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.

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 2017 - 07 September 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 same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 December 2017 - 06 July 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 required

Principles of method if other than guideline:

This study is used to determine the palatability and the systemic toxic potential of Gum turpentine oil in a 3-week dietary study in CD rats, to select a suitable high dose for a subsequent 13-week toxicity study.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: FAB-08082017
- Expiration date of the lot/batch: 07 August 2018
- Purity test date: 08 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2°C to 8°C) under nitrogen. Kept away from light and humidity

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 36-42 days
- Weight at study initiation: Males: 176-219 g; Females: 142-178 g
- Housing: Animals were housed up to 3/cage in polycarbonate cages with a stainless steel mesh lid
- Diet: Teklad 2014CM, powdered diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

Route of administration:

oral: feed

Vehicle:

other: Stabiliser: Corn oil (test material to corn oil ratio 5:1)

Details on oral exposure:

VEHICLE: corn oil to minimise evaporation

DIET PREPARATION
- Method of preparation: On each occasion of the preparation of the premix the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equaled the weight of test substance was added and the mixture stirred together. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet.
Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.

- Frequency of preparation: Weekly and subdivided into daily aliquots which were stored frozen prior to administration

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analyses of the formulated diets were scheduled on this study.

Duration of treatment / exposure:

Three weeks

Frequency of treatment:

Continuously

Dose / conc.:

15 000 ppm

Dose / conc.:

7 500 ppm

Dose / conc.:

3 500 ppm

No. of animals per sex per dose:

5

Control animals:

other: Untreated diet of the same batch (with corn oil)

Details on study design:

- Dose selection rationale: An acute oral toxicity study conducted in the rat similarly to OECD guideline 401 (Moreno, 1972) showed that the median lethal dose (LD50) of Gum turpentine oil was greater than 3700 mg/kg body weight. However, in a recent GLP study conducted according to OECD guideline 423, the LD50 of alpha-pinene multiconstituent was found to be between 300 and 2000 mg/kg bwt. Also, in a preliminary study on alpha-pinene multiconstituent (Envigo study Number: GR06DS), overall body weight gains for males exposed to 3000, 6000 and 12000 ppm were low at all treatment levels (range 77 to 84% of Control). It was confirmed in the following OECD guideline 421 study (Envigo study Number: CM17RD), in which animals were exposed at 12000 ppm for 36 days for males and during the first 22 days before pairing for mating for females that an overall decrease in bodyweight gain of 12% and 66% was observed, respectively. Pre-study chemistry results showed 20% loss of test item and as a result, the treatment levels were increased by 20% to account for the initial loss. As the test item contained about 75% of alpha-pinene multiconstituent and that palatability issues were anticipated at 15000 ppm, this dose was considered as the highest dose to be tested.
- Rationale for animal assignment: Randomly allocated on arrival.

Positive control:

Not applicable

Observations and examinations performed and frequency:

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 occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on Days -3, 1, 4, 8, 11, 15, 18 and 21 on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded daily throughout the study from Week -1 (Day -5) and before necropsy.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily throughout the study from Week -1 (Day -5).

WATER CONSUMPTION:
- Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 3 (at termination)
- Anaesthetic used for blood collection: Yes; under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes; overnight.
- Parameters checked:
HAEMATOLOGY: Haematocrit (Hct)*, Haemoglobin concentration (Hb)*, Erythrocyte count (RBC)*, Absolute reticulocyte count (Retic)*, Mean cell haemoglobin (MCH)*, Mean cell volume (MCV)*, Mean cell haemoglobin concentration (MCHC)*, Red cell distribution width (RDW)* Total leucocyte count (WBC), Differential leucocyte count, Platelet count (Plt), Abnormalities of the blood morphology, Prothrombin time (PT) and Activated partial thromboplastin time (APTT)
CLINICAL CHEMISTRY: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea, Bilirubin, Blood urea nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Total protein (Total Prot) and Albumin (Alb), Triglycerides, Albumin/globulin ratio, Chloride, Calcium, Phosphorus and Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE: Animals were killed following three weeks of treatment by carbon dioxide asphyxiation with subsequent exsanguination

GROSS PATHOLOGY: Yes; All 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:
The required organs were weighed for all animals (see Table 7.5.1/1). For bilateral organs, left and right organs were weighed together.

