Registration Dossier

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:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name of test substance: Tetrahydrogeraniol
Chemical identity: 3,7-dimethyloctan-1-ol
CAS No.: 106-21-8
Purity: 98.8 area-%
Homogeneity: given
Storage stability: Expiry date: 25 Jan 2018
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Physical state/appearance: liquid/ colorless, clear
Storage conditions: Room temperature, protect against humidity

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: Crl:WI(Han)
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 147.5-189.8 g
- Housing: individually in Polycarbonate cages type III with wooden gnawing blocks and dust-free wooden bedding
- Diet: Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: tap water ad libitum
- Acclimation period: between the start of the study (beginning of the experimental phase) and the first administration (GD 6)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The stability of the test substance in corn oil at room temperature over a period of 7 days had been verified prior to the start of the study. The oily test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.

PREPARATION OF DOSING SOLUTIONS: The specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.
Duration of treatment / exposure:
GD 6-19
Frequency of treatment:
once daily
Duration of test:
on GD 20, all females were sacrificed
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The high dose was selected based on signs of toxicity noted at dose levels of 300 and 1000 mg/kg bw/d in a previously conducted maternal toxicity range-finding study (BASF project 10R0234/04R034) which preceded this definitive prenatal developmental toxicity study. In the maternal toxicity range-finding study, 7 pregnant Wistar rats per group were administered the test substance by oral gavage from gestational day (GD) 6 through GD 19. At 1000 mg/kg bw/d 4 dams died or were sacrificed moribund on GD 7 and the remaining 3 dams were sacrificed in serious condition two days later, showing signs of salivation, piloerection, head shaking, unsteady gait, labored respiration, abdominal position and hypothermia. Dams treated with 300 mg/kg bw/d showed salivation (all) and one case of piloerection and semiclosed eyelids. For these reasons, the dose level of 1000 mg/kg bw/d was considered to be potentially lethal to the dams in the OECD 414 study.
The selected high dose for the present study represented almost half of this lethal dose. This approved procedure of decreasing a lethal dose by a factor of two to become the high dose in a subsequent regulatory study meets the principles of guidelines OECD 414 (adopted 2001) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in in compliance with EU Directive 86/609/EEC on animal protection. The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day on working days or once a day on Saturday, Sunday or on public holidays (GD 0-20)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20
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
- Number of dead fetuses: Yes
Fetal examinations:
- Weight of each fetus: Yes
- Sex: Yes
- Weight of placentas: Yes
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
Statistics:
- DUNNETT's test: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass
weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions
of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
- FISHER's exact test: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
- WILCOXON test: Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
Indices:
The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals) / (number of fertilized animals) x 100

The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of corpora lutea – number of implantations) / (number of corpora lutea) x 100

The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice was calculated according to the following formula:
(number of implantations – number of live fetuses) / (number of implantations) x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All females (25 out of 25) of the high-dose group (450 mg/kg bw/d), 24 females of the middose group (150 mg/kg bw/d) and 4 females of the low-dose group (50 mg/kg bw/d) showed transient salivation during the treatment period. Salivation occurred in the respective animals only within the 2-hour examination interval immediately after treatment and was initially observed on GD 6. During the subsequent 5-hour examination interval (i.e. >2h<5h after treatment), no clinical signs or changes of general behavior were detected in any female of all test groups.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights (BW) and the average body weight gains (BWC) of the low-, mid and high-dose dams (50, 150, 450 mg/kg bw/d) were generally comparable to the controls throughout the entire study period.
Food efficiency:
no effects observed
Description (incidence and severity):
The mean food consumption of the dams in test groups 1, 2 and 3 (50, 150 and 450 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period. The statistically significantly decreased food consumption value in test group 3 during GD 10-13 is assessed as being incidental.
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:
no effects observed
Description (incidence and severity):
The mean gravid uterus weights of the animals of test groups 1-3 (50, 150 and 450 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the concurrent control groups revealed no dose-dependency and were assessed to be without biological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No necropsy findings which could be attributed to the test substance were seen in any dam.
There occurred one spontaneous finding in test group 1 (50 mg/kg bw/d), i.e. dilated renal pelvis. This finding was detected in one low-dose animal and was therefore assessed as incidental.
Neuropathological findings:
not examined
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): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate reached 96% in the low-dose group (50 mg/kg bw/d) and 100% in the control, mid- and high-dose groups (0, 150 and 450 mg/kg bw/d).
Other effects:
no effects observed
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between the test groups 0-3 (0, 50, 150 and 450 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no test substance-related adverse effects up to the high dose

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1-3 were not influenced by the test substance. This statement includes the statistically significantly lower mean fetal body weight in test group 1 (all viable fetuses) and test group 3 (female fetuses and all viable fetuses). There was no dose-response relationship and the values exactly match the mean of the historical control range (both sexes combined: mean 3.5 g [2.5 - 5.1 g]; female fetuses: mean 3.5 g [2.3 - 4.9 g]). Therefore, these findings were regarded as incidental and not treatmentrelated.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (50, 150 and 450 mg/kg bw/d) was comparable to the concurrent control fetuses.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were recorded.
One external variation was detected in one fetus each of test group 1 and 2 (50 and 150 mg/kg bw/d), i.e. limb hyperextension. This variation was neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant.
One unclassified external observation, i.e. placentae fused, was recorded in one fetus of the high-dose group (450 mg/kg bw/d). This finding was not considered biologically relevant.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal malformations were recorded for one fetus each of test group 0, 1, 2 and 3 (0, 50, 150 and 450 mg/kg bw/d). The respective fetuses had more than one malformation affecting the fetal skeleton. The total incidence of skeletal malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeleton and appeared in most cases without a relation to dosing. The overall incidences of skeletal variations were covered by the historical control data. Two variations - supernumerary thoracic vertebra and ribs (14th) - were, at the top dose (450 mg/kg bw/d), present at statistically significant incidences slightly above the historical control range. Other findings were either not related to the dose or covered by the historical control range. They are in any case not considered as adverse events.
Additionally, some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the sternum and ribs and did not show any relation to dosing.
Visceral malformations:
no effects observed
Description (incidence and severity):
Soft tissue malformations occurred in two fetuses of the control and one fetus of the middose group (0 and 150 mg/kg bw/d). The total incidence of soft tissue malformations did not differ significantly from the concurrent control group and was covered by the historical control data.
Two soft tissue variations were detected, i.e. dilated renal pelvis and dilated ureter. These variations were neither significantly different from the concurrent control nor dosedependently altered. Therefore, they were not considered biologically relevant.
No unclassified soft tissue observations were recorded.
Details on embryotoxic / teratogenic effects:
The mean placental weights of the low-, mid- and high-dose groups were comparable to the concurrent control group.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test substance-related adverse effects up to the high dose

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion