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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2008 – 09 March 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD Guideline 408 with minor deviations: temperature and relative humidity recorded in the animal room were sometimes out of the target ranges
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other 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:
temperature and relative humidity recorded in the animal room were sometimes out of the target ranges
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
9022-76-8
Details on test material:
- Name of test material (as cited in study report): CHIMEXANE NB
- Physical state: Brownish viscous liquid
- Lot/batch No.: 0129336
- Purity: 98.5%
- Date of receipt: 9 April 2008
- Expiration date of the lot/batch: September 2008
- 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: 211-263 g (mean: 236 g); females: 185-213 g (mean: 201 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: 6 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). On each day of analysis, duplicate samples was taken at three different levels (top, middle, bottom) of the mixer (groups 2, 3 and 4) or at a single level of the container (control group) for the determination of the test item concentration.
- Results: Satisfactory homogeneity and stability (over 14 days at room temperature in closed bags or 8 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 obtained in the dietary admixtures was in an acceptable range of ± 10 % from the expected value. Over the whole dosing period, for the targeted dose-levels of 150, 450 and 1000 mg/kg bw/day, the mean achieved dosages were equivalent to 127, 366 and 807 mg/kg bw/day in males and 148, 434 and 961 mg/kg bw/day in females, respectively. The mean achieved dosages were out of the acceptable range of ± 10 % from nominal value, in males at 150, 450 and 1000 mg/kg bw/day, from weeks 5 to 13 (deviations between -18 and -28 %), and in females given 450 mg/kg bw/day from weeks 5 to 8 (deviation of -11 %).
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
150, 450 and 1000 mg/kg bw/day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Males: 127, 366 and 807 mg/kg bw/day; females: 148, 434 and 961 mg/kg bw/day
Basis:
actual ingested
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. 31261 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, the test item did not induce any mortality, any clinical signs of toxicity, any significant changes in body weights or food consumption, and any changes in hematological or urinalysis parameters. An increase plasma level in urea concentration was pbserved in mid and high-dose males and increased absolute and relative adrenal weights were reported in mid and high-dose females. These effects were considered to be related to the test item. No histopathological correlates were found for these changes. The NOAEL were established at 1000 mg/kg/day (corresponding to 740 and 806 mg CHIMEXANE NB/kg/day for males and females, respectively).
- 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 stratification 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 13.

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 epithelial 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 for the determination of the bone marrow differential cell count was prepared from the femur of each animal sacrificed on completion of the treatment period.
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
- the mesenteric lymph nodes in the males and females in 150 and 450 mg/kg bw/day treatment groups
- additional microscopic examination of mesenteric lymph nodes from all animals after additional staining (toluidine blue) in order to obtain more accurate characterisation of mast cells than in standard/routine Hematoxylin and Eosin stained sections
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
Description (incidence and severity):
at 1000 mg/kg/day in both males and females
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
at 1000mg/kg/day in both males and females
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Reduced total cholesterol concentration in both Males and females at 1000 mg/kg/day
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mesenteric lymph nodes
Histopathological findings: neoplastic:
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 1000 mg/kg bw/day, lower mean body weight gains and mean body weights were noted in both sexes, particularly from week 11 until the end of the study, resulting in an overall lower mean body weight gain in males and females (-13 % and -14 %, vs. control, respectively).
- Mean body weight and body weight gain were unaffected at 150 and 450 mg/kg bw/day.

FOOD CONSUMPTION:
- At 1000 mg/kg bw/day, mean food consumption in males tended to be slightly lower than in controls from week 6 to the end of the study, while in females, a slight difference was observed during weeks 6 and 10 only.
- Mean food consumption was unaffected at 150 and 450 mg/kg bw/day.

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

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

CLINICAL CHEMISTRY:
- At 1000 mg/kg/day, when compared to controls, males and females showed low total cholesterol concentration (1.3 and 1.5 mmol/L, vs. 1.6 and 1.9 mmol/L in controls, respectively). These differences were attributed to treatment with the test item but were considered of no toxicological importance.
Lower mean glucose level and mean creatinine values were noted in females given 1000 mg/kg/day and were attributed to the test item treatment. As these findings were of small amplitude, they were considered to be of no toxicological importance.

URINALYSIS:
- In females given 450 and 1000 mg/kg/day, an increased colour of urines and increased number of epithelial cells were observed when compared to controls. These findings were attributed to the test item treatment but were considered of no toxicological importance.

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

ORGAN WEIGHTS:
- No test item treatment-related effects were noted.

GROSS PATHOLOGY AND HISTOPATHOLOGY:
- The test item administration at 150, 450 and 1000 mg/kg/day induced non dose-related aggregates of macrophages/epithelioid histiocytes, sometimes with multinucleated giant cells, and plasma cell infiltrates in the mesenteric lymph nodes. This inflammatory infiltrate, most likely related to the viscous properties of the test item and its draining within the lymph nodes, was not considered to be adverse.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
807 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
961 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

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 NB was considered to be 807 and 961 mg/kg bw/day for males and females, respectively (target dose-level: 1000 mg/kg bw/day).
Executive summary:

In a subchronic repeated dose oral toxicity study conducted according to the OECD Guideline 408 and in compliance with GLP, CHIMEXANE NB 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), 150, 450 or 1000 mg/kg bw/day. The mean achieved dosages corresponded approximately to 127, 366 and 807 mg/kg bw/day for males, 148, 434 and 961 mg/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 1000 mg/kg bw/day, mean body weights and body weight gains were affected by the test item treatment, from week 11 until the end of the study. The mean food consumption was affected by the treatment from week 6 in males and in weeks 6 and 10 in females. Clinical pathology did not reveal any clinical pathology changes of toxicological significance. There were no necropsy findings or changes in organ weights attributed to the test item at 150, 450 and 1000 mg/kg bw/day. The test item administration at 150, 450 and 1000 mg/kg bw/day induced non dose-related aggregates of macrophages/epithelioid histiocytes, sometimes with multinucleated giant cells, and plasma cell infiltrates in the mesenteric lymph nodes. This inflammatory infiltrate, most likely related to the viscous properties of the test item and its draining within the lymph nodes, was not considered to be adverse.

 

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of CHIMEXANE NB was considered to be 807 and 961 mg/kg bw/day for males and females, respectively (target dose-level: 1000 mg/kg bw/day).