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.

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

other: Study plan

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS: as requested by ECHA in the final decision CCH-D-2114349164-51-01/F

Test material

Test material information

Constituent 1

Reference substance name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene; 3,7,7-trimethylbicyclo[4.1.0]hept-3-ene; 3-methylidene-6-(propan-2-yl)cyclohex-1-ene; 6,6-dimethyl-2-methylidenebicyclo[3.1.1]heptane; 7-methyl-3-methylideneocta-1,6-diene

EC Number:

932-349-8

Cas Number:

8006-64-2

Molecular formula:

C10H16

IUPAC Name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene; 3,7,7-trimethylbicyclo[4.1.0]hept-3-ene; 3-methylidene-6-(propan-2-yl)cyclohex-1-ene; 6,6-dimethyl-2-methylidenebicyclo[3.1.1]heptane; 7-methyl-3-methylideneocta-1,6-diene

Test material form:

liquid

Details on test material:

Name of test material (as cited in study report): Turpentine ; turpentine oil

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) Approximately 4-5 weeks
- Housing: up to 4 during pre-pairing period for both sexes, for males and females (after weaning) to termination and for cohorts 1A and 1B (until pairing). Females are housed individually after pairing
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

corn oil

Details on mating procedure:

F0 and F1 pairing: after 10 weeks of treatment
- M/F ratio per cage: 1/1
- Duration of pairing: Up to 2 weeks
- Proof of pregnancy: vaginal plug and sperm within vaginal smear = day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Week 1 (F0 generation), Week 1 (F1 generation), last week (F1 generation)

Frequency of treatment:

Daily

No. of animals per sex per dose:

F0 generation: 25
F1 generation: 20

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week for all F0 and selected F1 generation animals.
Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation for F0 and F1B females.

BODY WEIGHT: Yes
F0 Males: Day that treatment commences, each week, before necropsy.
F0 Females: Day that treatment commences, each week until mating detected, days 0,6, 13 and 20 after mating, days 1, 4, 7, 14, 21 and 28 post-partum and before necropsy.
F1 selected animals: Days 21, 22, 25 and 28 of age, then each week.
F1 Cohort 1B females on Days 0, 6, 13 and 20 after mating and Days 1, 4, 7, 14, 21 and 28 post-partum. Before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Hematology, Peripheral Blood (F0 and F1A generation):
10 animals/sex/group
All samples will be examined for the following characteristics:
1)Using EDTA as anticoagulant: Hematocrit, Hemoglobin concentration, Erythrocyte count, Reticulocyte count, Total leucocyte count, Differential leucocyte count, Platelet count, Mean cell hemoglobin, Mean cell volume, Mean cell hemoglobinconcentration
2)Using citrate as anticoagulant: Prothrombintime, Activated partial thromboplastin time

Blood Chemistry (F0 and F1A generation):
10 animals/sex/group
All samples will be examined for the following characteristics, using lithium heparin as anticoagulant: Alkaline phosphatase, Alanine amino-transferase, Aspartate amino-transferase, Gamma glutamyl transpeptidase, Glucose, Bilirubin, Bile acids, Cholesterol, Creatinine, Urea, Total protein, Albumin (by chemical assay), Albumin/globulin ratio, Sodium, Potassium, Chloride, Calcium, Phosphorus

TSH and T4 (F0 and F1 generation):
F0 adults: 10 animals/sex/group
F1 offspring: 10 litters/group (pooled - Day 4 of age, T4 only)
10 animals/sex/group on Day 22 of age
F1 adults (1A): 10 animals/sex/group (13 weeks of age)

Urinalysis (F0 and F1A generation):
10 animals/sex/group
Using manual methods: Clarity/colour (appearance), Volume, pH, Specific gravity
Using semi-quantitative methods (Clinitek): Glucose, Ketone, Bilirubin (bile pigments), Blood pigments
Using quantitative automated methods (Cobas 6000): Protein (total and concentration), Sodium (total and concentration), Potassium (total and concentration), Chloride (total and concentration)
The deposit, obtained from centrifugation, will be examined microscopically for epithelial cell, leucocyte, erythrocytes, crystals, casts, spermatozoa, other abnormal components

Oestrous cyclicity (parental animals):

Dry smears - F0 only: Daily for 15 days before pairing, using cotton swabs.
Wet smears - F0 and Cohort F1B: Daily after pairing until evidence of mating confirmed, using pipette lavage. For four days before scheduled termination (nominally Days 25 to 28 post-partum).

