Oligomerisation products of alpha-pinene and beta-pinene

Endpoint summary

Description of key information

In a preliminary study performed according to a protocol similar to OECD Guideline 407 but not under GLP, the No Observed Adverse Effect Level (NOAEL) was considered to be > 1500 and < 7500 ppm.

In a 90-day repeated dose toxicity study conducted according to OECD Guideline 408 under GLP, the No Observed Adverse Effect Level (NOAEL) was considered to be 5000 ppm (equivalent to 331 mg/kg bw/day in male and 388 mg/kg bw/day in female).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04 July 2014 - 12 February 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The study generally followed GLP principles, however no specific study-related Quality Assurance procedures were performed and the report may not contain all of the elements required by GLP.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted 03 October 2008

Deviations:

yes

Remarks:

Duration of the study was 21 days. No recovery period.

GLP compliance:

no

Remarks:

The study was conducted in accordance with the applicable sections of the United Kingdom Animals (Scientific Procedures) Act 1986, Amendment Regulations 2012 (the Act).

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at the Test Facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 40 to 47 days old
- Weight at study initiation: Males: 204 - 247 g; Females: 145 - 202 g
- Fasting period before study: No
- Housing: Five animals of the same sex per cage in polycarbonate cages with a stainless steel mesh lid
- Diet: Rat and Mouse No. 1 Maintenance Diet, ad libitum; Non-restricted (removed overnight before blood sampling for haematology or blood chemistry).
- Water: Potable water from the public supply, ad libitum
- Acclimation period: 12 days before commencement of treatment

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinised and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 20 August 2014 to 22 September 2014

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was chosen to simulate a condition of potential human exposure.

Vehicle:

corn oil

Remarks:

Supplied by Consumer’s Pride

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
On each occasion of the preparation of the premix the required amount of test substance and corn oil were combined and stirred. The mixture was added to an equal amount of plain diet and stirred until visibly homogenous. This doubling up process was repeated until half of the final weight of premix was achieved or the mixture appeared dry. This mixture was then ground using a mechanical grinder. The weight of the mixture was then made up to the final weight of the premix with plain diet. The mixture was then mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet.
Aliquots of this 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 Control diet the corn oil was added directly to the diet and then prepared as indicated for the premix.
The test material was known to be incompatible with plastic rubber and card and these materials were not used during storage, preparation or feeding of the diet.

- DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with plain diet: Yes
- Storage temperature of food: Frozen (nominally -20°C), until required for feeding

- VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil is the second vehicle recommended in the OECD Guidelines.
- Concentration in vehicle: test material to corn oil ratio 5:1
- Lot/batch no.: L2 3317/AF/13:30

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

21 days

Frequency of treatment:

Continuously

Dose / conc.:

0 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

7 500 ppm

Dose / conc.:

15 000 ppm

No. of animals per sex per dose:

5 animals

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dietary concentrations used in this study were selected in conjunction with the Sponsor. An acute oral toxicity study (conducted according to OECD guideline 423) showed that oral LD50 was higher than 2000 mg/kg in the rat. As no data on repeated toxicity were available and in order to have a broad spectrum of test item palatability, a dose level of 15000 ppm, equivalent to approximately 1000 mg/kg bw/day for a 90-day treatment period, was tested. 7500 ppm and 1500 ppm were also tested in this study.
- Rationale for animal assignment (if not random): Randomly allocated on arrival

Positive control:

None

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: A detailed physical examination was performed on each animal on Days 1, 8, 15 and 22 to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded daily during the week before treatment commenced (Week -1), on the day that treatment commenced (Day 1), daily throughout 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 throughout the study.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 3 .
- Anaesthetic used for blood collection: Yes (general anaesthesia induced by isoflurane)
- Animals fasted: Yes, after overnight withdrawal of food
- How many animals: All animals
- Parameters checked in table [7.5.1/1a] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 3 .
- Anaesthetic used for blood collection: Yes (general anaesthesia induced by isoflurane)
- Animals fasted: Yes, after overnight withdrawal of food
- How many animals: All animals
- Parameters checked in table [7.5.1/1a] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE: All animals were killed by carbon dioxide asphyxiation with subsequent exsanguination after 21 days of treatment.

GROSS PATHOLOGY: Yes; 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. For bilateral organs, left and right organs were weighed together.

HISTOPATHOLOGY: Yes
Fixation: Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of testes which were preserved in Davidson’s fluid.
Details of histopathology are specified in Table 7.5.1/2a.

Other examinations:

None

Statistics:

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

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs seen in association with the administration of Terpenic Oligomers at 1500, 7500 or 15000 ppm.

Mortality:

no mortality observed

Description (incidence):

There were no premature deaths seen in association with the administration of Terpenic Oligomers at 1500, 7500 or 15000 ppm.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males receiving Terpenic Oligomers at 1500 ppm showed reduced body weight gain during the 5 days of treatment, corresponding to 61% of Control. Then body weight gain values tended to control values (91% of Control), leading to an overall body weight gain during Days 1-22 of 84% of Control. Females receiving Terpenic Oligomers at 1500 ppm showed body weight stasis during Days 1-2 of treatment, and body weight gain similar to Control thereafter. Body weight gain during Days 1-22 was 104% of Control.

Males receiving Terpenic Oligomers at 7500 ppm initially showed body weight loss during Days 1-2 of treatment leading to 44% of Control body weight gain during the first 5 days of treatment. Thereafter, variable body weight change, generally lower than Control, was recorded. Overall body weight gain during Days 1-22 was 70% of Control. Females receiving Terpenic Oligomers at 7500 ppm showed variable body weight change. Body weight gain during the first 5 days of treatment attained only 27% of Control whereas values reached Control values thereafter (107% of Control between Days 5-22). Overall body weight gain during Days 1-22 was 89% of Control.

Males receiving Terpenic Oligomers at 15000 ppm initially showed body weight loss during Days 1-2 of treatment leading to only 16% of Control body weight gain during the first 5 days of treatment. Thereafter, body weight change was variable, with a tendency to improvement (51% of Control between Days 5-22). Overall body weight gain during Days 1-22 was 43% of Control. Females receiving Terpenic Oligomers at 15000 ppm initially showed body weight loss during Days 1-2 of treatment and only 18% of Control body weight gain during the first 5 days of treatment. Thereafter, body weight change improved, leading to 62% body weight gain of Control between Days 8-22. Overall body weight gain during Days 1-22 was 54% of Control.

