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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:
09 September 2020 to 20 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 414 Guideline without deviations.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
other: Draft report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(+)-pin-2(3)-ene
EC Number:
232-087-8
EC Name:
(+)-pin-2(3)-ene
Cas Number:
7785-70-8
Molecular formula:
C10H16
IUPAC Name:
(1R,5R)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene
Constituent 2
Chemical structure
Reference substance name:
(-)-pin-2(3)-ene
EC Number:
232-077-3
EC Name:
(-)-pin-2(3)-ene
Cas Number:
7785-26-4
Molecular formula:
C10H16
IUPAC Name:
(1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene
impurity 1
Chemical structure
Reference substance name:
(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane
EC Number:
227-336-2
EC Name:
(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane
Cas Number:
5794-03-6
Molecular formula:
C10H16
IUPAC Name:
(1R,4S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane
impurity 2
Chemical structure
Reference substance name:
(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane
EC Number:
227-337-8
EC Name:
(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane
Cas Number:
5794-04-7
Molecular formula:
C10H16
IUPAC Name:
(1S,4R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane
impurity 3
Chemical structure
Reference substance name:
(-)-pin-2(10)-ene
EC Number:
242-060-2
EC Name:
(-)-pin-2(10)-ene
Cas Number:
18172-67-3
Molecular formula:
C10H16
IUPAC Name:
(1S,5S)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
impurity 4
Chemical structure
Reference substance name:
1,7,7-trimethyltricyclo[2.2.1.02,6]heptane
EC Number:
208-083-7
EC Name:
1,7,7-trimethyltricyclo[2.2.1.02,6]heptane
Cas Number:
508-32-7
Molecular formula:
C10H16
IUPAC Name:
1,7,7-trimethyltricyclo[2.2.1.0~2,6~]heptane
impurity 5
Chemical structure
Reference substance name:
7,7-dimethyl-2-methylene-(1R,4S)-bicyclo[2.2.1]heptane
Cas Number:
116724-26-6
Molecular formula:
C10H16
IUPAC Name:
7,7-dimethyl-2-methylene-(1R,4S)-bicyclo[2.2.1]heptane
impurity 6
Chemical structure
Reference substance name:
7,7-dimethyl-2-methylene-, (1S,4R)-bicyclo[2.2.1]heptane
Cas Number:
7378-37-2
Molecular formula:
C10H16
IUPAC Name:
7,7-dimethyl-2-methylene-, (1S,4R)-bicyclo[2.2.1]heptane
impurity 7
Chemical structure
Reference substance name:
(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
Cas Number:
19902-08-0
Molecular formula:
C10H16
IUPAC Name:
(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
Test material form:
liquid
Details on test material:
Batch No. : 1000082993
Purity : 96.6% (sum of the two main constituents)
Name of test material (as cited in study report): alpha-pinene multiconstituent
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 10 June 2021

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals or test system and environmental conditions:
RATIONALE FOR ANIMAL MODEL:
The rat was chosen as the test species because of the requirement for a rodent species by
regulatory agencies. The Sprague Dawley Crl: CD (SD) rat strain was used because of the
historical control data available at this laboratory.

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 69 to 75 days old.
- Weight at study initiation: 203-289 g
- Housing: Acclimatization - up to four animals; During pairing - one (stock) male and one female; Gestation - one female
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: Five days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
Environmental Enrichment
- Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
- Plastic shelter: Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

IN-LIFE DATES: From: 09 September 2020 To: 08October 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% methylcellulose
Details on exposure:
RATIONALE FOR ROUTE OF ADMINISTRATION:
The oral gavage route of administration was chosen to simulate the conditions of potential
human exposure.

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: The required amount of test item was weighed.
Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous for a minimum of 20 minutes. The formulation was transferred to the final containers, via syringe, whilst magnetically
stirring. A series of formulations at the required concentrations were prepared in ascending order.

Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 6, 12 and 22 mg/mL
- Dose volume: 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Achieved concentration: Samples of each formulation were analyzed for the achieved
concentration of the test item.

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 5 to 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix in Covance study CF77KM. Formulations were demonstrated to be homogeneous and stable for up to one day when stored at ambient temperature (15-25 °C) and for 15 days when stored refrigerated (2-8 °C).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
- Day 0 of gestation: When positive evidence of mating was detected.

