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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
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): Bis-(2-(2-butoxyethoxy)-ethoxy)-methane
- Physical state: liquid
- Analytical purity: see certificate of analysis: 12L00383
- Lot/batch No.: Ch. 48399416K0, v. 8.10.12
- Expiration date of the lot/batch: 08-Oct-2014
- Stability under test conditions: given
- Storage condition of test material: Ambient temperature; avoid temperatures > 70°C

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy
- Age at study initiation: 9 weeks old on day of receipt
- Weight at study initiation: 210 to 237 g on day of receipt
- Housing: During the pre-pairing period, the animals were housed no more than 5 of one sex to a cage in polysulfone cages measuring 59.5x38x20 cm (Code 1354 G, Techniplast Gazzada S.a.r.l., Buguggiate, Varese); nesting material was provided inside suitable bedding bags and changed at least 2 times a week.
During the mating period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
After the mating period, the rats were housed individually in clear polysulfone cages measuring 42.5x26.6x18.5 cm. Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approximately 18 days

The male rats were from the same supplier, and were at least 11 weeks old (at least 361 g).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of Bis-(2-(2-butoxyethoxy)-ethoxy)-methane was dissolved in the vehicle (an aqueous solution of 0.5 % carboxymethylcellulose) and brought to the final volume appropriate for each concentration (concentrations of 4, 12 and 40 mg/mL) and kept under magnetic stirrer at room temperature prior to use and until the time of dosing of the last animal.
The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): CMC was a suitable vehicle
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check and homogeneity). Results of the analyses were within the limits of acceptance.
The formulations were determined to be stable for 24 hours at room temperature in the concentration range of 4 to 40 mg/mL.
Samples of the formulations prepared on Week 1 and last Week of treatment were also analysed to check the concentration. Results of the analyses were within the limits of acceptance.

Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 94840; BASF Project No.: 10Y0863/08X047), in the range from 1 to 100 mg/mL. The software used for this activity was the Empower® Pro build No. 2154.
Details on mating procedure:
The females were paired with male rats. Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.
Duration of treatment / exposure:
All animals were dosed from Day 6 through Day 19 post coitum.
Frequency of treatment:
once a day
Duration of test:
until Day 20 post coitum
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 120, 400 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a non GLP compliant preliminary study (RTC Study no.: 94850EXT; BASF Project No: 10R0863/08X046). In this study, administration of Bis-(2-(2-butoxyethoxy)-ethoxy)-methane was administered orally to groups of 10 pregnant rats at the dose of 0, 250, or 750 mg/kg bw/day from Day 6 through Day 19 post coitum. Besides clinical signs the dose of 750 mg/kg bw/day led to decreased body weight gain from Day 6 to 20 post coitum of 13% and to a decreased corrected body weight gain from Day 6 to 20 post coitum of 44%. At 250 mg/kg bw/day, the respective decreases were 9% for body weight gain and 23% for corrected body weight gain.

- Rationale for animal assignment (if not random): On the day of allocation (Day 0 post coitum), all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified by group with an ear notch and housed individually.
The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in the cage racks was such that cages from each treatment group were evenly distributed across the cage racks to minimise possible environmental effects.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.
All clinical signs were recorded for individual animals. Each animal was observed at least once daily and any clinical signs recorded starting from allocation until sacrifice.

BODY WEIGHT: Yes
All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

FOOD CONSUMPTION: Yes
Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum, starting from Day 0 post coitum.

POST-MORTEM EXAMINATIONS: Yes
Euthanasia

The animals were killed on Day 20 post coitum and necropsied as detailed below.
Animals were euthanised with carbon dioxide.
All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.
All animals were subjected to necropsy, supervised by a pathologist.

Necropsy

The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Ovaries and uterine content:
The ovaries and uteri were examined to determine:

• Gravid uterine weight;
• number of corpora lutea;
• number of implantation sites;
• number, sex and weight of all live foetuses;
• number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing);
• number of intra-uterine deaths;
• gross evaluation of placentae.

Intra-uterine deaths were classified as:

Early resorptions: only placental remnants visible.
Late resorptions: placental and foetal remnants visible.

Uteri or individual uterine horns without visible implantations were immersed in a 10% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Fetal examinations:
Examination of foetuses

All live foetuses were examined externally. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter was preserved in Bouin's solution for subsequent fixed-visceral examination according to Wilson’s slicing technique The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal examination after bone staining with Alizarin Red S. Skeletal and fixed-visceral examinations were performed in all groups.
Structural deviations were classified as follows:

Malformations : major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies : minor abnormalities that are detected relatively frequently.
Variants : a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analyses of non-continuous variables were carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.
Indices:
Pre-implantation loss, Post-implantation loss and Total implantation loss were calculated. Sex ratios of the foetuses were calculated as the percentage of males per litter.

