N,N'-hexane-1,6-diylbis(hexahydro-2-oxo-1H-azepine-1-carboxamide)

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:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

As none of the deviations were rated as critical, deviation notes were not issued. The study report reflects the deviations in an appropriate way.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-hexane-1,6-diylbis(hexahydro-2-oxo-1H-azepine-1-carboxamide)

EC Number:

227-563-7

EC Name:

N,N'-hexane-1,6-diylbis(hexahydro-2-oxo-1H-azepine-1-carboxamide)

Cas Number:

5888-87-9

Molecular formula:

C20H34N4O4

IUPAC Name:

N,N'-hexane-1,6-diylbis(2-oxoazepane-1-carboxamide)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): N,N'-hexane-1 ,6-diylbis(hexahydro-2-oxo-1 H-azepine-1-carboxamide)
- Substance type: organic substance
- Physical state: solid
- Analytical purity: 100%
- Lot/batch No.: 07112601
- Storage condition of test material: Room temperature (20°C ± 5°C), dry
- Other:
The test item arrived at NewLab BioQuality GmbH on 07 January 2008 at ambient
temperature and was stored at room temperature on a cJosed shelf upon arrival. Additional
test item of the same batch arrived at NewLab BioQuality GmbH on 11 March 2008 at
ambient temperature and was stored at room temperature on a closed shelf upon arrival.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkel mann GmbH, Gartenstraße 27, 33178 Borehen, Germany
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: males: 167 to 175 grams (mean), females: 151 to 159 grams (mean)
- Fasting period before study: no
- Housing: Clean conventional housing: aeration with approx. 10 air changes per hour, room climate 22 ± 3°C at 30-70% relative humidity, artificial lighting , 12 h Iight / 12 h dark
Eight groups of five animals each in open makroion cages type 2000P (TechniPlast)
Lignocel hygienic animal bedding (J. Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg)
- Diet (e.g. ad libitum): Maintenance diet rat/mouse, pellets, No. 1324 (Altromin GmbH & Co. KG, 32791 Lage), ad /ib.
- Water (e.g. ad libitum): Autoclaved community tap water, ad lib.
- Acclimation period: 8 days (male) / 9 days (female)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70% relative humidity
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with (Type of food): corn oil

VEHICLE
- Justification for use and choice of vehicle (if other than water): test item has low solubility in water
- Amount of vehicle (if gavage): The test solutions were intended for an application volume of 8 ml per kg body weight.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Prior to this study, an acute oral toxicity study according to OECD 401 was performed in rats, which resulted in an LD50 of more than 2000 mg/kg body weight.
In a previously performed dose range finding study with dose escalation, the test item was administered in doses up to 2000 mg/kg body weight over a time period of 19 days and produced no observable toxic effects in the test animals. For this study, a high dose of 1000 mg/kg body weight was determined on request of the sponsor.
- Rationale for animal assignment (if not random):
On the day of arrival, the animals were caged in groups of five according to the following
stratification protocol: rats were weighed individually and grouped into weight categories, the
main group consisting of animals weighing between 100 g and 120 g. These were placed
consecutively into prepared cages. Rats weighing more than 120 g, but not more than 140 g,
were caged similarly after the main weight group was placed. At the time point of
stratification, no rat weighed less than 100 g or more than 120 g.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Viability/fatalities: Daily
- General clinical signs/behaviour: Once workdays

Cage-side observations to detect signs of illness, reactions to treatment, moribundity, or
mortality were performed at least once daily throughout the study, up to the day of necropsy.
Data were recorded on checklists. In ca se of any findings, the individual animal was
examined by use of an extra "health status" form sheet.


DETAILED CLINICAL OBSERVATIONS: Yes
Once weekly (including once before beginning of application)

Individual monitoring of detailed clinical signs was performed once before the first
application, and once a week thereafter. Findings were noted on prepared checklists,
comprising:
-changes in skin, fur, eyes, mucous membranes
-occurrence of secretions and excretions
-autonomie activity (e .g. lacrimation, piloerection, unusual respiratory patterns)
-changes in gait, posture, and response to handling
-presence of clonic or tonic movements, stereotypies (e.g. excessive grooming,
repetitive circling) , bizarre behaviour (e.g. self-mutilation, walking backwards)
-and any other observed, unusual signs.

