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Toxicological information

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:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1978

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Version / remarks:
13 weeks feeding study in dogs
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Butane-1,3-diol
EC Number:
203-529-7
EC Name:
Butane-1,3-diol
Cas Number:
107-88-0
Molecular formula:
C4H10O2
IUPAC Name:
butane-1,3-diol
Test material form:
liquid
Details on test material:
purity: > 99.5%
slightly viscous, clear liquid with mild odour
Specific details on test material used for the study:
slightly viscous, clear liquid with mild odour
purity: at least 97%

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
20 male and 20 female pure-bred beagle dogs, about 7-8 weeks. Animals were divided into 5 groups (one control and 4 dose groups). Dogs were individually housed in indoor kennels.

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The test substance was thouroughly mixed into the basal diet at levels providing an intake of ,/3/6/9 ro 12 g(kg bw/d. The diets were supplemented with an instant wheat product, glucose and soya bean oil in such a way that all diets were theoretically isocaloric. The calculation of the amounts was based on a caloric value of glucose=4, soya bean oil=9, and butanediol=5.9 cal/g. The diets were freshly prepared one a wek and stored in closed containers at at temperature of 10-15°C.
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All diets were analysed for protein, fat, ash, moisture, crude fibre and butanediol.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
The dogs were fed a restricted portion of food twice daily. The amount of food/ kg bw/d was either 50 or 40 g on different days, but was equal for the different dogs on one day.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
3 000 mg/kg bw/day (nominal)
Dose / conc.:
6 000 mg/kg bw/day (nominal)
Dose / conc.:
9 000 mg/kg bw/day (nominal)
Dose / conc.:
12 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Behaviour and health of all dogs were checked daily.

Individual body weights were measured weekly.

Individual food consumption was measured daily.

Hematological investigations were carried out at the beginning and at week 2, 6 and 12 in all dogs. Blood samples were collected form the cephalic vein. All blood samples were examined for:

- Hemoglobin content
- packed cell volume
- Methemoglobin
- Erythrocyte fragility
- Count of erytrocytes
- Count of leucocytes
- Count of thrombocytes
- Differential white blood cell counts
- Reticulocytes
- Heinz bodies

Clinical chemistry of the blood: Each dog was examined at the beginning and in week 6 and 12. Examinations included:
- SGPT
- SGOT
- SAP
- Total serum protein
- Serum albumin
- Fasting blood glucose
- Blood urea
- Triglycerides
- beta-hydroxybutyric acid
- Acetoacetic acid
- Plasma free fatty acids
- Lactate

Clinical chemistry in urine
Urine analyses, incuding apperance, specific gravity, pH, sugar, protein, occult blood, ketones and microscopic examination of the sediment were conducted upon all dogs at the beginning and at week 6 and 12. Readings of pH, sugar, protein, occult blood and ketones were done with Haemacombistix from Ames Laboratories.

Analysis of Butanediol in feces was carried out in one male and one female of each group at week 4 by means of liquid chromatography.

Liver-function
A liver-function test (bromosulphophtalein method) was carried out upon all dogs of the control and highest dose group at week 13. Blood samples were taken at one and at thirthy minutes after intravenous injection of bromosulphophtalein (12.5 mg/kg bw). The retention after 30 minutes was calculated from the extinction values at one and at thirthy minutes.

Kdney-function
A kidney-function test (phenolred excretion method) was conducted upon all dogs of the control and highest dose group at week 13. One hour after intramuscular injection of phenolred (0.5 mg/kg bw) the urine bladder was emptied and the total amount of phenolred excreted in the urine was determined.
Sacrifice and pathology:
After 13 weeks all surviving dogs were anaethesised by intravenous injection of xxxbutal followed by exsanguination. Ten dogs (two of each group) were killed on four succesive workdays for each group.

A thourough autopsy was performed on each animal immediately after death. The following organs were weighted:
- heart
- kidneys
- liver
- spleen
- lungs
- testicles/ ovaries
- pituitary
- thyroids
- adrenals
- brain

Samples of these organs together with a wide range of other organs and tissues were fixed in a 4 % neutral, phosphate-buffered formaldehyde solution.
Detailed microscopic examination was done on all dogs. Haematoxylin-eosin stained paraffin sections of the organs weighed and also of the following organs and tissues were examined:
- spinal cord
- sciatic nerve
- salviary glands
- skeletal muscle
- thoratic aorta
- skin
- tonsils
- bladder
- oesophagus
- stomach
- duodenus
- jejunum
- ileum
- caecum
- colon
- pancreas
- trachea
- cirdumanal glands
- eyes
- epididymis
- prostate
- uterus
- gall
- bladder
- tongue
thymus

Special attention was paid to the possible occurence of lipofuscin in various tissues.
Statistics:
Wilcoxon test

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Epileptic seizure observed in one dog of the top-dose groupt at the end of week 3. From that time the number of dogs with epilepsy-like symptoms and the number of attacks increased gradually in the two high-dose groups in male and female dogs. Generally the condition lassted for about 1-2 minutes ans was followed by rapid recovery.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female dog 7-78 (9000 mg/kg bw) died during an an epilepic sizure. At autopsy, it appeared that this dog suffered from a congenital heart defect.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Most individual body weights showed normal variation both between different dogs and between successive weighins. All dogs gained weight during the study, but in the two highest dose groups (9000 and 12000 mg/kg bw) clearly and statistically significantly less than in the controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The daily portion was consumed completely by all dogs.
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The treatment with 1,3-butanediol solely affected the thrombocyte counts and the amount of methemoglobin. Increased amounts of thrombocytes were observed in the two high-dose groups at all stages, although in the group receiving 9000 mg/kg bw, the effect was statistically significant only at week 6.
The concentration of methemoglobin in the top-dose group receiving 12000 mg/kg bw was statistically signicant increased at week 12 only.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The activity of Serum glutamic pyruvic transaminase was slightly and dose-dependently increased in the two high dose groups at week 6 only. The serum glutamic oxalactic transaminase activity was also statistically significant increased in the top-dose at week 6 only. The amounts of free fatty acids were increased in a dose-dependent manner, but the difference was statistically significant in the top-dose only. Blood levels of beta-hydroxy butyric acid, aceto acetic acid and lactate were also increased with increasing feeding level of 1,3-butanediol. Small amounts of 1,3-butanediol were recovered form the feces of dogs of the two high dose-group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The values of pH of the two high dose-groups were lower than in the other groups at week 7. Small amounts of ketones were found in the top-dose group at week 12. The phenolred retention was very low at the top dose. No toxicological significance was attached to this finding.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
During the third week, epileptic seizure was observed in a dog of the top-dose. From that time the number of dogs with epilepsy-like symptoms and the frequency of attacks increased in the two highest dose group (9000 and 12000 mg/kg bw). Generally the symptoms lasted for about 1-2 minutes and was followed by a rapid recovery. Epileptic seizures were only observed in the 9000 and 12000 mg/kg bw dose groups. The NOEL is 6000 mg/kg bw.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The relative organ weights of kidney, liver brain, adrenals and lungs were significantly increased in the top-dose group. The relative organ weights of thymus and spleen were decreased in the top-dose group.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 6 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
clinical biochemistry
clinical signs
haematology
organ weights and organ / body weight ratios
urinalysis
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 9 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
haematology
organ weights and organ / body weight ratios
urinalysis

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
9 000 mg/kg bw/day (nominal)
System:
central nervous system
Organ:
brain
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
A subchronic (13-week) feeding study in dogs was carried out with 1,3-butanediol administered in diets with provided intake levels of 0, 3000, 6000, 9000 and 12000 mg/kg bw/d. Epilepsy-like seizures were frequently observed in dogs fed 9000 and 12000 mg/kg bw/d. Body weight gain was depressed in dogs fed with 9000 and 12000 mg/kg bw/d. Thrombocyte counts were increased at 6000 mg/kg bw/d and above. Methemoglobin was elevated in dogs of the top-dose only. Blood levels of free fatty acids, beta-hydroxy butyric acid, aceto-acidic acid and lactate increased with increasing levels of 1,3-butanediol. The excretion of phenol-red and bromosulphophtahalein did not indicate impaired function of the liver and the kidneys. Slight ketonuria was observed in dogs of the top-dose group (12000 mg/kg bw/d) at week 12. Small quantities of 1,3-butanediol were recoeverd from feces of dogs fed with 9000 and 12000 mg/kg bw/d. The relative weights of liver and adrenals showed dose-related increases in the two high dose groups. In addition, the top-dose group showed increased relative weights of kidney, brain and lungs and decreased weights of thymus and spleen. Gross and microscopic examination failed to reveal any changes that could be ascribed to treatment. It was concluded that 6000 mg/kg bw/d was no-untoward effect level in the present study.
Executive summary:

A subchronic (13-week) feeding study in dogs was carried out with 1,3-butanediol administered in diets with provided intake levels of 0, 3000, 6000, 9000 and 12000 mg/kg bw/d. Epilepsy-like seizures were frequently observed in dogs fed 9000 and 12000 mg/kg bw/d. Body weight gain was depressed in dogs fed with 9000 and 12000 mg/kg bw/d. Thrombocyte counts were increased at 6000 mg/kg bw/d and above. Methemoglobin was elevated in dogs of the top-dose only. Blood levels of free fatty acids, beta-hydroxy butyric acid, aceto-acidic acid and lactate increased with increasing levels of 1,3-butanediol. The excretion of phenol-red and bromosulphophtahalein did not indicate impaired function of the liver and the kidneys. Slight ketonuria was observed in dogs of the top-dose group (12000 mg/kg bw/d) at week 12. Small quantities of 1,3-butanediol were recoeverd from feces of dogs fed with 9000 and 12000 mg/kg bw/d. The relative weights of liver and adrenals showed dose-related increases in the two high dose groups. In addition, the top-dose group showed increased relative weights of kidney, brain and lungs and decreased weights of thymus and spleen. Gross and microscopic examination failed to reveal any changes that could be ascribed to treatment. It was concluded that 6000 mg/kg bw/d was no-untoward effect level in the present study.