Reaction mass of (4R)-isopropenyl-1-methylcyclohexene and (4S)-isopropenyl-1-methylcyclohexene and 1-isopropyl-4-methylcyclohexa-1,3-diene and 1-isopropyl-4-methylcyclohexa-1,4-diene and 4-isopropylidene-1-methylcyclohexene

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June to 29 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 June to 29 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited
- Age of F0 animals at study initiation: Approximately 10 weeks
- Weight at study initiation: Males: 338-385 g; Females: 225-274 g
- Housing: No. of animals per cage - Males: pre pairing and post pairing - up to 5 animals; Females: pre pairing - up to 5 animals; During pairing - one male and one female; Gestation - one female; Lactation: one female + litter. Toxicity phase and recovery phase - up to five animals of one sex. Cages comprised of a polycarbonate body with a stainless steel mesh lid. Solid (polycarbonate) bottom cages were used during the acclimatisation, gestation, littering and lactation and maturation periods. Grid bottomed cages were used during pairing.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 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

Route of administration:

oral: feed

Vehicle:

other: carrier diet, including corn oil

Details on exposure:

DIET PREPARATION
- Carrier diet: SDS VRF1 certified diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1)
- Method of preparation: 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.
- Frequency of preparation: Weekly
- Storage of preparation: Frozen (nominally -20 °C), until required for feeding

GROUPS
Groups 1, 2, 3 and 4: 0, 800, 2500 and 5000 ppm, respectively

Details on mating procedure:

- Toxicity phase and recovery phase males were paired with reproductive phase females. Recovery phase females were not paired for mating.
- M/F ratio per cage: 1:1 from within the same treatment groups
- Pairing commenced: After three weeks of treatment
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of pregnancy.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Homogeneity and stability of the test material in the carrier diet was determined from Study No. OAD0009. In that study the stability that was achieved over the range 500 to 20000 ppm was: One day ambient in open containers; Eight days ambient in closed containers; 22 days frozen

Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 9 of formulation were analysed for achieved concentration of the test substance.
Samples that were taken in Week 6 of treatment were discarded without analysis due to incorrect storage.

Duration of treatment / exposure:

Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of six consecutive weeks.
Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 6 of lactation.
Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14-day period without treatment.

Frequency of treatment:

Continuously (fresh diet given daily). During the recovery period, all animals were given untreated diet (without corn oil).

Dose / conc.:

800 ppm

Remarks:

nominal in diet

Dose / conc.:

2 500 ppm

Remarks:

nominal in diet

Dose / conc.:

5 000 ppm

Remarks:

nominal in diet

No. of animals per sex per dose:

Reproduction phase: 10 females/dose for all dose groups
Toxicity phase : 5 males/dose for vehicle and 5000 ppm dose groups; 10 males/dose for 800 and 2500 ppm dose groups
Recovery phase: 5 animals/sex/dose for vehicle and 5000 ppm dose groups

Control animals:

other: control group was provided with the carrier diet, including corn oil

Details on study design:

- Dose selection rationale: Dose levels were selected in conjunction with the Sponsor following the completion of the dose range finder study (Study number OAD0019). In the dose range finding study the doses of 1500, 7500 or 15000 ppm were well tolerated, there were no mortalities and no adverse clinical signs or macroscopic changes detected. The body weight effect observed in males and females receiving 15000 ppm was considered too marked for use in an OECD 422 study. The magnitude of body weight effect at 7500 ppm for females was also considered to preclude this dose for use during gestation, parturition and early lactation.
- Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each adult animal. In addition, detailed physical examination and arena observations were performed on reproductive phase females on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation.
During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery phase animals in Group 1 and 4 and the five lowest numbered toxicity males in Group 2 and 3 during Week 6 of treatment and on the five lowest numbered lactating females in each group at Day 4-6 of lactation.
- Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and the five lowest numbered surviving toxicity phase males and females in Groups 2 and 3 and during Day 4 to 6 of lactation for the five lowest numbered lactating females in each group.

