Oligomerisation products of alpha-pinene and beta-pinene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects were observed on reproductive organs (gross pathology and histopathological examinations) in a 90-day repeated dose toxicity study conducted in rats. Therefore no effects on reproductive performance are anticipated.

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, other

Remarks:

data from a 90-day repeated dose toxicity study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2014 - 08 March 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity 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

Vehicle:

corn oil

Details on 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

Details on mating procedure:

Not applicable

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.

Details on study schedule:

Not applicable

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

Parental animals: Observations and examinations:

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

Oestrous cyclicity (parental animals):

Not applicable

Sperm parameters (parental animals):

Not applicable

Litter observations:

Not applicable

Postmortem examinations (parental animals):

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.

Postmortem examinations (offspring):

Not applicable

Statistics:

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

Reproductive indices:

Not applicable

Offspring viability indices:

Not applicable

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

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

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

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

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Key result

Reproductive 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. As no effects on reproductive organs were observed after 90 days of exposure even at the histopathological level, no reproductive adverse toxicological effects are anticipated.

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. As no effects on reproductive organs were observed after 90 days of exposure even at the histopathological level, no reproductive adverse toxicological effects are anticipated.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

334.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Based on the absence of effects on reproductive organs in the 90-day repeated dose toxicity study conducted in rats (gross pathology and histopathological examinations), no effects on reproductive performance are anticipated.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the absence of effects on reproductive organs in the 90-day repeated dose toxicity study conducted in rats (gross pathology and histopathological examinations), no effects on reproductive performance are anticipated.

Therefore, the registered substance should not be classified for toxicity to reproduction according to CLP Regulation (EC) No. 1272/2008 and to the UN-GHS Regulation.