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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
Study period:
2012-06-11 to 2012-09-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(3E)-dec-3-en-2-one
Cas Number:
18402-84-1
Molecular formula:
C10H18O
IUPAC Name:
(3E)-dec-3-en-2-one
Test material form:
liquid
Specific details on test material used for the study:
- Name: AMV-1018 ((3E)-dec-3-en-2-one Technical)
- CAS: 10519-33-2
- Purity: 99.81% w/w
- Batch No: HA-2011/08
- Physical state: liquid
- Colour: clear, colorless to pale yellow
- Storage conditions: under nitrogen, room temperature

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: At the start of the study (Day 0 of gestation) selected animals were approximately 10 weeks old
- Weight at study initiation: bodyweights were in the range of 232 to 285 g
- Housing: The animals were checked twice daily for health and general condition and were allowed to acclimatise to the conditions described below for at least five days before in cages up to 4 animals. Afterwards, they were paired on a 1:1 basis with stock males from the same source. During gestation the animals were held individually in polycarbonate cages of the type 2154.
- Diet (e.g. ad libitum): ad libitum, the animals were allowed free access to a standard rodent diet (SDS VRF1 Certified diet). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum): ad libitum, potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 ± 23
- Humidity (%): 40 to 70

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance, AMV-1018, was prepared for administration as a series of graded concentrations in the vehicle. Starting with the highest concentration (200 mg/mL), the required amount of test material was weighed and sufficient vehicle added and stirred, using a magnetic stirrer, to obtain a solution. The solution was made up to the required volume by the addition of more vehicle, then transferred to a suitable container and stirred again for five minutes. The formulation was then subdivided in a series of containers suitable for each day of use. The procedure was repeated for the intermediate and low dose concentrations (60 and 20 mg/mL) in descending order of concentration. The test substance was used as supplied. All formulations were prepared freshly each week, up to seven days in advance of the first day of dosing, and stored at 2-8 °C for up to 15 days before administration.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At specified intervals during treatment, the test formulations were analysed for achieved concentration of the test substance.The homogeneity and stability of a solution of the test substance in the vehicle has been demonstrated over a period of up to 15 days refrigerated at 4 °C and at ambient temperature 10-25 °C for 24 hours performed as part of Huntingdon Life Sciences Study Number BDG0145. Stored samples were retained as contingency for analysis at the temperature determined by
the stability information if any results required confirmation. Samples (contingency/residual) were disposed of once satisfactory results were obtained.
Details on mating procedure:
The animals were checked twice daily for health and general condition and were allowed to acclimatise to the conditions described below for at least five days before they were paired on a 1:1 basis with stock males from the same source. Daily checks were made after pairing for evidence of mating, including ejected copulation plugs in cage trays and the presence of sperm in a vaginal smear. Animals were allocated to study on Day 0 of gestation, when positive evidence of mating was detected. Only females that showed at least two copulation plugs were selected. Females were allocated to group and cage position in sequence of mating, thus ensuring that animals mated on any one day were evenly distributed amongst the groups. The sequence of allocation was controlled to prevent stock males from providing more than one mated female in each treatment group.
Duration of treatment / exposure:
between gestation day 6 and gestation day 19 (inclusive)
Frequency of treatment:
daily between gestation day 6 and 19
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The 3 dose groups were selected based on the results of a previous dose-range finding study (see IUCLID chapter 7.8.2 in endpoint summary “Leggett 2012_sup_DRF”)
- Rationale for animal assignment (if not random): Females were allocated to group and cage position in sequence of mating, thus ensuring that animals mated on any one day were evenly distributed amongst the groups. The sequence of allocation was controlled to prevent stock males from providing more than one mated female in each treatment group.
- Administration: Females were treated from Day 6 to Day 19 (inclusive) after mating. Animals received the test material or vehicle control formulations orally at a volume-dose of 5 mL/kg/day, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure. All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time each day, seven days per week. The volume administered to each animal was calculated from the most recently recorded scheduled bodyweight. A daily record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly. On some occasions the daily amount of dose used for administration exceeded the stated limit, as specified in the Huntingdon Life Sciences Standard Operating Procedure, in the Control, low and intermediate dose group; however it was considered that these variances had no impact on the integrity of the study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment and the cage was inspected once each day for evidence of ill-health of the occupant(s). Throughout the treatment period, detailed observations were recorded daily at the following times in relation to dose administration:
- On completion of dosing of each group
- Between one and two hours after completion of dosing of all groups
- As late as possible in the working day


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: In addition, a more detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- The weight of each adult rat was recorded on Days 0, 3, and 6-20 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- The weight of food supplied to each adult rat, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues
were examined as appropriate. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.
Ovaries and uterine content:
The following reproductive assessment was made:
The gravid uterus was weighed prior to dissection; this weight included the weight of the cervix and ovaries. For each animal, the number of corpora lutea in each ovary and the number of implantation sites, the number and distribution of resorption sites (classified as early or late) and live and dead fetuses were recorded for each uterine horn. The retained tissues were checked before disposal of the carcass.
Fetal examinations:
All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each fetus and placenta were externally examined and any abnormalities were recorded. The sex of each fetus was recorded. Approximately half of the fetuses in each litter had their sex confirmed internally, were eviscerated and their skeletons were fixed in Industrial Methylated Spirit, prior to processing
and staining with Alizarin Red. The remaining fetuses were fixed whole in Bouin’s fluid.
Free-hand serial sections were prepared from the Bouin’s fixed fetuses and were examined under the microscope for visceral abnormalities. Fetuses stained with Alizarin Red were assessed for skeletal development and abnormalities.
Statistics:
The following data types were analysed at each timepoint separately:
- Bodyweight, using absolute weights and gains over appropriate study periods
- Gravid uterine weight and adjusted bodyweight
- Food consumption, using over appropriate study periods
- Litter data and survival indices
- Fetal, placental and litter weight
The following sequence of statistical tests was used for bodyweight, gravid uterine weight, food consumption, litter data and placental, litter and fetal weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p <0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other comparisons the F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F-distribution with 1 and EMS degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose-response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
Indices:
mean corpora lutea, implantations, early, late and total resorption counts, live young, sex ratio, and pre- and post-implantation loss
Historical control data:
yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was evident in all animals treated at 1000 mg/kg/day during the treatment period and in all animals receiving 300 mg/kg/day. Chin rubbing (associated with salivation) was seen in some animals at 1000 mg/kg/day following the second treatment and in two animals at 100 or 300 mg/kg/day on one occasion. These signs were attributed to the palatability of AMV-1018 and considered not to be toxicologically significant. One animal (No. 56) receiving 300 mg/kg/day showed salivation, post dose salivation and was underactive following its first dose which, as the animal received the intermediate dose level, was considered attributable to an individual animal reaction to the dosing procedure.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain in animals treated at 100 or 300 mg/kg/day was not affected by treatment with AMV-1018. When compared with the control, overall mean body weight gain was marginally low in animals receiving 1000 mg/kg/day during Days 6 to 20 of gestation and mean body weight gain during Days 18 to 20 was low.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment with AMV-1018 at 100 or 300 mg/kg/day. Overall food consumption of females receiving 1000 mg/kg/day was slightly low, when compared with Control. Food consumption in animals treated at 300 mg/kg/day was slightly low during Days 6-9 of gestation; however since the magnitude of the difference was the same as that apparent before the start of dosing, no effect of treatment was inferred.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination did not reveal any findings considered to be related to treatment. One female receiving 1000 mg/kg/day had pale areas on the liver.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for number of implantation sites and percent pre- and post-implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for number of total resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for number of early and late resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead foetuses were observed in any of the groups.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females were pregnant.
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for number of live foetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for sex ratio.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter weight, placental, male and female fetal weight and overall fetal weight were considered unaffected by treatment.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Formulation analysis:

The mean concentrations of AMV-1018 in samples of test formulations analysed during the study were within 3% of nominal concentrations, demonstrating the accuracy of formulation preparation.

Incidental findings:

At all doses, there was a slightly higher percentage incidence of fetuses with 13/14 and 14/14 ribs compared with the concurrent control, but these incidences were within historical control data and, in the absence of any other related finding, is considered not to be related to treatment.

Applicant's summary and conclusion

Conclusions:
In a prenatal developmental toxicity study (performed in accordance with OECD 414) the test item was prepared using corn oil as vehicle and orally administered to 24 female Wistar rats/dose group at dose levels of 0, 100, 300 and 1000 mg/kg bw/day from days 6 through 19 of gestation. On gestation day 20 the animals were sacrificed and examined. No test item-specific adverse effects were found in pregnant females and foetuses up to 1000 mg/kg body weight/day. Based on the results, the NOAEL for both maternal toxicity and foetal toxicity of the test item in this study is considered to be 1000 mg/kg body weight/day.
Executive summary:

In a prenatal developmental toxicity study (performed in accordance with OECD 414) the test item was prepared using corn oil as vehicle and orally administered to 24 female Wistar rats/dose group at dose levels of 0, 100, 300 and 1000 mg/kg bw/day from days 6 through 19 of gestation. On gestation day 20 the animals were sacrificed and examined. The treatment was well tolerated with no deaths and no toxicologically significant signs observed after dose administration. Salivation and/or chin-rubbing evident in two animals receiving 100 mg/kg/day and all animals at 300 or 1000 mg/kg/day were attributed to the palatability of AMV-1018 and considered not to be toxicologically significant. At 1000 mg/kg/day, overall food consumption was slightly low and resulted in marginally low overall body weight gain, especially during Days 18 to 20. There were no maternal macroscopic findings related to treatment. All treated animals were pregnant and the number of corpora lutea, implantations, early, late and total resorptions, live young, sex ratio, and pre- and post-implantation loss, litter weight, placental, male and female fetal weight and overall fetal weight were unaffected by treatment. Major and minor fetal abnormalities and skeletal variants showed no relationship to treatment. Based on the results, the NOAEL for both maternal toxicity and foetal toxicity of the test item in this study is considered to be 1000 mg/kg body weight/day.