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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:
supporting study
Study period:
2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well performed study, no standard method used
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Non-guideline study with special focus on testicular toxicity. In this non-guideline subacute study with special focus on testicular toxicity, dibutyl ether was administered by gavage to male Sprague-Dawley rats (7 per group) in doses of 2, 20, and 200 mg/kg bw, 5 days per week for 4 weeks. Negative control and positive control groups were treated with 10 ml corn oil/kg bw and 200 mg 1,6-dimethoxy hexane/kg bw, respectively. No post-exposure recovery group was included in this study.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
dibutyl ether (purity > 99 %)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 weeks old
- Weight at study initiation: 280 ± 20 grams

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Groups of male Sprague-Dawley rats (7/group) were administered 2, 20 and 200 mg dibutyl ether/kg body weight in corn oil, 5 days/week for 4 weeks. Negative control were administered corn oil (10 ml/kg body weight) and positive control animals were administered 200 mg 1,6-dimethoxy hexane/kg body weight.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not applicable
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily (5 days/week for 4 weeks)
Doses / concentrations
Remarks:
Doses / Concentrations:
2, 20 and 200 mg/kg bw
Basis:
other: nominal dose
No. of animals per sex per dose:
7 male rats/group
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: no
Positive control:
200 mg 1,6-dimethoxyhexane/kg body weight

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage side observations were made weekly. The battery observations included - posture, clonic movement, gait score, piloerection, ptosis, lacrimation, salivation, vocalization and ease of removal from the home cage

BODY WEIGHT: No data

FOOD CONSUMPTION: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination, blood was withdrawn from the abdominal aorta
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: erythrocyte count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, platelet count, white blood cell count and
percent lymphocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination, blood was withdrawn from the abdominal aorta
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: lactic dehydrogenase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, inorganic phosphate, urea nitrogen, creatinine, glucose, cholesterol, thyroxin (T4), and triiodothyronine (T3)), and detection of thiobarbituric acid-reactive substances (TBARS

URINALYSIS: Yes
- Parameters examined: Urine analysis at the end of the 4th week included the detection of 2-methoxy acetic acid (MAA, the proximal toxic metabolite of the positive control substance 1,6-dimethoxy hexane), protein, N-acetylglucosaminidase (NAGA) activity, ascorbic acid, creatine and creatinine
Sacrifice and pathology:
Brain, heart, thymus, liver, lung, kidneys, spleen, ventral prostate, epididymis and testis were weighed and prepared for histopathological examinations. No post-exposure recovery group was included in this study.


Other examinations:
At study termination blood was withdrawn from the abdominal aorta for preparation of plasma for 2-methoxy acetic acid analysis. Cell-free bronchoalveolar lavage fluid was prepared for the analysis of protein and N-acetylglucosaminidase activity (NAGA). In liver homogenates reduced glutathione, thiobarbituric acid-reactive substances (TBARS), protein carbonyl content, and activity of UDP-glucuronosyltransferase (UDPGT), and in liver S9 fraction the activities of ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD), benzyloxyresorufin-O-dealkylase (BROD), and glutathione-S-transferase (GST) were measured.
Statistics:
Standard statistical methods were employed.

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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
A marked increase in urinary ascorbic acid was observed in the high dose group receiving 200 mg dibutyl ether/kg bw
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
The animals treated with dibutyl ether did not show any significant testicular or epididymal effect. All animals survived the 4-week treatment with no abnormal cage-side observations. Food intake, final body weight, relative organ weights, measured hematological parameters and serum chemistry parameters were not significantly different from the control. At the high dose of 200 mg dibutyl ether/kg bw changes to the thyroid, liver and bone marrow that were mild and adaptive in nature were caused. Changes of minimal degree were observed in the thyroid (reduced follicle size and nuclear vesiculation) of dibutyl ether treated animals, some negative control animals and more prominent in positive control animals. In the absence of significant modulation in serum thyroxin (T4) and triiodothyronine (T3), the thyroid effects were discussed by the study authors as may be adaptive and reversible. Some bone marrow changes (increased granulocytes and myeoloid/erythroid ratio), minimal in severity, were seen in animals treated with 200 mg dibutyl ether/kg bw. In the liver, the high dose of dibutyl ether produced minimal to mild histopathological changes such as vesiculation of nuclei and increase in cytoplasmatic homogeneity. Urinary ascorbic acid, a biomarker of hepatic response to xenobiotics, was also elevated, suggesting that the hepatic glucuronic acid pathway, which is associated with the glucuronide pathway of detoxification, was stimulated. A hepatic response was further indicated by the increase in an hepatic xenobiotic enzyme activity (benzyloxyresorufin-o-dealkylase (BROD)). There were no significant treatment related changes in the activities of UDP-glucuronosyltransferase (UDPGT) in liver homogenates, and of ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD) and glutathione-S-transferase in liver S9 fraction. However, as the study authors discussed, in the absence of signs of necrosis or elevated level of aspartate aminotransferase in serum, the urinary ascorbic acid and hepatic enzyme changes and histopathological changes can be considered as mild metabolic responses. Brain, heart, thymus, lungs, kidneys and spleen were free of any histopathological changes. The lack of a kidney effects was supported by biochemical findings such as normal serum creatinine and urinary N-acetylglucosaminidase (NAGA) activity and protein levels. Unaltered bronchoalveolar levels of protein and NAGA also pointed to a lack of pulmonary effects. In contrast, the positive control substance 1,6-dimethoxyhexane caused decreased testis and thymus weights, degeneration of the seminiferous tubules and reduction of sperm density in the epididymides, an elevated creatine/creatinine ratio, an increase in plasma and urinary 2-methoxy acetic acid levels, histopathological thymus changes, mild dyserythropoiesis and dysthrombopoiesis in the bone marrow, thyroid changes of moderate severity and more pronounced adaptive liver changes.