HISTOPATHOLOGY: No
Fixation: Tissues were preserved in 10% Neutral Buffered Formalin with the exception of the testes which were retained in modified Davidson’s fluid.

Other examinations:

None

Statistics:

None

Clinical signs:

no effects observed

Description (incidence and severity):

Administration for 21 days at dose levels up to and including 15000 ppm was well tolerated. There were no premature deaths and no test item-related changes in clinical condition.

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths and no test item-related changes in clinical condition.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean bodyweight loss was observed on Day 2 in males and females receiving 15000 ppm, however by Day 3, this effect was no longer observed.
Overall mean bodyweight gains in males at 15000 ppm were slightly lower when compared to Control (93%).
No effect of treatment on mean overall bodyweight gain was observed at 3500 and 7500 ppm in males and at all dose levels in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption for males and females receiving Gum turpentine oil at 15000 ppm was lower than that of Control on Day 1 of treatment. Food consumption remained slightly low on Day 2 of treatment in males and females receiving 15000 ppm but was generally similar to that of the Controls and pre treatment intake in males and females thereafter.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute and adjusted liver weights were higher than that of Control for males and females receiving 15000 ppm (absolute weights 13% and 7% higher than Controls, respectively), with a dose-related increase in males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Distended stomachs were identified in two males receiving 15000 ppm and one female receiving 3500 ppm.
No other treatment-related findings at the macropathological examination were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

It was concluded that there were no findings that would preclude the use of 15000 ppm as the high dietary concentration to be tested on the main OECD 408 study.

Executive summary:

In a preliminary repeated dose toxicity study, three groups, each comprising five male and five female CD rats, received Gum turpentine oil at dietary concentrations of 3500, 7500 or 15000 ppm. A similarly constituted control group received the vehicle, basal diet with added corn oil. During the study, clinical condition, body weight, food consumption, visual water consumption, hematology (peripheral blood), blood chemistry, organ weight and macropathology investigations were undertaken.

The overall mean achieved dosages were 319, 705 and 1311 mg/kg bw/day in males and 306, 655 and 1310 mg/kg bw/day in females receiving 3500, 7500 and 15000 ppm, respectively.

Administration for 21 days at dose levels up to and including 15000 ppm was well tolerated. There were no premature deaths and no test item-related changes in clinical condition.

Mean bodyweight loss was observed on Day 2 in males and females receiving 15000 ppm, however on Day 3, this effect was no longer observed. Overall mean bodyweight gains in males at 15000 ppm were slightly lower when compared with Control (93%). No effect of treatment on overall mean bodyweight gain was observed at 3500 and 7500 ppm in males and at all dose levels in females.

The food consumption for males and females receiving Gum turpentine oil at 15000 ppm was lower than that of Control on Day 1 of treatment. Food consumption remained slightly low on Day 2 of treatment in males and females receiving 15000 ppm but was generally similar to that of the Controls and pretreatment intake in males and females thereafter.

The hematological examination of peripheral blood and the biochemical examination of plasma performed during week 3 of treatment for males and females did not reveal any toxicologically significant differences from Control.

Mean absolute and adjusted liver weights were higher than that of Control for males and females receiving 15000 ppm (absolute weights 13% and 7% higher than Controls, respectively), with a dose-related increase in males. All other organ weights for males and females were unaffected by treatment.

Distended stomachs were identified in two males receiving 15000 ppm and one female receiving 3500 ppm. No other treatment-related findings at the macropathological examination were observed.

Therefore, it was concluded that there were no findings that would preclude the use of 15000 ppm as the high dietary concentration to be tested in the main OECD 408 study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

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:

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)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 41-47 days
- Weight at study initiation: Males: 188-255 g; Females: 153-194 g
- Housing: Animals will be housed up to 3/cage in polycarbonate cages with a stainless steel mesh lid
- Diet: Teklad 2014CM, powdered diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: Stabiliser: Corn oil (test material to corn oil ratio 5:1).

Details on oral exposure:

VEHICLE: corn oil to minimise evaporation

DIET PREPARATION
- Method of preparation: On each occasion of the preparation of the premix the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equaled the weight of test substance was added and the mixture stirred together. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet.
Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.

