Gum turpentine oil, consisting of the volatile fraction resulting from the distillation of oleoresin collected by tapping softwoods from pinacea derivatives (genus: pinus). It consists of terpenes, mainly monoterpenes such as alpha-pinene and beta-pinene.

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March-May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

other: Study plan

Title:

Unnamed

Year:

2017

Report date:

2017

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:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pin-2(3)-ene

EC Number:

201-291-9

EC Name:

Pin-2(3)-ene

Cas Number:

80-56-8

Molecular formula:

C10H16

IUPAC Name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Constituent 2

Reference substance name:

Pin-2(10)-ene

EC Number:

204-872-5

EC Name:

Pin-2(10)-ene

Cas Number:

127-91-3

Molecular formula:

C10H16

IUPAC Name:

6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Constituent 3

Reference substance name:

Dipentene

EC Number:

205-341-0

EC Name:

Dipentene

Cas Number:

138-86-3

Molecular formula:

C10H16

IUPAC Name:

4-isopropenyl-1-methylcyclohexene

Constituent 4

Reference substance name:

Camphene

EC Number:

201-234-8

EC Name:

Camphene

Cas Number:

79-92-5

Molecular formula:

C10H16

IUPAC Name:

2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Constituent 5

Reference substance name:

p-mentha-1,4(8)-diene

EC Number:

209-578-0

EC Name:

p-mentha-1,4(8)-diene

Cas Number:

586-62-9

Molecular formula:

C10H16

IUPAC Name:

4-isopropylidene-1-methylcyclohexene

Constituent 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

Constituent 7

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

Constituent 8

Reference substance name:

7,7-dimethyl-2-methylenebicyclo[2.2.1]heptane

EC Number:

610-330-2

Cas Number:

471-84-1

Molecular formula:

C10H16

IUPAC Name:

7,7-dimethyl-2-methylenebicyclo[2.2.1]heptane

Constituent 9

Reference substance name:

p-mentha-1,4-diene

EC Number:

202-794-6

EC Name:

p-mentha-1,4-diene

Cas Number:

99-85-4

Molecular formula:

C10H16

IUPAC Name:

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

Constituent 10

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

Constituent 11

Reference substance name:

p-mentha-1(7),2-diene

EC Number:

209-081-9

EC Name:

p-mentha-1(7),2-diene

Cas Number:

555-10-2

Molecular formula:

C10H16

IUPAC Name:

3-isopropyl-6-methylenecyclohexene

Test material form:

liquid

Details on test material:

Batch No.: FAB-08082017
Name of test material (as cited in study report): gum turpentine oil
Physical state: colourless - slightly yellow liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiration Date: 07 August 2018

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) Limited
- Age at study initiation: Approximately 65 days old
- Weight at study initiation: 227 to 254 g
- Housing: After mating and allocation, females will be housed singly to permit collection of food consumption data individually for pregnant females.
Grid bottomed cages will be suspended above absorbent paper which will be changed daily during pairing. Solid bottomed cages will have bedding which will be changed at appropriate intervals. Cages, cage-trays, food hoppers and water bottles will be changed at appropriate intervals.
- Diet (ad libitum): SDS VRF1 Certified pelleted diet
- Water (ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes.
- Acclimation period: Five days before commencement of pairing.

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Method of preparation: The formulation procedure will be documented in the study data and included in the final report.
- Frequency of preparation: Weekly. If weekly formulation is not supported by the homogeneity and stability data, a more frequent mixing regime will need to be initiated
- Storage of preparation: Refrigerated (nominally 2-8 °C)

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Week 1 and the last week will be analyzed for achieved concentration of the test item.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: Ejected copulation plug / sperm in vaginal smear referred to as Day 0 of pregnancy.
- A colony of stud males is maintained specifically for the purpose of mating; these animals will not be part of the study and are maintained as stock animals

Duration of treatment / exposure:

14 days (Days 6-19 p.c.)

Frequency of treatment:

Once daily at approximately the same
time each day

No. of animals per sex per dose:

20 mated females/dose

Control animals:

yes

Details on study design:

Dose levels will be selected in conjunction with the Sponsor based on the results of the preliminary embryo-fetal toxicity study in Sprague Dawley rats (Envigo Study No. YH66TP).

Examinations

Maternal examinations:

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

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

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

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

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Animals will be killed by carbon dioxide asphyxiation on Day 20 after mating.
- All adult animals will be subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues will be performed. All external features and orifices will be examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) will be recorded and the required tissue samples preserved in appropriate fixative.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; Gravid uterine weight (including cervix and ovaries)
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorption sites: Yes
- Number of late resorption sites: Yes
- Number of live fetuses: Yes
- Number of dead fetuses: Yes

Fetal examinations:

Fetuses will be killed by chilling on a cool plate (approximately 0 °C).
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. The sex of each fetus will be recorded.
Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
Fixation: Fetuses eviscerated will be fixed in Industrial Methylated Spirit (IMS). Remaining fetuses will be fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed fetuses will be subject to free-hand serial sectioning for visceral abnormalities. IMS fixed fetuses will be processed and stained with Alizarin Red for skeletal development and abnormalities.

Statistics:

See "Any other information on materials and methods incl. tables"

Indices:

Reproductive assessment
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100
Post-implantation loss (%) = [(Number of implantations – Number of live fetuses)/ Number of implantations] x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Mortality:

no mortality observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Effect levels (maternal animals)

Remarks on result:

other: On-going study

Results (fetuses)

Changes in postnatal survival:

not examined

Description (incidence and severity):

not relevant for OECD 414

Effect levels (fetuses)

Remarks on result:

other: On-going study

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

A preliminary study is currently on-going.

Applicant's summary and conclusion

Conclusions:

A preliminary study is currently on-going.

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, gum turpentine oil will be administered by oral (gavage) to groups of mated female Crl:CD(SD) rats (20/dose) from Days 6 to 19 after mating at dose levels determined according to the results of the on-going preliminary study.

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