Registration Dossier

Administrative data

Endpoint:
developmental toxicity
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 according to OECD TG 414 and EU Method B.31 and in accordance with the Principles of Good Paboratory Practice (GLP)
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
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): n-dibutylether
- Physical state: colourless liquid
- Analytical purity: 99.7%
- Lot/batch No.: 0000093881
- Storage condition of test material: protected from sunlight; in a tightly closed container; stored in a cool, well ventilated place; protected from air/oxygen

Test animals

Species:
rat
Strain:
other: CD/Crl: CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Germany
- Age at study initiation: mature young adults (8-9 weeks)
- Weight at study initiation: 192-240 grams
- Housing: except during mating period, the dams (female rats) were kept singly in MAKROLON cages (type III) with a basal surface of approximately 39x23x15 cm.
- Diet (e.g. ad libitum): Sniff R-Z V1324 was offered daily ad libitum, however, food was removed from all dams at approximately 3:00 PM on gestation day 19
- Water (e.g. ad libitum): tap water (in drinking bottles) was offered daily ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 15%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: n-dibutylether was diluted in corn oil to the appropriate concentrations and administered orally at a constant volume of 2 ml/kg body weight once daily from the 6th-19th day of pregnancy. The dose of the item was adapted to the animal's body weight daily. The control animals received the vehilce (corn oil) at a constant volume of 2 ml/kg body weight orally once daily in the same way. Dosing solutions were prepared fresh every day and the male rats were not treated during the study period.

VEHICLE
- Justification for use and choice of vehicle (if other than water): n-dibutylether was insoluble in water and corn oil is a recommended vehicle
- Lot/batch no.: 31107483 (expiry date - April 2005)
- Purity: according to European Pharmacopeia
- Supplier: Caesar & Lorentz GmbH, 40721 Hilden, Germany
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
no information available
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 male:female
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0] of pregnancy
- Rats which did not get pregnant were excluded from the analysis of the results and replaced by the other animals. A post mortem negative staining according to Salwski was carried out in the replaced animals in order to confirm the non-pregnancy status
Duration of treatment / exposure:
day 6 to 19 of pregnancy
Frequency of treatment:
daily from days 6-19 of gestation
Duration of test:
entire gestation period (days 0-20)
No. of animals per sex per dose:
20-25 rats (female)/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of the dose range finding study in rats (LPT study no. 18302/04: Dose range finding study for a prenatal developmental toxicity study of n-dibutylether in rats by oral administration).

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded on day 0 of gestation, followed by daily weighings - at the same time of the day. The body weight was also calculated in intervals (i.e. 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20)

FOOD CONSUMPTION: Yes
- The quantity of food consumed by each rat was recorded daily with the exception of gestation day 20. Food intake per rat (g/rat/day) was calculated along with the relative food consumption (g/kg body weight/day)

WATER CONSUMPTION: No
- Time schedule for examinations: Daily monitoring by visual observation of the drinking water bottles was maintained throughout the study

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: all organs

OTHER: Plasma biochemistry was done on animals from the control and the high dose group and the following parameters evaluated - ALAT, ALP, ASAT and LDH.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: macroscopic inspection (gross evaluations) of the placentae for focal indurations
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
Standard statistical techniques were employed, malformation rate per group (%), retardation rate per group (%), pre-implantation loss and post-implantation loss
Indices:
Resorption rate (%),
Historical control data:
yes - result from the last 28 embryotoxicity studies in SD-rats performed at LPT from July 2000 - March 2005 were provided in the report