- Frequency of preparation: Weekly and subdivided into daily aliquots which were stored frozen prior to administration

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 12 of treatment were analysed for achieved concentration of the test substance.

Duration of treatment / exposure:

Main phase: Minimum of 90 days (13 weeks)
Recovery phase: 4 weeks

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 500 ppm

Doses / concentrations 2

Dose / conc.:

7 500 ppm

Doses / concentrations 3

Dose / conc.:

15 000 ppm

No. of animals per sex per dose:

Main phase: 10 animals/sex/dose
Recovery phase: 5 animals/sex/dose (2 groups only)

Control animals:

other: Untreated diet of the same batch (with corn oil)

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

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 occupants.
During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded daily for the week before treatment commenced, on the day that treatment commenced (Day 1), daily for the first two weeks of treatment (until Day 15) and twice weekly throughout the rest of the study and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily for the week before treatment started, daily for the first two weeks of treatment (until Day 15) and twice weekly throughout the rest of the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of the animals were examined by means of a binocular indirect ophthalmoscope and a slit-lamp biomicroscope
- Time schedule for examinations: Pre-treatment: all animals; Week 12: All animals of Groups 1 and 4
- The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 (at termination): All main phase animals
- Anaesthetic used for blood collection: Yes; under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes; overnight
- Parameters checked:
HAEMATOLOGY: Haematocrit (Hct)*, Haemoglobin concentration (Hb)*, Erythrocyte count (RBC)*, Absolute reticulocyte count (Retic)*, Mean cell haemoglobin (MCH)*, Mean cell volume (MCV)*, Mean cell haemoglobin concentration (MCHC)*, Red cell distribution width (RDW)* Total leucocyte count (WBC), Differential leucocyte count, Platelet count (Plt), Abnormalities of blood morphology, Prothrombin time (PT) and Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea, Bilirubin, Blood urea nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Total protein (Total Prot) and Albumin (Alb), Triglycerides, Albumin/globulin ratio, Chloride, Calcium, Inorganic phosphorus # and Bile acids #
# Samples collected in Recovery Week 4 were examined for these parameters only.

URINALYSIS: Yes
- Time schedule for collection of blood: Week 13 (at termination): All main phase animals
- Conditions: overnight in an individual metabolism cage with deprivation of food and water
- Parameters checked: Appearance, volume, pH, specific gravity, glucose, ketone, bilirubin, blood pigments, urobilinogen, protein

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity
Time schedule for examinations:
- Sensory reactivity and grip strength assessments were performed on the first five main study animals and all recovery phase animals in Groups 1 and 4 and all main study animals from Groups 2 and 3 during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.
- During Week 12 of treatment, the motor activity of the first five main study animals and all recovery phase animals in Groups 1 and 4 and all main study animals from Groups 2 and 3 was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo. In addition, all recovery phase females were tested during Week 4 of recovery.

Sacrifice and pathology:

SACRIFICE: Main phase animals were killed following 13 weeks of treatment. Recovery phase animals were killed following 13 weeks of treatment and 4 weeks of recovery. Animals were killed by carbon dioxide asphyxiation with subsequent exsanguination.

GROSS PATHOLOGY: Yes; All main study and recovery 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: For bilateral organs, left and right organs were weighed together, unless specified in Table 7.5.1/1.

HISTOPATHOLOGY: Yes
Fixation: Tissues were preserved in 10 % Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid; Eyes: In Davidson’s fluid.
Histology:
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required. Kidneys Main study males from Groups 2 and 3. All recovery males.
Full List: Main phase animals of Groups 1 and 4 killed at a scheduled termination.
Routine staining: Sections were stained with haematoxylin and eosin.
Light microscopy: Tissues preserved for examination were examined as specified in Table 7.5.1/1 for all Main phase animals of Groups 1 and 4
Males from Group 1, Group 2, Group 3 and Group 4, and males from Recovery Groups 1 and 4 were examined for alpha 2µ globulin nephropathy.