As a result of these decreases in body weight gain, dose-related lower body weights were observed in all groups of treated males and in 7500 and 15000 ppm female groups.

There was group mean body weight loss and very low food consumption for all groups during Days 17-18 as a result of food deprivation for collection of blood samples for haematology and blood chemistry.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption of males receiving Terpenic Oligomers at 1500 ppm was generally slightly lower (91% of Control) than that of the Control throughout treatment. Food consumption of females receiving Terpenic Oligomers at 1500 ppm was generally similar to that of the Control.

Food consumption of males receiving Terpenic Oligomers at 7500 and 15000 ppm was generally lower than that of the Control throughout treatment (85 and 73% of Control during Days 2-22, respectively), particularly during Days 1-2 of treatment (less than 50% Control). Food consumption of females receiving Terpenic Oligomers at 7500 and 15000 ppm was generally lower than that of the Control between Days 1-12 of treatment, particularly during Days 1-2 of treatment. Thereafter, the food consumption of females receiving Terpenic Oligomers at 7500 and 15000 ppm generally improved, leading to an overall food consumption of 89 and 85% of Control, respectively.

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

no effects observed

Description (incidence and severity):

No effect was observed on water consumption when assessed visually.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Amongst males receiving Terpenic Oligomers at 1500, 7500 or 15000 ppm, there were several changes from Control values which achieved statistical significance:
- For males receiving Terpenic Oligomers at 15000 ppm, red blood cell counts were statistically significantly higher than Control but without dose dependant trend and reticulocyte counts and mean cell volume were statistically significantly and dose dependently lower than Control.
- Red cell distribution width was statistically significantly lower than Control for males receiving Terpenic Oligomers at 7500 or 15000 ppm.
- Lymphocyte counts, monocyte counts and large unstained cell counts were statistically significantly lower than Control for males receiving Terpenic Oligomers at 1500, 7500 or 15000 ppm but there was no indication of dose dependant trend.
- Platelet counts were statistically significantly and dose dependently higher than Control for males receiving Terpenic Oligomers at 7500 or 15000 ppm.
- Prothrombin times were slightly but statistically significantly shorter than Control at 15000 ppm.
- Activated partial prothrombin times were longer for males receiving Terpenic Oligomers at 1500, 7500 or 15000 ppm, however statistical significance was achieved at 15000 ppm only.
Amongst females receiving Terpenic Oligomers at 1500 ppm, there were no statistically significant changes from Control.
Amongst females receiving Terpenic Oligomers at 7500 ppm, platelet counts were statistically significantly higher than Control, which was confirmed at 15000 ppm but without dose dependency.
Amongst females receiving Terpenic Oligomers at 15000 ppm, mean cell haemoglobin concentrations were statistically significantly lower than Control. White blood cell counts were statistically significantly lower than Control attributable to low neutrophil, lymphocyte, monocyte and large unstained cell counts. Variability of white blood cell populations was seen at the lower dose levels however these effects did not show a dose dependency or attain statistical significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Amongst males receiving Terpenic Oligomers at 1500 ppm, there were no statistically significant changes from Control, although phosphorus levels were slightly lower than Control, and creatinine levels were slightly higher than Control.
Amongst males receiving Terpenic Oligomers at 7500 ppm, phosphorus levels were slightly but statistically significantly lower than Control, and creatinine levels were slightly higher than Control. Total protein was significantly higher than Control, attributed to an increase in globulin detected by the statistically low albumin globulin ratio when compared with the Control.
Amongst males receiving Terpenic Oligomers at 15000 ppm, alkaline phosphatase levels were statistically significantly lower than Control. Phosphorus and albumin/globulin ratio, as observed at 7500 ppm, were statistically significantly lower than Control however this change was not dose related. Creatinine, total protein and albumin levels were statistically significantly higher than Control.
Amongst females receiving Terpenic Oligomers at 1500, 7500 or 15000 ppm, there were some changes from Control values which achieved statistical significance:
- Alanine amino-transferase and aspartate amino-transferase levels were lower than Control at 1500, 7500 or 15000 ppm, however statistical significance was achieved at 15000 ppm only.
- Creatinine levels were higher than Control at 1500, 7500 or 15000 ppm, with statistical significance achieved at both 7500 and 15000 ppm.
- Sodium and chloride concentrations were only slightly higher than Control at 1500, 7500 or 15000 ppm, but with statistical significance achieved at 15000 ppm.
- Calcium concentrations were significantly lower than Control at 15000 ppm only (a dose trend was not apparent).
- Albumin levels were statistically significantly lower than Control at 1500, 7500 and 15000 ppm. This change was associated with the statistically significantly low albumin/globulin ratio at all treatment levels. No dose trend was apparent.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No abnormalities were reported.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Amongst males receiving Terpenic Oligomers at 1500, 7500 and 15000 ppm, body weight at termination was lower than Control with a dose response apparent. The absolute and adjusted thymus and testes weights of males receiving Terpenic Oligomers at 15000 ppm were slightly lower than Control but without dose dependency or statistical significance. The absolute and adjusted heart and spleen weights were slightly lower than Control for males receiving Terpenic Oligomers at 1500, 7500 and 15000 ppm, with heart adjusted weights attaining statistical significance at 7500 and 15000 ppm. In addition, the adjusted liver weights were slightly higher than Control for males receiving Terpenic Oligomers at 7500 and 15000 ppm.

Amongst females receiving Terpenic Oligomers at 15000 ppm, body weight at termination was lower than Control. In addition, the absolute and adjusted liver weights were higher than Control for females receiving Terpenic Oligomers at 1500, 7500 and 15000 ppm, with a dose response apparent and statistical significance from 7500 ppm for adjusted weights. Adjusted mean brain weights were slightly higher than Control, with statistical significance at the 7500 and 15000 ppm levels but without dose dependency.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The macroscopic examination performed after 21 days of treatment revealed no test substance related lesions.
The incidence and distribution of all findings were consistent with the common background seen here.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 500 - < 7 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

haematology

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

In this study the systemic toxic potential and palatability of Terpenic Oligomers were assessed over a period of 21 days in Crl:CD (SD) rats at dietary concentrations of 1500, 7500 or 15000 ppm. This resulted in respective overall mean achieved dose levels (Days 1-22) of 130.7, 619.0 and 1137.6 mg/kg/day for males and 129.1, 610.0 and 1223.4 mg/kg/day for females.

