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Toxicological information

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:
25-10-2010 to 22-11-2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study performed similar to OECD 407. No analyses of the dose formulations was performed, so no information is available on stability of the test substance in the dose formulations and the actual exposure concentrations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: ICH Guidance for industry: M3 (R2) Non clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no analysis of the dose formulation samples was performed.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name: Ethanol, 2,2'-oxybis-, reaction products with ammonia, morpholine derivs. residues
Physical state: liquid
Appearance: brown liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Amine C-8
- Physical state: dark liquid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Lot/batch No.: not provided
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: room temperature, 10 to 25°C

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: ca. 7 weeks at start of dosing
- Weight at study initiation: 231-264 g for males and 166-189 g for females
- Fasting period before study: no
- Housing: Animals were group housed by sex upon receipt and individually housed upon assignment to study in compliance with National Research Council "Guide for the care and use of laboratory animals". The room in which the animals are kept is documented in the study records. No other species are kept in the same room.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Study animals are acclimated to their housing for a minimum of 7 days prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-22°C
- Humidity (%): 14-76%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark, except when room lights will be turned on during the dark cycle to accomodate blood sampling or other study procedures.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
deionized water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: the test article and vehicle control preparations are prepared weekly or additionaly as needed by diluting the test article in vehicle (w/v) to reach the proper concentrations.

Dose formulation samples: On the first and last day of dosing, duplicate 1-mL samples will be obtained from top, middle, and bottom of each formulation, including the vehicle control, to determine the concentration and homogeneity of the test article in vehicle. These samples are stored at room temperature, approximately 10 to 30°C.

Dose levels: 0, 100, 500, 1000 mg/kg bw/d
Concentration: 0, 10, 50, 100 mg/ml
Dose volume: 10 ml/kg
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the sponsor in consultation with the Study Director
- Rationale for animal assignment: at random
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once prior to scheduled sacrifices
- Cage side observations: each animal observed for evidence of death or impending death

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to each dose administration, at approximately 1-2 hours post dose, and additionally as needed; once prior to scheduled sacrifice

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded at the time of randomization/selection, prior to dose administration on Day 1 and weekly thereafter. A fasted body weight will be recorded before all scheduled sacrifices.

FOOD CONSUMPTION:
- Frequency: Full feeder weights and/or feeder weigh backs were recorded weekly for determination of food consumption.

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood: prior to terminal sacrifice on Day 29
- Anaesthetic used for blood collection: Yes (CO2 inhalation)
- Animals fasted: Yes (Animals will be fasted overnight (approximately 12-24 hours)
- Parameters checked: red blood cell count and morphology, white blood cell count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, hematocrit, hemoglobin, reticulocyte count, coagulation: activated partial thromboplastin time and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to terminal sacrifice on Day 29
- Anaesthetic used for blood collection: Yes (CO2 inhalation)
- Animals fasted: Yes (Animals will be fasted overnight (approximately 12-24 hours)
- Parameters checked: alanine aminotransferase, albumin, albumin/globulin ratio (calculated), alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatinine, globulin (calculated), glucose, phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides, urea nitrogen


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All surviving animals will be euthanized by CO2 asphyxiation following terminal blood collection on Day 29

Gross necropsy:
A complete gross necropsy was performed on all animals that were sacrificed or found dead during the study. The necropsy included examination of:
- the external body surface
- all orifices
- the cranial, thoracid and abdominal cavities and their contents

Organ weights: adrenals, brain, heart, kidneys, liver, testes, ovaries, spleen, thyroids/parathyroids

HISTOPATHOLOGY: Yes
For all animals necropsied, tissues were preserved in 10% neutral buffered formalin (except for the eyes, which were preserved in Davidson's fixative and the testes that were preserved in Bouin's fixed for optimum fixation). Tissues collected:
cardiovascular: aorta, heart;
digestive: salivary gland(s), tongue, esophagues, stomach, small intestine: duodenum, jejunum, ileum; large intestine: cecum, colon, rectum; pancreas, liver;
respiratory: trachea, larynx, lung with mainstem bronchus;
lymphoid/hematopoietic: sternum with bone marrow, thymus, spleen, lymph nodes, mandibular, mesenteric;
urogenital: kidneys, urinary bladder, ovaries, uterus, cervix, vagina, testes, epididymides, prostate, seminal vesicles;
endocrine: adrenals, pituitary, thyroid/parathyroid;
skin/musculoskeletal: skin, mammary gland, skeletal muscle (thigh), femur with articular surface;
nervous/special sense: eye with optic nerve, sciatic nerve, brain, spinal cord - cervical, spinal cord-midthoracic, spinal cord - lumbar, lacrimal glands
Statistics:
Statistical analysis was performed on in-life data, clinical pathology, and necropsy data when 3 or more animals are present in 2 or more dose group. Statistical analysis were be performed if N< 3 animals per group. For in-life and clinical pathology parameters, the homogeneity of the data was determined by Bartlett's Test. If the data was homogeneous, a one-way analysis of variance was performed to assess statistical significance. If statistically significant differences between the means were found, Dunnett's test was used to determine the degree of significance for the control means (p<0.05, p<0.01), the Mann-Whitney U-Test was used to determine the degree of significance from the control means (p<0.05 and p<0.01). If only 2 dose groups were present for evaluation, the Mann-Whitney U-test was used to assess statistical significance between the 2 groups. For necropsy organ weight data, the evaluation of the equality of means was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, Dunnett's test was used to determine the degree of significance from the control means (p<0.05 and p<0.01).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increases in absolute kidney weights (mid- and high-dose females), kidney-to-body weight ratio (high-dose females) and kidney-to-brain weight ratio (all dosed females).
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Rats (5 per sex per dose) were exposed once daily to 0, 100, 500, 1000 mg/kg bw/d of test substance for 28 consecutive days. The only statistically significant effects in absolute and relative female kidney weights could not be related to any macroscopic or microscopic findings or changes in clinical chemistry parameters. There is thus no indication of kidney toxicity. The NOAEL was therefore considered to be 1000 mg/kg bw/d.