N,N'-(2-chloro-1,4-phenylene)bis(3-oxobutaneamide)

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:

2000-01-13 to 2000-10-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted July 27, 1995

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-010-4

EC Name:

-

Cas Number:

53641-10-4

Molecular formula:

C14H15ClN2O4

IUPAC Name:

N-[3-chloro-4-(3-oxobutanamido)phenyl]-3-oxobutanamide

Test material form:

solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

HSD

Details on species / strain selection:

TEST ANIMALS
- Source: HARLAN WINKELMANN, Gartenstraße 27, 33178 Borchen, SPF breeding colony
- Age at study initiation: approx. 6 weeks
- Randomization: computer-generated algorithm
- Housing: in fully air-conditioned rooms in makrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet (e.g. ad libitum): ssniff R/M-H (V 1534), ad libitum, except for the period in which the animals were kept in diuresis cages
- Water (e.g. ad libitum): tap water in plastic bottles ad libitum, except for the period in which the animals were kept in diuresis cages
- Acclimation period: at least five days
- Animal identification: animal tattooing and cage numbering

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 50 +/- 20%
- Lighting time: 12 hours daily

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

No. of animals per sex per dose:

Male: 5 animals at 0 mg/kg bw/day + 5 animals at 0 mg/kg bw/day for recovery group
Male: 5 animals at 50 mg/kg bw/day
Male: 5 animals at 200 mg/kg bw/day
Male: 5 animals at 800 mg/kg bw/day + 5 animals at 800 mg/kg bw/day for recovery group
Female: 5 animals at 0 mg/kg bw/day + 5 animals at 0 mg/kg bw/day for recovery group
Female: 5 animals at 50 mg/kg bw/day
Female: 5 animals at 200 mg/kg bw/day
Female: 5 animals at 800 mg/kg bw/day + 5 animals at 800 mg/kg bw/day for recovery group

Control animals:

yes

Details on study design:

Rationale for dose-selection:
Testing of the acute oral toxicity of the test item in rats led to a median lethal dose (LD50)
of over 2000 mg/kg body weight in male and female animals. The rats showed
hypoactivity, irregular respiration, uncoordinated, stilted gait, and squatting posture on
the day of treatment. Due to these findings, a pilot study with doses of 500 and
1000 mg/kg body weight/day for a duration of two weeks was conducted in 3 animals
per dose and sex. High dose animals showed hypoactivity, irregular respiration,
increased salivation, reddish colored urine, and a swollen abdomen during the entire
study. Additionally, the body weight development was impaired in these animals. Low
dose animals did not show any clinical findings and a regular body weight gain. On
day 15 all animals were killed and dissected. In high dose animals an enlarged
stomach was found. All other organs and tissues, as well as low dose animals were
free of any pathologically gross findings.
As a result of these studies, doses of 0, 50, 200 and 800 mg/kg body weight/day were
chosen for the 28-day oral gavage study in rats.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Behavior and state of health:
The behavior and general health condition of the animals were observed once daily.

Neurotoxicological examinations:
Once before the first treatment and thereafter once a week detailed clinical observations
were performed in all animals outside the home cage in a standard arena ('open
field'). Each animal was assessed for changes in skin, fur, eyes, mucous membranes,
occurrence of secretions and excretions and autonomic activity such as lacrimation,
salivation, nasal discharge, pi/oerection, pupil size, and unusual respiratory pattern.
Changes in gait, posture, and response to handling as well as the presence of clonic
or tonic movements, tremor, and any other abnormal motor movements (such as excessive
grooming, repetitive circling or other stereotypes) or bizarre behavior (e.g.
self-mutilation, walking backwards) were also recorded. In addition, defecation and
urination were evaluated.
At the termination of the study sensory reactivity to stimuli of different types (auditory,
visual, and proprioceptive) was evaluated including startle reflex (click response),
response to approach with the finger to the nose of the animal, and righting reflex.
The presence and absence of pupil/ary constriction was assessed using a pen flashlight
directed into the eye. Assessments of motor function were performed including
measurement of motor activity, and forelimb and hindlimb grip strength. The animals
were evaluated for motor activity during a 60-minute period in an 16-station automated
motor activity monitoring device. Activity counts were recorded by the interruption of photocells in 3-minute-intervals to give a total of 20 intervals. Fore- and hindlimb grip strength were measured by a strain gauge device.

