Registration Dossier

Administrative data

Description of key information

A repeated dose toxicity study is available, which was performed under GLP conditions and according to standard guidelines.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
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 information:
Composition 1
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
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)
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
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.
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.
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
Critical effects observed:
not specified

None

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.

Endpoint conclusion
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted as per OECD TG 412, EC Method B.8 and in accordance with the Principles of Good Laboratory Practice (GLP).
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Deviations observed during the conduct of the range and the main study were not considered to have influenced the integrity and validity of the study results
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Deviations observed during the conduct of the range and the main study were not considered to have influenced the integrity and validity of the study results
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfield, Germany
- Age at study initiation: 7-9 weeks
- Weight at study initiation: mean male weight - 226 gram, mean female weight - 175 gram
- Housing: standard housing conditions
- Diet (e.g. ad libitum): ad libitum from the arrival of rats until the end of the study, except during exposure and during overnight fasting prior to necropsy
- Water (e.g. ad libitum): ad libitum from the arrival of rats until the end of the study, except during exposure and during overnight fasting prior to necropsy
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle
Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed to the test atmosphere in noe-only exposure units, each unit consisted of a cylindrical column, surrounded by a transparent cylinder. The column consisted of a top assembly with the inlet of the test atmosphere, one or two rodent tube sections and at the bottom the base assembly with the exhaust port.
- Method of holding animals in test chamber: Each rodent tube section had 20 ports for animal exposure. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column (males and females alternated). Several empty ports were used for test atmosphere sampling and measurement of temperature and relative humidity. The extra remaining ports were closed. Only the nose of the rats protruded into the interior of the column. Animals were rotated on a weekly basis in such a way that at the end of the exposure period, the animals were at about the initial location again. The units were illuminated externally by normal laboratory TL-lighting.
- Source and rate of air: The test atmosphere for each exposure level was generated by passing humidified air for each exposure unit through glass evaporators. The test material was pumped as a liquid in the evaporators using syringe pumps. The air streams measured by rotameters were directed to the exposure units. At the bottom of the units the test atmospheres were exhausted. The rotameters and the syringe pumps were read out and recorded at regular intervals (approximately bi-hourly, i.e. 3 times a day). The rotameters were calibrated at 3 settings in triplicate.
- Temperature, humidity, pressure in air chamber: 22 ± 3 °C, 30-70% relative humidity

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of n-dibutylether in the test atmosphere was measured by infrared analysis at 3.4 µm. The response of the analyser was recorded by an analogue recorder. Also, the concentration of n-dibutylether in the test atmospheres was measured by total carbon analysis for the low and mid concentration test atmospheres, the response was recorded by an analogue recorder.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mean concentrations were 151 ± 3, 497 ± 3 and 1497 ± 3 mg n-dibutylether/m3
Duration of treatment / exposure:
28 days followed by a 2-week recovery period
Frequency of treatment:
6 hours/day, 5 days/week for 4 weeks (a total of 20 exposures) daily during the exposure period.
Remarks:
Doses / Concentrations:
150, 500 and 1500 mg/m³ (actual concentration)
Basis:
other: mean actual concentrations were 151 ± 3, 497 ± 3 and 1497 ± 3 mg n-dibutylether/m3, mean nominal concentrations were 179, 538 and 1553 mg/m3
No. of animals per sex per dose:
5 males + 5 females/test group and 5 males + 5 females - control and 1500 mg/m3 reversal group
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: based on the results of the range finding study with exposure concentrations of 0, 1000, 3000 and 10000 mg/m3 (reduced to 6500 mg/m3 since ataxia was observed in the exposed males after first exposure to 10000 mg/m3 of n-dibutylether)
- Rationale for animal assignment: computer randomization taking body weights into account
- Post-exposure recovery period in satellite groups: 2 weeks - control and high dose reversal groups
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each animals was observed daily in the morning and if necessary handled to detect signs of toxicity. A group-wise observation was made halfway during exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: once during the acclimatization period, one day before the start of exposure, at initiation of treatment (day 0) and once per week thereafter. The animals were also weighed on the day before overnight fasting and on the day of sacrifice

FOOD CONSUMPTION: Yes
- Food consumption for each cage determined over successive periods of 7 days (and a 6-day period at the end of exposure and recovery period) and mean daily diet consumption calculated as g food/kg body weight/day

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: Yes - Nembutal
- Animals fasted: Yes
- How many animals: all rats of the main study group
- Parameters examined: haemoglobin, PCV, RBC count, reticulocytes, differential WBC count, prothrobin time, thrombocyte count. The following parameters were calculated - MCV, MCH and MCHC

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treament period
- Animals fasted: Yes
- How many animals: all rats of the main study group
- Parameters examined: ALP, ASAT, ALAT, GGT, total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, total bilirubin, cholesterol, triglycerides, phospholipids, calcium, sodium, potassium, chloride and inorganic phosphate

URINALYSIS: Yes
- Time schedule for collection of urine: on day 28
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: volume, appearanc, pH, glucose, ketones, occult blood, protein, bilirubin, urobilinogen, electrolytes (sodium, potassium, chloride), creatinine and microscopy of the sediment

NEUROBEHAVIOURAL EXAMINATION:No
Sacrifice and pathology:
ORGAN WEIGHTS: the following organs were weighed (paired organs together) as soon as possible after dissection to avoid dryong - adrenals, brain, heart, kidneys, liver, spleen , testes, lungs with trachea and larynx, epididymides, ovaries and uterus
GROSS PATHOLOGY: Yes (all animals of the main groups and recovery groups
HISTOPATHOLOGY: Yes (all animals of the control, and high dose groups, in the event of any treatment related changes, histology on the affected organs from the lower dose groups would be performed
Other examinations:
None
Statistics:
Standard statistical methods were employed
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
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:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY - No animal exposed to dibutyl ether died during the exposure period or the recovery period. Individual observations in the mornings, i.e. before the start of each day´s exposure, did not reveal treatment-related clinical signs. One male control animal was found dead in its inhalation tube in the morning of study-day 24. The animal had tried to turn around, got stuck and most probably died from suffocation. In another control male penile blood loss was observed on two successive days of the exposure period, when releasing the animal from the animal holder. This recovered thereafter. The group-wise observations carried out about halfway through each day´s exposure revealed no abnormalities. During the 2-week recovery period, treatment related clinical signs were not observed.

BODY WEIGHT AND WEIGHT GAIN - Body weights in high-concentration females were statistically significantly lower than in controls on day 7, 14 and 21 of the study. Body weights were also lower in females of the recovery group on day 28, but not in females of the main study group on day 27. The lower body weights
could be explained by a body weight loss during the first week of exposure; thereafter, weekly body weight gain was comparable among all groups of females. Mean body weight gain in treated males was normal when compared to control animals. Some statistically significant differences from controls were observed in low- and mid-concentration males towards the end of the treatment period, but in the absence of a concentration-response relationship, these differences
were considered to be incidental findings. No statistically significant differences in body weight were observed between control and high-concentration animals during the 2-week recovery period.

FOOD CONSUMPTION - Overall food consumption was similar among the groups during the treatment and the recovery period. Food conversion efficiency was slightly lower in mid-concentration males and lower in high-concentration females during the first week of the study, when compared to control animals. This recovered thereafter. There were no differences in food conversion efficiency between control and high-concentration animals during the 2-week recovery period. The weight loss during the first week of exposure was in line with the concentration-related weight loss observed in animals exposed during the range-finding study at higher levels (3000 and 6500 mg/m³) and at a slightly lower level of 1000 mg/m³.