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
All dams survived to sacrifice. At the high tested dose level of 1000 mg/kg bw, signs of toxicity in form of piloerection was noted in 5 of 20 dams. One animal showed pale eyes and ears on gestation days 7 to 12. Additionally, a reduced body weight (up to 7 % below the control), reduced food consumption (up to 36 % below the control value), increased drinking water consumption, slightly increased absolute and relative liver weights and an increased activity of plasma aspartate aminotransferase were noted. A slight reduction by 9 % was noted for the gravid uterus weight caused by slightly lowered fetal weights. The carcass weight was 6 % below the control value. Necropsy revealed no test item-related pathological changes in any of the dams treated with either 100, 300 or 1000 mg/kg bw/day. The treatment of dams with 100 or 300 mg dibutyl ether/kg bw/day had no effects on behaviour, external appearance, nature of the faeces, body weight, body weight change and body weight gain, food and drinking water consumption, uterus weight and net body weight change. The marginal increase of absolute and relative weights of the liver at 100 and 300 mg/kg bw/day were regarded to be an inductive effect but not an adverse effect

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions, placental weights, live fetuses, the values calculated for the pre- and post-implantation losses and the sex distribution of fetuses. Laparotomy revealed one dead fetus each (both of them were runts) at 100 and 300 mg/kg bw/day. In total, five runts were noted at laparotomy: two runts each at 100 and 1000 mg/kg bw/day and one runt at 300 mg/kg bw/day. One twin implant (one male fetus and one late resorption) was detected in one dam of the intermediate dose-group. All findings of dead fetuses, runts and twin implants are regarded to be spontaneous and are within the normal range of variation. At the high tested dose level of 1000 mg/kg bw, reduced fetal weights calculated for male and female fetuses and for all fetuses were slightly but statistically significantly below the control values. In addition, in this dose group skeletal examination according to Dawson revealed significantly increased fetal incidences for the following retardations: hyoid not ossified and absence of ossification in 5th metacarpalia.

Significantly increased litter incidences were noted for the caudal vertebral bodies, only one body ossified. These incidences were above the range of the test facility background data and are regarded to be test item-related. However, the incidence of total skeletal retardations in the high dose was not increased. The examination of the fetal organs (according to Wilson) revealed statistically significantly increased fetal and liter incidences for dilatation of the 4th cerebral ventricle. The mean fetal weights were not influenced by the administration of 100 or 300 mg/kg bw/day. Also no test item-related influence was noted for the incidence of skeletal retardations or soft tissue variations at the low and intermediate dose levels. At 100, 300 or 1000 mg/kg bw/day, no malformations were noted in the fetuses during external examination at laparotomy, skeletal examination (according to Dawson) or soft tissue examination (according to Wilson).

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Results of the dose range finding study in rats (LPT study no. 18302/04: Dose range finding study for a prenatal developmental toxicity study of n-dibutylether in rats by oral administration) -

In this range finding study, dibutyl ether was administered to 3 pregnant female CD/Crl: CD-rats/group once daily from the 6th to the 19th day of pregnancy (the ´critical` phase of organogenesis and fetal

development) in doses of 0 (control), 100, 300 and 1000 mg/kg bw. The day on which sperm was found in the vaginal lavage was considered as the day of conception (day 0 of pregnancy). The test item was diluted in corn oil to the appropriate concentrations and was administered orally at a constant volume of 2 ml/kg bw. The dose of the test item was adapted to the animal´s body weight daily. The test item preparations were freshly prepared every day. The control animals received the vehicle corn oil at a constant volume of 2 ml/kg bw orally once daily. Individual animals were observed daily for viability, behaviour, external appearance and nature of the faeces. Immediately after administration, any sign of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the day. Maternal body weights, food and drinking water consumption were recorded daily.

 

The dams were laparotomised under ether narcosis on day 20 of gestation. The ovaries and uteri were removed; the uteri (in toto) were weighed. A dissection with macroscopic examination of the internal organs and placentae of the dams was carried out. In case of macroscopical findings, the affected maternal tissues were preserved for possible further histopathological examinations. The fetuses were removed and the following examinations were performed: macroscopic inspection of the placentae, number of fetuses, sex and viability of fetuses, number and size of resorptions, corpora lutea in the ovaries, implantations and location of fetuses in uterus, weight of fetuses and weight of placentae, external inspection of fetuses for damages, especially malformations. 2 litters per group were evaluated. The fetuses were sacrificed by an ether atmosphere.

