Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May 2008 – 09 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

ophthalmological examinations were performed in week 12, instead of week 13

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

EC Number:

939-579-8

Molecular formula:

Empirical Formula : C11H21N3O3Na C2nH4n+1 (n=4 – 9), C11H21N3O3NaC18H35, C11H21N3O3NaC18H33 Molecular formula of the two main constituents : C23H46N3O3Na

IUPAC Name:

Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate

Test material form:

semi-solid (amorphous): gel

Details on test material:

- Physical state: Yellowish thick gel
- Storage condition of test material: At room temperature

Specific details on test material used for the study:

- Name of test material (as cited in study report): CHIMEXANE HB
- Physical state: Yellowish thick gel
- Analytical purity: 53.8 %
- Lot/batch No.: 0137998
- Date of receipt: 21 April 2008
- Expiration date of the lot/batch: April 2009
- Storage condition of test material: At room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: Males: 230-293 g (mean: 266 g); females: 198-237 g (mean: 222 g)
- Housing: Two rats of same sex and group were housed in suspended wire-mesh cages
- Diet: A04 C powdered maintenance diet (SAFE, Augy, France), ad libitum
- Water: Tap water (filtered with a 0.22 μm filter), ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: Approximately 12 cycles/h of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Appropriate amount of the test item was mixed with a dietary admixture, in A04C powdered maintenance diet (SAFE, Augy, France), batch No. 71219 (SAFE, Augy, France)
- Storage temperature of food: Stored in closed bags at room temperature and protected from light prior to use

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Concentration: Concentration of samples taken from each dietary admixture (control group included) prepared for use in weeks 1, 5, 9 and 13 was analysed by reversed phase chromatography (HPLC) with MS/MS detection (positive mode) after ionisation using an Electro-Spray Interface (ESI).
- Results: Satisfactory homogeneity and stability (over 14 days at room temperature in closed bags or 7 days in open feeders) of dietary admixtures prepared at 500 and 20000 ppm was demonstrated before the start of the study. Throughout the dosing period, the concentration values obtained in the dietary admixtures were within an acceptable range of ± 20 % of the expected value. Over the whole dosing period, for the targeted dose-levels of 50, 150 and 450 mg active content/kg bw/day, the mean achieved dosages corresponded respectively to approximately 43, 128 and 428 mg active content/kg bw/day in males and 47, 140 and 451 mg active content/kg bw/day in females.

Duration of treatment / exposure:

91 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Doses / concentrations 2

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were selected on the basis of the results of a 4-week toxicity study (Study No. 31265 TSR) in which the same test item was administered by dietary admixture at the nominal dose-levels of 150, 450 and 1000 mg/kg bw/day. In this study, low body weight gain was noted in the male during weeks 1 and 4 and in the females in week 1 at achieved 830/905 mg/kg bw/day (males/females). Over the study, males and females showed a lower body weight gain than controls. In the males, this was associated with low food consumption during the first week. Laboratory investigations revealed a high fibrinogen concentration in the males and low glucose concentrations in both sexes. At 120/130 and 350/390 mg/kg bw/day, no treatment-related findings were noted.
- Rationale for animal assignment: Animals were selected according to body weight and/or clinical condition and grouped (by sex) according to body weight, using a randomisation procedure, so that the average body weight of each group was similar.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- Mortality and morbidity: Twice a day
- Clinical signs: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the beginning of the treatment period and then once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: Once before group allocation, on the first day of treatment and then once a week until the end of the study and before sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal was recorded once a week and mean daily diet consumption was calculated as g food/animal/day