BODY WEIGHT: Yes
- Time schedule for examinations:
Toxicity phase males and recovery phase males and females: Before feeding on the day that treatment commenced (Day 1), weekly thereafter and before necropsy.
Reproductive females: Before feeding on the day that treatment commenced (Day 1), weekly before pairing, on Days 0, 6, 13 and 20 after mating, on Days 1, 4 and 7 of lactation and before necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations:
Toxicity phase males, recovery phase males and females and reproductive phase females before pairing: Daily. From these records the mean daily consumption per animal (g/rat/day) was calculated for each cage; Food consumption was not recorded for males during the period when paired for mating (Day 22 to 26). Food consumption recordings recommenced on Day 27.
Reproductive phase females: Daily, during the gestation and lactation periods. From these records the mean daily consumption per animal (g/rat/day) was calculated for each animal.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematology: Week 3 (prior to pairing) - Five lowest numbered reproductive phase females per group and recovery phase females; Week 6 - Five lowest numbered toxicity phase males per group; Recovery Week 2 - All surviving recovery phase animals
Clinical chemistry: Week 3 (prior to pairing) - Five lowest numbered reproductive phase females per group and recovery phase females; Week 6 - Five lowest numbered toxicity phase males per group
- Animals fasted: Yes, Blood samples were collected after overnight withdrawal of food.
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Percentage reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Morphology: Anisocytosis, Macrocytosis, Microcytosis, Hypochromasia, Hyperchromasia, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Oestrous cyclicity (parental animals):

- Dry smears: Daily smears were taken for 15 days before pairing, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the oestrous cycle.
- Wet smears: After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.

Litter observations:

PARAMETERS EXAMINED
- Clinical observations: Examined at approximately 24 h after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age. On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 (before and after culling) and Day 7 of age.
- Individual offspring bodyweights: Days 1, 4 (before and after culling) and Day 7 of age

Postmortem examinations (parental animals):

SACRIFICE
- Toxicity phase animals: Following completion of Week 6 investigations.
- F0 females: Scheduled kill - Day 7 of lactation; Failing to produce viable litter - Day 25 after mating.
- Recovery phase animals: After at least 14 days without treatment.
- Method of sacrifice: F0 animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination.

GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. 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. In addition the number of uterine implantation sites recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together.
Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
The five lowest numbered surviving toxicity phase males and the five lowest numbered reproductive phase females with live litter in Groups 1 and 4 at scheduled termination
Abnormalities only: All animals
Kidneys: All males from the toxicity (Group 2 and 3) and recovery (Group 1 and 4) phases which had tissues retained.
- Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid Schiff (PAS) method.
- Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of Testes in modified Davidson’s fluid; Eyes In Davidson’s fluid.

Postmortem examinations (offspring):

SACRIFICE:
- F1 offspring scheduled kill: Day 7 of age
- Method of sacrifice: Intraperitoneal injection of sodium pentobarbitone

GROSS NECROPSY
- Offspring at scheduled termination: All F1 offspring were examined externally; any offspring found to be externally abnormal were also examined internally.
- Day 4 cull: Up to two males and up to two females offspring per litter were examined, eviscerated and fixed in industrial methylated spirit.

This also included an assessment for the presence of milk in the stomach, where this was possible. Abnormal tissues retained.

Statistics:

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

Reproductive indices:

Mating Performance and Fertility:
- Percentage mating = (Number animals mating / Animals paired) x 100
- Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
- Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100

Gestation length
- Gestation index (%) = (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

Survival indices:
- Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
- Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
- Viability index (%) = (Number of live offspring on Day 4 before culling / Number of live offspring on Day 1 after littering) x 100
- Lactation index (%) = (Number of live offspring on Day 7 after littering / Number live offspring on Day 4) x 100

Sex ratio:
- Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

An increased incidence (7/15 compared to 0/15 in Control) of vocalisation was apparent for females receiving 5000 ppm; this is not an uncommon finding in this species but may indicate that females at this dietary concentration were more irritable. Brown staining affected the head or upper dorsal thorax of animals during the study, males and females were still affected during the recovery phase. These observations lacked a dose relationship indicating it is unlikely to be related to the administration of Multiconstituent Dipentene

