(1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

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:

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:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

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:

(+)-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

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:

(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 4

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 5

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 6

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. : 1000107238
Purity : 89%
Name of test material (as cited in study report): (-)-alpha-pinene
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 30 November 2021

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DRT / 1000107238
- Appearance: Colorless liquid
- Expiration date of the lot/batch: 30 November 2021

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C), under nitrogen. Protected from light and humidity.

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)) strain was used because of the historical control data available at this laboratory.

TEST ANIMALS:
- Source: Charles River (UK) Ltd.
- Sexe: Females
- Age at study initiation (Day 2 after mating): Minimum age 10 weeks.
- Weight at study initiation (Day 3 after mating): 212-326 g on arrival.
- Number of animals per cage: one
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid and solid (polycarbonate) bottom; changed at appropriate intervals. Cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Environmental enrichment: A soft white untreated wood block as aspen chew block was provided to each cage throughout the study and replaced when necessary and a plastic shelter was provided to each cage throughout the study and replaced at the same time as the cages.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum; the diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum. Bottles were changed at appropriate intervals.
- Acclimation period: 4 days from arrival on Day 2 after mating to commencement of treatment on Day 6 after mating.

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

IN-LIFE DATES: From: 01 December 2021 To: 23 December 2021.

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 possible human exposure.

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: Starting with the lowest concentration the required amount of the test item was weighed out into a beaker and 50% of the final volume of vehicle was added to the test item and magnetically stirred for two minutes to disperse the test item in the vehicle. The beaker was covered with foil during stirring. It was then made up to the required volume with vehicle. The suspension was transferred into a clear glass jar and mixed using a high shear homogenizer until it was visibly homogeneous. A lid was placed on the jar and it was mixed with a magnetic stirrer until homogeneous for a minimum of 20 minutes.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly, and have been prepared in advance of the first day of dosing within the known stability.
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 6, 10 and 20 mg/mL
- Dose volume: 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 the first week and last week of treatment were analyzed for achieved concentration of the test item.
- Analytical procedure: The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 µg/mL to 100 µg/mL and an internal standard prepared at 0.38 mg/mL.

Details on mating procedure:

Method: Time mated to identified males of the same strain and source at the supplier’s facility.
Day 0 of gestation: When positive evidence of mating was detected.
Delivery to Labcorp: On Day 2 after mating.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 20 (inclusive) after mating.

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

From mating until Day 20 (necropsy).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 1 - Control

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 2 - Test item

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 3 - Test item

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Group 4 - Test item

No. of animals per sex per dose:

22 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 30, 50 and 100 mg/kg bw/day) were selected in conjunction with the Sponsor.
In a preliminary study conducted with gestating females treated at 125 mg/kg bw/day, signs observed in association with the dosing procedures included piloerection (5/6 females), elevated gait (3/6 females), paddling of forepaws (2/6 females) and hunched posture (1/6 females). Two females were also found to be of thin build towards the end of the study (Day 18 or Day 20 of gestation). Mean body weight gain (Day 6-20 of gestation) was markedly low in females treated at 125 mg/kg bw/day when compared with Controls (37% of Control), leading to statistically significantly lower mean bodyweight at Day 20 than Controls (-20%) which correlated with low food consumption (63% of Control) recorded for these females throughout the treatment period. On Day 20 of gestation, mean gravid uterine weight was statistically significantly lower than Controls (-23%). Overall body weight gain (Day 6-20 of gestation) when adjusted for the weight of the gravid uterus revealed mean body weight loss in females treated at 125 mg/kg bw/day of 28 g when compared to a gain of 26 g in Controls. Total litter weight in females treated at 125 mg/kg bw/day was low (-24%) when compared with Controls. As the dose of 125 mg/kg bw/day led to high maternal toxicity, the dose of 100 mg/kg bw/day was considered more suitable as the high dose for this study. The subsequent low and intermediate dose levels selected were 30 and 50 mg/kg bw/day, respectively, in order to assess any dose-related effect.

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 when 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 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 acclimatisation 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 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 3, 5, 12, 18 and 21 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 3 and 6 to 21 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 3-5, 6-8, 9-11, 12-14, 15-17 and 18-20 after mating inclusive.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Animals surviving until the end of the scheduled study period were killed on Day 21 after mating by Carbon dioxide asphyxiation.
- 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.

ORGAN WEIGHTS: Yes
- The liver, kidneys and thyroid (after partial fixation) were weighed. For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.
Organ weights were presented both as absolute/unadjusted and adjusted for terminal bodyweight, using the weight recorded on the day of necropsy.

HISTOLOGY: Yes
For all adult female, thyroid were routinely preserved in 10% Neutral Buffered Formalin and then dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. Sections were stained with hematoxylin and eosin.

MICROSCOPY: Yes
Tissues preserved for examination were examined for all animals from all groups for premature deaths and scheduled kill.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 1.

Ovaries and uterine content:

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

The number of uterine implantation sites were checked after staining with ammonium sulphide.

Blood sampling:

THYROID HORMONE ANALYSIS: Yes
- Blood samples were collected at termination of the study in all surviving adults to scheduled termination on GD 21 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: Subcutaneous injection of sodium pentobarbitone

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. Fetuses examined externally with abnormalities recorded. Particular attention was paid to the external genital organs of male fetuses. The sex and ano-genital distance of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter: Sexed internally, examined for visceral abnormalities by fresh microdissection (Modified Staples technique) and subsequently fixed in Bouin’s solution.
Examination of nominally other 50% of fetuses in each litter: Sexed internally, eviscerated and fixed in Industrial Methylated Spirit (IMS).
Processing: Heads were removed from Bouin’s fixed fetuses and were subject to Wilsons free-hand serial sectioning. Torsos were retained in Bouin’s solution. 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:

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 anomaly 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
- C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
- Placental, litter and fetal weights
- Ano-genital distance, average for each litter adjusted for litter average fetal body weight
- Organ weights, absolute

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

A parametric or non-parametric analysis sequence 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.

For litter average ano-genital distance data, analysis of covariance was performed using the average fetal weight as covariate, unless non parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in fetal weight which might influence the ano-genital distance.

Significant differences between the groups compared were expressed at the 5% (p<0.05 = *) or 1% (p<0.01 = **) level.

Indices:

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 control data for fetal examinations were available on Rat/Crl:CD(SD)/Charles River UK for a period from november 2017 to April 2022 and included:
- 8 studies (3 preliminary and 5 main studies) for major and minor visceral fetal examinations
- 7 studies (2 preliminary and 5 main studies) for minor skeletal fetal examinations

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Among females surviving until scheduled termination, two females that received 100 mg/kg bw/day were thin between GD 12-14 and 19-21 and one female that received 100 mg/kg bw/day had abnormally pale feces on Day 15-21. Piloerection was observed on GD 11-21 in several females that received 30, 50 or 100 mg/kg bw/day. The number of females observed at 30 or 50 mg/kg bw/day was however similar to Control in the same period, therefore the relationship to treatment is unclear in those groups. For females at 100 mg/kg bw/day between 1 and 13 animals per day were observed with piloerection in relation to dosing in this period.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Female No. 87 that received 100 mg/kg bw/day was dispatched for reasons of animal welfare on GD 12 due to signs of decreased activity, abnormally cold to touch, piloerection and hunched posture. The animal had shown a weight loss of 22 grams between GD 8 and GD 12, and negligible food consumption from GD 9. Macroscopic examination did not reveal any abnormalities. The animal was pregnant with 12 live embryos.

Female No. 67 that received 100 mg/kg bw/day was dispatched for reasons of animal welfare on Day 17 after mating due to excessive body weight loss. Following commencement of treatment, this animal exhibited an overall weight loss of 40 grams (284 grams on Day 6 after mating; 244 grams on Day 17 after mating). The animal had shown a decrease in food consumption between Days 9 and 12 after mating, however, this increased between Days 12 and 15 after mating. Macroscopic examination did not reveal any abnormalities. This animal was found to be not pregnant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean bodyweight for females that received 100 mg/kg bw/day was consistently statistically significantly lower than Controls from GD 10 onwards, owing to low body weight gain and body weight losses in these animals on GD 6-15. Mean body weight gain in females receiving 100 mg/kg bw/day was lower than Control from GD 6 to GD 15, with a mean weight loss of 1 gram. Thereafter, on GD 15-17, body weight gain was statistically significantly higher than Control, being 36% higher, and on GD 17-21 mean body weight gain was similar to Control. Overall mean body weight gain (GD 6-21) for females receiving 100 mg/kg bw/day was subsequently statistically significantly low, at 68% of Control.

There was no effect on group mean body weight or mean body weight gain in females receiving 30 or 50 mg/kg bw/day.

When adjusted for the contribution of the gravid uterus, mean adjusted body weight was statistically significantly low in females receiving 100 mg/kg bw/day at 87% of Control, and was associated with an adjusted body weight loss between GD 6 and GD 21 of 6 grams (compared to a gain of 33 grams in Control).

There was no effect of treatment on mean adjusted body weight gain in females receiving 30 or 50 mg/kg bw/day or on the mean gravid uterine weight of female at any dose level.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Compared to Control, females receiving 100 mg/kg bw/day showed a reduction in group mean food consumption throughout the treatment period, resulting in a statistically significantly lower overall group mean food consumption, at 72% of Control.

There was no effect on group mean food consumption in females receiving 30 or 50 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:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences in serum TSH levels in pregnant rats when administered with 30, 50 and 100 mg/kg bw/day of (-)-alpha-pinene when compared to the control group.
Concerning the measurement of T3 and T4 concentrations in rat serum, there was a statistically significant decrease in T3 only (i.e. 323 pg/mL compared to the control data of 456 pg/mL ; p-value < 0.01) in the high dose group of 100 mg/kg bw/day when compared with the control group. However, this decrease is within the laboratory HCD range based on 4 similar studies performed in 2022 (i.e. 272 to 451 pg/mL) and is probably due to a high mean control value. Also, it is not associated to a decrease in T4 or an increase in TSH values. Therefore, this effect is considered to not be attributed to the treatment with no biological significance.
See results in the Table 7.8.1/1.

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):

There was no effect on absolute group mean kidney, liver or thyroid weight at any dose level investigated.

All differences in organ weight parameters were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: lack of a dose relationship or correlative findings; and/or the magnitude was considered.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no maternal macroscopic abnormalities attributed to treatment detected during necropsy at any dose level investigated.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic changes in the thyroid gland at any dose level investigated.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Daily oral gavage administration of (-)-alpha-pinene to pregnant Sprague Dawley rats at 30 or 50 mg/kg bw/day during the organogenesis phase and fetal phase of gestation (GD 6-20) was maternally well tolerated, with no effects to body weight gain, food consumption, TSH levels, fetal development and survival noted and there were no macro- or microscopic changes found that were attributed to treatment.

Two animals at 100 mg/kg bw/day were euthanized early, No. 87 on GD 12 and No. 67 on GD 17. Both were dispatched for reasons of animal welfare after both showed sustained body weight loss and decreased food consumption and No. 87 showed signs of decreased activity, abnormally cold to touch, piloerection and hunched posture. No abnormalities were recorded at necropsy and No. 87 was pregnant with 12 live embryos. No. 67 was not pregnant.

Overall mean body weight gain and overall food consumption were both statistically significantly low for females at 100 mg/kg bw/day, at 68% or 72% of Control, respectively. Mean body weight loss was recorded for these females on GD 6-15 (-1 gram), owing to low mean body weight gain (GD 6-9 and GD 9-13) and mean body weight losses (GD 9-13) in this period. At the individual level, 11 pregnant females showed some degree of weight loss during Day 6-15. Group mean food consumption was consistently statistically significantly low in the same period (46-85% of Control), with a period of marked reduction in food consumption observed in several females in this group, particularly in No. 85.
Group mean body weight gain was however statistically significantly higher than Control on GD 15-17 (136% of Control), after which it was similar to Control (GD17-21). These body weight gains coincided with an increase in group mean food consumption, although on GD 15-18 this was still statistically significantly lower than Control.

Among females surviving until scheduled termination at 100 mg/kg bw/day, two females were thin between GD 12-14 and 19-21, resulting from their low food consumption and body weight loss. One female that received 100 mg/kg bw/day had abnormally pale feces on Day 15-21.

At 100 mg/kg bw/day there was no effect on absolute group mean kidney, liver or thyroid weight, no maternal macroscopic abnormalities attributed to treatment detected during necropsy and no microscopic changes in the thyroid gland at any dose level investigated. TSH levels at 100 mg/kg bw/day were similar to Control.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

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

Changes in number of pregnant:

effects observed, non-treatment-related

Details on maternal toxic effects:

At scheduled termination on GD 21, two Control females (Nos. 2 and 3), four females receiving 30 mg/kg bw/day (Nos. 24, 25, 26 and 37) and three females receiving 50 mg/kg bw/day (Nos. 46, 47 and 66) were found not to be pregnant. Two females receiving 100 mg/kg bw/day were euthanised for welfare reasons. Reproductive assessment was therefore made using 20 Controls and 18, 19 or 20 females at 30, 50 or 100 mg/kg bw/day, respectively.

There was no effect of maternal treatment on the mean number of implantations, resorptions (early or late), live young, sex ratio, or the levels of pre- or post-implantation losses at any dose level investigated.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

50 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:

effects observed, non-treatment-related

Description (incidence and severity):

Placental weight, male and female weights were statistically significantly low at 100 mg/kg bw/day, being 92%, 93% and 93% of Control, respectively.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the live young at any dose level investigated.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the sex ratio at any dose level investigated.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Overall litter weights were statistically significantly low at 100 mg/kg bw/day, being 93% of Control.
There was no effect on overall litter weight at 30 or 50 mg/kg bw/day and no effect of maternal treatment on total litter weight at any dose level investigated.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The ano-genital distance of the male and female fetuses was unaffected by maternal treatment at any dose level.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor abnormalities showed no relationship to treatment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of major and minor abnormalities showed no relationship to treatment.
At 100 mg/kg bw/day there was an increase in fetal incidence of delayed/incomplete ossification/unossified 5th and/or 6th sternebrae compared to concurrent control and outside of fetal HCD range; the incidence of litters affected was within the HCD range.
Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the statistically significant decrease in mean fetal weight seen at this dose level. It is therefore not considered adverse.

Visceral malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor abnormalities showed no relationship to treatment.

Details on embryotoxic / teratogenic effects:

The incidence of major and minor fetal abnormalities showed no relationship to treatment and there were not affects to embryo-fetal survival at 30 or 50 mg/kg bw/day.

There was no effect on embryo fetal survival, as the mean number of implantations, resorptions (early or late), live young, sex ratio, or the levels of pre- or post-implantation losses were similar to Control.

Fetal development was, however, slightly affected, as placental weight and male, female, and overall litter weight were marginally statistically significantly low at 100 mg/kg bw/day, being 92%, 93%, 93% and 93% of Control, respectively, and there was an increase in fetal incidence of delayed/incomplete ossification/unossified 5th and/or 6th sternebrae compared to Control and outside of fetal Historical Control Data range. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the statistically significant decrease in mean fetal weight seen at this dose level. The marginal reduction in mean fetal weight together with the increase in delayed/incomplete ossification of the 5 th/6th sternebrae are expected observations due to the magnitude and persistence of the reduced maternal food consumption recorded (Fleeman et al 2005). It is therefore not considered adverse.

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:

The mean concentrations were within the applied limits of +10/-15% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis. 

 

Table 7.8.2/1. Mean serum T3 and T4 concentrations (pg/mL) within treatment groups

Group	Treatment	Dose (mg/kg bw/day)	Parameters	T3 conc. in Females at termination (pg/mL)	T4 conc. in Females at termination (pg/mL)
1	Control (vehicle^a)	0	Mean	456	16200
			SD	171	6110
			CV%	37.5	37.7
			N	22	22
2	Test item	30	Mean	496	19200
			SD	138	7580
			CV%	27.8	39.5
			N	22	22
3		50	Mean	470	19000
			SD	145	8410
			CV%	30.9	44.3
			N	22	22
4		100	Mean	323**	13400
			SD	100	3760
			CV%	31.0	28.1
			N	20	20

^a: 1% Methylcellulose

**: statistically significant difference (p-value < 0.01) was observed between the treatment group (Group 4) and control group (Group 1) by Williams test (two tailed).

Applicant's summary and conclusion

Conclusions:

Due to the magnitude of the treatment related body weight loss and low food consumption of females that received 100 mg/kg bw/day, it is concluded that, based on these data, the maternal No-Observed-Adverse-Effect-Level (NOAEL) is 50 mg/kg bw/day and the NOAEL for embryo-fetal survival, growth and development is 100 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 22 females received (-)-alpha-pinene at doses of 30, 50 or 100 mg/kg bw/day by oral gavage administration, from Day 6 to 20 after mating. A similarly constituted Control group received the vehicle, 1% methylcellulose at the same volume dose as treated groups. Animals were killed on Day 21 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 21 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight were recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

 

The mean concentrations of the dose formulations were within the applied limits of +10/-15% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis.

 

There were two test item related early deaths in the study at the high dose. Two females that received 100 mg/kg bw/day were euthanised, on GD 12 or Day 17 after mating, after one showed signs of decreased activity, abnormally cold to touch, piloerection and hunched posture and both had weight loss and low food consumption.

At 100 mg/kg bw/day, two females were thin between GD 12-14 and 19-21, one female had abnormally pale feces on Day 15-21. The group mean body weight at this dose level was consistently statistically significantly lower than Controls from GD 10 onwards, owing to low body weight gain and body weight losses in these animals on GD 6-15. Mean body weight gain in females receiving 100 mg/kg bw/day was lower than Control from GD 6 to GD 15, with a mean weight loss of 1 gram. Thereafter, on GD 15-17, body weight gain was statistically significantly higher than Control, being 36% higher, and on GD 17-21 mean body weight gain was similar to Control. Overall mean body weight gain (GD 6-21) of these animals was subsequently statistically significantly low, at 68% of Control. Group mean food consumption was also consistently lower than Control at 100 mg/kg bw/day throughout the treatment period, resulting in a statistically significantly lower overall group mean food consumption, at 72% of Control. When adjusted for the contribution of the gravid uterus, mean adjusted body weight was also statistically significantly low in females receiving 100 mg/kg bw/day, at 87% of Control, and was associated with body loss between GD 6 and GD 21 of 6 grams (compared to a gain of 33 grams in Control).

There were no statistically significant differences in serum Thyroid Stimulating Hormone (TSH) levels in pregnant rats when administered with 30, 50 and 100 mg/kg bw/day of (-)-alpha-pinene when compared to the control group. Concerning the measurement of T3 and T4 concentrations in rat serum, there is a statistically significant decrease (p-value < 0.01) observed in pregnant rats when administered with 100 mg/kg bw/day of (-)-alpha-pinene when compared to the control group. This effect is not attributed to the treatment as it is within the laboratory HCD range. Adult thyroid weight was unaffected by treatment at any dose and the tissue was macroscopically and microscopically normal.

 

There was no effect of treatment on embryo fetal survival, as the mean number of implantations, resorptions (early or late), live young, sex ratio, or the levels of pre- or post-implantation losses were similar to Control. Fetal development was, however, affected, as placental weight and male, female, and overall litter weight were marginally statistically significantly low at 100 mg/kg bw/day, being 92%, 93%, 93% and 93% of Control, respectively, and there was an increase in fetal incidence of delayed/incomplete ossification/unossified 5th and/or 6th sternebrae compared to Control and outside of fetal Historical Control Data range. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the statistically significant decrease in mean fetal weight seen at this dose level. It is therefore not considered adverse.

Maternal treatment at 30 or 50 mg/kg bw/day was well tolerated by the dams and there were no effects to embryo fetal survival or development.

 

Due to the magnitude of the treatment related body weight loss and low food consumption of females that received 100 mg/kg bw/day, it is concluded that, based on these data, the maternal No-Observed-Adverse-Effect-Level (NOAEL) is 50 mg/kg bw/day and the NOAEL for embryo-fetal survival, growth and development is 100 mg/kg bw/day.