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were performed on all animals, before the beginning of the treatment period and on control and high-dose animals on one occasion during Week 12.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from the orbital sinus of all the animals at the end of the treatment period.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight (at least 14 h)
- Parameters checked (haematology): Hemoglobin, erythrocyte, mean cell volume, packed cell volume, mean cell hemoglobin concentration (MCHC), mean cell hemoglobin (MCH), thrombocytes, leucocytes, differential white cell count (neutrophils, lymphocytes and large unstained cells, monocytes, eosinophils, basophils), reticulocytes, prothrombin time, activated partial thromboplastin time and fibrinogen.
- Parameters checked (clinical chemistry): Sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), inorganic phosphorus, urea, creatinine, glucose, total bilirubin, total proteins, albumin, albumin/globulin ratio, total cholesterol, triglycerides, alkaline phosphatase (ALP), aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT)

URINALYSIS: Yes
- Time schedule: Urinalysis was performed in all animals at the end of the treatment period.
- Metabolism cages used for collection of urine: Yes, animals were individually placed in metabolism cages
- Animals fasted: Yes, overnight (at least 14 h)
- Parameters checked: Appearance, color, volume, specific gravity, pH, proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, cytology of sediment (leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals and cells)

NEUROBEHAVIOURAL EXAMINATION / FUNCTIONAL OBSERVATION BATTERY (FOB): Yes
- Time schedule for examinations: All animals were evaluated once at the end of treatment period. All the animals were observed in the cage, hand and in the standard arena.
Battery of functions tested:
- In the cage: ‘Touch escape’
- In the hand: Fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- In the standard arena (two-minute recording): Grooming, palpebral closure, defecation, urination, tremors, twitches, convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypic behavior, breathing, ataxia and hypotonia.
In addition, the following parameters, reflexes and responses were recorded:
- touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay and rectal temperature at the end of observation
- Motor activity of each animal was also measured once with automated infra-red sensor equipment over a 1 h period.

Sacrifice and pathology:

- GROSS PATHOLOGY: On completion of the treatment period, after at least 14 h fasting, all surviving animals were deeply anesthetised by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination. A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.
- ORGAN WEIGHTS: Body weight of each animal was recorded before sacrifice and the organs specified in the table 7.5.1/1 were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.
- HISTOPATHOLOGY: For all animals, the tissues specified in the table 7.5.1/1 were preserved in 10 % buffered formalin (except for the eyes with optic nerves and Harderian glands, and the testes and epididymides which were fixed in Davidson's fixative). All tissues required for microscopic examination were embedded in paraffin wax, sectioned at a thickness of approximately 4 µm and stained with hematoxylin-eosin (except for testes and epididymides which were stained with hematoxylin/PAS). A bone marrow smear was also prepared from the femur of each animal.
Microscopic examination was performed on:
- all tissues listed in the table 7.5.1/1 for animals in control and 1000 mg/kg bw/day treatment groups sacrificed at the end of the treatment period
- all macroscopic lesions from all the animals in 150 and 450 mg/kg bw/day treatment groups sacrificed on completion of the treatment period

Other examinations:

None

Statistics:

Refer figure 7.5.1/1 for the details of statistical analysis

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

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):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
- No unscheduled deaths or test item treatment-related clinical signs were noted.

BODY WEIGHT AND WEIGHT GAIN:
- At 450 mg active content/kg bw/day, from the start of the treatment period, lower mean body weight gains were generally noted and mean body weights were always statistically significantly lower when compared to controls. The overall mean body weight gain was -29 % lower than in controls in males, and -27 % lower in females. At 50 and 150 mg active content/kg bw/day, although some isolated/transient low mean body weight gains were noted when compared to controls, no differences were attributed to the test item treatment.

FOOD CONSUMPTION:
- At 450 mg active content/kg bw/day, male mean food consumption tended to be slightly lower than in controls throughout the study, while in the females slightly low food consumption was observed during week 1 only. At 50 and 150 mg active content/kg bw/day, mean food consumption was unaffected by the test item treatment.

OPHTHALMOSCOPIC EXAMINATION:
- No test item treatment-related effects were noted.

HAEMATOLOGY:
- No test item treatment-related effects were noted.