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:
Females were treated from Day 6 to Day 19 (inclusive) after mating
Frequency of treatment:
Once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
110 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the preliminary study using dose levels of 100, 125 and 150 mg/kg bw/day, (Covance Study Number KC09YH), 150 mg/kg bw/day was not tolerated with two females found dead at this dose level. Body weight loss was also evident from Day 10 to Day 14 of gestation and mean body weight stasis was recorded at 125 mg/kg bw/day during this period. Body weight gain at 125 mg/kg bw/day was statistically significantly lower than control throughout treatment (-23% of Control), whilst, bodyweight gain at 100 mg/kg bw/day was unaffected by treatment. Adjusted body weight was reduced for all treatment groups that received alphapinene multiconstituent. In addition, there was a large variation in individual adjusted body weight gain in females receiving 125 mg/kg bw/day, with two animals showing body weight loss over Days 6-20. This also correlated with reduced food consumption in these animals. Also, mean total litter weight, male fetal weight, female fetal weight and overall fetal weight was slightly lower at 125 and 150 mg/kg bw/day when compared with Control. Considering the steep response curve, it was considered that 110 mg/kg bw/day would elicit signs of maternal toxicity without causing death. The subsequent doses of 60 and 30 mg/kg bw/day were selected as mid and low doses, respectively, and were not expected to show any toxicity due to the steep response curve.

- Rationale for animal assignment: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.
Method: To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Examinations

Maternal examinations:
MORTALITY: Yes
- A viability check was performed near the start and end of each working day. Animals were
killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the
occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

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

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and and 6-20 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, 6-7, 8-10, 11-13, 14-16 and 17-19 after mating inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Animals surviving until the end of the scheduled study period were killed on Day 20 after mating by Carbon dioxide asphyxiation.
- Necropsy: 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 examined 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 10% Neutral Buffered Formalin. The organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 1.
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.
Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Ovaries and uterine content:
For females surviving to term, the ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight (including cervix and ovaries): Yes
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of Fetuses (live and dead): Yes
Blood sampling:
THYROID HORMONE ANALYSIS: Yes
- Blood samples were collected at termination of the study in all surviving adults in sublingual vein after an isoflurane anaesthesia. Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation (2000 g for ten minutes at 4°C).
- Number of aliquots: Two per animal. Aliquot 1: 0.2 mL serum for T3/T4; Aliquot 2: residual serum for TSH.
Fetal examinations:
Method of kill for fetuses: 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. The sex and ano-genital distance 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.
IMS fixed fetuses were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses: Assessed for skeletal development and abnormalities.
Statistics:
See " Any other information on materials and methods incl. tables"
Indices:
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss
was considered to reflect losses due to non-fertilization of ova and failure to implant. It was
calculated from the formula:
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100

Where the number of implantations exceeded the number of corpora lutea observed,
pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to
have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%)= [(Number of implantations – Number of live fetuses) / Number of implantations] x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs at 30, 60, or 110 mg/kg bw/day.
There were no signs associated with dose administration at 30, 60, or 110 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
One animal (No. 61) receiving 110 mg/kg bw/day was euthanized for welfare reasons on Day 14 of gestation (following 8 days of treatment) due to marked body weight loss that correlated with low food consumption. The death was attributed to an exaggerated individual animal response to treatment because this animal exhibited more severe changes in body weight and food intake than the other animals at this dose.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- At 110 mg/kg bw/day there was modest weight gain, or weight loss, from Day 8 to Day 12. Thereafter there was a modest weight gain but with a rate that was less than the control group. Overall, from Day 6 to 20 this group gained significantly less weight than the control group (76 g vs 117 g). For the 30 and 60 mg/kg bw/day groups, there was no significant effect on bodyweight gain over the same period.
- Gravid uterine weight was unaffected by treatment at 30, 60 or 110 mg/kg bw/day. When adjusted for gravid uterine weight, overall body weight stasis was evident at 110 mg/kg bw/day (overall change of -1 g) and adjusted body weight change was 90% of Control. Adjusted body weight change was unaffected at 30 or 60 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was persistently and statistically significantly low during Days 8-20 of gestation at 110 mg/kg bw/day and was marginally, but statistically significantly low, during Days 8-11 of gestation (86% of Control) at 60 mg/kg bw/day. Food intake at 30 mg/kg bw/day was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Serum concentrations of Triiodothyronine, Thyroxine and Thyroid Stimulating Hormone were unaffected by treatment on Day 20 of gestation at 30, 60, or 110 mg/kg bw/day.
Clinical biochemistry findings:
not examined
Endocrine 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):
Absolute and adjusted thyroid weight was unaffected by treatment at 30, 60, or 110 mg/kg
bw/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination revealed pale contents in the esophagus, jejunum, colon, and rectum; and bedding material was observed in the esophagus and stomach.
There were no treatment-related macroscopic observations in the adults on Day 20 of gestation at 30, 60, or 110 mg/kg bw/day.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or were as expected for Sprague-Dawley rats of this age/sex; therefore, they were considered not test item related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Of all the maternal organs only the thyroid was examined microscopically and no abnormalities were found. There were no treatment-related microscopic observations in the thyroid gland in adults at 30, 60, or 110 mg/kg bw/day.
Other effects:
no effects observed
Details on results:
See table of results in the section "Attached background material".