Based on the results of this study, principally magnitude of the effect on body weight gain in males, it was concluded that 15000 and 7500 ppm were too high and would not be suitable for use on the subsequent study. There were some minor effects of treatment at 1500 ppm, particularly in the males, so a low dose below 1500 ppm should be considered for use as a low dose level on the subsequent main study.

It is concluded that a high dose level between 1500 ppm and 7500 ppm would be suitable for use on a subsequent 90-day repeated dose oral toxicity study and a dose level less than 1500 ppm should be considered for the low dose level.

Conclusions:

It is concluded that a high dose level between 1500 ppm and 7500 ppm would be suitable for use in a subsequent 90-day repeated dose oral toxicity study and a dose level less than 1500 ppm should be considered for the low dose level.

Observetions do not allow determining a No Observe Adverse Effect Level (NOAEL) as changes in body weight gain and food consumption for males receiving 1500 ppm were probably due to the low palatability of the preparation whereas changes observed in males and females receiving 7500 ppm can be considered as adverse effects.

Executive summary:

In a repeated dose toxicity study performed according to a protocol similar to OECD test Guideline No. 407, three groups, each comprising five male and five female Crl:CD(SD) rats, received Terpenic Oligomers orally, via the diet, at concentrations of 1500, 7500 or 15000 ppm. At dietary concentrations of 1500, 7500 and 15000 ppm overall mean achieved dose levels (Days 1-22) were 130.7, 619.0 and 1137.6 mg/kg/day for males and 129.1, 610.0 and 1223.4 mg/kg/day for females, respectively. A similarly constituted control group received untreated diet throughout the same treatment period. During the study, clinical condition, body weight, food consumption, visual water consumption, haematology, blood chemistry, organ weight and macropathology investigations were undertaken.

There were no premature deaths, and there were no clinical signs seen in association with the administration of Terpenic Oligomers at 1500, 7500 or 15000 ppm.

Overall (Days 1 to 22) body weight gain and food consumption for males and females receiving 7500 or 15000 ppm were generally low when compared with Controls; a dose response was apparent. No effect was observed on water consumption when assessed visually. Overall (Days 1 to 22) body weight gain and food consumption for males receiving 1500 ppm were slightly low when compared with Controls, while body weight performance and food consumption were considered unaffected by treatment for females at 1500 ppm.

Haematology effects were observed in males and females and included lower reticulocyte counts (statistically significantly different from Controls in males dosed at 15000 ppm), lower white blood cell populations including lymphocytes, monocytes and large unstained cells (statistically significantly different from Controls at 15000 ppm). Statistically significantly higher numbers of platelets were observed at 7500 or 15000 ppm in males and females with statistically significant disturbances in clotting time only in males receiving 15000 ppm.

Blood chemistry effects included higher creatinine concentrations at all dose levels although attaining statistical significance only at 15000 ppm in males and females and 7500 ppm in females. Creatinine concentrations showed a consistent relationship to treatment in males and females. Statistically significant lower albumin levels were observed in females at all dose levels (without relationship to treatment) and higher albumin levels were observed in males at 7500 and 15000 ppm (statistically significantly different from Controls at 15000 ppm only).

Consistent effects on organ weight included high adjusted liver weights in 7500 or 15000 ppm treated groups, with a dose response apparent for both males and females.

The macroscopic examination performed after 21 days of treatment revealed no test substance related lesions.

It is concluded that a high dose level between 1500 ppm and 7500 ppm would be suitable for use in a subsequent 90-day repeated dose oral toxicity study and a dose level less than 1500 ppm should be considered for the low dose level.

Observetions do not allow determining a No Observe Adverse Effect Level (NOAEL) as changes in body weight gain and food consumption for males receiving 1500 ppm were probably due to the low palatability of the preparation whereas changes observed in males and females receiving 7500 ppm can be considered as adverse effects.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2014 - 08 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 408 and in compliance with GLP.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Revised 1998

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK Department of Health (inspected on the end of 2015 / signed on 15 February 2016)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague Dawley [Crl:CD(SD)] strain was used because of the historical control data available at the Test Facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: ca. 6 to 7 weeks old
- Weight at study initiation: Males: 200 - 24_ g; Females: 163 - 20' g
- Fasting period before study: No
- Housing: Five animals of the same sex per cage in polycarbonate cages with a stainless steel mesh lid
- Diet: Rat and Mouse No. 1 Maintenance Diet, ad libitum; Non-restricted (removed overnight before blood sampling for haematology or blood chemistry).
- Water: Potable water from the public supply, ad libitum
- Acclimation period: 13 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinised and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 05 November 2014 to 17 March 2015

Route of administration:

oral: feed

Details on route of administration:

The dietary route of administration was chosen to simulate a condition of potential human exposure.

Vehicle:

corn oil

Remarks:

Supplied by Consumer’s Pride

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
On each occasion of the preparation of the premix the required amount of test substance and corn oil were combined and stirred. The mixture was added to an equal amount of plain diet and stirred until visibly homogenous. This doubling up process was repeated until half of the final weight of premix was achieved or the mixture appeared dry. This mixture was then ground using a mechanical grinder. The weight of the mixture was then made up to the final weight of the premix with plain diet. The mixture was then mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet.
Aliquots of this 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 Control diet the corn oil was added directly to the diet and then prepared as indicated for the premix.
The test material was known to be incompatible with plastic rubber and card and these materials were not used during storage, preparation or feeding of the diet.

- DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with plain diet: Yes
- Storage temperature of food: Diets in the food hopper were changed daily. The test substance was considered stable and homogenous in an open drum container for 24 hours at ambient temperature at 800, 1000, 1500 and 20000 ppm. In addition, frozen stability was confirmed for 8 days for the range 1000 to 20000 ppm. The results of additional stability investigations confirmed stability for 8 days frozen (nominally -20°C) at 800 ppm.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil is the second vehicle recommended in the OECD Guidelines.
- Concentration in vehicle: test material to corn oil ratio 5:1
- Lot/batch no.: L2 3317/AF/13:30 and L2 4105

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the preparation was verified using chromatographic analysis. The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection, limit of quantification, linearity of detector response, system precision, method accuracy and precision.
The homogeneity was confirmed for Terpenic Oligomers in Rat and Mouse No. 1 Maintenance diet formulations at nominal concentrations of 100 ppm and 20000 ppm. Stability and homogeneity was confirmed for 1 day in an open container at ambient temperature at nominal concentrations of 800 ppm to 20000 ppm. Stability was confirmed for frozen storage at nominal concentrations of 100 ppm to 20000 ppm for 8 days.

Samples of each formulation prepared for administration in Weeks 1 and 12 of treatment were analysed for achieved concentration of the test substance. The mean concentrations of Terpenic Oligomers in test formulations analysed for the study were within applied limits +10%/-15% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Continuously. During the recovery period, all animals were given untreated diet.

Dose / conc.:

0 ppm

Dose / conc.:

800 ppm

Dose / conc.:

2 000 ppm

Dose / conc.:

5 000 ppm

No. of animals per sex per dose:

Main study: 10 animals/sex/dose
Recovery phase: 5 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 800, 2000 and 5000 ppm) were selected in conjunction with the Sponsor.
Based on the results of the 3-week palatability/preliminary study (Envigo Study Number OAD0024), it was considered that 15000 ppm was too high and would not be suitable for use on this 13 week main study. The dose of 7500 ppm elicited an effect on body weight gain (overall body weight gain during Days 1-22 was 70% of Control in males), so a high dose just below this (5000 ppm) was selected for use as a high dose. There were some minor effects of treatment at 1500 ppm, particularly in males (overall body weight gain during Days 1-22 was 84% of Control), it was considered necessary to use a low dose of 800 ppm to achieve a NOAEL. The mid dose of 2000 ppm was selected because it was equally separated by a factor of 2.5 between low and high doses.
- Rationale for animal assignment (if not random): Randomly allocated on arrival
- Rationale for selecting satellite groups: Not specified
- Post-exposure recovery period in satellite groups: 4 week recovery period

Positive control:

None

Observations and examinations performed and frequency:

MORTALITY: Yes, a viability check was performed near the start and end of each working day. Animals were isolated or killed for reasons of animal welfare where necessary.

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, during each week of treatment and weekly during recovery, detailed physical examination and arena observations were performed on each animal. After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy. More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored.

FOOD CONSUMPTION: Yes; the weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No 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 on Pretreatment (all animals) and Week 12 (All main study animals of Groups 1 and 4). Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13 (all main study animals at termination) and Recovery Week 4 (recovery animals at termination).
- Anaesthetic used for blood collection: Yes (general anaesthesia induced by isoflurane)
- Animals fasted: Yes, after overnight withdrawal of food
- Parameters checked in table [7.5.1/1b] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 (all main study animals at termination) and Recovery Week 4 (recovery animals at termination).
- Anaesthetic used for blood collection: Yes (general anaesthesia induced by isoflurane)
- Animals fasted: Yes, after overnight withdrawal of food
- Parameters checked in table [7.5.1/1b] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced, during each week of treatment and weekly during recovery, detailed physical examination and arena observations were performed on each animal. After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena.
- Battery of functions tested: sensory activity / grip strength / motor activity
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all main study animals and all recovery phase animals during Week 12 of treatment. In addition, all recovery phase animals were assessed for grip strength during Week 4 of recovery.
Motor activity: During Week 12 of treatment, the motor activity of all main study animals and all recovery phase animals was measured using a Rodent Activity Monitoring System (Version 2.0.5), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo. In addition, all recovery phase animals were tested during Week 4 of recovery.

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE: All animals were killed by carbon dioxide asphyxiation with subsequent exsanguination after 13 weeks of treatment for the main study animals and after 13 weeks of tretatment and 4 weeks of recovery for recovery animals.

GROSS PATHOLOGY: Yes; 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 above. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals.

HISTOPATHOLOGY: Yes
Fixation: Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of testes which were preserved in Davidson’s fluid.
Full List: All animals killed or dying prematurely / Main study animals of Groups 1 and 4 killed at a scheduled interval.
Abnormalities only: All main study animals of Groups 2 and 3 killed at a scheduled interval.
Recovery phase animals: Tissues from Recovery phase animals were retained in fixative pending results from initial examinations of tissues from Main phase animals.
Details of histopathology are specified in Table 7.5.1/2b.

Other examinations:

None

Statistics:

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

Clinical signs:

no effects observed

Description (incidence and severity):

During the 13-week treatment period with the administration of Terpenic Oligomers at 800, 2000 or 5000 ppm, there were no test article related signs observed during the detailed physical examination and arena observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one unscheduled death in this study. Group 3 male number 7 was killed for welfare reasons on Day 36, displaying abnormal behavior (increased activity, circling around the cage, aggression, and the animal was irritable and vocalising), piloerection and pale eyes. At necropsy, scabs were noted on the head and extremities. Microscopic examination of the lungs revealed multiple foci of mixed inflammatory cell infiltrates associated with an eosinophilic granular material within the alveoli. Some of this material was noted within macrophages. While the aetiology of the pulmonary changes is uncertain, it is likely that they contributed towards the clinical condition of the animal, resulting in killing due to welfare reasons. Microscopic examination of the skin of the extremities revealed ulceration of the skin and infiltration of inflammatory cells. It is possible that these arose due to trauma in light of the aggression and hyperactivity displayed by the animal. All other changes seen in this animal were considered incidental and it is not considered that they were related to administration of the test substance.
CONCLUSION: No adverse effects

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males receiving Terpenic Oligomers at 2000 or 5000 ppm showed statistically significant lower body weight gain in Weeks 0-2 when compared with Control, and slightly low body weight gain in Weeks 2-5 when compared with Control. Thereafter, in Weeks 5-12 males receiving Terpenic Oligomers at 2000 ppm showed similar body weight gain to that of the Control, while males receiving Terpenic Oligomers at 5000 ppm continued to show slightly low body weight gain when compared with Control. These variations led to statistically significant lower overall bodyweight gain in males receiving 2000 and 5000 ppm, corresponding to 81% and 77% of Control, respectively.

The body weight gain of males receiving Terpenic Oligomers at 800 ppm was similar to that of the Controls in Weeks 1 - 13 (Days 1-92).

Females receiving Terpenic Oligomers at 800, 2000 or 5000 ppm showed low body weight gain in Week 1 when compared with Control (85%, 88% and 65% of Control, respectively), with statistical significance attained at 5000 ppm. The body weight gain for females receiving Terpenic Oligomers at 800, 2000 or 5000 ppm was similar to that of the Control in Weeks 1-12.

During the recovery period, the body weight gain of males and females which had been given Terpenic Oligomers at 5000 ppm was greater than that of the Controls (169% of Control for males).

CONCLUSION: No adverse effects.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption of males and females receiving Terpenic Oligomers at 5000 ppm was lower than that of the Control in Weeks 1 and 2 (88% and 91% of Control, respectively for males and females). Food consumption of males receiving Terpenic Oligomers at 5000 ppm remained slightly lower than that of the Control in Weeks 3 - 13, leading to an overall food consumption of 89% of Control, while food consumption of females receiving Terpenic Oligomers at 5000 ppm was similar to that of the Control in Weeks 3 - 13. Food consumption of males and females receiving Terpenic Oligomers at 800 or 2000 ppm was similar to that of the Control in Weeks 1 - 13.
Throughout the recovery period, the food consumption of males and females which had received Terpenic Oligomers at 5000 ppm was similar to that of the Control.
CONCLUSION: No adverse effects.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

No effect was observed and consequently quantitative measurements were not performed.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmoscopy assessment conducted prior to the commencement of dosing did not reveal any findings which were outside of the normal expectations for animals of this age and strain.
There were no test article-related ophthalmoscopy findings apparent during Week 12 of treatment.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of haematological parameters, including morphology, during Week 13 of treatment revealed reticulocyte counts which were statistically significantly lower in males receiving Terpenic Oligomers at 5000 ppm when compared with the Controls. In addition, prothrombin time was statistically significantly shorter, and activated partial prothrombin time was statistically significantly longer than when compared with the Controls. For females receiving Terpenic Oligomers at 2000 and 5000 ppm, platelet counts were statistically significantly high when compared with the Controls and prothrombin times were statistically significantly shorter than Controls.

After the 4 week recovery period, reticulocyte counts, prothrombin time and activated partial prothrombin time for males were measured, and prothrombin time for females were measured. All values were similar to those of the Controls.

CONCLUSION: No adverse effects. Observed changes are reversible.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical analysis of plasma after 13 weeks of treatment revealed several minor test article related differences from Control in the plasma composition of animals given Terpenic Oligomers at 800, 2000 and 5000 ppm; the magnitude of these differences were small, and the majority of individual values were within the 5-95% confidence limits of the Historical Control Data.

Alkaline phosphatase was slightly low for all groups of males, with statistical significance achieved for males receiving Terpenic Oligomers at 2000 and 5000 ppm. A dose response was not apparent, and there was no similar effect for females. Alanine amino-transferase was slightly low for all groups of males and females, with statistical significance achieved. A dose response was not apparent. Aspartate amino-transferase was slightly low in males, with statistical significance in males receiving Terpenic Oligomers at 5000 ppm, and females receiving Terpenic Oligomers at 2000 and 5000 ppm. A dose response was apparent. Creatinine concentrations were statistically significantly low in females receiving Terpenic Oligomers at 2000 ppm only, however all values were within the historical control data range. Potassium was slightly but statistically low and total protein concentrations were slightly but statistically high for all females, however all values were within the historical control data range. Albumin concentrations were marginally but statistically significantly low for males receiving Terpenic Oligomers at 5000 ppm; there was no similar effect in females. A/G ratio was slightly low in males and females receiving Terpenic Oligomers, with statistical significance in males and females receiving Terpenic Oligomers at 5000 ppm. A dose response was apparent in males only. The values for all treated groups were within the historical control data range, while the values for male and female control animals were higher than the historical control data range.

Biochemical analysis of plasma for selected parameters after the 4 week recovery period showed all values were similar to those of the Controls. For males which had received Terpenic Oligomers at 5000 ppm, albumin levels were statistically lower than the Controls (35 g/L vs 36 g/L in the Controls), and cholesterol levels were statistically significantly lower than the Controls (1.23 mmol/L vs 1.59 mmol/L in the Controls). These minor differences are considered not to be of toxicological significance.

CONCLUSION: No adverse effects.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Sensory reactivity and grip strength:
Sensory reactivity observations for all groups of animals during Week 12 were considered unaffected by treatment.
Grip strength values for all groups of females during Week 12 were considered unaffected by treatment.
Males given Terpenic Oligomers at 800, 2000 and 5000 ppm showed a slight reduction in mean forelimb grip strength when compared to Control, and males given Terpenic Oligomers at 2000 and 5000 ppm showed a slight reduction in mean hindlimb grip strength when compared to Control with differences in the 5000 ppm group attaining statistical significance for hindlimb grip strength.
For forelimb strength, mean values were just below the Historical Control Data range (1.08 1.25 kg), and for hindlimb strength, mean values were within the Historical Control Data range (0.44 0.53 kg). The hindlimb strength of Control males was only just within the Historical Control Data range. These minor differences in forelimb and hindlimb grip strength were considered to be a consequence of the slightly lower bodyweight of these animals.
Grip strength values for males during Week 4 of recovery which had been given Terpenic Oligomers at 5000 ppm were similar to those of the Controls.
Grip strength values for females during Week 4 of recovery which had been given Terpenic Oligomers at 5000 ppm were statistically significantly lower than those of the Controls (p<0.05), however, in the absence of any apparent effect of treatment in Week 12 of treatment, this small difference is considered incidental and not an effect of previous treatment with Terpenic Oligomers.
CONCLUSION: No adverse effects.

- Motor activity:
Group mean motor activity scores (high and low beam) for all treated females were similar to Controls.
For males in Group 4 (5000 ppm), the majority of group mean low beam activity scores, including the total score are slightly high compared with Controls with statistical significance attained at the first 6 minute interval score. This score is also above the Historical Control Data maximum, however, all other scores (including total score) are within the Historical Control Data range. Individually, only two males had a first 6-minute interval score which is above the highest 6-minute interval score of the concurrent Controls.
It is considered that the first half of the 1-hour recording period is the most important as this is when the highest levels of exploratory behaviour are recorded. The high beam score at the first 6-minute interval for Group 4 (5000 ppm) males was also slightly high compared with Controls (and also above the Historical Control Data maximum) but did not attain statistical significance. High beam scores are generally more sensitive than low beams scores as indicators of treatment-related changes in motor activity levels so with the absence of similar or more marked differences in high beam scores throughout the rest of the testing period, these low beam differences should be viewed with caution. Very few other 6-minute interval high beam scores are high compared with Controls and although the total score is, it is not the highest - Group 3 (2000 ppm) has a higher score.
During Week 4 of recovery, group mean motor activity scores (high and low beam) for males and females previously given Terpenic Oligomers at 5000 ppm were similar to Controls. Therefore, the differences seen in Week 12 of treatment are considered a result of natural variation, and not an effect of treatment with Terpenic Oligomers.

CONCLUSION: No adverse effects.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The analysis of organ weights following 13 weeks of treatment showed no clear effect of treatment with Terpenic Oligomers at 800, 2000 and 5000 ppm.

Adjusted liver weights were slightly high for males which received Terpenic Oligomers at 2000 ppm, and for males and females which received Terpenic Oligomers at 5000 ppm, and adjusted kidney weights were slightly high for males which received Terpenic Oligomers at 2000 or 5000 ppm. These statistical differences are likely related to the decrease in bodyweights, since the absolute values are within the concurrent control range and within the control data range from a similar study.

Adjusted testes weights were slightly high for males which received Terpenic Oligomers at 5000 ppm. These statistical differences are also likely related to the decrease in bodyweights, since the absolute values are similar to the concurrent control range and similar to the control data range from a similar study.

For males, the analysis of organ weights following 4 weeks of recovery showed no clear effect of previous treatment with Terpenic Oligomers at 5000 ppm.

For females, the analysis of organ weights following 4 weeks of recovery showed no clear effect of previous treatment with Terpenic Oligomers at 5000 ppm. Adjusted brain and kidney weights were slightly high for females which received Terpenic Oligomers at 5000 ppm, and adjusted ovary weights were slightly low for females which had received Terpenic Oligomers at 5000 ppm. Although statistical significance was achieved, these differences are considered not to be of toxicological significance.

CONCLUSION: No adverse effects.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination performed after 13 weeks of treatment revealed changes in the brain (dilated ventricles in one female from each group treated with 2000 and 5000 ppm) and stomach (stomach mass observed in one female treated with 5000 ppm, and depressions in the mucosal surface observed in one male treated with 5000 ppm).
The incidence and distribution of all the other findings were consistent with the background lesions typically seen in this species.

The macroscopic examination performed after 4 weeks of recovery revealed no test substance related lesions.

CONCLUSION: No adverse effects.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment related findings (main study animals):
Changes related to treatment with Terpenic Oligomers were not observed in any of the tissues examined.

Incidental findings (main study animals):
Minimal foci of inflammatory cell infiltrates were seen in the livers of male and female animals and were primarily perivascular in location. Minimal changes were additionally observed in the kidneys consisting of hyaline casts, basophilic tubules and foci of inflammatory cells. Additionally, in males more so than females, there were foci of inflammatory cell infiltration and myocardial degeneration in the heart. These changes are typically seen as background lesions in CD rats at our laboratories and are not considered to be related to administration of the test substance.
All other histological changes were considered to be unrelated to treatment.

CONCLUSION: No adverse effects.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: None of the findings were considered adverse in this study.

Key result

Critical effects observed:

no

Formulation analysis:

Procedural recovery values were within ±10% of the fortified concentration confirming the continued accuracy of the analytical procedure with the exception of two recoveries prepared in the Week 1 analysis.

The mean concentrations of Terpenic Oligomers in test formulations analysed for the study were within applied limits +10%/-15% of nominal concentrations, confirming accurate formulation.

Achieved dose:

At dietary concentrations of 800, 2000 and 5000 ppm mean achieved dose levels over Weeks 1-13 were 55, 151 and 331 mg/kg bw/day for males in Groups 2, 3 and 4, respectively, and 62, 158 and 388 mg/kg bw/day for females in Groups 2, 3 and 4, respectively.

Discussion:

The systemic toxic potential of Terpenic Oligomers, an industrial chemical, was assessed by dietary administration to Crl:CD(SD) rats over a period of 13 weeks. Groups of male and female rats received daily dietary administration at 0, 800, 2000 or 5000 ppm.

Administration of the test article was well tolerated. There was one premature death which was considered not related to treatment. There were no test article-related clinical signs observed or adverse effects on body weight, food consumption, sensory reactivity, grip strength, motor activity, ophthalmoscopy, haematology, blood chemistry, organ weights or macropathology. There were no target organs identified during the histopathological evaluations.

Males receiving Terpenic Oligomers at 2000 or 5000 ppm showed low body weight gain in Weeks 0-2 when compared with Control, and slightly low body weight gain in Weeks 2 -5 when compared with Control. Thereafter, in Weeks 5-12 males receiving Terpenic Oligomers at 2000 ppm showed similar body weight gain to that of the Control, while males receiving Terpenic Oligomers at 5000 ppm continued to show slightly low body weight gain in Weeks 5-12 when compared with Control. During the full 13-week dosing period, the weight gain of these males was 18 and 23% lower than Controls, respectively, however as there was no impact on the clinical condition of these animals and the weight gain observed in the 4-week recovery period was slightly higher than Control, the lower weight gain observed during the treatment period was considered not to be adverse. Also, lower food consumption, probably due to the low palatability of the preparation, was associated with lower weight gain throughout treatment at 5000 ppm.

The differences in food consumption of males and females receiving Terpenic Oligomers at 5000 ppm in Weeks 1 and 2 (88% and 91% of Control, respectively for males and females, and for males receiving Terpenic Oligomers at 5000 ppm in Weeks 3 – 13 were considered to represent an effect of treatment with Terpenic Oligomers, however were considered not adverse. Indeed, in the absence of any other relevant toxicological effect, this decrease is probably due to the low palatability of the preparations at this dose level rather than a toxicological effect, similarly to what had been observed to a higher extent, at higher concentrations, in the preliminary study.

At the end of the treatment period several minor test article-related changes in blood plasma composition were apparent, however, there were no changes in the general clinical condition of the animals and no histopathological abnormalities in organs which were examined. Assessments conducted following the 4-week off-dose period showed that full or partial recovery was evident for the haematology and blood chemistry changes. It was therefore concluded that these changes were of no toxicological significance and considered not to be adverse.

The analysis of organ weights following 13 weeks of treatment, and again after 4 weeks of recovery showed no clear effect of treatment with Terpenic Oligomers at 800, 2000 and 5000 ppm.

Administration of Terpenic Oligomers to CD Rats in this study was not associated with any test substance related effects. There was one unscheduled death and the macroscopic and microscopic examination did not reveal changes consistent with administration of the test substance. All other changes were considered to be background lesions that are commonly seen in CD rats at these laboratories.

It was concluded that dietary administration of Terpenic Oligomers to Sprague Dawley rats at doses of 800, 2000 or 5000 ppm for 13 weeks provided clear evidence of systemic exposure but none of the effects observed were deemed to be adverse. The No Observed Adverse Effect Level (NOAEL) was concluded to be 5000 ppm, equivalent to 331 mg/kg bw/day for males and 388 mg/kg bw/day for females

Conclusions:

The dietary administration of Terpenic Oligomers to Sprague Dawley rats at doses of 800, 2000 or 5000 ppm for 13 weeks provided clear evidence of systemic exposure but none of the effects observed were deemed to be adverse. Therefore, the No Observed Adverse Effect Level (NOAEL) was considered to be 5000 ppm, equivalent to 331 mg/kg bw/day for males and 388 mg/kg bw/day for females.