Body weight:
The body weights of all animals were determined before the start of the study and
then twice weekly throughout the study.

Food consumption was determined continuously (2 times per week).
Water consumption was determined once weekly over a period of 16 hours.

Hematological investigations
At the termination of the study and after the recovery period, hematological examinations were performed on all animals without previous withdrawal of food. Blood
samples were taken from the retrobulbar venous plexus in narcosis (intraperitoneal
injection of 67 + 6.7 mg/kg body weight Ketamine-Hydrochloride + Xylazine). In order
to prevent systematic errors, blood sampling was conducted in a randomized order.

Clinical chemistry
After blood sampling for hematological testing, the animals were killed by section of
the vena cava cranialis in deep narcosis and exsanguinated. In order to prevent systematic errors, exsanguination was conducted in a randomized order.

Sacrifice and pathology:

Necropsy and macroscopic examination:
After exsanguination, all animals were necropsied and checked for macroscopically
visible abnormalities. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.
All abnormal findings were recorded.

Endotracheal fixation of the lungs: The lungs, including part of the trachea, were
removed. The lungs were then fixed endotracheally with a formalin solution using a
needle inserted into the trachea. The instillation pressure was between 20 and 30 cm
water column. Following completion of the endotracheal fixation the lungs were fixed,
together with the other organs, in formalin solution.

Organ weights:
The following organs were weighed and the organ to body weight ratios calculated:
- Heart
- Adrenal glands
- Epididymides
- Liver
- Spleen
- Thymus
- Kidneys
- Testes
- Brain

Histopathology
The following tissues or organs (or pieces of them) were preserved in a suitable
fixative and processed for histopathological investigations:
- Brain
- Heart
- Lungs
- Trachea
- Thymus
- Liver
- Spleen
- Kidneys
- Adrenal glands
- Stomach
- Jejunum
- Colon
- Urinary bladder
- Testes
- Epididymides
- Prostate gland
- Ovaries
- Uterus
- Lymph nodes iliac
- Lymph nodes (mandibular)
- Thyroid
- Bone marrow (sternum)
- N. ischiadicus
- Spinal cord (cervical)

Optional endpoint(s):

urine analysis

Statistics:

The following parameters were compared statistically with the control group values at
the level of significance p = 0.05:
• Body weights at the designated measurement times
• Hematological data
• Clinical chemistry parameters
• Urine analysis (Volume, pH-value and specific weight)
• Absolute organ weights and organ to body weight ratios
• Neurotoxicological measurements (motor activity, forelimb and hindlimb
grip strength)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals of all dose groups showed sporadically scabby wounds at the dorsal neck,
from day 15 up to the end of recovery period. Increased salivation was observed
sporadically in all animals of the intermediate and high dose groups, from day 6 on up
to the end of the treatment period (day 29). Additionally, all animals of the high dose
group showed hypoactivity, irregular respiration, stilted gait, swollen abdomen,
squatting posture, and orange discolored urine, from day 9 on up to day 35 of the
study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal of the low dose group was found dead on day 23 of the study. The
death of this animal was due to its bad general condition. This bad condition was
incidental and not related to the treatment with CI-DAEP. The cause for the death
could neither macro- nor microscopically be detected.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The animals of the high dose group showed a statistically significant decreased body
weight gain in comparison to the control group animals throughout the whole study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The animals of the high dose group showed a decreased food consumption from start
of the study up to the end of treatment period. The food consumption of the remaining
high dose animals increased in the recovery period. It rose even higher as in the
control animals; probably a compensation effect as a consequence of the lower food
consumption in the treatment period.

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

effects observed, treatment-related

Description (incidence and severity):

The high dose animals showed slightly increased water consumption from day 6 up to
the end of recovery period when compared to the control group.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period female animals of the median dose group and
males and females of the high dose group showed statistically significant increased
reticulocyte counts. Additionally, a slightly, but statistically significant increased MCV
in females of the high dose group was found. Statistically significant decreases in
hemoglobin in both genders and in red and white blood cell counts and hematocrit in
females were observed in the high dose group. In males of the high dose group a
statistically not significant reduction of the red blood cell count could be observed too.
At the end of the recovery period high dose animals showed a statistically significant
increase in reticulocyte counts and MCV, and decrease in hemoglobin and red blood
cell count. Additionally, a statistically significant decrease in hematocrit and white
blood cell count was observed in female animals of the high dose group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period in males of the high dose group statistically
significant increased phosphorus serum levels were found. Female animals of the
intermediate and high dose groups showed statistically significant increased calcium
and cholesterol values. Additionally, in high dose females statistically significant
increased urea nitrogen, ALA T, y-GT, and alkaline phosphatase as well as decreased
phosphorus levels were observed.
At the end of the recovery period male animals of the high dose group showed
statistically significant decreased chloride and statistically significant increased
calcium, phosphorus and alkaline phosphatase levels. In female animals statistically
significant increased alkaline phosphatase levels were found.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

