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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July-November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to other study
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
other: Audited draft study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : 176025
Purity : 98.4%
Name of test material (as cited in study report): NEROL (CAS 106-25-2)
Physical state: colourless - slightly yellow liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 14 January 2018

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 68 to 75 days old
- Weight at study initiation: 240 to 288 g.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods; Grid bottomed cages were used during pairing
- Diet ( ad libitum): SDS VRF1 Certified pelleted diet.
- Water ( ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes.
- Acclimation period: Five days before pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: Approximately 50 % of the final volume of vehicle was added to the required amount of test substance and magnetically stirred to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogeniser. A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance.
- Frequency of preparation: Weekly
- Storage of preparation: Refrigerated (nominally 2-8 °C)

VEHICLE
- Concentration in vehicle: 20, 60 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in Week 1 and the last week were analyzed for achieved concentration of the test item.
Details on mating procedure:
- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: Ejected copulation plug / sperm in vaginal smear referred to as Day 0 of pregnancy.
- A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals
Duration of treatment / exposure:
14 days (Days 6-19 p.c.)
Frequency of treatment:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same
time each day
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 mated females/dose
Control animals:
yes
Details on study design:
Dose levels were selected in conjunction with the Sponsor based on the results of a preliminary embryo-fetal toxicity study in Sprague Dawley rats (Envigo Study No. TS59TC). At the high dose of 1000 mg/kg bw/day in the preliminary study there was evidence that some females were more susceptible to dosing with Nerol towards the end of gestation with two females showing signs including piloerection, and/or under-activity and flat posture. It was considered possible that susceptible females could show marked post dosing signs towards the end of gestation on a main study with a greater group size, which could necessitate humane kill of affected females. At dose levels of 500 or 1000 mg/kg bw/day on the TS59TC study, slightly low overall body weight gain and transient body weight loss at 1000 mg/kg bw/day, as well as lower food consumption than in Controls, were observed during the first few days of treatment. A lower high dose of 750 mg/kg bw/day was therefore selected for this main study to reduce the possibility of marked signs being observed during late gestation; this dose was anticipated to fulfil regulatory expectations for some signs of toxicity at the high dose, manifest as reduced body weight gain and food consumption, following the start of treatment.
Low and intermediate dose levels were selected as 100 and 300, respectively, in order to assess the response at lower dose levels.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of animal ill-health amongst the occupant(s).
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.
Detailed observations were recorded daily at the following times in relation to dose administration:
At the end of dosing of each group; one to two hours after completion of dosing of all groups; as late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and daily from Days 6 to 20 after mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Animals were killed by carbon dioxide asphyxiation on Day 20 after mating.
- All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examin ed visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; Gravid uterine weight (including cervix and ovaries)
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorption sites: Yes
- Number of late resorption sites: Yes
- Number of live fetuses: Yes
- Number of dead fetuses: Yes
Fetal examinations:
Fetuses were killed by chilling on a cool plate (approximately 0 °C).
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. The sex of each fetus was recorded.
Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning for visceral abnormalities. IMS fixed fetuses were processed and stained with Alizarin Red for skeletal development and abnormalities.
Statistics:
See "Any other information on materials and methods incl. tables"
Indices:
Reproductive assessment
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100
Post-implantation loss (%) = [(Number of implantations – Number of live fetuses)/ Number of implantations] x 100
Historical control data:
Historical background data was used to compare the incidences of developmental toxicity.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Signs associated with dose administration were limited to a dose related incidence of increased salivation for animals receiving Nerol, with associated chin rubbing for females receiving 750 mg/kg bw/day. These signs were transient and for the majority of animals were no longer apparent 1 to 2 hours after completion of dosing all groups. Increased salivation and chin rubbing is often observed in association with gavage administration and is considered to relate to the palatability of the test formulations rather than a behavioural effect of treatment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Following the onset of treatment animals receiving 300 or 750 mg/kg bw/day showed a slight but statistically significant mean weight loss during Days 6 to 7 of gestation compared with a Control weight gain of 2g; thereafter body weight gain from Days 7 to 19 of gestation was similar to the Controls. Body weight gain at 100 mg/kg bw/day was unaffected by treatment. (see attached results tables)
Maternal body weight gain following adjustment for the gravid uterine weight was unaffected by treatment with Nerol. (see attached results tables)