Sperm parameters (parental animals):

Cohort 1A:
Vas deferens (from left side): Sperm sample (where possible at least 200) assessed for motility using a computer assisted sperm analyzer (CASA). Each animal in each group. A manual assessment of sperm morphology will be performed. Each animal in Groups 1 and 4.

Cauda epididymis (from left side): the cauda epididymis will be weighed and homogenized and the number of sperm will be counted using a computer assisted sperm analyzer (CASA). Each animal in Groups 1 and 4.

Testis (from left side): the testis will be homogenized and the number of homogenization-resistant spermatids will be counted using a computer assisted sperm analyzer (CASA). Each animal in Groups 1 and 4.

Litter observations:

Clinical observations: Observed approximately 24 hours after birth and then daily for evidence of ill-health or reaction to treatment. Each offspring will be examined daily. On Day 1of age all offspring will receive a qualitative assessment of body temperature, state of activity and reaction to handling.

Litter size: Daily on Days 1-21 of lactation. Litters culled to 10 (where possible 5 males and 5 females) on Day 4 of age.

Sex ratio: Days 1, 4 (before and after culling) and 21 of age.

Individual offspring body weights: Days 1, 4, 7, 14, 21 and 22 of age (day of necropsy).

Ano-genital distance: Offspring on Day 1 of age

Nipple count: Male offspring on Day 13 of age

Sexual Maturation (Selected F1 Generation):
Males: Examined daily from Day 38 of age for the completion of balano-preputial separation. Body weight recorded on day of completion of separation.
Females: Examined daily from Day 28 of age until vaginal opening occurs. Body weight recorded on day of vaginal opening. For females in Cohort 1A, a wet smear will be taken daily from the day of vaginal opening until first estrus

Postmortem examinations (parental animals):

Macroscopic Pathology: All animals
F0 and F1B Females Implantation site count

Organ Weights (F0, F1A): adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thymus, thyroid with parathyroids, uterus with cervix and oviducts

Organs subjected to light microscopy (F0, F1A): Abnormalities, Adrenals, Brain (cerebellum, cerebrum, midbrain), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femurs - (longitudinal section through joint), Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Optic nerves, Ovaries, Pancreas, Pituitary, Prostate-dorsolateral and ventral combined, Rectum, Sciatic nerves, Seminal vesicles (with coagulating gland), Skeletal muscle, Skin with mammary glands (inguinal area), Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum -bone marrow, Stomach, Testes, Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus with cervix and oviducts, Vagina, Vas Deferens

Organ weights (F1B): epididymides, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, testes, thymus, uterus with cervix and oviducts

Organs bubjected to light microscopy (F1B): epididymides, ovaries, pituitary, prostate, seminal vesicles, testes, uterus with cervix and oviducts, vagina

Postmortem examinations (offspring):

Macroscopic Pathology: All animals, including surplus offspring culled on Day 4 of age. Where possible, decedent offspring ≤21 days of age (found dead or welfare kill) will be examined and carcass retained.

Statistics:

Data-types:
The following data types will be analyzed at each timepoint separately, where required, in support of interpretation:
body weight, using absolute weights and gains over appropriate study periods.
food consumption, over appropriate study periods
estrous cycles, vaginal opening to first estrus and pre-coital interval
mating performance and fertility
gestation length
litter size and survival indices
pre-weaning examination (ano-genital distance)
sexual maturation, age and body weight at completion
clinical pathology (hematology, blood chemistry, urinalysis)
thyroid hormones (TSH and T4)
immunophenotyping
organ weights, either absolute or adjusted for terminal body weight where appropriate
sperm analysis, motility, morphology and count
corpora lutea and ovarian primordial follicle counts

Methods:
For categorical data, the proportion of animals will be analyzed for each treated group (as appropriate) versus the control group.
For continuous data, Bartlett’s test will first be applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups will then be compared with the control group, incorporating adjustment for multiple comparisons where necessary.

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Remarks on result:

not determinable

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

not determinable

Results: F1 generation

Effect levels (F1)

Remarks on result:

not determinable

Results: F2 generation

Effect levels (F2)

Remarks on result:

not determinable

Overall reproductive toxicity

Reproductive effects observed:

not specified