Geraniol

Developmental toxicity / teratogenicity

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: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Geraniol

EC Number:

203-377-1

EC Name:

Geraniol

Cas Number:

106-24-1

Molecular formula:

C10H18O

IUPAC Name:

3,7-dimethylocta-2,6-dien-1-ol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Geraniol Extra
- Physical state: liquid/colorless, clear
- Homogeneity: given
- Analytical purity: 98.4 corr. area%
- Test substance No.: 10/0046-5
- Lot/batch No.: 00056777LO
- Expiration date of the lot/batch: 31 Oct 2016
- 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, under light exclusion

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system 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: 145.5–192.0 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:

PREPARATION OF DOSING SOLUTIONS: The specific amount of test substance was weighed, topped up with corn oil in a calibrated beaker and intensely mixed with a magnetic stirrer until it is 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 surviving females were sacrificed

Doses / concentrations

Remarks:

Doses / Concentrations:
30, 100 and 300 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

In a maternal toxicity rangefinding study dose levels of 300 an 1000 mg/kg bw/d were testet. The test substance was administered to pregnant female rats on GD 6 through GD 19. In this study a dose-related decrease of corrected body weight gain was noted, weight gain was approx. 9 and 12% below concurrent control, respectively. In addition increased relative weights of adrenals (116%), kidneys (117%) and liver (131%) were increased above concur-rent control. Thus dose levels of 30, 100 and 300 mg/kg bw/d were considered appropriate for the present definitive study.

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: at least once a day, or more often when clinical signs of toxicity were elicited (GD 0-20)

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross pathology

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
- MORTALITY:
There were no substance-related or spontaneous mortalities in any females of all test groups.

- CLINICAL SYMPTOMS:
All females of the high-dose group (300 mg/kg bw/d), nearly all (24 out of 25) females of the mid-dose group (100 mg/kg bw/d) and two females of the low-dose group (30 mg/kg bw/d) showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 20 minutes) and was initially observed on GD 7.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected.

- FOOD CONSUMPTION:
The mean food consumption of the dams in all test groups was generally comparable to the concurrent control throughout the entire study period.

- BODY WEIGHT:
The mean body weights (BW) and the average body weight gains (BWC) of the low-, mid- and high-dose dams (30, 100, 300 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. This includes the statistically significantly higher BW/BWC values in the low-dose group on GD 19 and 20 (BW), GD 17-19 and GD 6-19 (BWC).

- CORRECTED (NET) BODY WEIGHT GAIN:
The corrected body weight gain of test groups 1-3 (30, 100 and 300 mg/kg bw/d) revealed no difference of any biological relevance to the concurrent control group. Mean carcass weights remained also unaffected by the treatment.

- UTERUS WEIGHT:
No test-substance related findings.

- NECROPSY FINDINGS:
No test-substance related findings.

- REPRODUCTION DATA:
The conception rate was 100% in all test groups (0, 30, 100 and 300 mg/kg bw/d).
No test-substance related findings in conception rate, in the mean numbers of corpora lutea, implantation sites and viable fetuses or the number of resorptions and the value calculated for postimplantation loss.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- SEX DISTRIBUTION:
comparable to the concurrent control fetuses

- WEIGHT OF PLACENTAE:
comparable to the concurrent control group

- WEIGHT OF FETUSES:
no test substance related effects

- FETAL EXTERNAL MALFORMATIONS:
One fetus with multiple external malformations was recorded in test group 2 (100 mg/kg bw/d). The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was comparable to the histori-cal control data.

- FETAL EXTERNAL VARIATIONS:
no external variations were recorded

- FETAL EXTERNAL UNCLASSIFIED OBSERVATIONS:
no unclassified external observations were recorded

- FETAL SOFT TISSUE MALFORMATIONS:
One soft tissue malformation was recorded for two control fetuses and one mid-dose fetus (0 and 300 mg/kg bw/d). The overall incidences of soft tissue malformations were comparable to those found in the historical control data.