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
All animals surviving to scheduled termination were pregnant.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of implantations and the pre- and post-implantation losses (%) were similar to Control and were therefore unaffected by treatment at 30, 60, or 110 mg/kg bw/day.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of resorptions (early, late) were similar to Control and were therefore unaffected by treatment at 30, 60, or 110 mg/kg bw/day.
Dead fetuses:
no effects observed
Description (incidence and severity):
The numbers of live young were similar to Control and were therefore unaffected by treatment at
30, 60, or 110 mg/kg bw/day.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The female was pregnant with 18 live embryos.
Other effects:
no effects observed
Description (incidence and severity):
All animals surviving to scheduled termination were pregnant. The numbers of implantations,
resorptions (early, late), pre- and post-implantation losses (%), live young and the ratio of
male to female fetuses were similar to Control and were therefore unaffected by treatment at
30, 60, or 110 mg/kg bw/day.
Details on maternal toxic effects:
There was no effect of treatment at 30, 60, or 110 mg/kg bw/day on reproductive performance, as assessed by the numbers of resorptions, implantation losses, and live young, the ratio of male to female fetuses, total litter or overall fetal weights and there was no effect of treatment on fetal
ano-genital distance.
See table of results in the section "Attached background material".

Effect levels (maternal animals)

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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Placental weight was marginally low at 110 mg/kg bw/day (87% of Control); placental weight was unaffected by treatment at 30 or 60 mg/kg bw/day.
Total litter and overall fetal weights were unaffected by treatment at 30, 60, or 110 mg/kg
bw/day.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was unaffected by treatment.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The litter weight was unaffected by treatment.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
When compared with Control, anogenital distance was unaffected by maternal treatment at
30, 60, or 110 mg/kg bw/day.
External malformations:
no effects observed
Description (incidence and severity):
There were no major or minor treatment related fetal abnormalities at 30, 60, or 110 mg/kg
bw/day.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no major or minor treatment related fetal abnormalities at 30, 60, or 110 mg/kg
bw/day.
One fetus at 30 mg/kg bw/day had a ventricular septum membranous defect and one fetus at
both 60 and 110 mg/kg bw/day had ventricular septum muscular defects, and at 110 mg/kg
bw/day two fetuses from two litters had cleft palate/lip. As the incidence of these major
abnormalities was low and within the Historical Control Data (HCD), a relationship to treatment is not inferred.
There was an increase in incidence of malpositioned testes and brain haemorrhages, when
compared to Control that exceeded the Historical Control Data (HCD) range at 110 mg/kg
bw/day. At 60 mg/kg bw/day there was an increase in incidence of short 13 ribs and brain
haemorrhages, when compared to Control that exceeded the HCD range.
At 30 mg/kg bw/day there was an increase in incidence of malpositioned testes, when
compared to Control that exceeded the HCD range.
As the incidental findings showed no clear dose response they were considered not to be
treatment related.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
There was no effect of
treatment at 30, 60, or 110 mg/kg/day on reproductive performance, as assessed by the
numbers of resorptions, implantation losses, and live young, the ratio of male to female
fetuses, total litter or overall fetal weights and there was no effect of treatment on fetal
ano-genital distance. Mean placental weight was marginally low at 110 mg/kg bw/day;
however, subsequent pathological examination of the fetuses showed that the low incidence
of major or minor abnormalities showed no relationship to treatment at 30, 60, or 110 mg/kg/day; therefore a relationship to treatment is not inferred.
See table of results in the section "Attached background material".