Other examinations:

None

Statistics:

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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

15000 ppm was well tolerated and showed no test-item related changes in clinical condition.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean bodyweight loss was observed over Days 1-2 in males and females receiving
15000 ppm (-18 g and -11 g, respectively). This contributed to statistically low mean bodyweight gains over Days 1-8 of treatment for both males and females at 15000 ppm when compared with Control (69 and 57% of Control, respectively).
Statistically significant low mean bodyweight gains were also noted over Days 36-43 and Days 57-64 for males and over Days 8-15, 29-36, 36-43 and 57-64 for females receiving 15000 ppm when compared with Control (males: 75 and 68% of Control; females: 74, 56, 40 and 37% of Control, respectively). A mean bodyweight loss was observed over Days 85-92 in females receiving 15000 ppm (-3 g).
These low mean bodyweight gains contributed to the low overall bodyweight gains over Days 1-92 in males and females at 15000 ppm (88 and 76% of Control, respectively) and to the low bodyweights in males and females treated at 15000 ppm at the end of the treatment period (93% and 87% of Control, respectively).
There was no significant effect on mean bodyweight gains in males and females at 3500 and
7500 ppm during the treatment phase and mean bodyweight gains in males and females at 15000 ppm during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption for males and females receiving 3500, 7500 and 15000 ppm was lower than that of Control on Day 1 of treatment with a dose-dependent decrease (Males: 96, 79 and 17% of Control; Females: 82, 41 and 24% of Control, respectively).
Overall mean food consumption over Days 1-92 also showed a dose-dependent decrease in males and females receiving 3500, 7500 and 15000 ppm (Males: 98, 97 and 92% of Control; Females: 93, 90 and 83% of Control, respectively), with statistical signficiance attained in males receiving 15000 ppm and in females receiving 7500 and 15000 ppm.
Food consumption during the recovery phase was unaffected by treatment.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The haematological examination of peripheral blood performed during Week 13 of treatment for males and females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included an isolated statistically significantly low mean eosinophil counts in males at 15000 ppm (50% when compared with Control). In females treated at 15000 ppm there was a statistically significantly low mean red cell distribution width, high large unstained cell concentration and high prothrombin time (96, 157 and 111%, respectively) when compared with Control values.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma in Week 13 indicated high mean bile acid concentrations in males and a dose-dependent increase in females at 3500, 7500 and 15000 ppm when compared with Controls (Males: 173, 154 and 201% of Control; Females: 171, 284 and 595% of Control, respectively). The biochemical examination of the blood plasma in Week 4 of recovery showed mean bile acid concentrations at 15000 ppm in males and females similar to Control values, indicating reversibility of this isolated finding.
Males receiving 3500, 7500 and 15000 ppm, and females receiving 7500 and 15000 ppm showed an increase, compared with Controls, in mean phosphorus concentrations after 13 weeks of treatment (Males: 109, 106 and 110%; Females: 114 and 121% of Control, respectively). After 4 weeks of recovery, phosphorus concentrations were not significantly increased when compared to Control in males at 15000 ppm. In addition, Control values were higher than the phosphorus concentrations observed at 15000 ppm during Week 13 of treatment and therefore this finding is considered attributed to normal biological variation.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Sensory reactivity for males and females and grip strength for males was unaffected by treatment.
The hindlimb values for females receiving 7500 and 15000 ppm were lower than Controls but without statistical significance and individual mean values remained within Control mean values.
The forelimb values for males receiving 15000 ppm were lower than Control values but without statistical significance and with only one value clearly below the minimum Control mean value (0.81-0.87-0.88 and 0.89 vs. 0.91 at 15000 ppm and Controls, respectively).
The forelimb values for treated females were lower than Control values but without statistical significance. Individual mean values at 3500 and 7500 ppm remained above the minimum Control mean value. Only 2 individual mean values at 15000 ppm were lower than minimum mean value in females (0.67 and 0.72 vs. 0.78, at 15000 ppm and Controls, respectively).
Comparison with Historical Control Data (HCD) is not relevant as some Control values are also lower than the minimum value of the HCD.

