(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2018 - 24 September 2018

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

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

developmental toxicity

Remarks:

Preliminary study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 April 2018 - 24 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted to assess the influence of delta-3-carene on embryo-fetal survival and development in the Sprague-Dawley rat and to establish suitable doses for a main embryo-fetal toxicity study.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation (Day 0 of gestation):
Group 2: approximately 72 days old.
Group 3: approximately 72 days old.
Group 4: approximately 81 days old.
Group 1: age matched with either Group 2 or 4.

- Weight at study initiation (Day 0 of gestation): 239 to 280 g
- Housing: Number of animals per cage: Acclimatization: up to three 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 acclimatisation and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum; the diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period:
Group 1 and 2: six days before commencement of pairing.
Group 3: 27 days before commencement of pairing.
Group 1 and 4: 36 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

IN-LIFE DATES: From: 25 April 2018 To: 24 June 2018

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: Starting with the lowest concentration, the required amount of test item was weighed and then mixed with the vehicle (approximately 50% of the final volume). The mixture was 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. A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Frequency of preparation: Weekly
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 60, 120 and 90 mg/mL
- Dose volume: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No formulation analysis was performed on this study.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Proof of pregnancy: Ejected copulation plugs in cage tray and presence of sperm in the vaginal smears referred to as day 0 of pregnancy
- A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating.

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

6 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 300, 600 and 450 mg/kg body weight/day) were selected in conjunction with the Sponsor Representative. Group 2 was treated at 300 mg/kg body weight/day, based on an acute oral toxicity study in the rat similarly to OECD guideline 401 (Moreno, 1972) that demonstrated the acute median lethal dose (LD50) of delta-3-carene was 4800 mg/kg body weight. Treatment to any group would be stopped if there was evidence of poor maternal tolerability (toxicity) and re-commence at a lower dose, following at least two days off-dose. Selection of the dose level to Group 3 was based on the results of Group 2, following termination. Selection of the dose level to Group 4 was based on the results of Group 2 and following seven doses to the first mated animals in Group 3.

Three control animals were allocated to study with each of Groups 2 and 4.

- Rationale for animal assignment: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare when necessary. 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(s).
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration: Pre-dose observation; On return to home cage; One to two hours after completion of dosing; As late as possible in the working day.
A detailed physical examination was performed on each surviving animal on Days 0, 5, 12, 18 and 20 after mating (where applicable) to monitor general health.

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

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive (where applicable). No food consumption measurements were recorded for spare mated females until allocated to study as part of Group 6.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Animals surviving until the end of the scheduled study period were killed on Day 20 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.
- Organs examined: Organs weighed (Liver) and tissue samples fixed (Liver, Lungs and Trachea)
Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; Gravid uterine weight (including cervix and ovaries)
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorption sites: Yes
- Number of late resorption sites: Yes
- Other: Apparently non-pregnant animals: The number of uterine implantation sites were checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski, E, 1964).

Fetal examinations:

SACRIFICE: Method of kill for fetuses - Chilling on a cool plate (approximately 0 °C)

FETAL EXAMINATION: All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each fetus and placenta was externally examined and any abnormalities were recorded, sampled as appropriate and retained in appropriate fixative. The sex of each fetus was recorded. Grossly normal fetuses were discarded.

Statistics:

No statistical analysis was performed.

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Isolated incidences of chin rubbing and/or salivation was seen shortly after dose administration in three animals receiving 300 mg/kg body weight/day and five animals receiving 600 or 450 mg/kg body weight/day. This may be due to poor palatability of the test item.
Hairloss (forelimbs or ventral surface) was evident in one Control animal and one animal receiving 300, 450 or 600 mg/kg body weight/day; brown stained muzzle was evident in one animal receiving 450 mg/kg body weight/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