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: based on no significant testicular effects or epididymal effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
In conclusion, the animals treated with dibutyl ether did not show any significant testicular or epididymal effect. The reproductive organs were of normal weight and free of histopathological changes. The urinary creatine/creatinine ratio (as sensitive biomarker of testicular injury) was unchanged, and the amount of 2-methoxy acetic acid (the proximal toxic metabolite of the positive control substance 1,6-dimethoxy hexane) in urine was within the control levels and in plasma not detectable. At the high dose of 200 mg dibutyl ether/kg bw changes to the thyroid, liver and bone marrow that were mild and adaptive in nature were caused. In contrast, the positive control substance 1,6-dimethoxy hexane caused decreased testis and thymus weights, degeneration of the seminiferous tubules and reduction of sperm density in the epididymides, an elevated creatine/creatinine ratio, an increase in plasma and urinary 2-methoxy acetic acid levels, histopathological thymus changes, mild dyserythropoiesis and dysthrombopoiesis in the bone marrow, thyroid changes of moderate severity and more pronounced adaptive liver changes.
Executive summary:

In this non-guideline sub-acute study with special focus on testicular toxicity, dibutyl ether was administered by gavage to male Sprague-Dawley rats (7 per group) in doses of 2, 20, and 200 mg/kg bw, 5 days per week for 4 weeks. Negative control and positive control groups were treated with 10 ml corn oil/kg bw and 200 mg 1,6-dimethoxy hexane/kg bw, respectively. No post-exposure recovery group was included in this study.

The animals treated with dibutyl ether did not show any significant testicular or epididymal effect. All animals survived the 4-week treatment with no abnormal cage-side observations. Food intake, final body weight, relative organ weights, measured hematological parameters and serum chemistry parameters were not significantly different from the control. At the high dose of 200 mg dibutyl ether/kg bw changes to the thyroid, liver and bone marrow that were mild and adaptive in nature were caused. Changes of minimal degree were observed in the thyroid (reduced follicle size and nuclear vesiculation) of dibutyl ether treated animals, some negative control animals and more prominent in positive control animals. In the absence of significant modulation in serum thyroxin (T4) and triiodothyronine (T3), the thyroid effects were discussed by the study authors as may be adaptive and reversible. Some bone marrow changes (increased granulocytes and myeoloid/erythroid ratio), minimal in severity, were seen in animals treated with 200 mg dibutyl ether/kg bw. In the liver, the high dose of dibutyl ether produced minimal to mild histopathological changes such as vesiculation of nuclei and increase in cytoplasmatic homogeneity. Urinary ascorbic acid, a biomarker of hepatic response to xenobiotics, was also elevated, suggesting that the hepatic glucuronic acid pathway, which is associated with the glucuronide pathway of detoxification, was stimulated. A hepatic response was further indicated by the increase in an hepatic xenobiotic enzyme activity (benzyloxyresorufin-o-dealkylase (BROD)). There were no significant treatment related changes in the activities of UDP-glucuronosyltransferase (UDPGT) in liver homogenates, and of ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD) and glutathione-S-transferase in liver S9 fraction.

However, as the study authors discussed, in the absence of signs of necrosis or elevated level of aspartate aminotransferase in serum, the urinary ascorbic acid and hepatic enzyme changes and histopathological changes can be considered as mild metabolic responses. Brain, heart, thymus, lungs, kidneys and spleen were free of any histopathological changes. The lack of a kidney effects was supported by biochemical findings such as normal serum creatinine and urinary N-acetylglucosaminidase (NAGA) activity and protein levels. Unaltered bronchoalveolar levels of protein and NAGA also pointed to a lack of pulmonary effects. In contrast, the positive control substance 1,6-dimethoxyhexane caused decreased testis and thymus weights, degeneration of the seminiferous tubules and reduction of sperm density in the epididymides, an elevated creatine/creatinine ratio, an increase in plasma and urinary 2-methoxy acetic acid levels, histopathological thymus changes, mild dyserythropoiesis and dysthrombopoiesis in the bone marrow, thyroid changes of moderate severity and more pronounced adaptive liver changes.

In conclusion, the animals treated with dibutyl ether did not show any significant testicular or epididymal effect. The reproductive organs were of normal weight and free of histopathological changes. The urinary creatine/creatinine ratio (as sensitive biomarker of testicular injury) was unchanged, and the amount of 2-methoxy acetic acid (the proximal toxic metabolite of the positive control substance 1,6-dimethoxy hexane) in urine was within the control levels and in plasma not detectable. At the high dose of 200 mg dibutyl ether/kg bw changes to the thyroid, liver and bone marrow that were mild and adaptive in nature were caused. In contrast, the positive control substance 1,6-dimethoxy hexane caused decreased testis and thymus weights, degeneration of the seminiferous tubules and reduction of sperm density in the epididymides, an elevated creatine/creatinine ratio, an increase in plasma and urinary 2-methoxy acetic acid levels, histopathological thymus changes, mild dyserythropoiesis and dysthrombopoiesis in the bone marrow, thyroid changes of moderate severity and more pronounced adaptive liver changes.