weight was measured at zero days (lactation Day 0) and at four days after birth. At four days after birth, all pups were exsanguinated under ether anesthesia, necropsies were performed, and the presence of internal organ abnormalities was checked.

Statistics:

9. Statistical processing
Statistical analysis was performed using the following methods and the two-sided significance level was 5 and 1%. For the delivery rate, birth rate, and viability rate, the mean value and the rate were obtained for each dam and compared.
1) Multiple comparison tests
A variance uniformity assay was performed for each group using the Bartlett method. As a result, when the variance was uniform, a one-way analysis of variance was performed and if a significant difference between groups was observed, a paired comparison test was performed for the control group and each administration group using the Dunnett method. If the variance was not uniform, a Kruskal-Wallis rank test was performed, and if it was significant, a Dunnett’s test was performed for the difference in the average of the ranks for the control group and each administration group.
Weight, food consumption, frequency of estrus images, estrous cycle, number of days of cohabitation, gestation period, organ weight, number of corpora lutea, number of implantation sites, number of live born pups, sex ratio, implantation rate, delivery rate, birth rate, viability rate

2) x2 test
Copulation rate, fecundation rate, delivery rate

3) Mann-Whitney U test
This was performed for the frequency of occurrence and intensity of degree for findings in the histopathological testing.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Mortality:

mortality observed, treatment-related

Description (incidence):

see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

I. Repeated dose toxicity
1. Effects on males (P)
1) Clinical signs
No changes were observed in clinical signs in groups with dosages of 40 mg/kg or lower. Salivation was observed temporarily or continuously immediately post-administration in 11 out of 12 cases (all except No. 4008) in the 80 mg/kg dosage group starting at seven days of administration. However, other neurological indications were not observed, so salivation was not determined to be an effect of administration of the test substance.

2) Body weight
The weight on each measurement day and the amount of weight gain in the groups with dosages of 40 mg/kg or lower were equivalent to that of the control group and no effect due to the administration of the test substance was observed. Weight gain was inhibited in the 80 mg/kg dosage group and a significant difference was observed in the measurement values at 29 days and at the day after final administration (50 days after the start of administration) as well as in the amount of weight gain between this group and the control group.

3) Food consumption
Significantly lower values were observed for food consumption at 22 days in the 80 mg/kg dosage group, but food consumption in the test substance administration groups was equivalent to the control group overall and no effect was observed due to administration of the test substance.

4) Necropsy findings
Other than dark red spots that were observed on the liver of one case in the 80 mg/kg dosage group (No. 4001) and that were considered incidental findings, macroscopic abnormalities were not observed in the internal organs or tissue in any of the cases.

5) Organ weight
No significant difference was observed between the control group and the test substance administration groups in the absolute weight of the testes or epididymis. In terms of relative weight, the testes weight in the 80 mg/kg dosage group was high and a significant difference was observed in the weight of the right side and in the bilateral weight. The high relative weight of the testes was considered to be a change caused by the low final body weight.
 
6) Histopathological testing
In an examination of the testes and epididymis performed in the control group and the 80 mg/kg dosage group, minor sperm retention and decreased sperm and cell debris in the lumen of the epididymis were observed in one case of the control group and one case of the 80 mg/kg dosage group (control group: No. 1005, 80 mg/kg dosage group: No. 4012), and mild sperm granuloma was observed in the epididymis of one case in the 80 mg/kg dosage group (No. 4006), but these were considered incidental changes from the histological properties.

