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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sept - Oct 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Hostavin N 20
IUPAC Name:
Hostavin N 20
Specific details on test material used for the study:
Batch No.: DEFD097971
Puritiy: > 99.9%
Expiry date: 26.07.2012

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone, Italy
- Age at study initiation: 9 weeks
- Weight at study initiation: 200-225 g
- Housing: no more than 5 per cage (clear polycarbonte with stainless steel mesh and floor)
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Settimo Milanese, Italy), ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2°C
- Humidity (%): 55+/-15%
- Air changes (per hr): 15 - 25
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: 25.09. To:23.10.2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Solubility
- Concentration in vehicle: 1.00, 2.50 and 6.25 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
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. Stability data at room temperature up to 24 hours were available from RTC Studies.
Samples of the formulations prepared during the first and the last week of treatment were analysed to check the homogeneity and concentration of the test item.
The results were within the limits of acceptance.
Details on mating procedure:
Females were paired one to one in the home cage of the male and left overnight. The day of mating, as judged by the presence of sperm on the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum).
Duration of treatment / exposure:
gestation days 6 to 19
Frequency of treatment:
once daily
Duration of test:
21 days
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 mated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on previously performed dose range finding study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice dauly

BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 6, 9, 12, 15 and 20 post partum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, on days 6, 9, 12, 15, and 20 post partum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus, ovaries
The clinical history of the animal was studied and a detailed post motem examination was conducted including examination of the external surface and orifices

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: gross evaluation of placentae
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
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 analysis of non-continuous variables was 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 was calculated as a percentage from the formula:
(no. of corpora lutea-no. of implantations)/no. of corpora lutea x 100

Post-implantation loss was calculated as a percentage from the formula:
(no. of implantations-no. of live young)/no. of implantations x 100

Total implantation loss was calculated as a percentage from the formula:
(no. of corpora lutea-no. of live young)/no. of corpora lutea x 100

Sex ratios of the foetuses were calculated as the percentage of males per litter.
All derived values (e.g. means, percentages, ratios) first were 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

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs of toxicity such as cold to touch, hunched posture, emaciation and piloerection were noted in the high dose females receiving 62.5 mg/kg/day towards the end of the treatment period.
Hairloss was also reported in the other treated groups, but due to the low incidence of this sign, it was considered to be incidental.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in body weight was noted in the high dose females from gestation Day 9 to gestation Day 20 and in the mid- dose females on gestation Day 20, when compared to controls.
A lower body weight gain was observed in mid- and high dose females compared to controls starting from gestation Day 9. The differences were statistically significant from gestation Day 12 to gestation Day 20 for mid-dose females and from gestation Day 9 to gestation Day 20 for high dose females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in food consumption was noted in all treated females compared to controls from gestation Day 9.
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:
effects observed, treatment-related
Description (incidence and severity):
Gravid uterus weight of the high dose females was statistically significantly lower than controls by approximately 20%.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Small size of the thymus was reported in 5 females allocated to the high dose group. This change could be considered related to the general conditions of the high dose group animals. Also hairloss, which was detected on several regions of the body in 9/24 high dose animals and rated from mild to marked, was considered to be treatment-related.
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:
no effects observed
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:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Foetal weight and consequently litter weight of the high dose females were statistically significantly lower than controls by ca. 20%.
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): Foetal weight and consequently litter weight of the high dose females were statistically significantly lower than controls by ca. 20%.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Foetal weight and consequently litter weight of the high dose females were statistically significantly lower than controls by ca. 20%.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One low dose foetus and 3 mid-dose foetuses from three different litters showed lower foetal weight (less than 2.7g) and were classified as small. Due to the low incidence of this finding, it was considered to be incidental. A total of 82 small foetuses out of 364 foetuses from 14 different litters were noted in the high dose group. The lower foetal weight of some litters was highly pronounced (i.e. the weight of foetus no. 5 from litter 74240163 was 1.18g, which was 69% less than the mean foetal weight of the control group foetuses).
One control foetus (exencephalia (protrusion of the brain from the skull)), and two high dose foetuses, , showed external malformations (acaudia/anus imperforate and anus imperforate/tail rudimentary) . Both high dose foetuses showed lower foetal weight (2.35g and 1.75g) .
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal examination was comparable between the control and the low and mid-dose group. Skeletal malformations were described in one high dose foetus the same foetus showed malformations at the external examination. The foetus showed all sacral arches absent, thoracic centra 8th to 13th absent, all lumbar arches absent, ribs (left and right) 8th to 13th absent.
An increased incidence in incomplete or no ossification of the skeleton was noted in high dose foetuses, compared to control foetuses. The delay in the ossification was present in most parts of the skeleton such as skull, sternebrae, thoracic, cervical and sacral vertebrae, hind and forepaws and pelvic girdle.
No ossification of the pubis, bilateral, classified as a major anomaly (malformation), was described in 10 foetuses, 9 from one litter. All foetuses showed marked lower foetal weight.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Exencephalia observed at external examination in control foetus was confirmed during visceral examination. In addition, the same foetus showed malformations such as cleft palate and macroglossia (enlarged tongue). Anomalies such as testis and displaced lens were also observed.
Acaudia and anus imperforate observed at external examination in high dose foetus were confirmed during visceral examination. During the examination this foetus also showed unilateral kidney, ureter aplasia and persistent truncus arteriosus of the great vessels. In addition, anomalies such as heart septal defect and presence of an abnormal mass in the abdominal cavity were also described.
Unilateral testis, not descended, was observed in one foetus of the low dose group and in two mid-dose foetuses. These findings were considered to be incidental and not dose-related.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
60 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes

Any other information on results incl. tables

For clarification on the substance`s mode of action please refer to attached document "Assessment of toxicological mode of action_Hostavin N 20"

Applicant's summary and conclusion

Conclusions:
On the basis of the results of this prenatal developmental study the dosage of 25 mg/kg/day could be considered the NOAEL (No Observed Adverse Effect Level) for dams and offsprings.
Executive summary:

The effects of the test item during pregnancy and embryo-foetal development were investigated in the rat after oral administration from Day 6 to Day 19 of gestation. Three groups of females with positive identification of mating received the test item at dosages of 10, 25 and 62.5 mg/kg/day. One group received the control item (sesame oil) during the same treatment period and acted as a control.

Cold to touch, hunched posture, emaciation and an increase in piloerection and hairloss were noted in the high dose females receiving 62.5 mg/kg/day. In addition, in this group marked maternal toxicity was present as demonstrated by the moderate reduction in food consumption, body weight, body weight gain, uterus weight and absolute weight gain. As a consequence of the marked maternal toxicity, foetal toxicity was present as lower foetal weight (82 out of 364 foetuses were small) and delay in the ossification. The delay in the ossification was observed in most parts of the skeleton, such as skull, sternebrae, thoracic, cervical and sacral vertebrae, hind and forepaws and pelvic girdle. Malformations were observed only in the high dose group, which showed marked maternal toxicity. These malformations could be an expression of the lower foetal weight observed rather than a direct effect of the test item on foetuses.

Moderate maternal toxicity was noted in mid-dose females receiving 25 mg/kg/day as demonstrated by reduction on body weight and body weight gain. In addition a reduction in food consumption was observed. Terminal body weight, uterus weight and absolute weight gain were statistically significantly reduced compared to controls.

Low dose females receiving the dosage of 10mg/kg/day showed a slight reduction in food consumption.