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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21-Sept-2015 to 20-Oct-2015
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Octa-1,7-diene
EC Number:
223-054-9
EC Name:
Octa-1,7-diene
Cas Number:
3710-30-3
Molecular formula:
C8H14
IUPAC Name:
octa-1,7-diene
Test material form:
liquid

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Strain: Rat / CD® / Crl:CD(SD)
- Number and sex: 100 females
- Breeder: Charles River Laboratories
- Body weigth: 179 - 252.4 g
- Age: 59 days on day 0 of pregnancy
- Adaptation period: 4 days, followed by 1 mating day and 5 gestation days

ENVIRONMENTAL CONDITIONS:
- Diet: a certified commercial diet (ssniff® R/Z- V1324) ad libitum
- Water: tab water ad libitum
- Housing singly in MAKROLON cages (type III plus)
- Temperature: 22°C ± 3°C
- Rel. Humidity: 55% ± 15%
- Light/dark period: 12/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration (immediately after preparation and at study termination): 93.2% - 97.5%
Stability at room temperature for 8 or 24h: 94.4% - 96.3%
Homogeneity (at start of administration, during administration and before administration to the last animal): 94.7% - 99.7%

These results indicated correctly prepared and homogenised test item vehicle mixtures, which were stable for at least 24 hours.
Details on mating procedure:
Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy).
Duration of treatment / exposure:
Day 6 to 20 of gestation
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females per dose
20 evaluated litters per dose
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Individual animals were observed daily for behavioural changes, reaction to treatment, or illness. Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the day. Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. Animals showing signs of abortion or premature delivery would have been sacrificed on the same day.
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.
The quantity of food consumed by each rat was recorded daily. Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.

On gestation day 21, the rats were laparotomised under ether narcosis. The ovaries and the uteri of the dams were removed; the gravid uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations.

EVALUATION PARAMETERS:
Corpora lutea:
- number per dam
- absolute number per group
- mean per group

Implantations:
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group

Resorptions:
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
- early resorptions < 2 mm
- late resorptions > 2 mm

Weight of placentae
- individual data per fetus
- mean per litter
- mean per group
- mean per sex and group
Ovaries and uterine content:
On gestation day 21, the rats were laparotomised under ether narcosis. The ovaries and the uteri of the dams were removed; the gravid uteri (in toto) were weighed.
Fetal examinations:
The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(g) All fetuses (dead and alive) were inspected externally for damages, especially for malformations.
(h) The fetuses were sacrificed by an ether atmosphere.
(i) Examination of fetuses and determination of number and kind of retardations, variations or malformations:
1) 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
2) The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON. The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.

EVALUATION PARAMETERS:
Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per group
- mean per sex and group

Fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive + dead) per sex and dam
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- male/female ratio (alive + dead)

Runts
- number per dam
- number per group

Malformed fetuses, fetuses with variations and with retardations
- type of malformation
- individual data per fetus
- number and incidence (%) per group and litter


Statistics:
Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings: Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test: FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01) or Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01). The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group was done using StatXact 4.0.1 software.
Indices:
Calculation of group indices:
Pre implantation loss [%] = (Corpora lutea (per group) - Implantations (per group) / Corpora lutea (per group)) x 100

Post implantation loss [%] = (Implantations (per group) - living fetuses (per group) / Implantations (per group)) x100

Calculation of mean indices per litter:
Pre implantation loss [%] = sum of pre-implantation losses per litter in a group [%] / number of litters in a group

Post implantation loss [%] = sum of post-implantation losses per litter in a group [%] / number of litters in a group

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At the high-dose level slight to moderate salivation was noted for 8 of 20 dams, which might be a secondary reaction on the physico-chemical characteristics of the test item. One of 20 dams showed piloerection, and a possible increased drinking water consumption was noted for 4 of 20 dams of the high dose group observed by visual appraisal only between gestation days 13 and 21.
No test item-related changes in behaviour, the external appearance or the faeces were noted for the dams of the control group and the dams of the low- and the mid-dose dose group.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related differences in body weight were noted.

Body weight gain for the whole study period was 10.2% (p ≤ 0.05) below the value of the control group for the dams of the high-dose group. The 3-day interval of body weight gain revealed statistically significant (p ≤ 0.05) reductions in body weight gain after the start of treatment between gestation days 6 and 9 and gestation days15 and 18 at the high dose level corresponding to the observed reductions in food consumption.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the high-dose group statistically significant (p ≤ 0.01) reductions in food consumption were noted after the start of treatment between gestation days 6 and 9 (between 23.3% and 26.4% below the values of the control group) and again between gestation days 15 and 17 (between 10.1% and 11.8% below the values of the control group) corresponding to the decreased body weigth gain.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
A possible increased drinking water consumption was noted for 4 of 20 dams of the high-dose group observed by visual appraisal only between gestation days 13 and 21.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A slight and statistically not significant reduction in the carcass weight (4.0% below the value of the control group) was noted at the high-dose level corresponding to the decrease in body weigth gain. No reduction was noted for the gravid uterus weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No changes were noted during the macroscopic inspection of the dams at necropsy.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
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): not examined
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
In the high-dose group, food consumption and body weigth gain were reduced.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: decreased food consumption and body weigth gain
Description (incidence and severity):
dams of the high-dose group: food consumption 23.3% and 26.4% and body weigth gain 10.2% below below the values of the control group

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the high-dose group slight but statistically significant reductions (p ≤0.01) were noted for the placental weight (10.2% below the value of the control group) and the fetal body weight (8.9% below the value of the control group).
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): In the high-dose group slight but statistically significant reductions (p ≤0.01) were noted for the placental weight (10.2% below the value of the control group) and the fetal body weight (8.9% below the value of the control group).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No dead fetuses were noted in any of the test groups.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
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 organs and tissues for gross lesions).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The skeletal examination according to DAWSON revealed no test item-related malformations, variations and retardations.
Non-treatment related, spontaneous skeletal retardations were observed in similar incidences in treatment and control groups.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
A test item-related increase in the incidence of variations was noted at the high-dose level during the soft tissue examination according to WILSON in the form of an increased incidence of fetuses with one or more dilated cerebral ventricle (12.0% fetuses with one or more dilated cerebral ventricle in the high dose group in comparison to 1.7% in the control group).
The incidence of fetuses with a dilated cerebral ventricle in the high-dose group is still in the upper range of background data of the contract research institute, however, in this study the incidence of the finding in the high-dose group is distinctly increased in comparison to the concurrent control group.
Details on embryotoxic / teratogenic effects:
The test item did not show any teratogenic potential up to a dose of 1000 mg/kg b.w./day; test-item related malformations and retardations were not obeserved in all dose groups. However, in the high-dose group an increased incidence of fetuses with one or more dilated cerebral ventricles was observed which was classified as variation. The incidence of those fetuses was still in the upper range of background data of the contract research institute, however, in this study the incidence of the finding in the high-dose group is distinctly increased in comparison to the concurrent control group.
Dams in the high-dose group also showed sign of maternal toxicity (reduced food consumption and body weigth gain).
As a conclusion, the test item did not show teratogenic potential.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
visceral/soft tissue: central nervous system
Description (incidence and severity):
Increased incidence of fetuses with one or more dilated cerebral ventricle (12.0% fetuses with one or more dilated cerebral ventricle in the high dose group in comparison to 1.7% in the control group).

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 occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The test item did not show any teratogenic potential under the conditions of the study up to a dose of 1000 mg/kg b.w./day; test-item related malformations and retardations were not obeserved in all dose groups. However, in the high-dose group a developmental variation (increased incidence of fetuses with one or more dilated cerebral ventricles) was observed. The incidence of those fetuses was still in the upper range of background data of the contract research institute, however, in this study the incidence of the finding in the high-dose group is distinctly increased in comparison to the concurrent control group. Furthermore, dams in the high-dose group also showed sign of maternal toxicity (reduced food consumption and body weigth gain).