2. Effects on females (P)
1) Clinical signs
No changes in clinical signs were observed throughout the pre-mating administration period, the mating period, the gestation period, or the lactation period in the groups with dosages of 40 mg/kg or lower. One case died at 21 days of gestation and one died at 22 days of gestation (No. 4102, 4110) in the 80 mg/kg dosage group. Salivation was observed immediately after administration in all of the cases starting at eight days and it was observed temporarily or continuously during the period up to three days of lactation, and unkempt fur was recorded prior to death (22 days of gestation) in one of the cases that died (No. 4102). However, no neurological symptoms other than salivation were observed in that group, so the salivation was not determined to be an effect of the administration of the test substance. In addition, clinical signs including a prone position, oligopnea, hypothermia, and poor pup retrieval were recorded in one case (No. 4108) two hours after administration after delivery had been completed at 22 days of gestation. During the lactation period of the same group, two cases of unkempt fur (No. 4108, 4111) were observed as clinical sign changes, and poor pup retrieval was observed in seven cases (No. 4101, 4104, 4106, 4107, 4108, 4109, 4112).
It is not indicated in the table, but one case of wheezing (No. 2106) was observed in the 20 mg/kg dosage group from 12 days to 15 days and the accumulation of white matter in the chest cavity was observed in the necropsy, so incorrect administration was believed to have occurred in that group.

2) Body weight
In the groups with dosages of 40 mg/kg or lower, the weights on each measurement day and the amount of weight gain were equivalent with those of the control group during the pre-mating period, the gestation period, and the lactation period and no effects from the administration of the test substance were observed. Weight gain was inhibited in the 80 mg/kg dosage group and a significant difference was observed between that group and the control group in the measurement values at four days, eight days, and 11 days, as well as at 21 days of gestation and at four days of lactation. In addition, the weight gain in each period was low and a significant difference was observed between that group and the control group in the weight gain amount in the gestation period.

3) Food consumption
Significantly lower values were observed in food consumption at one day of gestation in the groups with dosages of 40 mg/kg or lower, but overall, the values were equivalent to those of the control group throughout the pre-mating administration period, the gestation period, and the lactation period, and no effects from administration of this test substance were observed. Food consumption in the 80 mg/kg dosage group was lower than in the control group and significant differences were observed between the two groups at four days, 11 days, and 15 days during the pre-mating administration period, at one day and 21 days of gestation in the gestation period, and at one day and at four days of lactation in the lactation period. The decrease in food consumption of that group was remarkable during the lactation period.

4) Necropsy findings
A diaphragmatic hernia and dark red spots in the glandular stomach were observed in one case (No. 4102) of the 80 mg/kg dosage group that died at 22 days of gestation and unkempt fur was recorded in the external findings. No other macroscopic abnormalities were observed in the internal organs or tissue in any of the cases. The findings from the aforementioned fatal cases were not observed in other cases of the same group and were congenital abnormalities, so they were determined to be incidental findings.

5) Histopathological testing
There were no abnormal findings in any of the cases in the examination of the ovaries that was performed in the control group and the 80 mg/kg dosage group.

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

Critical effects observed:

no

Conclusions:

determined the general toxicity no observed effect level for males and females to be 40 mg/kg/day. No effects were observed on the estrous cycle, copulation, or fertility, or in the gestation period or delivery in any of the administration groups due to administration of the test substance. However, many dams with poor pup retrieval were observed in the 80 mg/kg dosage group during lactation and effects were observed on pup survival and development due to administration of the test substance. Consequently, the reproductive development no observed effect level was determined to be 80 mg/kg/day for males, 40 mg/kg/day for females, and 40 mg/kg/day for pups.

Executive summary:

A preliminary reproduction toxicity screening test of dicyclohexylamine was performed in rats by oral administration. Dicyclohexylamine was administered orally at dosages of 0 (corn oil: control group), 20, 40, and 80 mg/kg with 12 males and 12 females Sprague-Dawley SPF rats [Crj; CD (SD)] per group, and the effect on reproduction, such as the male and female animals’ gonadal function, mating behavior, gestation, and delivery, was examined.

 

I. Repeated dose toxicity

1. Effects on males (P)

1) No effects due to administration of the test substance were observed in clinical signs, body weight, food consumption, necropsy findings, or testes and epididymis weight for the groups with dosages of 40 mg/kg or lower.

2) An increase in body weight was inhibited in the 80 mg/kg dosage group. However, no effects due to administration of the test substance were observed for that group in clinical signs, food consumption, necropsy findings, or testes and epididymis weight or in the histopathological findings of these organs.

 

1. Effects on females (P)

1) No effects due to administration of the test substance were observed in clinical signs, body weight, food consumption, or necropsy findings for the groups with dosages of 40 mg/kg or lower.

2) In the 80 mg/kg dosage group, one case died at 21 days of gestation and one case died at 22 days of gestation. Unkempt fur and poor retrieving were changes in clinical signs that were observed during the lactation period. Weight gain was inhibited in the pre-mating, gestation, and lactation periods and food consumption was lower. However, no effects due to administration of the test substance were observed in the necropsy findings or the histopathological findings of the ovaries.

           From the above results, the general toxicity no observed effect level was found to be 40 mg/kg/day in both males and females.

 

II. Reproduction toxicity

1. Effect on parent animal (P)

1) No effects due to administration of the test substance were observed on the female estrous cycle, male and female mating, and fertilization, or the gestational period and delivery.

 

 

2) Lactation abnormalities were not observed in the groups with dosages of 40 mg/kg or lower. However, many dams with poor retrieving were observed in the 80 mg/kg dosage group and only four dams had surviving pups up to four days of lactation.

3) No effects due to administration of the test substance were observed in the number of dams with live pups, the birth rate, the number of corpora lutea, the number of implantation sites, or the implantation rate.

 

2. Effects on pups (F1)

1) No effects due to the administration of the test substance were observed on the number of live births, the number of stillborn, or the birth rate in the groups with dosages of 40 mg/kg or lower. There were many stillbirths in the 80 mg/kg dosage group and the number of live births was low, so the birth rate was significantly lower. However, no effects due to administration of the test substance were observed on the delivery rate in any of the dosage groups.

2) No effects due to administration of the test substance were observed on the sex ratio of the pups and no external malformations caused by administration of the test substance were observed.

3) No effects due to administration of the test substance were observed on the viability rate of the pups in the groups with dosages of 40 mg/kg or lower. In the 80 mg/kg dosage group, there were many pup deaths during the lactation period and the pup viability rate of that group was demonstrably low.

4) No effects due to administration of the test substance were observed on pup body weight in the groups with dosages of 40 mg/kg or lower. The pup body weight in the 80 mg/kg dosage group was low for both males and females at zero days and four days after birth.

5) No abnormalities caused by administration of the test substance were observed in the necropsies at four days after birth.

 

     From the above results, the reproductive development no observed effect level was found to be 80 mg/kg/day for males, 40 mg/kg/day for females, and 40 mg/kg/day for pups.

 

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4 Jan 1993 - 5 April 1993

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

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

L-706,631-001M021 (factor 1.04) was used
throughout the study. The purity of this material
was documented to be 99.3% (area %) via HPLC.

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Mouse, Crl:CD-1(R) (ICR) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age at Initiation: 36 days
Weight at Initiation:
Males: 23.9 to 31.9 grams
Females: 19.5 to 28.0 grams
Source: Charles River Laboratories, Raleigh, NC
Identification Method:
Tattoos and ear notching
Assignment to Dosage Groups:
Animals were assigned to dosage groups using a
balanced random allocation scheme.

b. Environmental Conditions:
Housing: Animals were housed 2 to 3/box in plastic boxes in
clean-air rooms with a 12-hour light cycle based on
a balanced random allocation scheme.
Diet: Purina Certified Rodent Chow and drinking water
were provided ad libitum. Animals were fasted
overnight prior to scheduled venipunctures and
necropsies.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on oral exposure:

The appropriate amount of test article was
suspended in 0.5% methylcellulose to achieve the
desired dosages at a standard dosing volume of
10 ml/kg. The dosing suspensions used for the
1600 mg/kg/day dosage group and the 800 mg/kg/day
dosage group were prepared separately; all other
dosing suspensions were prepared via dilution of the
dosing suspension used for the 800 mg/kg/day dosage
group.
Analysis of the dosing suspensions demonstrated
them to be of appropriate concentration and
uniformity.
The test article has been demonstrated
to be stable under the conditions of use in this
study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Not described

Duration of treatment / exposure:

91 days

Frequency of treatment:

once daily

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Dose / conc.:

1 600 mg/kg bw/day (actual dose received)

Remarks:

The 1600 mg/kg/day dosage group was terminated
early (following 19 doses) due to high mortality.