HAEMATOLOGY - At the end of the exposure period, there were no treatment related changes in the haematological parameters examined

CLINICAL CHEMISTRY - Mean clinical chemistry values in plasma collected at the end of the exposure period showed no treatment related differences between the groups. The albumin/globulin ratio in plasma was significantly increased in males of the mid-concentration group but in the absence of a similar change in the high dose group, this can be considered to be not treatment related. The high dose group females showed a tendency towards lower urea levels when compared to control animals, however, the values were well within the range of historical contol values.

URINALYSIS - At the end of the exposure period, except for some non statistiscally significant changes in creatinine and electrolytes concentration, there were no treatment related changes in the urological parameters examined

ORGAN WEIGHTS - No treatment-related changes in absolute or relative organ weights, including the reproductive organs were observed at the end of the exposure period. Differences in organ weights recorded in low- and mid-concentration males were considered fortuitous findings not related to treatment because of the absence of similar changes in organ weights in animals of the high-concentration group. In females, no changes in absolute or relative organ weights were observed at the end of the exposure period. After 2 weeks of recovery, a slight, but statistically significant decrease in absolute (but not relative) brain weight was observed in high-concentration females. At the end of the treatment period, absolute testes weight, absolute epididymides weight and absolute liver weight were statistically significantly decreased in males of the mid-concentration group. These findings could be explained by the low terminal body weight of animals of this group. In addition, the lower testes and epididymides weights could be attributed to the observation of "small testes and small epididymides" in two animals of this group. Absolute brain weight was statistically significantly decreased in males of the mid-concentration group, and relative brain weight was statistically significantly increased in males of the low- and mid-concentration groups. Because of the absence of similar changes in organ weights in animals of the high-concentration group, the differences in organ weights recorded in low- and mid-concentration males were considered fortuitous findings not related to treatment. The changes in absolute and relative organ weights as observed in low- and mid-concentration males at the end of the exposure period (i.e.,significant decreases in absolute testes, epididymides, liver and brain weight), were not observed after 2 weeks of recovery in high concentration males

GROSS PATHOLOGY - Treatment-related macroscopical or histopathological changes were absent in organs and tissues examined, including the reproductive organs (testes, epididymides, prostata, seminal vesicles, coagulating glands, ovaries, uterus, vagina and mammary gland).

HISTOPATHOLOGY - Treatment-related macroscopical or histopathological changes were absent in organs and tissues examined, including the reproductive organs (testes, epididymides, prostata, seminal vesicles, coagulating glands, ovaries, uterus, vagina and mammary gland).
Dose descriptor:
NOAEL
Effect level:
1 500 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: based on overall effects
Dose descriptor:
NOAEL
Effect level:
500 mg/m³ air
Based on:
test mat.
Sex:
female
Basis for effect level:
other: based on body weight reduction noted in the first exposure week at the highest dose of 1500 mg/m3
Dose descriptor:
LOAEL
Effect level:
1 500 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on overall effects
Critical effects observed:
not specified

None

Conclusions:
Overall, the results of the study, it can be concluded that exposure to concentrations of 150, 500 and 1500 mg of n-dibutylether/m3 concentrations did not result in treatment related effects in male rats and only in limited effects in females exposed to 1500 mg/m3. Exposure to 1500 mg/m3 resulted in a temporary body weight loss in these females during the first exposure week. After this week, including the 14-day recovery period, growth was comparable to the control group. This weight loss can be considered as a temporaray effect and the highest expsoue of 1500 mg/m3 can therefore be considered as the Low observed adverse effect level (LOAEL) for both the sexes. The No observed adverse effect level (NOAEL) for male rats can be considered as 1500 mg/m3 and for female rats the mid dose of 500 mg/m3.
Executive summary:

The 28 -day inhalation toxicity study was conducted according to OECD TG 412, EC Method B.8 and in accordance with the Principles of Good Laboratory Practice (GLP). Groups of 5 male and 5 female rats were exposed to n-dibutylether at concentrations of 150, 500 and 1500 mg/m3 (based on the results of a 7 -day inhalation toxicity study at concentrations of 1000, 3000 and 10000 mg/m3 (later reduced to 6500 mg/m3 as all animals of 10000 mg/m3 exhibited signs of ataxia). Also, a concurrent control was used in the study along with the control and the 1500 mg/m3 reversal group.