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male that previously received 5000 ppm (Group 4 Male animal number 40) was found dead on Day 8 of recovery. None of the macroscopic or microscopic findings were considered to be related to treatment or have contributed to the death of this animal; the cause of death was undetermined. In the absence of any other deaths in this dose group an association to treatment is considered unlikely.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain up to pairing (Days 1 to 22) and for the complete treatment period was low among animals receiving 2500 or 5000 ppm although statistical significance (p<0.05) was only attained for recovery phase females receiving 5000 ppm during Days 1 to 43; a dose dependant trend was not apparent for females. During recovery animals that had previously received 5000 ppm displayed statistically higher body weight gain when compared with Controls (p<0.01 for males and p<0.05 for females).

At the start of gestation body weights were similar across all groups, however gains were slightly low for animals receiving 5000 ppm with an indication of similar trend at 2500 ppm.
During lactation mean body weight loss was recorded at 5000 ppm during Days 1 to 4 and body weight gain was subsequently low during Days 4 to 7, the overall bodyweight gain (Day 1-7) was significantly low (0.30X Control) when compared with Controls (p<0.05). No effects were apparent at 2500 or 800 ppm.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Weekly mean food consumption data indicated males receiving 2500 or 5000 ppm throughout the dosing period and all females receiving 5000 ppm before pairing was marginally lower than Controls this difference was not considered to be adverse at the degree observed. The food consumption during recovery was noted to be slightly higher than Control for animals that previously received 5000 ppm.
No effect on food consumption during gestation or lactation was apparent at 2500 or 5000 ppm.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

During Week 6 of study, males receiving 5000 ppm were recorded to be slightly but statistically significantly low haematocrit, haemoglobin concentration and large unstained cell count. However these data were within historical control data. During recovery these parameters for males which had previously received 5000 ppm were similar to Control.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings considered to be related to treatment with Multiconstituent Dipentene were observed in the male kidney. Minimal hyaline droplets within the cortical tubules were observed in all kidneys examined microscopically from males that received 5000 or 2500 ppm and the majority of males that received 800 ppm. A similar change was not observed in the kidneys of any females.
Microscopically there was minimal hyaline droplet accumulation in the cortical tubules of the kidneys of males only at all dose levels and this is consistent with the accumulation of alpha 2u globulin. This common pathological change has been associated with the exposure to a variety of hydrocarbons-containing chemicals, and is restricted to male rats. The minimal levels of droplet accumulation observed were considered to be non-adverse. In addition, following a 2-week recovery period hyaline droplets were not observed in the kidneys of any males previously receiving 5000 ppm, indicating complete recovery. No pathological changes observed in the reproductive tissues examined were considered to be related to the administration of Multiconstituent Dipentene. This observation is not relevant to humans.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- No adverse signs were observed in association with dose administration and there were no signs at routine physical examination that could be related to treatment.
- An increased incidence (7/15 compared to 0/15 in Control) of vocalisation was apparent for females receiving 5000 ppm; this is not an uncommon finding in this species but may indicate that females at this dietary concentration were more irritable. Brown staining affected the head or upper dorsal thorax of animals during the study, males and females were still affected during the recovery phase. These observations lacked a dose relationship indicating it is unlikely to be related to the administration of test item.
- On Day 8 of recovery one male that previously received 5000 ppm was found dead. The cause of death was undetermined but considered of unlikely relationship to treatment.