The majority of group mean high beam scores and to a lesser extent low beam scores for all treated females were high compared with Controls. Total high beam scores for females receiving 7500 and 15000 ppm attained statistical significance along with a few of the individual 6-minute scores during the first half of the 1-hour recording period. However, these values remained within the HCD. Also, motor activity was not significantly different from Controls during the recovery phase.
Motor activity for males was unaffected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The analysis of organ weights after 13 weeks of treatment indicated statistically significantly high mean body weight adjusted kidneys in males at 3500, 7500 and 15000 ppm (13, 16 and 22% higher than Controls, respectively), high mean body weight adjusted liver weights in males and statistically significantly high mean body weight adjusted liver weights in females at 3500, 7500 and 15000 ppm (Males: 4, 8 and 8%; Females: 11, 15 and 18% higher than Controls, respectively).
After 4 weeks of recovery, kidney weights in males and liver weights in males and females at 15000 ppm were comparable with Control.
All other organs were unaffected by treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Pale color of the kidneys was seen in all treated male animals. Pale areas were seen in kidneys of one male treated at 3500 ppm and one male treated at 15000 ppm.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minimal to moderate hyaline droplets (positively stained for alpha 2µ-globulin by immunohistochemistry) in the cortical tubules were seen in all males given 3500 ppm, 7500 ppm or 15000 ppm of Gum turpentine oil, with a dose dose-dependent increase on incidence and severity. All other changes were secondary to the hyaline droplets accumulation and consisted of minimal to slight basophilia of the tubular epithelium, corticomedullary/cortical granular casts, and interstitial mononuclear cell infiltrate.
Following a 4 -week recovery period, similar findings were observed in the kidney of the males, but reduced in terms of incidence and/or severity. Minimal hyaline droplets (positively stained for alpha 2µ-globulin by immunohistochemistry) in the cortical tubules were seen in one male only. Tubular basophilia was seen in all males and granular casts were seen in almost half of males, while minimal mononuclear inflammatory cell infiltrate was recorded for one male.
Focal tubular basophilia and minimal mononuclear inflammatory cell infiltrate were also seen in a few control males. Based on its focal nature, the constellation of these lesions is characteristic of chronic progressive nephropathy (CPN), a common spontaneous lesion in kidneys of rodents

Histopathological findings: neoplastic:

no effects observed

Details on results:

The homogeneity and stability was confirmed for Gum turpentine oil in Teklad 2014C diet with corn oil stabilizer formulations at nominal concentrations of 500 ppm and 20000 ppm at ambient temperature (15 to 25ºC) for 8 days and frozen temperature (-10 to -30ºC) for 15 days.
The mean concentrations of Gum turpentine oil in test formulations analyzed for the study were within the applied limits of +10/-15% of nominal concentrations, confirming accurate formulation. The difference from mean value remained within 1%, confirming precise analysis. Procedural recoveries remained with the validated range, confirming the continued accuracy of the analytical methodology.

The overall mean achieved dosages were 215, 461 and 942 mg/kg bw/day in males and 249, 522 and 1033 mg/kg bw/day in females receiving 3500, 7500 and 15000 ppm, respectively.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

food consumption and compound intake

haematology

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Effect level:

15 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

3

Dose descriptor:

NOAEL

Effect level:

3 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3 500 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other parameters, the analyses were carried out using the individual animal as the basic experimental unit.

 

The following data types were analyzed at each timepoint separately:

Grip strength and motor activity

Body weight, using gains over appropriate study periods

Food consumption, over appropriate study periods

Hematology

Blood chemistry

Urinalysis

Organ weights, absolute or adjusted for terminal body weight

 

The following comparisons were performed:

Group 1 vs 2, 3 and 4

Group 1 vs 4 (recovery phase only)

 

The following sequence of statistical tests was used for grip strength, motor activity, body weight, food consumption, organ weight and clinical pathology data:

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test wasapplied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F distribution with 1 and error degrees of freedom. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead. Where there were only two groups, comparisons were made using t-tests.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. This test is designed to be used when the main test for comparison of the means is a non-parametric monotonic trend test, such as Shirley's test (Shirley 1977). The test statistic compares the non-monotonicity sums of squares, NRSS, for the deviations of the observed mean ranks from the maximum likelihood mean ranks with the non-parametric equivalent of the error sums of squares, ERSS = N(N+1)/12. The test statistic is H1 = NRSS/ERSS which can be compared to standard tables of theχ2-distribution with 1 degree of freedom. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate

test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel 1959) was performed instead. Where there were only two groups, comparisons were made using Wilcoxon rank sum tests (Wilcoxon 1945).

