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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

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).

Data source

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

Materials and methods

Test guidelineopen allclose all
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): n-dibutylether
- Physical state: colourless volatile liquid
- Analytical purity: range finding - > 99.3%, main study - > 99.76%
- Lot/batch No.: range finding - 0000093881, main study - 0000100437
- Expiration date of the lot/batch: range finding - 19 december 2004, main study - 31 July 2005
- Storage condition of test material: ambient temperature, dark, protected from air

Test animals

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

Administration / exposure

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.
Doses / concentrations
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

Examinations

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

Results and discussion

Results of examinations

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).

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

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.