BODY WEIGHT (PARENTAL ANIMALS)
- Body weight gain up to pairing (Days 1 to 22) and for the complete treatment period was low among animals receiving 2500 or 5000 ppm although statistical significance (p<0.05) was only attained for recovery phase females receiving 5000 ppm during Days 1 to 43; a dose dependant trend was not apparent for females. During recovery animals that had previously received 5000 ppm displayed statistically higher body weight gain when compared with Controls (p<0.01 for males and p<0.05 for females).
- At the start of gestation body weights were similar across all groups, however gains were slightly low (0.90X Control) for animals receiving 5000 ppm with an indication of similar trend at 2500 ppm (0.94X Control).
- During lactation mean body weight loss was recorded at 5000 ppm during Days 1 to 4 and body weight gain was subsequently low during Days 4 to 7, the overall bodyweight gain (Day 1-7) was significantly low (0.30X Control) when compared with Controls (p<0.05). No effects were apparent at 2500 or 800 ppm.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Weekly mean food consumption data indicated males receiving 2500 or 5000 ppm throughout the dosing period and all females receiving 5000 ppm before pairing was marginally lower than Controls this difference was not considered to be adverse at the degree observed. The food consumption during recovery was noted to be generally slightly higher than Control for animals that previously received 5000 ppm.
- Food consumption throughout gestation was essentially similar to the Control, with only statistically significantly lower food consumption than Controls at Days 5 and 7 in females receiving 5000 ppm. During lactation (Days 2 to 6 inclusive) food consumption for females of this latter group was significantly low when compared with Controls (0.81X Control). No effect on food consumption during gestation or lactation was apparent at 2500 or 5000 ppm.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- Overall mean achieved doses at 800, 2500 or 5000 ppm were 50, 148 or 298 mg/kg bw/day for males, respectively. Overall mean achieved dose levels for reproductive phase females prior to paring and recovery females throughout receiving 800, 2500 or 5000 ppm were 53, 174 or 316 mg/kg bw/day, respectively. Overall mean achieved dose levels for females during gestation receiving 800, 2500 or 5000 ppm were 56, 172 or 333 mg/kg bw/day and during lactation were 97, 301 or 529 mg/kg bw/day, respectively.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- There was considered to be no effect of treatment on oestrous cycles.
- The distribution of cycles for females receiving 5000 ppm attained statistical significance, the majority of females showed regular cycles, one female had an irregular cycle and one female had an extended cycle. The irregular cycle was within Historical Control Data range and the extended oestrous for a single female in the absence of cycle disruption in the majority of females is considered to have arisen by chance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Pre-coital interval and fertility were unaffected by the dietary administration of test item.
- All the females gave birth within the expected range of 22-23.5 days after mating therefore this parameter was not considered to be adversely affected. A slight shift in gestation length was apparent in comparison with the concurrent Control the difference for females receiving 5000 ppm attained statistical significance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Organ weights for toxicity phase males killed after 6 weeks of treatment, females killed on Day 7 of lactation and recovery males and females killed following 2 weeks of recovery indicated no adverse effects of dosing.
- Males at 5000 ppm terminated after Week 6 of study had slightly low adjusted mean prostate and seminal vesicle weights (0.85X and 0.86X Control, respectively) but these differences did not attain statistical significance. At the end of the 2-week recovery period prostate and seminal vesicle weights were similar to Controls for males which had previously received 5000 ppm.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- Examination of males after 6 weeks of dosing, of females on Day 7 of lactation and males and females following a 2 week recovery period did not reveal any findings that could be attributed to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Males killed after 6 weeks of dosing and females killed on Day 7 of lactation:
Treatment related findings: Minimal hyaline droplets within the cortical tubules were observed in all kidneys examined microscopically from males that received 5000 or 2500 ppm and the majority of males that received 800 ppm. A similar change was not observed in the kidneys of any females.
Incidental findings: All other findings were considered to be incidental and unrelated to treatment and were consistent with common background microscopic changes previously seen in control Sprague Dawley rats.

Animals killed after 2 weeks of recovery
Treatment related findings: The kidneys from all recovery males and any abnormalities identified at the macroscopic examination were examined microscopically. No findings were considered to be related to treatment.
The incidence and distribution of all findings were consistent with common background changes previously seen in control Sprague Dawley rats.