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females receiving 750 mg/kg bw/day showed statistically significantly low food consumption from Days 6-9 (p<0.01) which marginally improved over Days 10-13 (p<0.05). From Day 14 onwards, food consumption at 750 mg/kg bw/day was similar to Control values.
Food consumption at 100 and 300 mg/kg bw/day was unaffected by treatment. (see attached results tables)
Food efficiency:
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:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Litter data as assessed by the post-implantation loss showed no adverse effect of maternal treatment with Nerol.
The extent of the pre-implantation loss was high in the treated groups when compared with concurrent controls. A dose response was apparent in mean values but the mean pre-implantation loss in the high dose group was mainly due to one female (No. 79) with an incidental pre-implantation loss of 75%; the mean pre-implantation loss without this outlier is 6.17% which is comparable to control value and within historical control data. In addition to the absence of a real dose response, the difference did not attain statistical significance and the resultant mean live litter size was considered unaffected by treatment. (See attached results tables)
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 specified
Details on maternal toxic effects:
One Group 4 animal (No. 78) was not pregnant. The assessment of litter data is therefore based on a total of 20, 20, 20 and 19 litters at 0, 100, 300 and 750 mg/kg bw/day.
Placental, litter and fetal weights were essentially similar to Controls at all dose levels and were considered to be unaffected by maternal treatment.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
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
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
Description (incidence and severity):
not relevant for OECD 414
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment. (see attached results tables)
Skeletal malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment. (see attached results tables)
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment. (see attached results tables)
An increase of abdominal cavity haemorrhages in fetuses, but not in litters, slightly above historical control data was observed. However, it is an isolated effect, that was considered to have no toxicological significance and to be an artefact of the necropsy process. (see attached results tables)

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for the maternal and fetal toxicity was considered to be 750 mg/kg bw/day.
Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, Nerol was administered by oral (gavage) to groups of mated female Crl:CD(SD) rats (20/dose) at the dose levels of 0, 100, 300 and 750 mg/kg bw/day from Days 6 to 19 after mating.

A similarly constituted Control group received the vehicle, corn oil, at the same dose volume. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

 

There were no unscheduled deaths and clinical condition was unaffected by treatment.

Signs associated with dose administration were limited to a dose related incidence of increased salivation and chin rubbing; these were considered to relate to palatibility rather than an effect of treatment.  

During Days 6 to 7 of gestation animals receiving 300 or 750 mg/kg bw/day showed a slight but statistically significant mean weight loss compared with a Control weight gain; thereafter body weight gain from Days 7 to 19 of gestation was similar to the Controls. Body weight gain at 100 mg/kg bw/day was unaffected by treatment.

Females receiving 750 mg/kg bw/day showed statistically significantly low food consumption from Days 6-13 of gestation. From Day 14 onwards, food consumption at 750 mg/kg bw/day was similar to Control values. Food consumption at 100 and 300 mg/kg bw/day was unaffected by treatment.

There were no maternal macroscopic findings that could be related to treatment.

Litter data as assessed by the number of corpora lutea, implantations, resorptions, live embryos and the extent of pre- and post-implantation loss showed no adverse effect of maternal treatment with Nerol.

Placental, litter and fetal weights were unaffected by maternal treatment.

Detailed fetal examination did not reveal any skeletal or visceral findings that could be related to maternal treatment.

 

Oral gavage administration of Nerol to pregnant Sprague-Dawley rats at dose levels of 100, 300 and 750 mg/kg bw/day during the organogenesis and fetal phase of gestation was well tolerated with no unscheduled deaths, no adverse effect on clinical condition and no treatment-related maternal macropathology. There was a slight initial effect on body weight gain and food consumption however there was no overall effect on maternal bodyweight gain.

Maternal treatment did not affect fetal survival, weight or development.

It was therefore concluded that in this study both the maternal and fetal no observed adverse effect level was 750 mg/kg bw/day.