One animal (No. 15) receiving 600 mg/kg body weight/day, was killed for humane reasons approximately six hours after dosing on Day 7. The animal had flattened swaying gait or prostrate posture, unresponsive behaviour, shallow respiration and piloerection. Macroscopic examination revealed no findings.
One animal (No. 17) receiving 600 mg/kg body weight/day had flattened swaying gait or prostrate posture on Day 7, approximately six hours after dosing and had piloerection at 1-2 hours after dosing on Day 8.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gain (Days 6-20) was low at 450 or 600 mg/kg body weight/day (78 or 74% of Control, respectively) and was marginally low at 300 mg/kg body weight/day (92% of Control). These differences at 600 or 450 mg/kg/day were largely due to initial mean bodyweight losses (10 g and 6 g respectively) compared with mean body weight stasis in the Control, together with low mean weight gains during Days 14-17 of gestation.
Gravid uterine weight was low at 450 or 600 mg/kg body weight/day (87 or 89% of Control, respectively), but was unaffected at 300 mg/kg body weight/day. Adjusted body weight gain (Days 6-20) was low or markedly low at 300, 450 or 600 mg/kg body weight/day (79, 51 or 38% of Control, respectively).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake at 300, 450 or 600 mg/kg body weight/day was moderately or marginally low (range 75 to 85% of Control) during Days 6-14 and was marginally low during Days 6-20 at 600 mg/kg body weight/day (85% of Control).

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

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):

Mean liver weight was unaffected by treatment at 450 mg/kg body weight/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One adult treated at 450 mg/kg body weight/day had a mammary mass; dorsocranial, right, firm, pale and dark, 40 49mm; there were no other findings in any adult.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The mean number of corpora lutea at 600 mg/kg body weight/day was 0.7 higher than Control. However due to an apparent increase in pre-implantation losses (4 litters contained 2 or more losses compared with none in Control), the mean numbers of implantations and live fetuses were slightly lower than Control. There was no conclusive increase in pre implantation losses at 450 mg/kg body weight/day.
Post-implantation loss was unaffected by the treatment.

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:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

other: no NOAEL identified

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean litter weight was marginally low at 450 or 600 mg/kg body weight/day (85 or 87% of Control, respectively) that was attributed in part, to marginally low overall fetal weights (90 and 93% of Control, respectively) at these doses; placental weight was unaffected by treatment. Mean placental weight was unaffected by treatment at 300 mg/kg body weight/day.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Mean litter weight was marginally low at 450 or 600 mg/kg body weight/day (85 or 87% of Control, respectively) that was attributed in part, to marginally low overall fetal weights (90 and 93% of Control, respectively) at these doses; placental weight was unaffected by treatment. Mean placental weight was unaffected by treatment at 300 mg/kg body weight/day.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

other: no NOAEL identified

Key result

Abnormalities:

not examined

Key result

Developmental effects observed:

not specified

None

Conclusions:

Based on the results of this study it is concluded that the high dose for the main embryo-fetal study (MP11SV) should not exceed, and perhaps should be slightly lower than 450 mg/kg body weight/day. Although treatment at this dose was tolerated, adjusted mean body weight gain was only 52% of Control with one female showing an overall adjusted weight loss of 14 g.

Executive summary:

Oral gavage administration of delta-3-carene to pregnant Sprague-Dawley rats at dose levels of 300 or 450 mg/kg body weight/day during the organogenesis and fetal phase of gestation (Days 6-19) was generally well-tolerated maternally, with no unscheduled deaths or effect on clinical condition. Treatment at 600 mg/kg body weight/day, the highest dose tested, resulted in several post dosing signs that indicated marked toxicity in one animal (flattened swaying gait or prostrate posture, unresponsive behavior, shallow respiration and piloerection) following the second dose and the animal was humanely killed. Similar signs were evident in a second animal following the second and third doses; however the general condition of this animal remained good and the animal was retained on the study. Macroscopic examination of the decedent animal revealed no findings. 

Other post dosing signs were limited to isolated and sporadic incidences of salivation, with associated chin rubbing in some animals at 300, 450 or 600 mg/kg body weight/day that may have been attributable to poor palatability of treated formulations. 

Slight mean body weight loss was evident at 450 or 600 mg/kg body weight/day, following the first or first two administrations and overall and adjusted body weight gains (Days 6-20) were low at 300, 450 or 600 mg/kg body weight/day, both correlated with low food intake (Days 6-14 and Days 6-20); mean gravid uterine weight was low at 450 or 600 mg/kg body weight/day. 

There were no treatment related macroscopic findings and all animals at 300, 450 or 600 mg/kg body weight/day were pregnant. 

Mean pre-implantation loss (%) appeared high at 600 mg/kg body weight/day and resulted in a slightly low litter size. This may be associated with treatment; however it may also be attributable to the low number of animals investigated and mean litter and fetal weights were marginally low at 450 or 600 mg/kg body weight/day. All other reproductive parameters were unaffected by treatment.

Based on the results of this study it is concluded that the high dose for the main embryo-fetal study should not exceed, and perhaps should be slightly lower than 450 mg/kg body weight/day. Although treatment at this dose was tolerated, adjusted mean body weight gain was only 52% of Control with one female showing an overall adjusted weight loss of 14 g.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

other: Raw data

Title:

Unnamed

Year:

2018

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

EC Number:

207-856-6

EC Name:

(1S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

Cas Number:

498-15-7

Molecular formula:

C10H16

IUPAC Name:

3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

impurity 1

Reference substance name:

(1R,6S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

Cas Number:

20296-50-8

Molecular formula:

C10H16

IUPAC Name:

(1R,6S)-3,7,7-trimethylbicyclo[4.1.0]hept-3-ene

impurity 2

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 3

Reference substance name:

7-methyl-3-methyleneocta-1,6-diene

EC Number:

204-622-5

EC Name:

7-methyl-3-methyleneocta-1,6-diene

Cas Number:

123-35-3

Molecular formula:

C10H16

IUPAC Name:

7-methyl-3-methyleneocta-1,6-diene

impurity 4

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-isopropenyl-1-methylcyclohexene

impurity 5

Reference substance name:

(S)-p-mentha-1,8-diene

EC Number:

227-815-6

EC Name:

(S)-p-mentha-1,8-diene

Cas Number:

5989-54-8

Molecular formula:

C10H16

IUPAC Name:

(4S)-isopropenyl-1-methylcyclohexene

impurity 6

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

impurity 7

Reference substance name:

p-mentha-1,3-diene

EC Number:

202-795-1

EC Name:

p-mentha-1,3-diene

Cas Number:

99-86-5

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,3-diene

impurity 8

Reference substance name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

2243-33-6

Molecular formula:

C10H16

IUPAC Name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

impurity 9

Reference substance name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

EC Number:

224-167-6

EC Name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

4221-98-1

Molecular formula:

C10H16

IUPAC Name:

(5R)-isopropyl-2-methylcyclohexa-1,3-diene

impurity 10

Reference substance name:

3-isopropyl-(6S)-methylenecyclohexene

Cas Number:

6153-16-8

Molecular formula:

C10H16

IUPAC Name:

3-isopropyl-(6S)-methylenecyclohexene

impurity 11

Reference substance name:

3-isopropyl-(6R)-methylenecyclohexene

Cas Number:

6153-17-9

Molecular formula:

C10H16

IUPAC Name:

3-isopropyl-(6R)-methylenecyclohexene

impurity 12

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

impurity 13

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 14

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 15

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 16

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 17

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No. : 203192
Purity : 82.2%
Name of test material (as cited in study report): delta-3-carene
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 18 February 2019

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approximately 71 days old
- Weight at study initiation: 229-283 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.

- 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: 6 days

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: 27 June 2018 To: 24 September 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: Starting with the lowest concentration, the required amount of test item was weighed and then mixed with the vehicle (approximately 50% of the final volume). The mixture was 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.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 18, 35 and 70 mg/mL
- Dose volume: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. 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).
Achieved concentration: Samples of each formulation prepared for the first and last formulation were analyzed for achieved concentration of the test item.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Proof of pregnancy: Presence of ejected copulation plug / sperm in vaginal smear referred to as day 0 of pregnancy.

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 / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

350 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: The doses used in this study (0, 90, 175 and 350 mg/kg/day) were selected in conjunction with the Sponsor and based on the results of the preliminary study (Envigo Study Number: XW55FP). In that study, one female receiving 600 mg/kg/day was killed for humane reasons on Day 7, due to toxicity, and another animal showed flattened swaying gait or prostrate posture on Day 7 and piloerection on Day 8. These findings lead to the exclusion of this dose for the main study. At 300 and 450 mg/kg/day, individual animals showed small body weight losses following the first or first two administrations. Overall body weight gain, adjusted body weight gain (Day 6 to 20) and food consumption were also marginally low. Gravid uterine weight was low at 450 mg/kg/day, but was unaffected at 300 mg/kg/day. Mean litter weight was marginally low at 450 mg/kg/day, attributed in part, to marginally low overall fetal weight. Other reproductive parameters were unaffected, there was no effect on adult liver weight and there were no adult or fetal macroscopic findings.
The high dose of 350 mg/kg/day, was expected to elicit slightly reduced body weight gain and food consumption, with the intermediate and low doses (175 mg/kg/day and 90 mg/kg/day respectively) chosen to reveal any dose related trends.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).
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: At the end of dosing each group; 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 daily from Day 6 after mating.

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

POST-MORTEM EXAMINATIONS: Yes
- Animals 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 appropriate fixative.

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

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 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:

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One animal recieving 175 mg/kg/day had salivation on Day 20, immediately before dispatch for scheduled termination.
Transient chin rubbing was evident shortly after dose administration during Days 15-19 at 90, 175 or 350 mg/kg/day, the incidences and occurence of which were related to dose; one other animal recieving 90 mg/kg/day had salivation on Day 18. These signs are normally associated with poor palatability of the test item.

Mortality:

no mortality observed

Description (incidence):

On Day 18 of gestation, one Control animal (No. 10) was found with red staining of the vaginal area, whole body pallor and piloerection and was killed for humane reasons. Macroscopic examination revealed pale liver, abnormal firm and dark contents in the stomach and caecum, abnormal dark fluid in the uterus that correlated with the red staining of the vaginal area and two dark areas (both 2 9 mm) in the lungs.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The majority of animals receiving 90, 175 or 350 mg/kg/day showed body weight loss following the first administration (mean losses of -2 g, -5 g or -8 g, respectively). Thereafter, body weight gain at 90 or 175 mg/kg/day was similar to Control, but was low during Days 14-20 at 350 mg/kg/day (81% of Control).
Gravid uterine weight was marginally, but statistically significantly low at 350 mg/kg/day (93% of Control). Adjusted body weight change (Days 6-20) was statistically significantly low or markedly low at 175 or 350 mg/kg/day (68 or 26% of Control, respectively).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

When compared with Control, mean food intake was marginally low during Days 6-10 at 90 mg/kg/day and low during Days 6 14 at 175 mg/kg/day and Days 6 20 at 350 mg/kg/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

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:

effects observed, non-treatment-related

Description (incidence and severity):

The liver of one adult treated at 175 mg/kg/day was pale and the contents of the ileum, caecum and colon were yellow and abnormal in one adult treated at 350 mg/kg/day. A relationship with treatment was not clear.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

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

Description (incidence and severity):

One female treated at 350 mg/kg/day was not pregnant

Other effects:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

175 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)

Description (incidence and severity):

On-going

Description (incidence and severity):

On-going

Description (incidence and severity):

On-going

Description (incidence and severity):

On-going

Changes in postnatal survival:

not examined

Description (incidence and severity):

On-going

Description (incidence and severity):

On-going

Description (incidence and severity):

On-going

Effect levels (fetuses)

Remarks on result:

other: On-going

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Formulation analysis:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and limit of quantification, linearity of detector response, repeatability, method accuracy and precision.

Homogeneity and stability was confirmed for delta-3-carene incorn oilformulations at nominal concentrations of 1 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (+15 to +25ºC) for 1 day and refrigerated storage (+2 to +8ºC) for up to 15 days. 

The mean concentrations of delta-3-carene in test formulations analyzed for the study were within ±8% of nominal concentrations, confirming accurate formulation.

Applicant's summary and conclusion

Conclusions:

Oral gavage administration of delta-3-carene resulted in small body weight loss following the first dose at 90, 175 or 350 mg/kg/day and subsequent low weight gain (-24%) and food intake at 350 mg/kg/day. Therefore, the No Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was 175 mg/kg/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 delta-3-carene at doses of 90, 175 or 350 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil, 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 and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

The mean concentrations of delta-3-carene in test formulations analyzed for the study were within ±8% of nominal concentrations, confirming accurate formulation.

Transient chin rubbing was evident at 90, 175 or 350 mg/kg/day and was attributed to poor palatability of delta-3-carene.

A treatment related loss in body weight was evident after the first administration at 90, 175 or 350 mg/kg/day and subsequent body weight gain during Days 14-20 of gestation at 350 mg/kg/day and food intake at 175 or 350 mg/kg/day were low. Food intake was marginally low at 90 mg/kg/day during Days 6-10 of gestation.

Adjusted body weight change was low or markedly low at 175 or 350 mg/kg/day and gravid uterine weight was marginally low at 350 mg/kg/day.

Oral gavage administration of delta-3-carene resulted in small body weight loss following the first dose at 90, 175 or 350 mg/kg/day and subsequent low weight gain (-24%) and food intake at 350 mg/kg/day. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity was 175 mg/kg/day.

Examination of the foetuses is on-going.