Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

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:
2012

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): Hydroxypivalic acid neopentylglycolester
- Analytical purity: see certificate of analysis: 12L00328
- Lot/batch No.: 95078568E0
- Expiration date of the lot/batch: July-2014
- Storage condition of test material: Ambient temperature; avoid temperatures > 40°C, protext against moisture

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy
- Age at study initiation: female rats: 10 weeks old on day of receipt; male rats: 11 weeks old
- Weight at study initiation: 177 to 190 g on day of receipt; male rats at least 291g
- 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 24 days


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:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of Hydroxypivalic acid neopentylglycolester was dissolved in the vehicle (deionised water).
The formulations were prepared daily (concentrations of 10, 30 and 100 mg/mL) and the concentrations were calculated and expressed in terms of test item as supplied

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 checkand stability). Results of the analyses were within the limits of acceptance stated in RTC SOPs and the formulations were determined to be stable for 24 hours at room temperature in the concentration range of 10 to 100 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 stated in RTC SOPs.
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 94870; BASF Project No: 10Y0084/03X024) in the range from 1 to 120 mg/mL.
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, 100, 300, 1000 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.: 95250EXT; BASF Project No: 10R00084/03X027).In this study the test item was administered orally, by gavage. The oral route was selected as it is apossible route of exposure of the test item in man. One group of 10 mated female rats was used in the study and received the test item at the dose of 1000 mg/kg/day. A similarly constituted group received the vehicle alone (purified water).
- 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 starting from Day o 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 20% 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.

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 animals died during the study. One low dose female and one high dose female were found not pregnant at necropsy.
The number of females with live foetuses on gestation Day 20 was 24 in each of the control and mid-dose groups and 23 in each of the low and high dose groups.
Clinical signs
Hairloss in different regions of the body surface was recorded in few control and treated females.
Rales were detected in one high dose female (animal no. 94880155) starting from Day 14 post coitum.
These signs were not considered of toxicological significance.

Body weight and body weight gain
No differences in body weight and body weight gain were noted between control and treated groups.

Food consumption
No differences in food consumption were detected between control and treated groups.

Terminal body weight, uterus weight, corrected body weight and corrected body weight gain of pregnant females
Gravid uterus weight was statistically higher in the high dose group. Consequently, corrected maternal body weight gain was statistically significantlylower in this group. Since gravid uterus weight was higher compared to controls and not decreased, a compound-related effect is highly unlikely. The statistically significant differences observed in the gravid uterus weight and corrected weight gain in the high dose group with respect to the
controls were therefore considered to be incidental and not of toxicological relevance.
No other differences were detected between control and treated groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 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
The statistically significant differences observed in the corpora lutea of the mid-dose group and in the number of males of the high dose group with respect to the control were not considered of toxicological relevance, due to the absence of a dose-relationship. The same applies to the statistically significantly higher number of male foetuses in the high dose group, since the sex ratio was well within the expected range of the rat strain used.
No other differences in litter data, mean foetal weight and sex ratios were detected between control and treated groups.

Macroscopic observations of females
Detailed macroscopic observations were reported for individual animals in all groups. No relevant changes were observed at post mortem examination in treated female animals when compared with the controls.

External examination of foetuses
Multiple malformations such as oedema, exencephalia and malrotated hindlimbs with polysyndactylia of the hindlimb were detected in one foetus of a control female
A total of 6 small foetuses (<2.7 g) were detected: 2 foetuses in each of the control and low dose groups and 1 foetus in each of the mid- and high dose groups.
Skeletal examination of foetuses
One foetus in the low dose group showed additional vertebra as malformation at skeletal examination. This malformation was considered incidental due to the absence of dosedependency.
No treatment-related changes were seen at skeletal examination of foetuses performed in control and treated groups, since the findings and their incidences were comparable. In addition, in our experience they can be considered as a common spontaneous alteration in
animals of this strain.

Fixed visceral examination of foetuses
Malformations detected at the external examination of the foetuses of the control female were confirmed and others recorded such as: malformed brain, anotia,anophtalmia and syndactylia of the forelimb.
No findings that could be considered treatment-related were noted at visceral examination of foetuses of the treated groups.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion