Pin-2(3)-ene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 September 2020 to [TBC]

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

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

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

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

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

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

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

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

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

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. : 1000030769
Purity : 96.9% (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: 25 March 2020

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

RATIONALE FOR ANIMAL MODEL
The rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.

TEST ANIMALS
- Source: Envigo RMS (UK)
- Age at study initiation: approximately 21 to 24 weeks old.
- Weight at study initiation: 2.43 to 4.36 kg
- Housing: One animal per cage. Suspended cages fitted with perforated floor panels and mounted in batteries. Undertray lined with absorbent paper which is changed at least 3 times a week. Cages were also fitted with a plastic resting platform.
- Environmental enrichment: A soft white untreated wood block as aspen chew block was provided to each cage throughout the study and replaced when necessary. In addition, a stainless steel key ring was attached to the cage and a cage paper was provided to each cage from Day 20 after mating to allow expression of nesting behavior and was replaced as necessary.
- Diet (e.g. ad libitum): Teklad 2930, pelleted diet, restricted (200 g/animal/day).
In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.
- Water (e.g. ad libitum): Ad libitum, potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Water bowls were given to some animals as required.
- Acclimation period: For five days before the start of treatment (Days 1-5 of gestation).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21ºC.
- Humidity (%): 45-70%
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light):14 hours light : 10 hours dark

IN-LIFE DATES: From: 29 September 2020 to 30 October 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% Methylcellulose

Details on exposure:

DETAILS ON ROUTE OF ADMINISTRATION:
Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Rationale for route of administration: The oral (gavage) route of administration was chosen to simulate the conditions of possible human exposure.

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: Starting with the lowest concentration, approximately 50% of the final volume of vehicle (1% methylcellulose) was added to the test item and magnetically stirred until uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was transferred into a beaker and mixed using a high shear homogenizer until visibly homogenous. The formulation was then returned to the container with a lid and mixed using a magnetic stirrer until homogenous for a minimum of 20 minutes.
Formulations were then transferred to their final containers via syringe while being magnetically stirred. Formulations were prepared in ascending order of concentration.
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Frequency of preparation: Weekly, and divided into 7 daily aliquots.
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 15, 30 and 60 mg/mL.
- Dose volume: 5 mL/kg bw/day for all groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS of STABILITY
The stability of a homogenous suspension of the test item in the vehicle was demonstrated as part of another study (Covance Study No. CF77KM) which demonstrated that prepared formulations in the concentration range of 5 to 200 mg/mL in the vehicle were stable for up to 15 days following refrigerated storage (2 to 8°C), and for one day following ambient storage (15 to 25°C).

FORMULATION ANALYSIS
Samples of each formulation prepared for administration in the first and last week of treatment were analyzed for achieved concentration of the test item

Details on mating procedure:

Method: Natural mating with New Zealand White bucks of established fertility at the supplier’s facility. Males and females were not closely related
Male/female ratio: 1 : 1 using identified stock New Zealand White bucks.
Checks: natural mating observed.
After mating: each female injected intravenously with 25 i.u. luteinizing hormone.
Day 0 of gestation = Day of mating.
Delivery to Covance: Day 1 after mating.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 28 (inclusive) after mating.

Frequency of treatment:

Once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE
The dose levels for investigation in this study (0, 75, 150 and 300 mg/kg bw/day) were selected in conjunction with the Sponsor and were based on the results of the preliminary embryo-fetal study in the rabbit, Covance Study No WT37TF.
In the preliminary study, where doses of 87.5, 175 or 350 mg/kg bw/day were tested, there were no unscheduled deaths and no treatment related clinical signs. Two females that received 350 mg/kg bw/day were observed with decreased activity following dose administration (one female on Day 7, the other female on Day 9 after mating), however, this observation was transient in nature. Slight body weight loss was observed following the commencement of treatment, at 350 and 175 mg/kg bw/day (-0.13 and -0.08 kg, respectively). Overall group mean body weight gain for all groups of treated females was lower than control, especially at 350 mg/kg bw/day. Maternal body weight loss adjusted for the contribution of the gravid uterine weight was greater in all treated groups and statistically significant at 350 mg/kg bw/day. Food intake of all groups of treated females was generally lower than control from Day 6 of gestation at 350 mg/kg bw/day or from Day 8 at 175 or 87.5 mg/kg bw/day, with the greatest and statistically significant reduction observed at 350 mg/kg bw/day (Days 6-29: -24% of Control). The lowest food intake was observed at 350 mg/kg bw/day following the commencement of treatment on Days 6 to 9 of gestation when intake was markedly lower than control. Despite a slight increase in liver weights at the high dose, there was no adverse effect of treatment on liver weights at any dose level investigated and no treatment related macroscopic abnormalities were detected among the maternal females or fetuses at scheduled termination. At 350 mg/kg bw/day, male, female and overall fetal weights at 350 mg/kg bw/day were lower than control (about 90% of controls).

Based on these results, the high dose level for investigation in this main embryo-fetal development study was set at 300 mg/kg bw/day with effects on body weight and food intake expected. The intermediate and low dose levels were set at 150 and 75 mg/kg bw/day, respectively, to fulfill the 2-fold to 4-fold dosing internal specified in the test guideline.

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
- 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. 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
- Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration: Pre-dose observation; One to two hours after completion of dosing; As late as possible in the working day.
- A detailed physical examination was performed on each animal on Days 1, 6, 12, 18, 24 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- The weight of each adult was recorded on Days 1, 3 and 6-29 after mating.

FOOD CONSUMPTION : Yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 2 after mating.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Animals surviving until the end of the scheduled study period were killed on Day 29 after mating, by intravenous injection of sodium pentobarbitone.
- 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 appropriate fixative.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
For females surviving to term, the following uterine content was recorded:
Gravid uterine weight (including cervix and ovaries).

For each ovary/uterine horn, the following were recorded for all animals (including those prematurely sacrificed, where possible):
- 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

For apparently non-pregnant animals and for apparently empty uterine horns, the absence or number of uterine implantation sites was confirmed.

Blood sampling:

Not examined

Fetal examinations:

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. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Approximately 50% of eviscerated fetuses were decapitated; heads were initially stored in Bouin’s fluid and subject to free-hand serial sectioning. Serial sections were examined for soft tissue abnormalities.
Remaining eviscerated fetuses and torsos were fixed in Industrial Methylated Spirit and processed and stained with Alizarin Red for skeletal development and abnormalities assessment.

Statistics:

For adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anormaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Caesarian section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
Placental, litter and fetal weights

The following comparisons were performed:
Group 1 vs 2, 3 and 4

A parametric or a non-parametric analysis of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights data.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level given as following:
* p<0.05
** p<0.01

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

Historical control data:

Historical Control data were routinely supplied for selected observations where this information was considered to assist interpretation of study data, and normally include both fetal and litter incidences.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related clinical signs observed from Day 6 to Day 29 after mating or post-dose observations observed at any dose level investigated.
Clinical signs of decreased fecal pellets, fecal pellets reduced in size were observed in individual animals on occasion throughout the treatment groups, including Control.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female 4F 90 that received 300 mg/kg bw/day was euthanized for welfare reasons on Day 21 of gestation due to persistent body weight loss and several days of little/no dry diet intake. There were no findings at macroscopic examination and this female was found to be pregnant with 4 live fetuses (5 early resorptions were noted). This premature death was of uncertain relationship to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Following the commencement of treatment on Day 6 after mating, slight group mean body weight loss was observed in all groups, including Control, until Day 11-12 of gestation. Thereafter, slight group body weight gain was observed in all groups. Overall (Day 6-29 of gestation) body weight gain was observed in all groups of treated females, however, this gain was slightly lower than Control for females receiving 300 mg/kg bw/day (-33% of Control) but the difference did not attain statistical significance.