BODY WEIGHT: Yes
- Time schedule for examinations:
Once weekly (including once before beginning of application)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual body weight and group food intake was monitored on a weekly basis, group water intake was monitored semiweekly during the in Iife phase, including day 1.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all
- Parameters checked in table below were examined:
Leukocytes
Erythrocytes
Reticulocytes
Hemoglobin concentration (HB)
Hematocrit (HKT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin conc. (MCHC)
Sodium
Potassium
Chloride
Calcium
Glucose (in serum)
Total cholesterol
Urea
Creatinine
Platelets/thrombocytes
Lymphocytes
Monocytes
Band neutrophils
Segmented neutrophils
Eosinophils
Basophils
Total protein
Albumin (in serum)
Globulin
Albumin/Globulin Ratio
Alanine aminotransterase (ALT)
Aspartate aminotransterase (AST)
Alkaline phosphatase
Gamma glutamyl transterase (GGT)


CLINICAL CHEMISTRY: Yes
see under "HEMATOLOGY"


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before application and once during the last
exposure week
- Dose groups that were examined: all
- Battery of functions tested:
On day 1 and in the last exposure week, additional recordings were made of grip strength
and reactivity to stimuli (Iimb placing test).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
The weight of organs denoted by "W" in was recorded; tissues denoted by "P" were preserved in the appropriate fixatives.
1 Gross lesions P
2 Oesophagus P
3 Trachea and thyroid P
4 Stomach P
5 Thymus P, W
6 Liver P, W
7 Spleen P,W
8 Duodenum P
9 Jejunum P
10 Ileum (with Peyer's patches)
11 Cecum P
12 Adrenals P,W
13 Urinary bladder P
14 Testes/ovary P,W
15 Epididymides P,W
16 Prostate/uterus P
17 Heart P, W
18 Lungs P
19 Peripheral nerve P
20 Bane marrow P
21 Sternum P
22 Spinal cord P
23 Colon P
24 Rectum P
25 Lymph nodes (intestinal area)
26 Kidney P,W
27 Whole brain W
28 Cerebrum P
29 Cerebellum P
30 Pans P


HISTOPATHOLOGY: Yes
Histological preparation was performed of the organs and tissues denoted by "P" in the table above.
The selected tissues were embedded in paraffin wax, sectioned, and stained with hemalaum
and eosin. The stained sections were transferred to the principal investigator 2 for
histopathological examination. The results were returned in a pathology expert report to the
hands of the study director.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the course of this study, no signs of illness (e.g. changes in skin, secretions), autonomic activity (e.g. lacrimation, piloerection), stereotypies, or behavioural reactions (e.g. selfmutilation) in response to the treatment or otherwise was observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

In the course of this study, no signs of illness (e.g. changes in skin, secretions), autonomic activity (e.g. lacrimation, piloerection), stereotypies, or behavioural reactions (e.g. selfmutilation) in response to the treatment or otherwise was observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The weight increase of the male and female animals from all animal groups was within normal range for rats of this strain and age.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of all animals was within normal range for rats of this strain and age.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

The water consumption of all animals was within normal range for rats of this strain and age.

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, treatment-related

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
No abnormalities in the general clinical signs or the behaviour were observed.
No animal of the test item groups or the vehicle groups died during the in-life phase of this
study.
In the course of this study, no signs of illness (e.g. changes in skin, secretions), autonomic
activity (e.g. lacrimation, piloerection), stereotypies, or behavioural reactions (e.g. selfmutilation)
in response to the treatment or otherwise was observed.

BODY WEIGHT AND WEIGHT GAIN
The weight increase of the male and female animals from all animal groups was within
normal range for rats of this strain and age. The weight gain of
male rats that received the high dose of the test item was slightly but significantly lower than
the control group during the fourth week of the study.
The low dose group of the female animals showed a similar weight gain when compared to
the vehicle group, animals from the medium and high dose groups gained significantly more
weight during the course of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption of all animals was within normal range for rats of this strain and age.

FOOD EFFICIENCY
no data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The water consumption of all animals was within normal range for rats of this strain and age.

OPHTHALMOSCOPIC EXAMINATION
yes

HAEMATOLOGY
Analysis of hematology and serum biochemistry showed normal results in most instances.
The blood parameters of male animals treated with the test
item showed erratic and few significant alterations when compared to the vehicle control
group. Chloride and the blood clotting time were slightly raised in the high dose group. In the
medium dose group, MCHC was slightly raised and reticulocytes and Albumin was slightly
lowered in comparison to their control group. The low dose group showed slightly elevated
numbers of segmented neutrophiles and a slightly decreased number of Iymphocytes. All
parameters measured in the blood of the male animals were without pathological findings.

The most prominent effects of the females of the high dose group and the medium dose
group were a significantly raised level of cholesterol and of globulin, indicating liver stress.
The raised levels of total protein and of the albumin/globulin ratio of the high dose group and,
milder, in the medium dose group when compared to the vehicle control group also point to
the liver as main target of the test item. A slight increase of monocytes and leucocytes was
restricted to the female high dose group and possibly indicates a mild effect of liver stress.