 

All dams survived to sacrifice. No clinical signs of systemic toxicity were noted. At 300 and 1000 mg/kg bw/day, the body weight was in a dose-related way slightly below that of the control dams from the 2nd administration onwards until laparotomy (up to 10 % at 1000 mg/kg bw/day). A slight reduction was also noted for the net weight change from day 6 (carcass weight minus day 6 body weight) for the dams treated with 1000 mg/kg bw/day. At 1000 mg/kg bw/day, a distinctly reduced food intake was noted, predominantly during the first treatment days. Drinking water consumption was not influenced. No test item-related pathological findings were noted. The gravid uterus weight and the carcass weight of the animals treated with 100, 300 or 1000 mg/kg bw/day were not influenced. 

 

No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions, number of live fetuses, fetal or placental weights as well as the values calculated for the pre- and post-implantation loss. No dead fetuses were noted at laparotomy. External examination of the fetuses revealed no malformations and no variations.

 

At 300 mg/kg bw/day, a marginal reduction was noted for body weight, at 1000 mg/kg bw/day, reductions were noted for both body weight and food consumption. There was no increase in the incidence of external malformations or external variations at the tested doses, not even at materno-toxic dose levels. The no-observed-effect level (NOEL) for toxic effects on the dams was 100 mg/kg bw/day. The no-observed-effect-level (NOEL) for toxic effects on the fetuses was 1000 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
At 1000 mg/kg bw/day, signs of toxicity were noted in form
of pilo-erection in dams. Additionally, a reduced body
weight and food intake and increased drinking water consumption, absolute and relative liver weights and an
increased plasma ASAT activity were noted at 1000 mg/kg
bw/day in dams.
At the high tested dose level of 1000 mg/kg bw/day, reduced
mean fetal weights were noted. In addition, statistically
increased incidences were recorded for skeletal retardations (according to Dawson) in form of missing or incomplete
ossification of the hyoid, caudal vertebral bodies and 5th
metacarpalia and for soft tissue variations (according to Wilson) in form of dilatation of the 4th cerebral ventricle.
Dibutyl ether possessed no teratogenic properties. There was no test item-related increase in the incidence of fetal malformations, external or skeletal variations, not even at
materno-toxic dose levels. An increase in retardations and variations was only noted in the materno-toxic dose of 1000
mg/kg bw/day. The no-observed-effect level (NOEL) for toxic effects on the dams was 300 mg/kg bw/day. The no-observed-effect-level (NOEL) for fetal skeletal retardations and soft tissue variations was 300 mg/kg
bw/day. The no-observed-effect-level (NOEL) for teratogenic properties was 1000 mg/kg bw/day (highest dose tested).
Executive summary:

This study was conducted according to OECD TG 414 and EU Method B.31 and in accordance with the Principles of Good Laboratory Practice (GLP). Dibutyl ether was administered to 25 pregnant female CD/Crl: CD-rats/group once daily from the 6th to the 19th day of pregnancy (the ´critical` phase of organogenesis and fetal development) in doses of 0 (control), 100, 300 and 1000 mg/kg bw. The day on which sperm was found in the vaginal lavage was considered as the day of conception (day 0 of pregnancy). Dose selection was based on the results of a dose-range-finding study (LTP Report No. 18302/04, also cited in this IUCLID). The test item was diluted in corn oil to the appropriate concentrations and was administered orally at a constant volume of 2 ml/kg bw. The dose of the test item was adapted to the animal´s body weight daily. The test item preparations were freshly prepared every day. The control animals received the vehicle corn oil at a constant volume of 2 ml/kg bw orally once daily. Male rats for mating remained untreated.

 

Individual animals were observed daily for viability, behaviour, external appearance and nature of the faeces. Immediately after administration, any sign of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the day. Maternal body weights, food and drinking water consumption were recorded daily. Early in the morning of gestation day 20 blood was taken from all animals of the control and the high dose group to determine the plasma activity of alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT) and lactate dehydrogenase

(LDH).