There were no exposure related clinical signs, haematology, clinical chemistry and urinalysis and organ weights. In addition treatment realted changes were not evident in gross pathology of all the animals of the main study and the reversal group and no evidence of any treatment related histopathjological changes in any organs. The only exposure related change noted was a decrease in the body weight gain of the high dose group during the first week of exposure, thereafter the weekly body weight gain was comparable to the other groups including control.

Overall, the results of the study, it can be concluded that exposure to concentrations of 150, 500 and 1500 mg of n-dibutylether/m3 concentrations did not result in treatment related effects in male rats and only in limited effects in females exposed to 1500 mg/m3. Exposure to 1500 mg/m3 resulted in a temporary body weight loss in these females during the first exposure week. After this week, including the 14-day recovery period, growth was comparable to the control group. This weight loss can be considered as a temporaray effect and the highest expsoue of 1500 mg/m3 can therefore be considered as the Low observed adverse effect level (LOAEL) for both the sexes.

The No observed adverse effect level (NOAEL) for male rats can be considered as 1500 mg/m3 and for female rats the mid dose of 500 mg/m3.

Endpoint conclusion
Dose descriptor:
NOAEC
500 mg/m³
Study duration:
subacute
Species:
rat

Mode of Action Analysis / Human Relevance Framework

Additional information

Inhalation

In a 28-day inhalation study performed according to directive 92/69/EEC, B.8 (1992) and OECD TG 412 (1981) and under GLP conditions, four groups (5 rats/sex/group) of Wistar rats were exposed in nose-only exposure units to target concentrations of 0 (control), 150, 500 and 1500 mg dibutyl ether/m³ for 6 hours/day, 5 days/week (a total of 20 exposure days, purity of the test substance: > 99.76 %). In addition, two groups of 5 male and 5 female rats each, one control group and one high-concentration group, were equally exposed and were kept for a subsequent post-exposure period of 2 weeks (recovery groups). Data on clinical observations, body weight gain, food consumption, food conversion efficiency, hematology, clinical chemistry, urinanalysis, organ weights, gross examination at necropsy and microscopic examination of various tissues and organs including the gonads were used as criteria for disclosing possible harmful effects. No animal exposed to dibutyl ether died during the exposure period or the recovery period. Individual observations in the mornings, i.e. before the start of each day´s exposure, did not reveal treatment-related clinical signs. The group-wise observations carried out about halfway through each day´s exposure revealed no abnormalities. During the 2-week recovery period, treatment related clinical signs were not observed. Body weights in high-concentration females were statistically significantly lower than in controls on day 7, 14 and 21 of the study, but not on day 27. Body weights were also lower in females of the recovery group on day 28. The lower body weights could be explained by a body weight loss during the first week of exposure; thereafter, weekly body weight gain was comparable among all groups of females. Mean body weight gain in treated males was normal when compared to control animals. Some statistically significant differences from controls were observed in low- and mid-concentration males towards the end of the treatment period, but in the absence of a concentration-response relationship, these differences were considered to be incidental findings. No statistically significant differences in body weight were observed between control and high-concentration animals during the 2-week recovery period. Overall food consumption was similar among the groups during the treatment and the recovery period. Food conversion efficiency was slightly lower in mid-concentration males and lower in high-concentration females during the first week of the study, when compared to control animals. At the end of the exposure period, there were no treatment-related changes in red blood cell variables, coagulation variables, total white blood cell counts and differential white blood cell counts. Mean clinical chemistry values in plasma collected at the end of the exposure period showed no treatment-related differences between the groups. The albumin/globulin ratio in plasma was statistically significantly increased in males of the mid-concentration group, this change was not considered to be related to treatment. There were no statistically significant changes in urinary volume, urine density and electrolyte concentrations in the urine of dibutyl ether-exposed males or females at the end of the exposure period. A tendency towards lower creatinine and electrolytes concentrations was observed in high-concentration females. This observation may partly be explained by the statistically not significant higher urine volumes in high-concentration females, when compared to urine volumes in control animals. Microscopic observations in urine revealed no statistically significant differences between the groups. No treatment-related changes in absolute or relative organ weights, including the reproductive organs were observed at the end of the exposure period. Differences in organ weights (testes, epididymides, liver and brain) recorded in low- and mid-concentration exposed males were considered accidental findings not related to treatment because of the absence of similar changes in organ weights in animals of the high-concentration group. The changes in absolute and relative organ weights as observed in low- and mid-concentration males at the end of the exposure period, were also not observed after 2 weeks of recovery in high concentration males. In females, no changes in absolute or relative organ weights were observed at the end of the exposure period. After 2 weeks of recovery, a slight, but statistically significant decrease in absolute (but not relative) brain weight was observed in high-concentration females. Treatment-related macroscopical or histopathological changes were absent in organs and tissues examined, including the reproductive organs (testes, epididymides, prostata, seminal vesicles, coagulating glands, ovaries, uterus, vagina and mammary gland) and the respiratory tract.

NOAEL:        = 500 mg/m³ in females, and 1500 mg/m³ (highest concentration tested) in males

LOAEL:         = 1500 mg/m³ in females

The LOAEL is based on the temporary body weight loss during the first week of exposure.

In the 7-day range-finding study to this 28-day study, clear toxicity, consisting of ataxia, piloerection and blepharospasm, was observed in males dosed with 10 000 mg/m³ immediately after the first exposure. Based on this observation, the highest concentration level was decreased to 6500 mg/m³. At 6500 mg/m³, males and females showed slight labored breathing after the first exposure. Females only had blepharospasm and sluggishness after the second and third exposure. No treatment-related clinical signs were seen in animals of the control, low- (1000 mg/m³) and mid- (3000 mg/m³) concentration groups. Effects on body weight were slight in low-concentration females, moderate in mid-concentration females and severe in high-concentration males and females. In addition, effects on food consumption and food conversion efficiency were found in a dose-dependent manner at all concentration levels (TNO, 2005).

Oral

In a non-guideline 4-week 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. 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. 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 probably 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, dibutyl ether caused in this non-guideline 4-week study with special focus on testicular toxicity at the high dose of 200 mg/kg bw changes to the thyroid, liver and bone marrow that were mild and adaptive in nature, but no testicular or epididymal effects (Poon et al., 2005).

Justification for classification or non-classification

No target organ appeared up to and including the highest tested concentration of 1500 mg/m³ in a 28-day inhalation study in rats, performed under GLP according to OECD TG 412 with test concentrations of 0, 150, 500 and 1500 mg dibutyl ether/m³. The repeated exposure to 1500 mg dibutyl ether/m³ caused only a temporary body weight loss in females during the first exposure week. After the first week of exposure and during the 14-day recovery period, growth of high-dose females was comparable to the control group. The treated male rats showed changes in testes, epididymides, liver and brain weights in the low- and mid-dose groups that were not confirmed by data of the high-dose group of 1500 mg/m³. In addition, There were no effects on male reproductive organs reported in an oral repeated dose study focussing on this endpoint.

Therefore the findings regarding the low- and mid-dose groups were considered as fortuitous and thus the no observed adverse effect level (NOAEL) was 1500 mg/m³. The temporary effect on body weight seen in female rats is not considered to be a serious adverse effect, and the high-concentration level of 1500 mg/m³ can be regarded as a minimum observed adverse effect level in female rats. Due to the absence of treatment-related changes, the next lower level tested, viz. 500 mg/m³, was considered to be the NOAEL in females.

Therefore, the substance does not need to be classified and labelled for repeated dose toxicity, according to the Regulation 1271/2008 and the Directive 67/548/EEC.