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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 12, 1997 - November 4, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted according to the principles of GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: (Kanpogyo Notification No. 39, Yakuhatsu Notification No. 229, and 59 Kikyoku Notification No. 85 issued on March 31, 1984)and amended on November 18, 1988 (Kankiken Notification No. 233, Eisei Notification No. 38, and 63 Kikyoku Notification No. 823)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(2R)-2-methyloctane-1,8-diamine; (2S)-2-methyloctane-1,8-diamine
EC Number:
700-111-0
Cas Number:
148528-05-6
Molecular formula:
C9H22N2
IUPAC Name:
(2R)-2-methyloctane-1,8-diamine; (2S)-2-methyloctane-1,8-diamine
Details on test material:
- Name of test material (as cited in study report): 2-methyl-1,8-octanediamine (MODA)
- Substance type: technical material
- Physical state: colourless fluid
- Analytical purity: 99.94%
- Impurities (identity and concentrations): 1,9-nonanediamine (0.06%)
- Composition of test material, percentage of components: N/A
- Isomers composition: N/A
- Purity test date: Not stated
- Lot/batch No.: 093538
- Expiration date of the lot/batch: March 2000
- Stability under test conditions:
- Storage condition of test material: in a tight container in the cool and dark
- Other:

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 Laboratories Japan, Inc.
- Age at study initiation: 6 weeks
- Weight at study initiation: Males 179 - 200 grammes; Females 147 - 175 grammes
- Fasting period before study: N/A
- Housing: Animals were individually housed in wire-mesh cages (W 254 x D 350 x H 170 mm; Lead Engineering Co., Ltd.)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 50-64% (momentary deviations to 72% in two instances)
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs per day (from 07:00 to 19:00)


IN-LIFE DATES: From: June 11th 1997 To: July 30th 1997

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: 0.6, 3, 15 mg/ml
- Amount of vehicle (if gavage): 10 mls per kg bodyweight per rat
- Lot/batch no. (if required): water for injection (JP) Otsuka Pharmaceutical Factory, Inc., Lot Nos. 7C86 and 7D72
- Purity: Not reported but is water for injection
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dosing solutions used in the study were analyzed by high performance liquid chromatography (HPLC) at Bozo Research Center Inc. before
starting the study. Concentrations found ranged between 96% and 105% of the labeled concentration (CV 0.7-2.5%) and were within the acceptable
range (labeled concentration ±5%; CV 10% or less)
Duration of treatment / exposure:
Dosed for 28 days for groups 1 to 4. Followed by a two week (dose free) recovery period for further 6 animals per sex in groups 1 and 4.
Frequency of treatment:
Once daily (between 08:30 am and 12:00 midday)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.6 mg/mL
Basis:
nominal in water
Remarks:
Doses / Concentrations:
3 mg/mL
Basis:
nominal in water
Remarks:
Doses / Concentrations:
15 mg/mL
Basis:
nominal in water
No. of animals per sex per dose:
12 per sex in the control and at 150 mg/kg; 6 per sex in the 6 and 30 mg/kg group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral administration route was selected in accordance with the Toxicity Study Guidelines; At first, the dosage levels were set at 25, 80 and 250 mg/kg based on the results of the 14-day toxicity study previously conducted by the sponsor). However, 2 males and 2
females died in the first week after starting administration in the 250 mg/kg group, and further deaths were expected if the study was continued. In
addition, body weight gain showed a tendency to be inhibited slightly in females from the 80 mg/kg group. The study was then terminated at that
stage, and the high dose was set at 150 mg/kg, nearly in the middle of 80 and 250 mg/kg. Mid and low doses were set at 30 and 6 mg/kg with a
common ratio of 5.
- Rationale for animal assignment (if not random): Assignment to groups was made by combining the block placement method and the random
extraction method using a computer. (The necessary groups were formed by the block placement method, and the dose and intra-group individual
No. were randomly assigned.)
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random):
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations were included daily.


