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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:
May 2 - 31, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
January 22, 2001
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 3001545196
- Expiration date of the lot/batch: At least one year upon receipt (December 03, 2015) of the test item, that is Dezember 03, 2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At + 10°C to +25°C, in a tightly closed container.
Blanketing with nitrogen before recapping is recommended.
- Stability under test conditions: At least one year upon receipt of the test item
- Solubility and stability of the test substance in the solvent/vehicle: An analysis of the test item-formulation indicated correctly prepared and homogenised test item vehicle mixtures,
which were stable for at least 24 hours.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item formulations were freshly prepared every day.The test item was diluted in the vehicle to the appropriate concentrations using a magnetic stirrer and was administered orally at a constant volume once daily from the 6th to the 20th day of gestation. Stirring of the formulations was continued until the last animal of the dose group had been dosed.

Test animals

Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sulzfeld, Germany
- Age at study initiation:
- Weight at study initiation:
- Housing: The animals are kept singly in MAKROLON cages (type III plus).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature of 22°C ± 3 °C (maximum range)
- Humidity (%): relative humidity of 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle

IN-LIFE DATES: From: May 3, 2016 To:TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 59 days
- Weight at study initiation: 180.8 - 250.8 g
- Housing: Except during the mating period, the dams were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm.
- Diet (e.gad libitum): Commercial diet ssniff® R/Z V1324 ad libitum
- Water (e.g. ad libitum): Drinking water (in drinking bottles) was offered ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): The ventilation rate of the animal room was between fifteen to twenty air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle

IN-LIFE DATES: From:18 May 2016 To: 26 May 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency):Commercial diet was offered daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test laboratory has extensive experience with the vehicle corn oil and also in combination with the test item oxooil. Furthermore the test item mixture with the vehicle was verified regarding stability, homogenicity and concentration.
- Concentration in vehicle: The measured actual concentrations of the test item in the test item vehicle mixtures were between 99.8% and 109.1% of the nominal concentrations, indicating correctly prepared and homogenized formulations which were stable at room temperature for at least 24h.
- Amount of vehicle (if gavage): 2 mL/kg b.w.
- Lot/batch no. (if required): 15296403
-Source: supplier: Caesar & Loretz GmbH, 40721 Hilden, Germany
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: cohoused
- Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
- M/F ratio per cage: 1 M/1 F per cage
- Length of cohabitation: Cohabitation during the dark period; If findings were negative, mating was repeated with the same partner.
- Further matings after two unsuccessful attempts: no; Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Treatment period: Day 6 to 20 of gestation
Frequency of treatment:
Once daily
Duration of test:
Start of mating May 2, 2016
First mating results May 3, 2016
First administration May 9, 2016
Termination of the in-life part May 31, 2016
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day
Remarks:
low dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
intermediate dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
high dose
No. of animals per sex per dose:
100 females in order to have at least 20 pregnant females per group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels have been selected in agreement with the Sponsor based on the results of a preliminary dose-range-finding study in rats
- Rationale for animal assignment (if not random):

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: early in in the morning and again in the afternoon of each working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Each day the body weight will be registered always at the same time

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION : Yes
- Time schedule for examinations: Daily monitoring by visual appraisal of the drinking water bottles will be maintained throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21th
- Organs examined:
Examination of the dams:
Heart, kidney and liver were weighed
Examination of the fetuses:
Macroscopic inspection (gross evaluation) and weighting of the placentae.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Weight of placentae: Yes
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: No
Statistics:
Toxicology (and Pathology, if applicable) data will be captured, whenever possible,
using the departmental computerized systems (Provantis®8 Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems will be maintained on paper according to appropriate SOPs.
Historical control data:
The relationship between 'Variations and Retardations' and 'Malformations' should be seen under the aspect of cumulative historical data.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (1000 mg Oxooil LS9/kg b.w./day) several dams were noted with signs of clinical toxicity on several test days (e.g. a reduced motility (17 of 22), salivation (21 of 22), prone position (3 of 22), piloerection (3 of 22), pale faeces (9 of 22) and an increased (4 of 22) or decreased water consumption (2 of 22)).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (1000 mg Oxooil LS9)/kg b.w./day) a statistically significantly reduced body weight was noted from gestation day 10 until laparotomy on gestation day 21 (max.: 15.0% below the value of the control group at the day of laparotomy). Body weight gain for the whole study period was 49.7% for the dams of the high dose group in comparison to 78.4% for the dams of the control group (p ≤ 0.01).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant (p ≤ 0.01) reductions in food consumption were noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day) on several gestation days after the start of treatment (max. 36.4% below the value of the control group between gestation days 6 and 7).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (1000 mg Oxooil/kg b.w./day) 4 dams with an increased water consumption and 2 other dams with a decreased water consumption were noted by visual appraisal.
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:
effects observed, treatment-related
Description (incidence and severity):
Uterus and carcass weights: At the high dose level (1000 mg Oxooil LS9/kg b.w./day) statistically significant reductions were noted for the gravid uterus weight (22.4% below the value of the control group) and the carcass weight (12.5% below the value of the control group).

Further organ weights: The weighing of the heart, the kidneys and the liver revealed no test item-related differences between the dams of the control group and the treatment groups.
Gross pathological findings:
no effects observed
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
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
A total resorption of all implants (a postimplantation loss of 100%) was noted for 2 of 22 dams of the high dose group (1000 mg Oxooil LS9/kg b.w./day), leading to a statistically significantly (p ≤ 0.01) increased post-implantation loss (10.9% in comparison to 4.7% in the control group).
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
pre and post implantation loss

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly (p ≤ 0.01) decreased fetal weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day). The fetal weight 13.4% below the value of the control group (male and female fetuses together).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): A statistically significantly (p ≤ 0.01) decreased fetal weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day).
The fetal weight was 13.4% below the value of the control group (male and female fetuses together).
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No malformation was noted during the macroscopic examination at laparotomy (external inspection and inspection of the inner organs and tissues for gross lesions).
Skeletal malformations:
no effects observed
Description (incidence and severity):
No malformation was noted during the skeletal examination according to DAWSON.
Visceral malformations:
no effects observed
Description (incidence and severity):
No malformation was noted during the soft tissue examination according to WILSON.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly (p ≤ 0.01) decreased placental weight was noted at the high dose level (1000 mg Oxooil LS9/kg b.w./day). The placental weight at the high dose level was 9.9% below the value of the control group (male and female placentae combined).

Retardations: At the high dose level (1000 mg Oxooil LS9/kg b.w./day) the skeletal examination according to DAWSON revealed an increased incidence of delays in ossification for the metacarpalia and the sternebra(e) that are considered to be test item related.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: retardations: Increased incidence of delayed ossifications of the metacarpalia and the sternebra(e) at the high dose level

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg Oxooil LS9/kg b.w./day for the dams.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was 300 mg Oxooil LS9/kg b.w./day.