- FETAL SOFT TISSUE VARIATIONS:
no soft tissue malformations were recorded

- FETAL SOFT TISSUE UNCLASSIFIED OBSERVATIONS:
no unclassified soft tissue observations were recorded

- FETAL SKELETAL MALFORMATIONS:
Two fetuses bearing skeletal malformations were detected in test groups 0 and 2 (0 and 100 mg/kg bw/d) affecting the skull, vertebral column (with or without involving the ribs) and humerus. An association of these malformations to the treatment is not assumed.

- 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 skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.

- FETAL SKELETAL UNCLASSIFIED CARTILAGE OBSERVATIONS:
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. The incidence of branched rib cartilage was significantly increased in test group 2 (100 mg/kg bw/d). However, this finding showed no dose-dependency and was therefore assessed to be without biological relevance

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at highest tested dose

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results obtained in the study, the NOAEL for maternal toxicity and for developmental toxicity could be set at 225 mg/kg bw/day.

Executive summary:

The effects of the test item were investigated in rat during pregnancy and embryo-foetal development.

Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. All femaleswere administered by oral gavage during the gestation period fromDay 6 through Day 19 post coitum at 75, 225 and 750 mg/kg bw/day and 10 mL/kg body weight as the dose volume. Control females received the vehicle (0.5% carboxymethylcellulose in softened
water (0.5% CMC)) at the same dose volume during the same treatment period.

No mortality of females occurred during the study. The final number of pregnant females on Day 20 post coitum was 25 in the control group,low- (75 mg/kg bw/day) and top level (750 mg/kg bw/day) and 23 in the mid-dose group (225 mg/kg bw/day).

Piloerection was the treatment-related clinical sign noted in all females receiving 750 mg/kg bw/day. Other clinical sign, such as hunched posture was also noted. No other relevant clinical signs were noted in females receiving 75 and 225 mg/kg bw/day.

Reduction in maternal body weight (not greater than 10%) was observed at 750 mg/kg bw/day between Days 12 to 20 post coitum, when compared to the control group. The maternal growth retardation was better evident on body weight gain which was decreased from Days 9 to 20 post coitum, with significance, at statistical analysis, when compared to the control group.

Statistically significant reduction in food consumption was noted in the high-dose females receiving 750 mg/kg bw/day starting from Day 9 post coitum until termination. Some differences (decrease) observed in the remaining treated groups against to control, was transient and with a trend of recovery.

There was no treatment-related effect on thyroid hormone determination.

A decrease in body weight gain, corrected for gravid uterus weight, terminal body weight and body weight at Day 6 post coitum was recorded in treated females receiving 750 mg/kg bw/day (approximately 20%). Terminal body weight and corrected maternal body weight were also statistically significant decreased in females dosed at 750 mg/kg bw/day, when compared to the control group.

Litter data, mean foetal weight and sex ratios did not show relevant differences between control and treated groups.

Compared to the control, no alterations were seen in the anogenital distance performed in foetuses maternally exposed to the test item.

Statistically significant increase in absolute and relative mesenteric nodes weight was observed in females receiving the dose levels ≥ 225 mg/kg bw/day, when compared to the controls.

Females that completed the treatment period did not show relevant macroscopic changes.

No treatment-related changes were noted in thyroid when examined microscopically.

The overall incidences of foetuses or litters with findings did not indicate test item-related effect.

No relevant changes that could be considered treatment-related were observed at visceral examination of foetuses between the control and treated groups. Consequently, under the conditions described for this study, the test item did
not reveal teratogenic potential up to and including the dose level of 750mg/kg bw/day.

The skeletal assessment revealed instances of an increase incidence of irregular sternebral ossification in the high dose foetuses (750 mg/kg bw/day) compared to control. The alterations noted can be considered as a transient delay of ossification that will resolve shortly after birth rather than as teratogenicity sign.