DETAILED CLINICAL OBSERVATIONS:
- Time schedule: All animals were observed with respect to general signs and symptoms, such as the external appearance
of the body, nutritional status, and behavior, 3 times a day (before, immediately after and 2 hrs after administration except on Saturdays and
holidays when observation was made twice, before and immediately after administration) during the treatment period.


BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed once before administration on Day 1 and during the subsequent administration period,
twice a week, i.e., every 3 or 4 days, before administration (between 08:30 and 10:00). During the recovery period, animals were weighed
in the morning (between 08:30 and 10:00) on Days 1, 3, 7, 10, and 14 of recovery. Animals were also weighed after fasting on the day of necropsy in
order to calculate organ weight relative to body weight.



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
Daily feed consumption, i.e., the amount of feed consumed from the previous day, was determined before administration on Day 1 of treatment.
During the treatment period thereafter, cumulative 3 or 4-day feed consumption was measured twice a week, i.e., every 3 or 4 days, before
administration (between 09:00 and 10:30), and the daily feed consumption per animal was calculated. During the recovery period, cumulative 2-day
feed consumption from Days 1 to 3 of recovery, cumulative 4-day feed consumption from Days 3 to 7 of recovery, and thereafter, cumulative 3
or 4-day consumption were determined, and daily feed consumption per animal was calculated.


OPHTHALMOSCOPIC EXAMINATION: Not performed


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Laparotomy was conducted under anesthesia on all animals fasted overnight (for about 16 hrs) when
animals were sacrificed the day after completion of the treatment period and the recovery period.
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: All
- The following parameters were examined.


RBC: Changes in electric resistance determination method
Hemoglobin (Hb): Cyanmethemoglobin method
Hematocrit (Ht): Calculated from RBC and MCV
Mean cell volume (MCV): Changes in electric resistance determination methodc)
Mean cell hemoglobin (MCH): Calculated from RBC and Hb pg
Mean cell hemoglobin concentration (MCHC): Calculated from Hb and Ht
Reticulocytes (%): Brecher method
Platelet count: Changes in electric resistance determination method
WBC: Changes in electric resistance determination method
Differential WBC count: May-Giemsa microscopic method
Prothrombin time (PT): Clot method
Activated partial thromboplastin time (APTT): Clot methodd) sec


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Laparotomy was conducted under anesthesia on all animals fasted overnight (for about 16 hrs) when
animals were sacrificed the day after completion of the treatment period and the recovery period.
- Animals fasted: Yes
- How many animals: All
- The following parameters were examined.

GOT: UV-rate method
GPT: UV-rate method
LDH: UV-rate method
gamma-GTP: gamma-glutamyl-3-carboxy-4-nitroanilide
Cholinesterase (ChE): DTNB method
ALP: Bessey-Lowry method
Total cholesterol (T.cho): CEH-COD-POD method
Triglyceride (TG): CEH-COD-POD method
Total bilirubin (T. bilirubin): Azobilirubin method
Glucose: Hexokinase-G6PD method
Blood urea nitrogen (BUN): Urease-GLDH method
Creatinine: Jaffé method
Sodium (Na): Ion selective electrode method
Potassium (K): Ion selective electrode method
Chloride (Cl): Ion selective electrode method
Inorganic phosphorus (P): molybdate method
Total protein (TP): Biuret method
Albumin (Alb): BCG method
A/G Ratio: calculated from TP and Alb


URINALYSIS: Yes
- Time schedule for collection of urine: Week 4 (days 27-28) of treatment and week 2 (days 13-14) of recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- The following parameters were examined:

pH: Uriflet 7A test paper (Kyoto Daiichi Kagaku Co., Ltd.a))
Protein: Uriflet 7A test paper (Kyoto Daiichi Kagaku Co., Ltd.a))
Keton bodies: Uriflet 7A test paper (Kyoto Daiichi Kagaku Co., Ltd.a))
Glucose: Uriflet 7A test paper (Kyoto Daiichi Kagaku Co., Ltd.a))
Occult blood: Uriflet 7A test paper (Kyoto Daiichi Kagaku Co., Ltd.a))
Bilirubin: Uriflet 7A test paper (Kyoto Daiichi Kagaku Co., Ltd.a))
Urobilinogen: Uriflet 7A test paper (Kyoto Daiichi Kagaku Co., Ltd.a))
Color: Naked eye
Sediments: Microscopic examination
Urine volume (24hrs): Volumetric measurement
Osmotic pressure (mOsm/kg): Freezing point depression methodb)
Water consumption: Gravimetric method


NEUROBEHAVIOURAL EXAMINATION: Not performed

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Numerical data (body weight, feed and water consumption, quantitative data obtained in urinalysis, data obtained in hematological and blood
biochemical examinations, and organ weight) were first analyzed using Bartlett’s method to determine whether or not the variance in each group was
homogeneous. If the variance was found to be homogeneous, one-way layout variance analysis was conducted. If significant inter-group differences were observed, further analyses were conducted using Dunnett's test. If the variance was not found to be homogeneous, Kruskal-Wallis’ rank test wasconducted. If significance was found, differences in the mean rank were compared between the control group and each treated group using
a Dunnett-type method (mean rank test method)2). Qualitative data obtained by urinalysis were analyzed by the cumulative Chi squared test.
Differences were regarded as significant if p<0.05 or P<0.01 on both sides.

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 examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
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 examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Treatment period:
One male and 2 females from the 150 mg/kg group died. The male (No. 4021) was sacrificed in extremis because it became moribund on Day 27
showing deep breathing, abdominal distention, unkempt fur, and decreases in spontaneous movements. This animal sporadically showed marked
salivation, deep breathing and abdominal distention immediately after administration while it was alive.
One female (No. 4121) died on Day 28 of treatment. Although it showed no abnormal general signs or symptoms shortly before death, it had shown marked salivation on Day 4 of treatment. The other female (No. 4129) died on Day 26 of treatment. It showed deep breathing, abdominal distention,
and unkempt fur shortly before death in addition to marked salivation on Day 13 of treatment.
No male or female from either the 6 or 30 mg/kg group showed any abnormality.
In the 150 mg/kg group, females showed no abnormality, but 3 males showed marked salivation shortly after administration on Day 1 of treatment.
One of these animals also showed marked salivation shortly after administration on Day 5. Another male showed deep breathing from Days 2 through 6 and abdominal distension on Day 5. These symptoms disappeared transiently on Day 7 and onward, but deep breathing recurred on Day 15 and
lasted until Day 27.
Recovery period:
Males showed no abnormality in the 150 mg/kg group, but 1 female showed wheezing from Day 10 of recovery and abdominal distension from
Day 13 of recovery.

BODY WEIGHT AND WEIGHT GAIN
Treatment period:
In the 6 and 30 mg/kg groups, both males and females showed changes similar to those seen in the control group, and no significant differences
were noted. In the 150 mg/kg group, females showed no significant differences compared to the control. However, body weight of males remained
significant smaller than in the control group throughout the treatment period.
Males also showed significant decreases in weight gain during the treatment period.

Recovery period:
In the 150 mg/kg group, females showed no significant differences compared to the control. Body weight of males remained significant smaller than
in the control group throughout the recovery period. However, their weight gain during the recovery period was similar to the control group.

FOOD CONSUMPTION
Treatment period:
In the 6 and 30 mg/kg groups, both males and females showed changes similar to those seen in the control group, and no significant differences
were noted. In the 150 mg/kg group, females showed no significant differences compared to the control. However, feed consumption by males
remained smaller than in the control group throughout the treatment period, and differences were significant on Days 4, 24 and 28.

Recovery period:
In the 150 mg/kg group, both males and females showed changes similar to those seen in the control group, and no significant differences were
noted.