For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values <c versus values ≥c, and for ii) values ≤c versus values >c, as applicable.

For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.

 

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level. The key to the annotation used on the tables that contain statistical results is given below.

1: Data were log transformed for the statistical analysis

Av: Pretreatment comparison of all groups using Analysis of variance followed by pairwise t-tests

Du: Treated groups compared with Control using Dunnett’s test

Sh: Treated groups compared with Control using Shirley’s test

Wi: Treated groups compared with Control using Williams’ test

Tt: Group 4 compared with Control using t-test

Wc: Group 4 compared with Control using Wilcoxon rank sum test

* p<0.05

** p<0.01

Applicant's summary and conclusion

Conclusions:

In conclusion, dietary administration of Gum turpentine oil to Sprague-Dawley rats at concentrations of 3500, 7500 and 15000 ppm for 13 weeks was well tolerated but induced a dose-dependent decrease in bodyweight and bodyweight gains associated with a decrease in food consumption, probably due to the low palatability of the preparations with the test item. The treatment elicited adaptative/reversible and isolated changes in forelimb grip strength, high mean bile acid concentrations and high mean body weight adjusted liver weights. Histopathological changes in the kidneys in the males associated with high kidney weights, were observed and considered indicative of the species and sex specific alpha 2μ-globulin nephropathy.
When excluding the species- and sex-specific histopathological findings observed in male kidneys, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity is 15000 ppm (Males: 942 mg/kg bw/day; Females: 1033 mg/kg bw/day), corresponding to the highest dose tested.

Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 408 and in compliance with GLP, three groups, each comprising ten male and ten female Sprague Dawley rats, received Gum turpentine oil at dietary concentrations of 3500, 7500 or 15000 ppm. A similarly constituted control group received untreated basal diet for the same duration. A further five male and five female Sprague Dawley rats were assigned to each of the control and high dietary concentration groups. These animals were treated for 13 weeks, followed by a four-week period without treatment to assess the potential for any treatment-related change to recover.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by daily visual observation), ophthalmic examination, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

The overall mean achieved dosages were 215, 461 and 942 mg/kg bw/day in males and 249, 522 and 1033 mg/kg bw/day in females receiving 3500, 7500 and 15000 ppm, respectively.

Administration of Gum turpentine oil at dietary concentrations up to and including 15000 ppm was well tolerated and showed no test-item related changes in clinical condition.

Sensory reactivity for males and females and grip strength for males were unaffected by treatment. The hindlimb values for females receiving 7500 and 15000 ppm were lower than Controls but without statistical significance and individual mean values remained within Control mean values. Forelimb values for males and females were lower than Controls, especially at 15000 ppm but this decrease was not statistically significant and limited to few individuals lower than Control values.

The majority of group mean high beam scores and to a lesser extent low beam scores for all treated females were high compared with Controls. Total high beam scores for females receiving 7500 and 15000 ppm attained statistical significance along with a few of the individual 6-minute scores during the first half of the 1-hour recording period. However, these values remained within the HCD. Also, motor activity was unaffected in females during the recovery phase and motor activity for males was unaffected by treatment.

Mean bodyweight loss was observed over Days 1-2 in males and females receiving 15000 ppm (-18 g and -11 g, respectively). This contributed to statistically low mean bodyweight gains over Days 1-8 of treatment for both males and females at 15000 ppm when compared with Control (69 and 57% of Control, respectively). Statistically significant low mean bodyweight gains were also noted over Days 36-43 and Days 57-64 for males and over Days 8-15, 29-36, 36-43 and 57-64 for females receiving 15000 ppm when compared with Control (males: 75 and 68% of Control; females: 74, 56, 40 and 37% of Control, respectively). A mean bodyweight loss was observed over Days 85-92 in females receiving 15000 ppm (-3 g). These low mean bodyweight gains contributed to the low overall bodyweight gains over Days 1-92 in males and females at 15000 ppm (88 and 76% of Control, respectively) and to the low bodyweights in males and females treated at 15000 ppm at the end of the treatment period (93% and 87% of Control, respectively). There was no significant effect on mean bodyweight gains in males and females at 3500 and 7500 ppm during the treatment phase and mean bodyweight gains in males and females at 15000 ppm during the recovery period

The food consumption for males and females receiving 3500, 7500 and 15000 ppm was lower than that of Control on Day 1 of treatment with a dose-dependent decrease (Males: 96, 79 and 17% of Control; Females: 82, 41 and 24% of Control, respectively), with a dose-dependent decrease. Overall mean food consumption over Days 1-92 also showed a dose-dependent decrease in males and females receiving 3500, 7500 and 15000 ppm (Males: 98, 97 and 92% of Control; Females: 93, 90 and 83% of Control, respectively). Food consumption during the recovery phase was unaffected by treatment.

No treatment related ophthalmic changes were evident in Week 12 of treatment.

The haematological examination of peripheral blood and the urinalysis examination performed during Week 13 of treatment for males and females did not reveal any toxicologically significant differences from control.

The biochemical examination of the blood plasma in Week 13 indicated high mean bile acid concentrations in males and a dose-dependent increase in females at 3500, 7500 and 15000 ppm when compared with Controls (Males: 173, 154 and 201% of Control; Females: 171, 284 and 595% of Control, respectively). The biochemical examination of the blood plasma in Week 4 of recovery showed mean bile acid concentrations at 15000 ppm in males and females similar to Control values, indicating reversibility of this isolated finding. Males receiving 3500, 7500 and 15000 ppm, and females receiving 7500 and 15000 ppm showed an increase, compared with Controls, in mean phosphorus concentrations after 13 weeks of treatment (Males: 109, 106 and 110%; Females: 114 and 121% of Control, respectively). After 4 weeks of recovery, phosphorus concentrations were not significantly increased when compared with Control in males at 15000 ppm. In addition, Control values were higher than the phosphorus concentrations observed at 15000 ppm during Week 13 of treatment and therefore this finding is considered attributed to normal biological variation.

The analysis of organ weights after 13 weeks of treatment indicated statistically significantly high mean body weight adjusted kidneys in males at 3500, 7500 and 15000 ppm (13, 16 and 22% higher than Controls, respectively), high mean body weight adjusted liver weights in males and statistically significantly high mean body weight adjusted liver weights in females at 3500, 7500 and 15000 ppm (Males: 4, 8 and 8%; Females: 11, 15 and 18% higher than Controls, respectively). After 4 weeks of recovery, kidney weights in males and liver weights in males and females at 15000 ppm were comparable with Control.

Pale color of the kidneys was seen in all treated male animals. Pale areas were seen in kidneys of one male treated at 3500 ppm and one male treated at 15000 ppm. Minimal to moderate hyaline droplets (positively stained for alpha 2µ-globulin by immunohistochemistry) in the cortical tubules were seen in all males given 3500 ppm, 7500 ppm or 15000 ppm of Gum turpentine oil, with a dose dependent increase on incidence and severity. All other changes were secondary to the hyaline droplets accumulation and consisted of minimal to slight basophilia of the tubular epithelium, corticomedullary/cortical granular casts, and interstitial mononuclear cell infiltrate. Following a 4-week recovery period, similar findings were observed in the kidney of the males, but reduced in terms of incidence and/or severity. Minimal hyaline droplets (positively stained for alpha 2µ-globulin by immunohistochemistry) in the cortical tubules were seen in one male only. Tubular basophilia was seen in all males and granular casts were seen in almost half of males, while minimal mononuclear inflammatory cell infiltrate was recorded for one male.

In conclusion, dietary administration of Gum turpentine oil to Sprague-Dawley rats at concentrations of 3500, 7500 and 15000 ppm for 13 weeks was well tolerated but induced a dose-dependent decrease in bodyweight and bodyweight gains associated with a decrease in food consumption, probably due to the low palatability of the preparations with the test item. The treatment elicited adaptative/reversible and isolated changes in forelimb grip strength, high mean bile acid concentrations and high mean body weight adjusted liver weights. Histopathological changes in the kidneys in the males associated with high kidney weights were observed and considered indicative of the species and sex‑specific alpha 2μ-globulin nephropathy.

Therefore, when excluding the species- and sex-specific histopathological findings observed in male kidneys, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity is 15000 ppm (Males: 942 mg/kg bw/day; Females: 1033 mg/kg bw/day), corresponding to the highest dose tested.