Although some significant changes were observed, none of the observed average data
points were overall extremely out of range for rats of this strain and age. Other biochemical
parameters and the blood cell counts showed no significant differences among the groups.

URINALYSIS
no data available

NEUROBEHAVIOUR
Motor activity and reactivity to visual and proprioceptive stimuli were not altered by the
administration of the test item. At the end of the observation
period, no differences in grip strength were observed in relation to the vehicle control group,
and none of the animals showed significant differences to the vehicle group during the limb
placing test.

ORGAN WEIGHTS
The organ weights of the male animals of the test item groups showed no difference to those
of the vehicle group. In the female dose groups, a significant increase in liver weight was
noted in the high dose group.

HISTOPATHOLOGY: NON-NEOPLASTIC
At histopathologic examination, a small number of microscopic findings were recorded in the
organs examined in this study. The type, incidence, and severity of almost all microscopic
findings noted did not indicate a relationship to the treatment with the test item.
In the liver, aminimal, centrilobular hepatocellular hypertrophy was observed in all males and
4 of the 5 females of the high dose group.
All other alterations were regarded to be spontaneous in nature and within the normal
background pathology commonly seen in rats of this strain and age.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed until 1000 mg/kg bw/day

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

The following table summarises the results of the treatment groups compared to the vehicle control group:

Parameter	Females	Males
	Low dose	Medium dose	High dose	Low dose	Medium dose	High dose
Detailed clinical signs	NAD	NAD	NAD	NAD	NAD	NAD
Body weight	NAD	elevated	elevated	NAD	NAD	NAD
Food consumption	NAD	NAD	NAD	NAD	NAD	NAD
Water consumption	NAD	NAD	NAD	NAD	NAD	NAD
Grip strength	NAD	NAD	NAD	NAD	NAD	NAD
Limb placing test	NAD	NAD	NAD	NAD	NAD	NAD
Hematology	NAD	cholesterol and globulin elevated	cholesterol and globulin elevated	NAD	NAD	NAD
Necropsy	NAD	NAD	NAD	NAD	NAD	NAD
Organ weights	NAD	NAD	liver weight increased	NAD	NAD	NAD
Histology	n.e.	n.e.	hepatoeellular hypertrophy	n.e.	n.e.	hepatoeellular hypertrophY

NAD: No abnormality detected

n.e.: not examined

Applicant's summary and conclusion

Conclusions:

General and detailed clinical signs, food and water consumption, and motor activity and
reactivity to sensory stimuli showed no abnormalities.

The weight gain of male animals was within normal range. The low dose group of the female
animals showed no difference in comparison to the vehicle group, the medium and high dose
groups gained mildly but significantly more weight during the course of the study.

Hematology and serum biochemistry parameters measured in the blood of the male animals
treated with the test item were not altered in comparison to their reference group. In
comparison to the vehicle control group, blood cholesterol and globulin levels were higher in
female rats of the high dose and of the medium dose groups. Other parameters were not
altered.

At macroscopic examination, no pathological findings were observed. The organ weights of
the male animals of the test item groups showed no difference to those of the vehicle group.
In the female high dose group, a significant increase in liver weight was noted.

Histopathological examination revealed aminimal, centrilobular hepatocellular hypertrophy in
the livers of all males and most females of the high dose groups.

The repeated daily oral administration of N,N'-hexane-1 ,5-diylbis(hexahydro-2-oxo-1 Hazepine-
1-carboxamide) to Wistar-Unilever rats at dose levels of 1000, 250, and 63 mg/kg
bodyweight for a treatment period of 28 days produced mild treatment-related alterations in
the liver of the high dose groups.

Microscopically, a minimal hepatocellular hypertrophy was noted in the liver of all male and
most female animals of the high dose group. A large number of medicinal agents with
different chemical structures and therapeutic activities produce liver enlargement when given
in high doses to species used in toxicity studies. Findings of this nature are in favour of an
adaptive response rather than a direct toxic effect of the test item. This finding, not present in
the control animals, was considered to be related to the treatment with the test item.

Executive summary:

The repeated daily oral administration of N,N'-hexane-1 ,6-diylbis(hexahydro-2-oxo-1H-azepine-

1-carboxamide) to Wistar-Unilever rats at dose levels of 1000, 250, and 63 mg/kg

bodyweight for a treatment period of 28 days produced mild treatment-related alterations in

the liver of the high dose groups.

Microscopically, a minimal hepatocellular hypertrophy was noted in the liver of all male and

most female animals of the high dose group. A large number of medicinal agents with

different chemical structures and therapeutic activities produce liver enlargement when given

in high doses to species used in toxicity studies . Findings of this nature are in favour of an

adaptive response rather than a direct toxic effect of the test item. This finding, not present in

the control animals, was considered to be related to the treatment with the test item.