WATER CONSUMPTION
Neither males nor females showed any significant differences in water consumption compared to the control group regardless of the dose during the treatment period or the recovery period.


HAEMATOLOGY
Upon completion of the treatment period:
In the 150 mg/kg group, females showed significant increases in activated partial thromboplastin time. Other significant changes observed included significant decreases in % lymphocytes in females from the 6 mg/kg group and significant decreases in the mean cell volume (MCV) and %
lymphocytes and significant increases in % segmented neutrophils in females from the 30 mg/kg group. However, these changes did not show any
dose responses.

Upon completion of the recovery period:
In the 150 mg/kg group, neither males nor females showed any significant differences compared to the control group.

CLINICAL CHEMISTRY
Upon completion of the treatment period:
In the 150 mg/kg group, males showed significant increases in GPT levels. However, neither males nor females from any group showed any other
significant differences compared to the control group.

Upon completion of the recovery period:
In the 150 mg/kg group, females showed significant decreases in triglyceride levels, but this change was not observed at the end of the treatment
period. Males showed significant differences compared to the control group.

URINALYSIS
Week 4 of treatment:
In the 150 mg/kg group, the number of females with urinary protein showed a significant increase. The number of females with urinary protein also
showed a significant increase in the 6 mg/kg group, but no dose responses were observed.

Week 2 of recovery:
In the 150 mg/kg group, neither males nor females showed any significant differences in any observation parameter compared to the control group.

NEUROBEHAVIOUR
No investigations performed

ORGAN WEIGHTS
Upon completion of the treatment period:
In the 150 mg/kg group, males showed significant decreases in absolute weight of the heart, liver and kidneys and significant increases in relative
weight of the lungs and testes, but all these changes were thought to be attributable to decreases in body weight determined before necropsy.

Upon completion of the recovery period:
In the 150 mg/kg group, females showed significant increases in absolute and relative weight of the adrenals, but these changes were not observed
at the end of the treatment period. Other changes observed included significant decreases in absolute weight of the kidneys (males) and the brain
(females) in the 150 mg/kg group, but relative weight showed no significant changes and the changes observed were thought to be attributable
to decreases in body weight determined before necropsy.

GROSS PATHOLOGY
Animals found dead or sacrificed in extremis:
Necropsy findings obtained in one male (No. 4021) and 2 females (Nos. 4121 and 4129) from the 150 mg/kg group are shown below.
No. 4021: Unkempt fur, atrophy of the spleen, and white spots in forestomach were observed in addition to dilatation of the GI tract from the stomachthrough the rectum due to gas retention.
No. 4121: This animal showed dark red spots in the glandular stomach and dilatation of the GI tract from the duodenum through cecum due to gas
retention.
No. 4129: This animal showed atrophy of the thymus and spleen and dilatation of the GI tract from the jejunum through colon due to gas retention.

Surviving animals:
No changes attributable to the test substance were observed upon completion of the treatment period. Upon completion of the recovery period,
however, one female (No. 4128) from the 150 mg/kg group showed undernourishment, as well as dilatation of the esophagus, stomach, jejunum,
ileum, and cecum due to gas retention.
Other changes described below were observed, but these were thought to be incidental because of their onset pattern.

Upon completion of the treatment period:
Lung: Dark red patches were observed in one male (No. 4024) from the 150 mg/kg group.
Ileum: Diverticulum was observed in one male (No. 3025) from the 30 mg/kg group.

Upon completion of the recovery period: Testes and epididymides: Small testes and epididymides (bilateral) were observed in one male (No. 4031) from the 150 mg/kg group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Animals found dead or sacrificed in extremis:
Histological findings obtained in one male (No. 4021) and 2 females (Nos. 4121 and 4129) from the 150 mg/kg group are shown below.
No. 4021: This animal showed slight ulcers (corresponding to white spots observed with the naked eye) in the forestomach and minimal vacuolizationof the mucosal epithelium in the glandular stomach, as well as minimal vacuolization of the mucosal epithelium in the duodenum, jejunum and ileum.
No. 4121: This animal showed slight erosions and hemorrhage (corresponding to dark red spots observed with the naked eye) in the glandular
stomach.
No. 4129: This animal showed slight hemorrhage in the adrenal cortex, as well as slight follicular atrophy in the spleen and slight atrophy of the
thymus.

Surviving animals:
Upon completion of the treatment period:
Changes attributable to the test substance were observed in the stomach as follows:
Slight mucosal hyperplasia in the forestomach associated with edema and cellular infiltration in 1 female from the 150 mg/kg group. The following changes were also observed in the lungs (including bronchi), but these changes were thought to be incidental because of their
onset pattern and pathological characteristics:
Slight hemorrhage in 1 male from the 150 mg/kg group and minimal focal pneumonia in 1 female from the control group. The following organs of males and females from the 150 mg/kg group showed no abnormal findings: cerebrum, cerebellum, heart, duodenum,
jejunum, ileum, cecum, colon, rectum, liver, pituitary, thyroid (including parathyroids), adrenals, spleens, kidneys, urinary bladder, testes, ovaries, eyeballs, sternum (bone marrow), and femur (bone marrow). The stomach, duodenum, jejunum, ileum, cecum, colon, and rectum also showed no
abnormality in the 6 and 30 mg/kg group.
Upon completion of the recovery period: Changes attributable to the test substance were observed in the jejunum and colon as follows:
Minimal vacuolization of mucosal epithelium in 1 female from the 150 mg/kg group. The stomach, duodenum, cecum, colon, and rectum showed no other abnormal findings.

Sites with gross abnormalities (upon completion of the recovery period)
Testes and epididymides: The atrophy observed with the naked eye in one male (No. 4031) from the 150 mg/kg group was found to be severe
atrophy of the testes and seminiferous tubules and associated marked decreases in the number of sperms in the epididymides. However, these
changes were thought to be incidental because they were observed in only 1 animal and were not observed at the end of the treatment period.
Esophagus: Dilation due to gas retention was observed in 1 female (No. 4128) from the 150 mg/kg group, but this was not associated with any
histological change.


HISTORICAL CONTROL DATA (if applicable)
NOT APPLICABLE


OTHER FINDINGS
NO OTHER FINDINGS

Effect levels

Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; histopathology;

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No effects of the test substance were observed in the 6 or 30 mg/kg group. Based on these findings, the no-observed-effect level (NOEL) for MODA was estimated to be 30 mg/kg/day under the conditions of the present study.
Repeated exposure resulted in mortality at 150mg/kg/d, this is sufficient evidence for classification as STOT-RE 2 H373
Executive summary:

A study was performed at Gotenba Research Center, Bozo Research Center Inc., Japan, on behalf of Kuraray Co., Ltd., to investigate the changes caused by 2-methyl-1,8-octanediamine (MODA) and their reversibility in Sprague-Dawley rats when administered daily by oral gavage over a 28 day period followed by a two week recovery period. The study was conducted according to the relevant Japanese Guidelines and in accordance with the principles of GLP. The number of animals per group was 12 males and 12 females each in the control and 150 mg/kg groups and 6 males and 6 females in the 6 and 30 mg/kg groups. Six males and 6 females each from the control and 150 mg/kg groups were used in the 2-week recovery study after treatment for 28 days. One male and two females died after administration of 150 mg/kg of MODA, and both males and females from this group showed changes mainly attributable to the irritating effects of the test substance. Animals from this group also showed increases in GPT activity (males) and increases in activated partial thromboplastin time (females). However, no effects of the test substance were observed in the 6 or 30 mg/kg group. Based on these findings, the no-observed-effect level (NOEL) for MODA was estimated to be 30 mg/kg/day under the conditions of the present study.