After 28 days of treatment with CI-DAEP male and female animals of the high-dose
group showed sporadically reddish discolored urine. Bilirubin was found in the urine of
almost all intermediate (9/10) and high (10/10) dose animals.
At the end of the recovery period, bilirubin was still present in the urine of two females
of the high dose group.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Neurotoxicological examinations:
Forelimb and hindlimb grip strength were statistically significant decreased in animals
of the high dose group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the 28-day treatment period animals of the high dose group showed
statistically significant decreased absolute heart weights. Additionally, in female
animals of the high dose group a statistically significant increase in absolute liver
weights were observed.
Female animals of the low and males and females of the intermediate and high dose
groups exhibited a statistically significant increase in relative liver weights.
Additionally, in males statistically significant increases in relative kidney (intermediate
and high dose), testes and brain weights (high dose) were noted. Female animals of
the high dose group showed furthermore a statistically significant decrease in relative
thymus weights.
At the end of the recovery period high dose males showed statistically significant
decreased absolute heart, kidney, and epididymis weight. In females a statistically
significant decrease in absolute kidney, spleen, and adrenals weight were noted.
Statistically significant increases in relative organ weights were noted in testes and
brain weights in males and liver weight in females

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In animals of the high dose group enlarged thyroid glands were observed.
Furthermore, scabbed wounds at the neck were observed sporadically in animals of
all groups.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Relevant histopathological findings could be seen in the thyroid and the spleen. The
thyroid glands of four female rats of the high dose group showed a follicular
hyperplasia with reduced amount of colloid. This follicular hyperplasia reflect the
thyroid gland as target organ under the treatment of the test substance. This finding
correlates with the gross pathology findings. Male animals, however, who also
exhibited an enlarged organ, did not develop follicular hyperplasia. Removal of the
test substance for two weeks (recovery group) led to a normal histopathological
pattern indicating the reversibility of the findings. No significant abnormal histological
findings were observed in the low and intermediate dose groups.

In the spleen of all animals of the high dose group a slight to moderate erythroid
hyperplasia was observed. This increase of erythropoietic foci reflects a higher
consumption or disintegration of erythrocytes and should be discussed as a
physiological answer to the loss of red blood cells.
Further histopathological findings could be seen in the subcutis of the skin, where
acanthosis/dermatitis were observed in single male and female animals of the control,
intermediate, and high dose groups. Other sporadically occurring findings observed in
various organs of different animals were not relevant for the assessment of the test
article. These spontaneously observed findings are interpreted as non compound
related.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

thyroid gland

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

Classified as: Not classified

Executive summary:

Oral gavage of the test item for 28 days caused unequivocal signs of toxicity in the high

dose group. Body weight gains, food and water consumption, general health and

behavior, hematology, clinical chemistry and urine parameters, organ weights, and

two target organs were impaired by repeated administration of the test substance at a

dose level of 800 mg/kg body weight per day.

Animals of the intermediate dose group did not show relevant clinical findings.

However, minor changes in hematology, clinical chemistry, urine parameters, and in

organ weights were found.

Low dose males did not show any sign of substance toxicity at all. However female

animals of the low dose group showed a slight increase in relative but not in absolute

liver weight.

High dose females showed an increase in liver weights and a very small increase in

liver enzymes. The intermediate and low dose group showed only an increase in liver

weights. In none of these groups a histopathological deviation in liver morphology

could be found.

Owing to the fact that the effect in the liver weights caused by the test substance in

the low dose group is only very small and is not accompanied by any clinical findings

or deviations in laboratory or histopathological investigations, this effect is of no

toxicological relevance in the current study.

With regard to the present study, a No Observed Adverse Effect Level (NOAEL) of

50 mg/kg body weight per day is determined.