Bicyclo[2.2.1]heptanebis(methylamine)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 1991 - 12 August 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to Guidelines for Toxicity Studies of Drugs (Notification No. 24 of the First Evaluation and Registration Division, 1989) and in compliance with GLP; on this basis the study is considered reliable without restriction.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Guidelines for Toxicity Studies of Drugs (Notification No. 24 of the First Evaluation and Registration Division, 1989)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bicyclo[2.2.1]heptanebis(methylamine)

EC Number:

260-280-7

EC Name:

Bicyclo[2.2.1]heptanebis(methylamine)

Cas Number:

56602-77-8

Molecular formula:

C9H18N2

IUPAC Name:

1-[3-(aminomethyl)bicyclo[2.2.1]heptan-2-yl]methanamine

Test material form:

liquid

Details on test material:

Details on test material
- Name of test material (as cited in study report): 2,5 (or 2,6)-bicyclo[2,2,1] heptanebis (methylamine),
NBDA
- Physical state: clear and colorless liquid
- Analytical purity: 99.3%
- Lot/batch No.: 900327
- Stability under test conditions: Stable
- Storage condition of test material: Not reported

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 5 weeks old
- Weight at study initiation: 148 - 171 g (males); 121 - 137 g (females)
- Fasting period before study: no
- Housing: two animals per polycarbonate cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% CMC-Na solution

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Preparation was conducted from just before dosing until 9 days after the start of administration, and once a week thereafter. The dosing preparations were stored in a refrigerator until use. The dosing solutions were confirmed to be stable for 14 days after preparation.

VEHICLE
- Concentration in vehicle: 1.5, 10 and 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 15, 100 and 600 mg/kg bw/day (450 mg/kg bw/day - additional test) or 0, 1.5, 10, 60 mg/mL/day (45 mg/kg bw/day -additional test)
Basis:
actual ingested

No. of animals per sex per dose:

6 males and 6 females at 15 and 100 mg/kg bw/day
12 males and 12 females at 0 and 600 mg/kg bw/day
6 males and 6 females at 450 mg/kg bw/day (additional test)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: According to the results of a one-week preliminary study at dose levels of 60, 450, 600 and 750 mg/kg, toxic effects such as decreases in body weight due to the test substance were observed in the 600 mg/kg group or above. In contrast, there were no changes at 450 mg/kg or below. Based on these results, dose levels were set at 600 mg/kg as the highest dose and at 15 mg/kg as the lowest dose. The middle dose was set at 100 mg/kg from the approximate geometric mean between these doses. In addition, the control group receiving the vehicle was eslablished.
- Rationale for animal assignment (if not random): weight-stratified randomization
- Rationale for selecting satellite groups: only females were subjected to the recovery period, since death or moribund was obserwed in more than half males at the high dose group during the administration period.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Animals were checked for mortality, appearance and behaviour.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of first dosing and once a week thereafter.

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

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day after final dosing or after a 14-day recovery period for the surviving animals and for moribund sacrificed animals
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: No data
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day after final dosing or after a 14-day recovery period for the surviving animals and for moribund sacrificed animals
- Animals fasted: No data
- How many animals: all
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 5 days before the end of administration
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4) Organ weights
HISTOPATHOLOGY: Yes (see table 5)

Other examinations:

In this study, since death occured in a half of males and a few females at the high dose group, the reversibility of toxicity were not fully assessed. Accordingly, a supplemental study was conducted under the same conditions of this study at a dose level of 450 mg/kg, at which it was considered producing toxicity and saving the number of evalualed animals were assessed. According to the results, it was confirmed to have reversibility.

Statistics:

For the numerical data, homogeneity was tested by Bartlett's test. When the variance of data in the group was homogenous, one-way analysis of variance was tested, while when the variance or data in the group was heterogeneous, Kruskal-Wallis test was performed. If there were stalistical significances in data between groups, Dunnett's test or Dunnett type rank-sum test was conducted. Moreover, the data of urinalysis was analyzed by Armitage's chi-square test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
From the beginning of dosing, decreases in locomotor activity, salivation and ptosis were observed continuously in both sexes at the high dose group. These symptoms disappeared during the recovery period. The dead and moribund sacrifice animals in males of the same group revealed the following signs on the day before: or the day of death and moribund sacrifice; ralc, hypothermia, diarrhea, soiled peritoneal region, irregular respiration, bradypnea, lateral position in males and anemia, irregular respiration, bradypnea and crouching position in females.
Death was observed in males at the high dose group from 6 days after dosing and 7 out of 12 animals died or were moribund sacrificed by the end of the dosing period. Death was also observed in females at the high dose group from 8 days after dosing and 4 out of 12 animals died or were moribund sacrifice by the end of the dosing period. No treatment-relaled death occurred in the other groups.

BODY WEIGHT AND WEIGHT GAIN
Suppressed body weight gains were observed continuously in males at the high dose group during the dosing period. The body weights of females at each dose group gained similarly aS those of the control group.

HAEMATOLOGY
At the end of dosing, decreases in red blood cell count, hematocrit and hemoglobin concentration were observed in bolh sexes at the high dose group. The following changes were also noted at the high dose group; increases in white blood cell count and segmented neutrophil and decreases in lymphocyte and MCH in males, an increase in reticulocyte count in females. Moreover, decreases in red blood cell count, hemoglobin concentration, MCHC and a decrease in MCV were observed in females at the middle dose. A decrease in MCH was observed in females at the high dose group at the end ofrecovery.

CLINICAL CHEMISTRY
At the end of dosing, increases in GOT and GPT in males at the high dose group and increases in GPT in females at the middle and high dose groups were observed. Increases of inorganic phosphorus were observed in both sexes at the high dose group. An increase in total cholesterol in males and a decrease in glucose in females were noted at the high dose group. Besides, a decrease of urea nitrogen was observed in males at the middle dose group. At the end of recovery, a decrease in A/G ralio was noted in females at the high dose group.

URINALYSIS
Before the end of dosing, decreases in protein and ketones were observed in both sexes at the high dose group.

ORGAN WEIGHTS
At the end of dosing, the following changes were observed at the high dose group; an increase in absolute adrenal weight in males, increases in relative adrenal weight in both sexes, an increase in relative brain weight in males and an increase in relative liver weight in females.

GROSS PATHOLOGY
Dead and moribund sacrifice animals
The main changes observed in the dead/moribund sacrifice animals were hemorrhage or mucosal roughness in the stomach and dilatation of the small intestine. Hemorrhage of mucosa in the stomach, which was spread focally and diffusely to mucosa in the forestomach Of glandular stomach, was seen in nearly all dead/scheduled sacrifice animals. TWo animals showed mucosal roughness in the fOrestomach or glandular stomach. In the stomach, congestion of mucosa in the glandular stomach and dilalation were seen in a few animals. Dilalation of the small intestine was observed in nearly all animals, however it mostly emerged in the duodenum and its cavity was in the catarrhal state. In addition, mucosal roughness, hemorrhage of mucosa, adhesion in the small intestine and omentum were observed in a few animals. The following other changes were observed; enlargement or atrophy of the cecum, enlargement of the kidney or discoloration of cortex in the kidney, atrophy or opacity of the thymus, atrophy or discoloralion of the spleen, dilatation of atrium in the heart, congestion or edema of the lung, enlargement of the adrenal and alopecia in the left inguinal region of skin.

Scheduled sacrifice animals
In the scheduled sacrifice animals after 28 days, changes attributed to the test substance were observed in the stomach, small intestine and cecum in both sexes at the high dose group. Mucosal roughness in the forestomach and/or glandular stomach was observed in all animals. The following changes were seen sporadically in a few animals; hemorrhage of mucosa in the stomach, congestion of mucosa in the glandular stomach, thickening of mucosa in the glandular stomach, dilatation of the stomach,adhesion of the stomach and liver. In 1 male with adhesion of the stomach and liver abo had an adhesion of the liver, diaphragm and left and caudale lobe of the liver. Dilatation of the small intestine that was mainly in the duodenum was seen in all males and 2 females. Enlargement of the cecum was observed in 3 animals of each sex. In addition, hemorrhage of the lung in 1 male at the low dose group and 1 female at the high dose group, and pyelectasis in the kidney in 1 female at the high dose group were observed. However, each change Was considered not treatment-related.

