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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:
1993-09-13 - 1995-05-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study owner prepared study while seeking authorisation of the substance as active incredient for medical treatment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1991-07-19
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
solid: bulk
Details on test material:
This composition is the usual techical grade of AlzChem AG. It will be used when test substance has this composition (or is very closed) or no specific information is available

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
Age: m 7 month, f 6-7 month
B.w. m 10.5 - 17.1 kg, f 7.7 - 14.5 kg
Caging: Boxes 6.0 m x 1.2 m
No of animals per box: 3
Diet: Standard dog diet ad libitum
Water: ad libitum in drinking water quality
Room temperature: 23.5 - 26 °C
Relative humidity: 30 - 55 %
Lighting: 6 a.m. - 6 p.m. artificial lighting, 6 p.m. - 6 a.m.natual dark rhythm

Administration / exposure

Route of administration:
oral: capsule
Details on route of administration:
gelatine capsules (12 x 30 mm)
Vehicle:
unchanged (no vehicle)
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
once daily (a.m.)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
14.7 mg/kg bw/day (nominal)
Remarks:
low dose
Dose / conc.:
31.6 mg/kg bw/day (nominal)
Remarks:
intermediate dose
Dose / conc.:
68.1 mg/kg bw/day (nominal)
Remarks:
high dose
From day 3 of administration onwards: 56.2 mg/kg b.w. (m)
From day 16 of administration onwards: 56.2 mg/kg b.w. (f)
From day 17 of administration onwards: 46,4 g/kg b.w. (m + f)
No. of animals per sex per dose:
3 m + 3 f
Control animals:
yes

Examinations

Observations and examinations performed and frequency:
Mortality: twice daily (a.m. and p.m.)
Behaviour and general condition: daily
Food consumption: daily, staring with 2nd pretest week
B.w.: Once a week, staring with 2nd pretest week
Reflexes: Pain, pupil, corneal and patella reflexes, once a week staring with 2nd pretest week
Heart rate, body temperature: once a week staring with 2nd pretest week, before administration each
Ophthalmology: before first administration and in week 4, all animals
ECG: before first subtance administrationand in week 4 before administration
Clinical chemistry and hematology: before first substance administration and in weeks 1 and 4
Urine: week 5 by punctation of the bladder during autopsy
Sacrifice and pathology:
Anesthesia: Rompun(R) and T61(R), exsanguination

Gross Necropsy: all animals -> external surface of body, orifices, cranial, toracic and abdominal cavities and their contents.
All gross lesions, adrenal glands (r/l), aorta, axillary lymph node, bone (femur and sternum), bone marrrow smear (from sternum), brain (cerebrum, cerebellum, medulla/pons), caecum, cervix, colon, duodenum, epididymides (r/l), gallbladder, heart, ileum, injection sites, jejunum, kidneys (r/l), liver, lungs, main stem bronchi, mammary gland, mesenteric lymph node, oesophagus, ovaries (r/l), pancreas, parathyrois glands (r/l), peripheral nerve, pituitary gland, prostate, rectum, salivary glands (parotid, submaxillary, sublingual), skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar segments), spleen, stomach, testes (r/l), thymus, thyroid gland (both lobes), tongue, trachea, urinary bladder, uterus, vagina.

Organ weigths: Adrenals (r/l), brain, heart, kidneys (r/l), liver, lungs, ovaries (r/l), pituitary, prostate, spleen, testes (r/l), thyroids (r/l).
Expression as absolute values and relative to b.w. recoreded in week 4.

Fixation: All organes and tissues exept eyes and bone marrow smears -> 10 % formalin
Eyes -> SUSA fixative 4 - 16 h, thereafter 10 % formalin
Bone marrow smears -> air dried
Staining: All organs and tissues -> embedded in parafine wax, sectioned at 4 µm and stained with H&E
Additional sections of kidneys -> periodic acid Schiff reaction
Cryostate sections of liver and one kidney -> Oil red 0
Statistics:
Mean and SD: each group and sex
B.w. and organ weigts: DUNNET-Test
Clinical Parameters, ECG, hematology, clinical chemistry: DUNNETT-Test (normal distribution), otherwise STEEL-Test
Significances:
DUNNETT: * p < 0.05, ** p < 0,01
STEEL: + p < 0.05

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Behaviour and general condition were dose dependently influenced in mid and high dose animals in extend, incidence, and severity during the treatment period.

Low dose animals were free of toxic symptoms.

Intermediate dose males had serve hypokinesia, decrease of muscle tone (abdominal position), salivation, vomitus and sunken sides. Females of this group showed salivation and vomitus only.

High dose animals of both sexes had in addition th mid dose males symptoms like coordination disturbances, loss if righting reflex (lateral position), and diarrhea. The animals were in a poor general condition. One out of three females had serve clonic convulsions temprorarily.
Mortality:
mortality observed, treatment-related
Description (incidence):
One male animal of group 3 and two male animals of group 4 had to be killed in extremis.
One female animal or group 4 had to be killed in extremis.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased b.w. in high dose animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Dose depßendent reduced.
After dose reduction in the high dose group food intake normalized to control group level.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Changes in some white blood cell parameters in individual high dose animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Substance related changes in individual high dose animals.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Immunological findings:
effects observed, treatment-related
Description (incidence and severity):
Changes in white blood cell parameters may show an immunologicel effect.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
High dose male: increased wight of liver and thyroid gland, decreased weight of prostate
Hight dose females: increased weights of lung and liver.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Pale discolored liver.
Ulcerations in pyloric mucosa of stomach.
Pale discolored kidneys.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Other effects:
not specified

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 14.7 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 31.6 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
urinalysis
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
histopathology: neoplastic
Key result
Dose descriptor:
other: lethal dose range
Effect level:
>= 56.1 - <= 68.1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
31.6 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
31.6 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of the present 4-week oral toxicity study with alpha-Lipoic acid it can be concluded that most of the observed clinical, clinical pathological and pathological findings are substance related.
Executive summary:

The low dose of 14.7 mg/kg b.w. was free of toxic symptoms, clinical pathological and histopathological changes.

Further findings in mid and high dose groups give an indication that liver and kidney are clearly involved als target organs for the test subatance.

The results of the macro- and microscopic examinations inclusive organ weight determination did distinctly confirm the results of the clinical chemistry investigations and they are in good correspondence to these findings.

Under the present experimental conditions it can be stated, that alpha-Lipoic acid exerts distinct toxic effects in doses of 31.6 and 68.1/56.2/46.4 mg/kg b.w. A dose range between 68.1 and 56.2 mg/kg b.w. represents the beginning lethal dose range.

A dose of 14.7 mg/kg b.w. represents the NOAEL in the present dog study.