All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.
Historical control data:
Historical control data is available

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
Mortality and fate of females:
No mortality of females occurred during the study.
Two females proved not to be pregnant at necropsy: one in the control group (94860027) and one in Group 3 (94860129) receiving 120 mg/kg/day.
The number of females with live foetuses on Day 20 post coitum was 23 in the control group, 24 in the low dose group (40 mg/kg/day), 23 in the mid-dose group (120 mg/kg/day) and 24 in the high dose group (400 mg/kg/day).

Clinical signs:
The following clinical signs were recorded in females during the treatment period:
- at 400 mg/kg bw/day, most of the females showed ataxia. In addition, salivation and lethargic aspect were also noted. A few females showed tremors and two females showed bradypnoea and piloerection on one occasion, respectively. In general, these signs appeared within 15-30 minutes after dosing and disappeared 24 hours later and all treated females recovered.
- at 120 mg/kg bw/day, salivation and prone position was observed in one dam on Day 8 and 10 post coitum, respectively. Ocular discharge occurred in another animal form Day 11 to 19. Since the latter finding was not observed at 400 mg/kg bw/day, it was considered to be fortuitous and not related to treatment.
- at 40 mg/kg bw/day, no clinical signs were observed throughout the study.

Body weight and body weight gain:
A statistically significant decrease in body weight gain was observed on Day 9 post coitum in treated females receiving 400 mg/kg bw/day (-34%).
No relevant differences were recorded in body weight between control and the other groups.

Food consumption:
A slight decrease in food consumption (about -5%) was recorded in females receiving 400 mg/kg bw/day on Day 9 post coitum, without statistical significance.

Terminal body weight, uterus weight, corrected body weight and corrected body weight gain of pregnant females:
A decrease in corrected body weight gain (terminal body weight at necropsy minus gravid uterine weight, minus body weight at Day 6 post coitum) was noted in treated females receiving 400 mg/kg bw/day. This change was -18% without statistical significance.
Terminal body weight, uterus weight and corrected body weight were similar between control and treated groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Litter data and sex ratios:
Litter data, mean foetal weight and sex ratios were not affected by treatment.

Macroscopic examination of females:
No changes were observed at post mortem examination in treated animals when compared to the control group.

External examination of foetuses:
A total of 6 small foetuses (< 2.7 g) were detected: 1 out of 341 in the control group, 3 out of 357 in females receiving 40 mg/kg bw/day, 2 out of 345 in females receiving 120 mg/kg bw/day. One dead foetus was observed in a low dose litter (40 mg/kg bw/day) and a foetus showing cranioschisis was noted in another low dose litter. This malformation was considered incidental due to absence of dose-dependency.

Fixed visceral examination of foetuses:
No relevant changes that could be considered treatment-related were observed at visceral examination of foetuses between the control and treated groups. Blackish mass (named haemorrhage in the tables) in the thoracic cavity was observed in seven foetuses of seven litters of the high dose group compared to one foetus of the control group. This observation, which is also commonly observed in control rat foetuses is considered to represent an artificial finding due to procedures during C-section and/or euthanasia of the foetuses. Therefore, this finding is considered not to be of toxicological significance.

Skeletal examination of foetuses:
The following findings were observed both in control and treated groups with similar incidence: short 14th rib, incomplete ossification or no ossification of the sternal elements (particularly 5th and 6th); incomplete ossification of the skull bones (parietal and interparietal); no ossification of hyoid; no ossification of the 4th metacarpal; incomplete ossification of the sacral arch(es) and thoracic centra.
Wavy ribs occurred in the treated groups at a low incidence. Since wavy ribs are a common finding in rat foetuses, and there was no dose-dependency, these findings were considered as incidental. Rudimentary 14th ribs were noted at a higher incidence at 400 mg/kg bw/day. However, this is also a common finding in rat foetuses, which is known to be inducible by non-specific maternal stress. Furthermore, rudimentary 14th ribs generally become integrated in the lumbar process of the 1st lumbar vertebrae after birth. Therefore, this finding is considered to be of no toxicological relevance.
Several malformations were noted in the low dose foetus showing cranioschisis (animal no.: 94860055), weighing 2.06 g and therefore considered as small (foetal weight < 2.7 g). Major findings included: no ossification of the caudal vertebrae, fused cervical and thoracic vertebral arch(es), no ossification of the pubis, fused ribs, parietal fissure, general no ossification of the skull bones, no ossification of the sternebrae and no ossification of the thoracic arch(es). Another low dose foetus (animal no.: 94860063) showed no ossification of the pubis. Also this foetus was a small foetus with a foetal weight of 2.42 g. Considering that these findings were noted in small foetuses, without dose relation, they were considered incidental.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion