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:
other: study according to guideline and GLP

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
Details on test material:
- Name of test material (as cited in study report): Citronellol
- IUPAC name: 3,7-Dimethyl-octen-6-ol-1
- Physical state: Liquid / colorless, clear
- Analytical purity: GC: 96.8 corr. area-%
- Homogeneity: given
- Lot/batch No.: 45640336W0
- Expiration date of the lot/batch: 25 Oct 2015
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: time-mated Wistar rats (Crl:WI[Han]), supplied by Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10 - 12 weeks
- Weight at study initiation: 151.1 – 195.6 g
- Housing: individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²)
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, tap water
- Acclimation period:

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:
PREPARATION OF DOSING SOLUTIONS:
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.
For the test substance preparations, 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.
During administration, the preparations were kept homogeneous with a magnetic stirrer.

The volume administered each day was 4 ml/kg bw.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance was completely miscible with corn oil.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verifications of 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 in a similar batch.
Analytical investigations of the test substance preparations were performed.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
GD 6 to GD 19
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
75, 250, 750 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for mortality was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily for any signs of morbidity, pertinent behavioral changes and 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: on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION: Yes
- Time schedule: GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20

WATER CONSUMPTION: Yes
Drinking water consumption was monitored by visual inspection of the water bottles for any changes in volume.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Gross pathology, special attention being given to the reproductive organs

CLINICAL PATHOLOGY AND HEMATOLOGY: Yes

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:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
Statistics:
- DUNNETT-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, pro-portions 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 mal-formations, variations and/or un-classified observations in each litter
- KRUSKAL-WALLIS test and WILCOXON-test: Blood parameters
- KRUSKAL-WALLIS test and WILCOXON-test: Weight parameters
Indices:
conception rate (in %) = (number of pregnant animals) / (number of fertilized animals) * 100
preimplantation loss (in %) = (number of corpora lutea – number of implantations) / (number of corpora lutea) * 100
postimplantation loss (in %) = (number of implantations – number of live fetuses) / (number of implantations) * 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- The following females were excluded from the calculation of mean gravid uterine weights, corrected (net) body weight gain and summary of reproduction data:
Test group 1 (75 mg/kg bw/d): female No. 27 – not pregnant
Test group 3 (750 mg/kg bw/d): female No. 94 – not pregnant

- Mortality: no test substance-related or spontaneous mortalities in any females of all test groups

- Clinical symptoms: All females of the high-dose group (750 mg/kg bw/d), 24 out of 25 females of the mid-dose group (250 mg/kg bw/d) and two females of the low-dose group (75 mg/kg bw/d) showed transient salivation (i.e. up to 15 minutes and initially observed on GD 6) during the treatment period. One animal of the high-dose group (750 mg/kg bw/d) showed piloerection on GD 16-19. No further clinical signs or changes of general behavior were detected in any dam at dose levels.

- Water consumption: no test substance-related, adverse changes

- Food consumption: The mean food consumption of the high-dose dams (750 mg/kg bw/d) was statistically significantly decreased on GD 6-10 (up to -16%). Afterwards it recovered and was comparable to the concurrent control throughout the remaining study period. If calculated for the entire treatment period or study period, the high-dose dams consumed a comparable amount of food than the concurrent control group.

- Body weight: The mean body weights and the average body weight gain of the high-, mid- and low-dose dams (750, 250 or 75 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. This includes the statistically significantly decreased body weight change value in test group 1 during GD 10-13. For details please refer to "Table 1: Mean maternal body weights during gastation [g]" and "Table 2: Mean maternal body weight change during gastation [g]" in the attached document "Detailed information on evaluated parameters".
The corrected body weight gain of test groups 1-3 revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.

- Clinical pathology: No treatment-related changes among hematological and clinical chemistry parameters were observed.

- Uterus weight: The mean gravid uterus weights of the animals of test group 1-3 (75, 250 and 750 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance. For details please refer to "Table 3: Mean gravid uterine weights and net maternal body weight change [g]" in the attached document "Detailed information on evaluated parameters".

- Reproduction data: The conception rate reached 96% in the low- and high-dose groups (75 and 750 mg/kg bw/d) and 100% in the control and mid-dose groups (0 and 250 mg/kg bw/d). For details please refer to "Table 3: Mean gravid uterine weights and net maternal body weight change [g]" in the attached document "Detailed information on evaluated parameters".
There were no test substance related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 75, 250 and 750 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 post-implantation losses, the number of resorptions and viable fetuses. For details please refer to "Table 4: Summary of reproduction data" in the attached document "Detailed information on evaluated parameters".

- Pathology
Absolute organ weights: When compared to control group 0 (set to 100%), the mean absolute liver weights were significantly increased in the high-dose group (126%, statistically significant).
Relative organ weights: When compared to control group 0 (set to 100%), the mean relative weights of following or-gans were significantly increased in the mid- and high-dose groups (108% and 128% respectively, statistically significant).
The increase in absolute and/or relative liver weights in test group 2 and 3 were regarded as treatment-related but non-adverse.

- Gross lesions: All findings occurred only individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Sex distribution of the fetuses: The sex distribution of the fetuses in test groups 1-3 (75, 250 and 750 mg/kg bw/d) was comparable to the control fetuses. For details please refer to "Table 4: Summary of reproduction data" in the attached document "Detailed information on evaluated parameters".

- Weight of the placentae: The mean placental weights of test groups 1-3 (75, 250 and 750 mg/kg bw/d) were comparable to the corresponding control group. For details please refer to "Table 5: Mean placental and fetal body weights (on a litter basis)" in the attached document "Detailed information on evaluated parameters".

- Weight of the fetuses: The mean fetal weight of test group 3 (750 mg/kg bw/d) was statistically significantly decreased (6% below control, if both sexes were combined). However, they were close to the mean of the historical control and well within the historical control range (3.1 – 4.0 in the 95% spread). For details please refer to "Table 5: Mean placental and fetal body weights (on a litter basis)" in the attached document "Detailed information on evaluated parameters".

- External examination of the fetuses
Fetal external malformations: The total incidence of external malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data. For details please refer to "Table 6: Total external malformations" in the attached document "Detailed information on evaluated parameters".
Fetal external variations: No external variations were recorded.
Fetal external unclassified observations: No external unclassified observations were recorded.

- Soft tissue examination of the fetuses
Fetal soft tissue malformations: No soft tissue malformations were recorded.
Fetal soft tissue variations: Two soft tissue variations were detected in all test groups including the control (0, 75, 250 or 750 mg/kg bw/d), i.e. dilated renal pelvis and dilated ureter. The incidences of these variations were not statistically significantly different from control and therefore, not considered biologically relevant. They are present in the historical control data. For details please refer to "Table 7: Total soft tissue variations" in the attached document "Detailed information on evaluated parameters".
Fetal soft tissue unclassified observations: No soft tissue unclassified observations were recorded.

- Skeletal examination of the fetuses
Fetal skeletal malformations: The total incidence of skeletal malformations in treated ani-mals did not differ significantly from the control group and was comparable to the historical control data. For details please refer to "Table 8: Total skeletal malformations" in the attached document "Detailed information on evaluated parameters".
Fetal skeletal variations: 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 skeletons and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable between treated groups and concurrent control as well as with the historical control. Thus it is not considered as an adverse event. For details please refer to "Table 9: Total fetal skeletal variations" and "Table 10: Occurrence of statistically significantly increased skeletal variations (expressed as mean percentage of affected fetuses/litter)" in the attached document "Detailed information on evaluated parameters".
Fetal skeletal unclassified cartilage observations: Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing. For details please refer to "Table 11: Total unclassified cartilage observations" in the attached document "Detailed information on evaluated parameters".

The assessment of all fetal external, soft tissue and skeletal observations is summarized in "Tabel 12: Total fetal malformations" and "Table 13: Total fetal variations" in the attached document "Detailed information on evaluated parameters".

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

For details please refer to the tables in the attached document "Detailed information on evaluated parameters".

Applicant's summary and conclusion