Executive summary:

The systemic toxic potential of Terpenic Oligomers was assessed in a 13 week dietary study in rats performed according to OECD Guideline 408 under GLP compliance. Recovery from any effects was also evaluated during a 4 week recovery period.

Three groups, each comprising ten male and ten female Crl:CD (SD) rats, received Terpenic Oligomers via the diet at concentrations of 800, 2000 or 5000 ppm. A similarly constituted control group received untreated diet, with vehicle (corn oil) only, throughout the same treatment period. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for 13 weeks, followed by a 4 week period without treatment to assess the potential for any treatment-related change to recover.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

The mean concentrations of Terpenic Oligomers in test formulations analysed for the study were within applied limits +10%/-15% of nominal concentrations, confirming accurate formulation. The overall achieved doses during Weeks 1-13 at 800, 2000 and 5000 ppm were 55, 151 and 331 mg/kg/day for males and 62, 158 and 388 mg/kg/day for females, respectively.

Dietary administration of Terpenic Oligomers at doses up to and including 5000 ppm for 13 weeks was well tolerated. There was one unscheduled death, it was considered not to be related to the dietary administration of Terpenic Oligomers.

There were no test article-related signs observed during the detailed physical examination and arena observations, sensory reactivity assessment or ophthalmic examination. Minor differences in grip strength and motor activity scores were detected for males during Week 12 of treatment; however when reviewed against historical data and re-assessed during Week 4 of recovery, these were considered not to represent an adverse effect of treatment.

Males receiving Terpenic Oligomers at 5000 ppm showed low body weight gain in Weeks 0-2 and slightly low body weight gain in Weeks 2-13, when compared with Control. Males receiving Terpenic Oligomers at 2000 ppm showed low or slightly low body weight gain in Weeks 0-2 or Weeks 2-5 respectively, thereafter body weight gain was similar to that of the Control. The body weight gain of males receiving Terpenic Oligomers at 800 ppm was similar to that of the Control in Weeks 0 - 13.

Females receiving Terpenic Oligomers at 800, 2000 or 5000 ppm showed low body weight gain in Weeks 0-1 (85%, 88% and 65% of Control, respectively). The body weight gain for females at all levels was similar to that of the Control in Weeks 1-13.

Food consumption of males and females receiving Terpenic Oligomers at 5000 ppm was lower than that of the Control in Weeks 1 and 2 (88% and up to 90% of Control, respectively for males and females in Week 1, and 88% and 92% of Control, respectively for males and females in Week 2). Food consumption of males receiving Terpenic Oligomers at 5000 ppm remained slightly lower than that of the Control in Weeks 3 - 13, while food consumption of females receiving Terpenic Oligomers at 5000 ppm was similar to that of the Control in Weeks 3 - 13. Food consumption of males and females receiving Terpenic Oligomers at 800 or 2000 ppm was similar to that of the Control in Weeks 1 - 13.

During the 4 week recovery period food consumption and body weight gain of males and females which had previously received Terpenic Oligomers at 5000 ppm, were similar or greater than that of Control.

Analysis of haematological parameters, including morphology, after 13 weeks of treatment revealed the following statistical changes; in males receiving 5000 ppm, low reticulocyte counts, shorter prothrombin time and longer activated partial prothrombin time, when compared with the Controls; in females receiving 2000 and 5000 ppm, high platelet counts and shorter prothrombin times, than Controls.

After the 4 week recovery period, reticulocyte counts, prothrombin time and activated partial prothrombin time for males, and prothrombin time for females were all similar to those of the Controls.

Biochemical analysis of plasma after 13 weeks of treatment revealed several minor test article related differences from Control in the plasma composition of animals given Terpenic Oligomers at 800, 2000 and 5000 ppm. Alkaline phosphatase was slightly low for all groups of males only, with statistical significance achieved for males receiving Terpenic Oligomers at 2000 and 5000 ppm. Alanine amino-transferase was slightly low for all groups of males and females, with statistical significance achieved. Aspartate amino-transferase was slightly low in males, with statistical significance in males receiving 5000 ppm, and females receiving 2000 and 5000 ppm. Potassium were slightly but statistically low and total protein concentrations were slightly but statistically high for all females, however all values were within the historical control data range. Albumin concentrations were marginally but statistically significantly low for males receiving 5000 ppm. A/G ratio was slightly low in males and females receiving Terpenic Oligomers, with statistical significance in males and females receiving Terpenic Oligomers at 5000 ppm.

Biochemical analysis of plasma for selected parameters after the 4 week recovery period showed most values were similar to those of the Control. Albumin levels remained statistically lower than the Controls in males previously receiving Terpenic Oligomers at 5000 ppm, although this minor difference is considered not to be of biological significance.

Adjusted liver weights were slightly but statistically high for males and females which received 2000 and/or 5000 ppm, and adjusted kidney weights were slightly high for males which received Terpenic Oligomers at 2000 and 5000 ppm. Males receiving 5000 ppm also had low adjusted mean testes weights and for females at this level high absolute brain weight was recorded.

For males, the analysis of organ weights following 4 weeks without treatment showed complete recovery. The liver weights of recovery females was no longer statistically different to the Control although adjusted brain and kidney weights were slightly high and adjusted ovary weights were slightly low for females which had previously received Terpenic Oligomers at 5000 ppm.

There were no macroscopic abnormalities detected at scheduled termination after 13 weeks of treatment or after 4 weeks of recovery and histopathological evaluation of the tissues from animals killed after 13 weeks of treatment did not reveal any test article-related changes.

Therefore, the dietary administration of Terpenic Oligomers to Sprague Dawley rats at doses of 800, 2000 or 5000 ppm for 13 weeks provided clear evidence of systemic exposure but none of the effects observed were deemed to be adverse. Therefore, the No Observed Adverse Effect Level (NOAEL) was considered to be 5000 ppm, equivalent to 331 mg/kg bw/day for males and 388 mg/kg bw/day for females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

334.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is GLP-compliant and of high quality (Klimisch score = 1).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A preliminary study was performed according to a protocol similar to OECD Guideline 407 in order to assess the palatability and the systemic toxic potential of the test item, Terpenic Oligomers, in a 22-day dietary study in Sprague Dawley rats and to select a suitable high dose for a subsequent 13-week repeated dose toxicity study.

It has been concluded from this study that a high dose level between 1500 ppm and 7500 ppm would be suitable for use in a subsequent 90-day repeated dose oral toxicity study and a dose level less than 1500 ppm should be considered for the low dose level.

Following the preliminary study, the systemic toxic potential of Terpenic Oligomers was assessed in a 13-week dietary study in rats performed according to OECD Guideline 408 under GLP. Recovery from any effects was also evaluated during a 4-week recovery period.

Three groups, each comprising ten male and ten female Crl:CD (SD) rats, received Terpenic Oligomers via the diet at concentrations of 800, 2000 or 5000 ppm. A similarly constituted control group received untreated diet, with vehicle (corn oil) only, throughout the same treatment period. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for 13 weeks, followed by a 4 week period without treatment to assess the potential for any treatment-related change to recover.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

The mean concentrations of Terpenic Oligomers in test formulations analysed for the study were within applied limits +10%/-15% of nominal concentrations, confirming accurate formulation. The overall achieved doses during Weeks 1-13 at 800, 2000 and 5000 ppm were 55, 151 and 331 mg/kg/day for males and 62, 158 and 388 mg/kg/day for females, respectively.

Dietary administration of Terpenic Oligomers at doses up to and including 5000 ppm for 13 weeks was well tolerated. There was one unscheduled death, it was considered not to be related to the dietary administration of Terpenic Oligomers.

There were no test article-related signs observed during the detailed physical examination and arena observations, sensory reactivity assessment or ophthalmic examination. Minor differences in grip strength and motor activity scores were detected for males during Week 12 of treatment; however when reviewed against historical data and re-assessed during Week 4 of recovery, these were considered not to represent an adverse effect of treatment.

Males receiving Terpenic Oligomers at 5000 ppm showed low body weight gain in Weeks 0-2 and slightly low body weight gain in Weeks 2-13, when compared with Control. Males receiving Terpenic Oligomers at 2000 ppm showed low or slightly low body weight gain in Weeks 0-2 or Weeks 2-5 respectively, thereafter body weight gain was similar to that of the Control. The body weight gain of males receiving Terpenic Oligomers at 800 ppm was similar to that of the Control in Weeks 0 - 13.

Females receiving Terpenic Oligomers at 800, 2000 or 5000 ppm showed low body weight gain in Weeks 0-1 (85%, 88% and 65% of Control, respectively). The body weight gain for females at all levels was similar to that of the Control in Weeks 1-13.

Food consumption of males and females receiving Terpenic Oligomers at 5000 ppm was lower than that of the Control in Weeks 1 and 2 (88% and up to 90% of Control, respectively for males and females in Week 1, and 88% and 92% of Control, respectively for males and females in Week 2). Food consumption of males receiving Terpenic Oligomers at 5000 ppm remained slightly lower than that of the Control in Weeks 3 - 13, while food consumption of females receiving Terpenic Oligomers at 5000 ppm was similar to that of the Control in Weeks 3 - 13. Food consumption of males and females receiving Terpenic Oligomers at 800 or 2000 ppm was similar to that of the Control in Weeks 1 - 13.

During the 4 week recovery period food consumption and body weight gain of males and females which had previously received Terpenic Oligomers at 5000 ppm, were similar or greater than that of Control.

Analysis of haematological parameters, including morphology, after 13 weeks of treatment revealed the following statistical changes; in males receiving 5000 ppm, low reticulocyte counts, shorter prothrombin time and longer activated partial prothrombin time, when compared with the Controls; in females receiving 2000 and 5000 ppm, high platelet counts and shorter prothrombin times, than Controls.

After the 4 week recovery period, reticulocyte counts, prothrombin time and activated partial prothrombin time for males, and prothrombin time for females were all similar to those of the Controls.

Biochemical analysis of plasma after 13 weeks of treatment revealed several minor test article related differences from Control in the plasma composition of animals given Terpenic Oligomers at 800, 2000 and 5000 ppm. Alkaline phosphatase was slightly low for all groups of males only, with statistical significance achieved for males receiving Terpenic Oligomers at 2000 and 5000 ppm. Alanine amino-transferase was slightly low for all groups of males and females, with statistical significance achieved. Aspartate amino-transferase was slightly low in males, with statistical significance in males receiving 5000 ppm, and females receiving 2000 and 5000 ppm. Potassium were slightly but statistically low and total protein concentrations were slightly but statistically high for all females, however all values were within the historical control data range. Albumin concentrations were marginally but statistically significantly low for males receiving 5000 ppm. A/G ratio was slightly low in males and females receiving Terpenic Oligomers, with statistical significance in males and females receiving Terpenic Oligomers at 5000 ppm.

Biochemical analysis of plasma for selected parameters after the 4 week recovery period showed most values were similar to those of the Control. Albumin levels remained statistically lower than the Controls in males previously receiving Terpenic Oligomers at 5000 ppm, although this minor difference is considered not to be of biological significance.

Adjusted liver weights were slightly but statistically high for males and females which received 2000 and/or 5000 ppm, and adjusted kidney weights were slightly high for males which received Terpenic Oligomers at 2000 and 5000 ppm. Males receiving 5000 ppm also had low adjusted mean testes weights and for females at this level high absolute brain weight was recorded.

For males, the analysis of organ weights following 4 weeks without treatment showed complete recovery. The liver weights of recovery females was no longer statistically different to the Control although adjusted brain and kidney weights were slightly high and adjusted ovary weights were slightly low for females which had previously received Terpenic Oligomers at 5000 ppm.

There were no macroscopic abnormalities detected at scheduled termination after 13 weeks of treatment or after 4 weeks of recovery and histopathological evaluation of the tissues from animals killed after 13 weeks of treatment did not reveal any test article-related changes.

Therefore, the dietary administration of Terpenic Oligomers to Sprague Dawley rats at doses of 800, 2000 or 5000 ppm for 13 weeks provided clear evidence of systemic exposure but none of the effects observed were deemed to be adverse. Therefore, the No Observed Adverse Effect Level (NOAEL) was considered to be 5000 ppm, equivalent to 331 mg/kg bw/day for males and 388 mg/kg bw/day for females.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification according to Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data, no self-classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure according to CLP Regulation (EC) No. 1272/2008 and to the UN-GHS Regulation.

There were no data regarding the dermal and inhalation route.