No. of animals per sex per dose:

10 (ten)

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Physical Examinations:

Daily observation for mortality and clinical signs of
treatment effects with less detailed examinations on
weekends and holidays.

Food Consumption and Body Weights:

Mice were weighed pretest,once during Drug Week 1,
and twice weekly thereafter; food consumption was
measured weekly over a three-day time interval for all
animals on study, except during weeks involving
holidays, bleedings, or necropsies.

Ophthalmoscopic Examinations:

Performed during Drug Weeks 6 and 11 on all surviving
animals in the control and 800 mg/kg/day dosage groups.
Examination performed using an
indirect ophthalmoscope. One or two drops of Mydriacyl
(1% tropicamide, Alcon Laboratories) were used to
facilitate inspection.

Hematologic Examinations
Performed during Drug Week 14 at necropsy on all
surviving animals. Under ether anesthesia, blood was
withdrawn from the vena cava for the determination of
the following parameters (sample size permitting):
erythrocyte count hemoglobin
hematocrit mean corpuscular volume
leukocyte count mean corpuscular hemoglobin
differential leukocyte count mean corpuscular Hgb concentration
platelet count

h. Serum Biochemical Determinations|:
Performed during Drug Week 14 at necropsy on all
surviving animals. Under ether anesthesia, blood was
withdrawn from the vena cava for the determination of
the following parameters (sample size permitting):
total protein albumin
glucose urea nitrogen
creatinine A/G ratio
AST ALT
alkaline phosphatase cholesterol
triglycerides sodium
potassium chloride
phosphorous calcium (total)

Sacrifice and pathology:

Terminal body weights and weights of selected
organs (see below) were recorded from survivors at
the scheduled termination.
Brain Heart
Kidney Liver
Spleen

Mice were killed by exsanguination while under
carbon dioxide anesthesia.
A complete gross necropsy was performed on all
mice except mice from the 1600 mg/kg/day group
killed and discarded without examination in Drug
Week 3. (Mice from this group which died or were
killed prior to this time were necropsied.)

Tissue Fixation:

Testes and epididymides from all male rats were
fixed in Bouin's solution. The remaining tissues
from all animals were fixed in ten percent neutral
buffered formalin.

Histomorphologic Examination:

Detailed examination (as shown below) on all
non-survivors to termination and on 5 surviving mice
per sex from the control group and the 800 mg/kg/day
group. (This was made necessary because the
1600 mg/kg/day group was terminated early due to
treatment-related mortality.)
Tissues routinely examined as part of detailed
examination were:
Salivary gland
Stomach
Small intestine
Large intestine
Liver
Gallbladder
Pancreas
Adrenals
Pituitary
Thyroids (and parathyroids when present in the
section)
Kidneys
Urinary bladder
Ovaries and uterus (or testes, epididymides and
prostate)
Skin
Mammary gland (when present in skin section)
Lung
Heart
Spleen
Lymph nodes
Thymus
Bone marrow
Bone
Skeletal muscle
Brain
Spinal cord
Peripheral nerve
Eye
Optic nerve
Harder's gland
Also, gross lesions of uncertain character were
examined from all mice.

Statistics:

i. Statistical Methods:
Body weight data were analyzed using the estimation of
linear and quadratic coefficients method. The level of
significance chosen was P = 0.05.