 

The dams were laparotomised under ether narcosis on day 20 of gestation. 20 dams/group were evaluated. In order to obtain the required number of pregnant animals/group, additional animals (5/group) were used at the start of study. The ovaries, the uteri and the liver of the dams were removed; the uteri (in toto) and the liver were weighed. A dissection with macroscopic examination of the internal organs and placentae of the dams was carried out. In case of macroscopical findings, the affected maternal tissues were preserved for possible further histopathological examinations. The fetuses were removed and the following examinations were performed: macroscopic inspection of the

placentae, number of fetuses, sex and viability of fetuses, number and size of resorptions, corpora lutea in the ovaries, implantations and location of fetuses in uterus, weight of fetuses and weight of placentae, external inspection of fetuses for damages, especially malformations. The fetuses were sacrificed by an ether atmosphere and 50 % of the number of fetuses in each litter were examined for skeletal anomalies. The skeleton was double-stained with Alician blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to Dawson). The remaining 50 % of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to Wilson.

 

All dams survived to sacrifice. At the high tested dose level of 1000 mg/kg bw, signs of toxicity in form of piloerection was noted in 5 of 20 dams. One animal showed pale eyes and ears on gestation days 7 to 12. Additionally, a reduced body weight (up to 7 % below the control), reduced food consumption (up to 36 % below the control value), increased drinking water consumption, slightly increased absolute and relative liver weights and an increased activity of plasma aspartate aminotransferase were noted. A slight reduction by 9 % was noted for the gravid uterus weight caused by slightly lowered fetal weights. The carcass weight was 6 % below the control value. Necropsy revealed no test item-related pathological changes in any of the dams treated with either 100, 300 or 1000 mg/kg bw/day. The treatment of dams with 100 or 300 mg dibutyl ether/kg bw/day had no effects on behaviour, external appearance, nature of the faeces, body weight, body weight change and body weight gain, food and drinking water consumption, uterus weight and net body weight change. The marginal increase of absolute and relative weights of the liver at 100 and 300 mg/kg bw/day were regarded to be an inductive effect but not an adverse effect. 

 

No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg/kg bw/day, with respect to the number of corpora lutea, implantation sites, resorptions, placental weights, live fetuses, the values calculated for the pre- and post-implantation losses and the sex distribution of fetuses. Laparotomy revealed one dead fetus each (both of them were runts) at 100 and 300 mg/kg bw/day. In total, five runts were noted at laparotomy: two runts each at 100 and 1000 mg/kg bw/day and one runt at 300 mg/kg bw/day. One twin implant (one male fetus and one late resorption) was detected in one dam of the intermediate dose-group. All findings of dead fetuses, runts and twin implants are regarded to be spontaneous and are within the normal range of variation. At the high tested dose level of 1000 mg/kg bw, reduced fetal weights calculated for male and female fetuses and for all fetuses were slightly but statistically significantly below the control values. In addition, in this dose group skeletal examination according to Dawson revealed significantly increased fetal incidences for the following retardations: hyoid not ossified and absence of ossification in 5th metacarpalia.

 

Significantly increased litter incidences were noted for the caudal vertebral bodies, only one body ossified. These incidences were above the range of the test facility background data and are regarded to be test item-related. However, the incidence of total skeletal retardations in the high dose was not increased. The examination of the fetal organs (according to Wilson) revealed statistically significantly increased fetal and liter incidences for dilatation of the 4th cerebral ventricle. The mean fetal weights were not influenced by the administration of 100 or 300 mg/kg bw/day. Also no test item-related influence was noted for the incidence of skeletal retardations or soft tissue variations at the low and intermediate dose levels. At 100, 300 or 1000 mg/kg bw/day, no malformations were noted in the fetuses during external examination at laparotomy, skeletal examination (according to Dawson) or soft tissue examination (according to Wilson).

The no-observed-effect level (NOEL) for toxic effects on the dams was 300 mg/kg bw/day. The no-observed-effect-level (NOEL) for fetal skeletal retardations and soft tissue variations was 300 mg/kg bw/day. The no-observed-effect-level (NOEL) for teratogenic properties was 1000 mg/kg bw/day (highest dose tested).