CLINICAL CHEMISTRY:
- At 450 mg active content/kg bw/day, in the males, slightly low mean glucose (-15 %) and a minimally lower albumin/globulin ratio were noted when compared to controls. These changes were considered of limited toxicological importance.

URINALYSIS:
- At 450 mg active content/kg bw/day, changes in urine pH (slightly low in the males), turbidity and color (more marked in both sexes) were noted when compared to controls. Trace levels of bilirubin (males) or proteins (females) in the urine were noted with a higher incidence than in controls.
- At 150 mg active content/kg bw/day, in females, trace levels of proteins in the urine, and more marked turbidity of the urine were noted when compared to controls. Changes noted at urinalysis were considered to be treatment-related.
- At 50 mg active content/kg bw/day, no test item treatment-related changes were observed.

NEUROBEHAVIOUR / FUNCTIONAL OBSERVATION BATTERY (FOB):
- No treatment-related changes were noted.

ORGAN WEIGHTS:
- At 450 mg/kg bw/day, higher relative kidney weights were noted in males and females when compared to controls.

GROSS PATHOLOGY AND HISTOPATHOLOGY:
- No macroscopic post-mortem test item treatment-related findings were observed.
- At 450 mg/kg bw/day, microscopic evaluation of the kidneys revealed minimal to marked tubular vacuolar degeneration in the inner cortex of all animals. Females were more severely affected than males. Inflammatory mononuclear cell infiltration and increased tubular basophilia were also recorded.
- At 150 mg/kg bw/day, tubular vacuolar degeneration in the inner cortex was recorded in 1/10 males and in 7/10 females.
- At 50 mg/kg bw/day, no test item treatment-related changes were observed.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: see remarks

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: See remarks

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of CHIMEXANE HB was considered to be 43 and 47 mg active content/kg bw/day for males and females, respectively (target dose-level: 50 mg active content/kg bw/day).

Executive summary:

In a GLP-compliant subchronic repeated dose oral toxicity study conducted according to the OECD Guideline 408, CHIMEXANE HB was administered daily in the diet to groups of Sprague-Dawley, Rj Han: SD, rats (10/sex/dose), for 13 weeks at the nominal dose-levels of 0 (vehicle), 50, 150 or 450 mg active content/kg bw/day. The mean achieved dosages corresponded approximately to 43, 128 and 428 mg active content/kg bw/day for males, and 47, 140 and 451 mg active content/kg bw/day for females. Examinations during the study included: mortality, clinical signs, functional observation battery (including motor activity), body weight change, food consumption, ophthalmology, hematology, blood chemistry, urinalysis, organ weights, macroscopic examination and histopathology.

 

No unscheduled deaths occurred and no test item treatment-related clinical signs were observed during the study. In addition, no adverse effects were observed during the Functional Observation Battery or at ophthalmology examinations. At 450 mg active content/kg bw/day, mean body weights, body weight gains, and to a lower degree mean food consumption were reduced by test item treatment. Minor differences in glucose concentration and albumin/globulin ratio were noted in males at terminal blood sampling, when compared to controls. Signs of nephrotoxicity were observed. They consisted of lower urine pH, differences in urine turbidity and color, and of trace levels of bilirubin and proteins in urine. At necropsy, higher relative kidney weights were noted in both sexes, associated with microscopic changes (vacuolar degeneration in the tubules of the inner cortex, inflammatory mononuclear cell infiltration and/or increased tubular basophilia). At 150 mg active content/kg bw/day, signs of nephrotoxicity were observed in females (trace levels of proteins in the urine marked urine turbidity). These findings corresponded microscopically to vacuolar degeneration in the tubules of the inner cortex (1/10 males and 7/10 females). At 50 mg active content/kg bw/day, no effects of toxicological importance were observed and there were no test item treatment-related changes in any of the organs microscopically examined.

 

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of CHIMEXANE HB was considered to be 43 and 47 mg active content/kg bw/day for males and females, respectively (target dose-level: 50 mg active content/kg bw/day).