Mean gravid uterine weights at termination on Day 29 of gestation was lower than Control (-10%) for females receiving 300 mg/kg bw/day, but without statistical significance. A dose-dependent slight adjusted maternal body weight loss was observed in all groups when taking into account the gravid uterine weight but differences did not attain statistical significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Following the commencement of treatment and throughout the treatment period, food consumption at 300 mg/kg bw/day was generally lower than controls and differences attained statistical significance on Days 6-8, 14-20, 23 and 25-27 of gestation, leading to a statistically significantly lower mean food consumption between Days 6 and 29 (-20% of Control). Food intake was unaffected by treatment at 150 or 75 mg/kg bw/day.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

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:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related abnormalities detected among the maternal females at macroscopic examination on Day 29 of gestation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Administration of alpha-pinene multiconstituent at doses of 75, 150 and 300 mg/kg bw/day was generally well tolerated. At 300 mg/kg bw/day one female which had shown high food consumption prior to the start of treatment was killed for welfare reasons on Day 21 of gestation due to marked body weight loss and a prolonged period of low food intake; however, this was an atypical response (also when considering the absence of mortality at 350 mg/kg bw/day in the preliminary study) and no effect of treatment was inferred. There were no treatment related clinical signs at any dose level investigated. A slight reduction in group mean maternal body weight gain was apparent at 300 mg/kg bw/day (-33% of Control) but the difference did not attain statistical significance. Food consumption at 300 mg/kg bw/day was generally lower than controls and differences attained statistical significance on Days 6-8, 14-20, 23 and 25-27 of gestation, leading to a statistically significantly lower mean food consumption between Days 6 and 29 (-20% of Control). The extent of the reduction in body weight gain and food intake was considered not to be adverse as it was only slight and not associated with any change in maternal clinical condition or adverse effect on pregnancy outcome.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was considered to be no effect of treatment on the mean number of implantations, pre or post implantation loss.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One female in each of the 75 or 150 mg/kg bw/day groups had a total litter resorption but no effect of treatment was inferred. It was noted that the mean number of resorptions and thus post implantation loss at 300 mg/kg bw/day was slightly higher than in controls but no effect of treatment was inferred as differences did not attain statistical significance and live litter size was similar to controls.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Two females from each group, including controls (1F 4 and 20, 2F 27 and 44, 3F 61 and 69, 4F 80 and 93) were found not to be pregnant at macroscopic examination.

Details on maternal toxic effects:

Litter data was unaffected by maternal treatment at 75 or 150 mg/kg bw/day.

Effect levels (maternal animals)

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 and 150 mg/kg bw/day, male, female and overall fetal weights were slightly low when compared to controls with statistical significance attained for female and overall fetal weights (90% of Control) at 300 mg/kg bw/day and for overall fetal weights at 150 mg/kg bw/day. Total litter weight was also low at 300 mg/kg bw/day compared to Control.
There was no effect of treatment on group mean placental weights. Total litter, male, female and overall fetal weights were unaffected by maternal treatment at 75 mg/kg bw/day.
At 150 and 300 mg/kg bw/day there was also a decrease in overall mean fetal weight compared to concurrent control. However, all the lower values were within the historical control data.

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

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no major abnormalities or minor skeletal or visceral structural abnormalities considered to be related to treatment.
Across all treated groups there was an increase in incidence of incompletely ossified sternebrae compared to concurrent control but within historical control data (HCD) range.
At 150 and 300 mg/kg bw/day there was also an increase in incidence of incomplete ossified epiphyses, and metacarpals compared to concurrent control but also within HCD range. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the slight decrease in mean fetal weight seen at these dose levels. It was therefore not considered adverse.

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryo-fetal survival and live litter size were unaffected by treatment.

Mean male and female fetal weights, and consequently overall fetal weights, were slightly lower than Control in litters derived from females given 150 or 300 mg/kg bw/day (overall fetal weight was 10% lower than Control at 300 mg/kg bw/day); total litter weights were also slightly low at 300 mg/kg bw/day, but did not show a dose-related response. As differences were slight and not associated with any structural fetal abnormalities they were not deemed to represent an adverse effect of treatment.

There were no major abnormalities or minor skeletal or visceral structural abnormalities considered to be related to treatment.

At 150 and 300 mg/kg bw/day there was also an increased incidence of incomplete ossified epiphyses, and metacarpals compared to concurrent control but within HCD range: incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the slight decrease in mean fetal weight seen at these dose levels. It was therefore not considered adverse. 

Effect levels (fetuses)

Key result

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
The mean concentrations were within 7% of the nominal concentration, confirming the accuracy of formulation. The difference from mean and coefficient of variation remained within 4%, confirming precise analysis.

Applicant's summary and conclusion

Conclusions:

Oral gavage administration of alpha-pinene multiconstituent at doses up to and including 300 mg/kg bw/day to pregnant New Zealand White rabbits from Day 6-28 of gestation inclusive, was associated with slight reductions in body weight gain and mean food consumption in females given 300 mg/kg bw/day, which were judged as non-adverse. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore concluded to be 300 mg/kg bw/day. The NOAEL for embryo-fetal survival and development was also concluded to be 300 mg/kg bw/day.

Executive summary:

In a study conducted according to OECD guideline 414 and in GLP conditions, three groups of 24 females received alpha-pinene multiconstituent at doses of 75, 150 or 300 mg/kg bw/day by oral gavage administration at a dose volume of 5 mL/kg bw, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, 1% methylcellulose by oral gavage over the same treatment period and at the same volume dose as the treated females. 

The mean concentrations were within 7% of the nominal concentration, confirming the accuracy of formulation. The difference from mean and coefficient of variation remained within 4%, confirming precise analysis.

Animals were killed on Day 29 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 29 after mating and the gravid uterine weight was recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Oral gavage administration of alpha-pinene multiconstituent at doses up to and including 300 mg/kg bw/day were generally well tolerated, with no test item-related premature deaths or changes in general clinical condition. Females which received 300 mg/kg bw/day showed a slight reduction in body weight gain throughout the treatment period (-33% of Control) and a statistically significantly lower mean food consumption between Days 6 and 29 (-20% of Control). At scheduled termination on Day 29 of gestation, no test item-related macroscopic abnormalities were detected at any dose level investigated.

Embryo-fetal survival and live litter size were unaffected by treatment. At 300 and 150 mg/kg bw/day, mean fetal weights were lower than Control (10% and 9% respectively) with male, female and total litter weights also slightly lower than control; this was associated with a lower group mean placental weight at 300 mg/kg bw/day. Group mean placental, litter and fetal weights were unaffected at 75 mg/kg bw/day. As differences were slight, within historical control data and not associated with any structural fetal abnormalities they were not deemed to represent an adverse effect of treatment. There were no major fetal abnormalities or minor skeletal or visceral structural abnormalities considered to be related to treatment. At 150 and 300 mg/kg bw/day there was also an increased incidence of incomplete ossified epiphyses, and metacarpals compared to concurrent control but within historical control data  range:  incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the slight decrease in mean fetal weight seen at these dose levels. It was therefore not considered adverse.

Oral gavage administration of alpha-pinene multiconstituent at doses up to and including 300 mg/kg bw/day to pregnant New Zealand White rabbits from Day 6-28 of gestation inclusive, was associated with slight reductions in body weight gain and in mean food consumption in females given 300 mg/kg bw/day, which were judged as non-adverse.

The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore concluded to be 300 mg/kg bw/day. The NOAEL for embryo-fetal survival and development was also concluded to be 300 mg/kg bw/day.