Absolute and relative splenic weights were
analyzed for significant drug-related trend
(P S 0.05). References to statistical methods are
shown in Appendix I.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The most prominent physical sign attributable to
test article administration was abdominal/gastro-
intestinal distention. This sign, the incidence of
which was dose-dependent, was observed in at least
one animal from all drug-treated groups except the
50 mg/kg/day dosage group.

Mortality:

mortality observed, treatment-related

Description (incidence):

high incidence of treatment-related
physical signs and subsequent mortality
in the 1600 mg/kg/day dosage group during the first
two weeks of the study (5 out of 20 animals dead or
in moribund condition; group terminated early). Nine animals at 800 mg/kg/day that died
as a result of treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant (P < 0.05) decrease in
body weight gain was observed in males and females
from all treated groups except the 50 mg/kg/day
dosage group. These body weight gain changes
appeared not to be dose-dependent; decreases for
females (males) were approximately 41% (35%), 28%
(31%), and 57% (31%), relative to the concurrent
control group, in the 200, 400, and 800 mg/kg/day
dosage groups, respectively

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In general, decreases
in food consumption paralleled these body weight
gain effects in females; these decrements in food
consumption, although present, were not as striking
in males.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Variations in mean weights of organs among various
groups were considered incidental. Statistical
analysis of weights of spleens showed a
statistically significant (P < 0.05) decrease in
mean absolute splenic weight of 800 mg/kg/day
males. Relative weights were not significantly
different (P > 0.05) compared to controls. In
addition, both absolute and relative splenic weights
of females were statistically significantly
(P < 0.05) decreased at the 400 and 800 mg/kg/day
level. There was no histologic correlate to account
for this change.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Distention of the gastro-intestinal tract, evident
grossly, was the only treatment-related change
seen. When present in non-survivors to
termination, it was considered the cause of death or
reason sacrificed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

mortality

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

intestine

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Please see appended report for detailed results

Conclusions:

The oral administration of L-706,631 to mice for 14 weeks was associated with abdominal/gastrointestinal distention,
body weight gain and/or food consumption decreases, and mortality at all dosages levels tested above 50 mg/kg/day.
The high incidence of treatment-related physical signs and subsequent mortality in the 1600 mg/kg/day dosage group during the first
two weeks of the study (5 out of 20 animals dead or in moribund condition) resulted in the surviving animals in
this dosage group being sacrificed during Drug Week 3. Each of these 5 animals at 1600 mg/kg/day was found to have
moderate to marked gastrointestinal tract distention at necropsy.
There were nine animals at 800 mg/kg/day that died as a result of treatment; 4 of these animals had
gastrointestinal tract distention. The cause of death/reason for sacrifice of the remaining
5 animals was undetermined. There was one treatment-related death at
400 mg/kg/day; the cause of death was undetermined.
At 200 mg/kg/day, there was I death considered treatment-related with abdominal distention.
The mortality in drug-treated animals for which the cause of death/reason for sacrifice was
undetermined was considered related to MK-0476 administration. This conclusion is based on the
fact that spontaneous death in mice of this age is uncommon.
Gastro-intestinal distention, of sufficient magnitude to cause death or be a reason for sacrifice, was the only postmortem change attributed to treatment.
The no-effect level for treatment-related changes in this study was the lowest dosage level, 50 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

84.6 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Adequate oral repeat dose studies are available for both of the constituent components of the substance.

System:

nervous system

Organ:

not specified

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The systemic effects noted in the studies conducted on dicyclohexylamine and sodium Montelukast did not fulfill the criteria for classification for Specific Organ Toxicity.

Adverse Effects seen in the DCHA studys were either: only present at concentrations significantly higher than the guidance values set out in table 3.9.3 of 1272/2008/EC when adjusted for molar equivalence in the registered substance; or findings were reversible following a recovery period and did not result in a significant functional change to the central /peripheral nervous system after administration was discontinued.

The significant treatment related adverse effect noted in animals treated with sodium Montelukast for 14 weeks was mortality/morbidity associated with abdominal distension due to gas. This is not of toxicological relevance in humans.