Effect levels (fetuses)

Key result
Sex:
male/female
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

Any other information on results incl. tables

Formulation analysis:


Mean concentrations of Alpha-pinene multiconstituent in the first and last formulations were within the applied limits of +10/-15% of nominal concentration, confirming accuracy of the formulations. The difference from mean remained within 7%, confirming precise analysis.


 


Table 2. Analyzed Concentrations for Alpha-pinene multiconstituent in 1% Methylcellulose

































































































































OccasionGroupNominal conc. (mg/mL)Analyzed concentration (mg/mL)RME (%)Difference from mean (%)
Analysis 1Analysis 2Mean
First preparation10NDND---
265.285.445.36-10.7±1.47
31211.011.511.2-6.7±2.33
42219.722.521.1-4.1±6.68
Second preparation10NDND---
265.485.565.52-8.0±0.74
31212.212.212.2+1.7±0.25
42221.321.521.4-2.7±0.38
Third preparation10NDND---
265.515.525.52-8.0±0.05
31211.111.711.4-5.0±2.84
42220.619.820.2-8.2±1.86

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 60 mg/kg bw/day (based on reduced food intake and reduced bodyweight gain) and the embryo-fetal No-Observed-Effect-Level (NOEL) was 110 mg/kg bw/day.
Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, three groups of 20 females received alpha-pinene multiconstituent at doses of 30, 60, or 110 mg/kg bw/day by oral gavage administration at a volume dose of 5 mL/kg bw, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 1% methylcellulose, at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.


Clinical observations, body weight, and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating. Gravid uterine and thyroid weights were recorded, and the thyroid, kidney and liver were retained. A microscopic pathology investigation was performed on the thyroid from all pregnant animals.
The ano-genital distance, individual body weight, and placental weights were recorded for all fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. Blood samples were taken at necropsy for subsequent thyroid hormone analysis in all pregnant animals.


Mean concentrations of alpha-pinene multiconstituent in the first and last formulations were within the applied limits of +10/-15% of nominal concentration, confirming accuracy of the formulations. The difference from mean remained within 7%, confirming precise analysis.


One animal receiving 110 mg/kg bw/day was euthanized for welfare reasons on Day 14 after 8 days of treatment due to marked body weight loss that correlated with low food consumption. The death of this animal was attributed to an exaggerated individual animal response to treatment because this animal exhibited more severe changes in body weight and food intake than the
other animals at this dose.
There were no treatment related clinical signs and there were no signs associated with dosing. Food intake was persistently low at 110 mg/kg bw/day during Days 8-20 of gestation. At 110 mg/kg bw/day, overall body weight gain (Days 6-20) was markedly low (76 g vs 117 g in control) and adjusted body weight change was -1 g (versus +34 g in Control). The reduced bodyweight gain was considered to be correlated with the reduced food consumption.
Gravid uterine weight was unaffected by treatment at any dose.
On Day 20 of gestation, serum concentrations of Triiodothyronine, Thyroxine, and Thyroid Stimulating Hormone were unaffected by treatment at 30, 60, or 110 mg/kg bw/day. Adult thyroid weight was unaffected by treatment at any dose and the tissue was macroscopically and microscopically normal.


Reproductive performance (numbers of resorptions, implantation losses, and live young, the ratio of male to female fetuses, placental, total litter or overall fetal weights) was considered to be unaffected by treatment at 30, 60, or 110 mg/kg bw/day. Fetal ano-genital distance was unaffected by treatment at 30, 60, or 110 mg/kg bw/day. There were no major or minor fetal abnormalities that showed a relationship to treatment at 30, 60, or 110 mg/kg bw/day.


Based on the results of this study, the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 60 mg/kg bw/day (based on reduced food intake and reduced bodyweight gain) and the embryo-fetal No-Observed-Effect-Level (NOEL) was 110 mg/kg bw/day.