HISTOPATHOLOGY: NON-NEOPLASTIC
Dead and moribund sacrifice animals
Common changes in Ihe dead/moribund sacrifice animals were necrosis of mucosa in the forestomach, glandular stomach and/or small intestine, hyaline droplet of hepatocyte, vacuolation off the proximal tubular epithelium in the kidney, atrophy oflymph follicle in the spleen. The extensive coagulation necrosis of the stratified squamous epithelium in the forestomach or glandular stomach was observed in all animals. Hemorrhage and marked in filtration of neutrophil were induced around the necrotic area, moreover, edema, exudation of fibrin, infiltration of inflammatory cells and an increase in fibroblast were observed under its submucosa. In additioo, the glandular stomach of 1 male and 2 females was exfoliated or partially defected and formed ulcer. These 3 animals showed squamous hyperplasia and hyperkeratosis. In the small intestine, similar coagulation necrosis as seen in the glandular stomach was observed in all animals. In 1 female having severe change, a lesion spread to muscular coat and chorionic membran and accompanied peritonitis. Hyperplasia of mucosal epithelium in the small intestine was also seen in 1 female. Hyaline droplet of hepatocyte was observed in nearly all animals. In the hepatocyte showing this change, granular hyaline droplet with acidophil Was frequently observed. This change was negative according to PAS staining. As for the other changes in the liver, a focal necrosis and congestion were seen in 2 males, 4 males and 2 females, respectively. In the kidneys, bilateral vacuolation of the proximal tubular epithelum was observed in nearly all animals. The epithelium was pervaded with various sizes of vacuolation, which led to enlarge the epitheliocyte. These changes were negative according to both PAS staining and Oil red O staining. In the spleen, atrophy of lymph follicle arising from a decrease in lymphocyte was observed in nearly all animals. Increases of neutrophil at the red pulp were noted in 1 female. Other changes were seen as follows; atrophy of the thymus arising from a decrease in conical thymocyte in 5 males and 1 female, congestion or hemorrhage of the thymus in 1 or 3 animals in males, congestion of the adrenal in 1 animal of each sex, congestion of the lung in 4 males and 2 females and pulmonary edema in 2 males and 1 female. Focal alopecia of the left inguinal skin was observed in 1 male at necropsy, however the hair follicle atroph

Scheduled sacrifice animals
In the scheduled sacrifice animals after 28 days, the following changes considered attributed to the test substance were observed at the high dose group; ulcer of the forestomach or squamous hyperplasia, hyperkeratosis, hyperplasia of mucosal epithelium in the small intestine, increases of neutrophils in the spleen and increases of fatty vacuole in the adrenals. Ulcer of the forestomach or squamous hyperplasia was observed in all aninmals of both sexes and hyperkeratosis was observed in 4 males and 3 females. The changes detected in the forestomach were similar to that of the dead/scheduled sacrifice animals. However, a lesion spread to muscular coat and chorionic membrane, had SignS of peritonitis around the affected area. In the liver of 1 animal, capsulitis caused by those effects was noted. Moreover, congestion of mucosa of the glandular stomach was observed in 1 animal of each sex. Hyperplasia of mucosal epithelium in the small intestine was oberved in all males and 3 females. In the spleen, increases of neutrophils at the red pulp were observed in 2 males and 3 females. In the adrenals, increases of fatty vacuole were observed in 1 animal of each seX. The cytoplasm of spongiocyte was pervaded with micro vacuolation by HE staining.
This vacuolation was lipid droplet, which was positive by Oil red O staining. These above changes of the scheduled sacrifice animals after 28 days disappeared in the recovery animals. In addition to the above-mentioned changes, focal hemorrhage of the lung, hyaline droplels in the tubular epithelium and pyelextais in the kidneys in the scheduled sacrifice animals and foreign body granuloma under submucosa in the forestomach were observed in a few animals. However, they were judged not treatrment-related.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed at this dose level

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The low dose group (15 mg/kg) demonstrated no treatment-related changes. Therefore, the no-observed-effect-level is considered to be 15 mg/kg under the conditions of this study.

Executive summary:

The test material was repeatedly administered to male and female SD rats for 28 days. A recovery period of 14 days was set for females of the control and high dose groups. The test material dissolved in the vehicle (0.5% CMC-Na solution) was administered orally by gavage with a metal gastric tube. Dose levels were set at 15, 100 and 600 mg/kg, and the control group was treated with the vehicle only. The dosing volume was set at 10 mL/kg. Death or moribundity occurred in 7 males and 4 females at the high dose group. In the clinical observations, decreases in locomotor activity, salivation and ptosis were observed in both sexes of the high dose group. Among dead and moribund sacrifice animals, lateral position, rale, hypothermia, diarrhea, soiled perineal region, irregular respiration and bradypnea in males and crouching position, anemia, irregular respiration, bradypnea in females were observed sporadically on the day before, or the day of death and moribundity. Suppression of body weight gain was observed continuously in males at the high dose group during the dosing period. A decrease in food consumption was observed continuously in males at the high dose group during the dosing period.

In the hematology test, it was revealed that red blood cell count, hematocrit value and hemoglobin concentration decreased in both sexes at the high dose group. Moreover, males revealed increases in white blood cell count and segmented neutrophil and decreases in MCH and lymphocyte and females showed an increase in reticulocyte count. No other in the report mentioned changes at the middle dose female rats like increases in red blood cell count, hemoglobin concentration and MCHC or decrease of MCV may be considered as non adverse since of the inconsistency or responses observed in the high dose treated rats.

In the blood chemistry test increases in GPT in males at the high dose group and in females at the middle dose group or above and increases in inorganic phosphorus in both sexes at the high dose group were revealed. However, the increases in GPT in females at the middle dose group were within normal biological variation (Table 18.7, Handbook of Toxicology, Ed. Derelanko; Hollinger, 2002). Moreover, at the high dose group, increases in GOT and total cholesterol in males, and a decrease in glucose in females were observed.

In urinalysis, there were no changes atributed to the test substance.

In organ weights, a high value of absolute adrenal weight was observed in males at the high dose group. As for the relative organ weight, an increased adrenal weight was noted in both sexes at the high dose group and increased liver weight was observed in females at the high dose group.

In the histopathological examination, the following findings were detected in nearly all dead/moribund sacrifice animals; necrosis of mucosa in the forestomach, glandular stomach and/or small intestine, hyaline droplet of hepatocyte, vacuolation of the proximal tubular epithelium in the kidney and atrophy of lymph follice in the spleen. Besides, the following findings were observed in both sexes at the high dose group necropsied after the 28-day dosing period; ulcer of the forestomach, squamous hyperplasia, hyperkeratosis, hyperplasia of mucosal epithelium in the small intestine, an increase of neutrophils in the spleen and an increase of lipid vacuole of spongiocyte in the adrenal. Since death occurred in about half or males and a few females at the high dose group (600 mg/kg), the reversibility or the toxicity could not be evaluated properly. Accordingly, a supplemental study was established anew at a dose level of 450 mg/kg, in which it was considered that toxicity would be produced, but enough animals would be survive to assess the reversibility of any induced changes.

The above-mentioned changes were considered reversible due to a recovery tendency or recovery by drug withdrawal.

From the results obtained, the no observed effect level of the test material was considered to be 15 mg/kg bw/day under the condition of this study and the no observed adverse effect level of the test material was considered to be 100 mg/kg bw/day.