OTHER FINDINGS (PARENTAL ANIMALS)
- Sensory reactivity and grip strength: Sensory reactivity and grip strength examined during Week 6 of dosing and Days 4 to 6 of lactation (females only) were considered unaffected by the administration of test item.
- Motor activity: There was no effect on either rearing (high beam) or ambulatory (low beam) motor activity.
- Haematology: Analysis of haematological parameters during Week 3 of study for females and Week 6 of study for males revealed low haematocrit and haemoglobin and large unstained cell counts, in males given 5000 ppm. Females receiving 5000 ppm had low reticulocytes (absolute count and percentage), mean cell haemoglobin concentration, and red cell distribution width and a slight increase in mean cell volume. There were no statistical differences to the Control 14 days after the cessation of dosing.
- Biochemistry: Analysis of biochemical parameters during Week 6 of study for males and Week 3 of study for males indicated there were no adverse effects of the administration of test item. In males, a statistical significance was attained for low alanine amino transferase activity at all dose levels (0.81X to 0.84X Control), a dose response was not apparent and a change of this degree was considered not to be adverse. Females receiving 2500 or 5000 ppm showed slightly low alanine amino transferase activity (0.78X Control for both), with 5000 ppm attaining statistical significance and females at 5000 ppm showed significantly low cholesterol levels (0.90X Control). The degree of change recorded was considered not to be adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

298 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effect observed at the highest concentration tested

Key result

Dose descriptor:

NOAEL

Effect level:

333 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effect observed at the highest concentration tested

Key result

Dose descriptor:

NOAEL

Effect level:

301 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Test item intake for females during lactation at 2500 ppm

Critical effects observed:

no

Lowest effective dose / conc.:

2 500 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

no

Clinical signs:

not examined

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Offspring body weight on Day 1 of age and the subsequent body weight gain up to Day 4 of age was not affected by parental administration of Multiconstituent Dipentene.
Offspring body weight change at 2500 or 5000 ppm, during Days 4 to 7 were lower when compared with Controls (0.85X or 0.79X Control for females and 0.87X Control for males respectively); statistical significance was attained for females only. As the offspring at 5000 ppm remained in good clinical condition, body weight was not significantly impacted and survival was not affected, the effect on body weight change within the context of this study was not considered to be adverse.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

CLINICAL SIGNS (OFFSPRING)
The distribution of neonatal signs at physical examination showed no relationship to parental administration of test item.

LITTER SIZE, SURVIVAL INDICES AND SEX RATIO
- Overall survival litter size and sex ratio was unaffected by parental administration of test item.
- The mean post implantation survival and litter size on Day 1 at 5000 ppm was marginally low compared with Controls, but this was attributed to litter no. 92 that had a post implantation survival index of 67% and a litter size of 10 offspring.

BODY WEIGHT (OFFSPRING)
- Offspring body weight on Day 1 of age and the subsequent body weight gain up to Day 4 of age was not affected by parental administration of test item.
- Offspring body weight change at 2500 or 5000 ppm, during Days 4 to 7 were lower when compared with Controls (0.85X or 0.79X Control for females and 0.87X Control for males respectively); statistical significance was attained for females only.

SKELETAL ASSESSMENT (OFFSPRING)
- There were no differences in structure or ossification at skeletal assessment of culled offspring on Day 4 of age that could be attributed to the test item.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 7 of age did not reveal any findings that could be attributed to the administration of test item.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Basis for effect level:

other: no effect observed at the highest concentration tested

Remarks on result:

not measured/tested

Critical effects observed:

no

Clinical signs:

not examined

Key result

Reproductive effects observed:

no

Formulation analysis:

The mean concentrations of test item in test formulations analysed for the Week 1 were within applied limits of +10 %/-15 % of nominal concentrations, confirming accurate formulation. The investigation into Week 9 showed that the concentration of Alpha‑terpinene in both the formulations and procedural recoveries had reduced by approximately 50 % due to the oxidization of the sub-sample of Multiconstituent Dipentene used for the analyses over the course of the study. When the mean Week 1 procedural recovery is used to correct the Week 9 formulations, Group 2 is outside acceptable limits with an RME of -20.6 % from the nominal concentration, Groups 3 and 4 are both within applied limits.

Table 7.8.1/4: Analysed concentrations for Multiconstituent Dipentene in SDS VRF1 certified diet

 

Occasion	Group	Nominal inclusion (ppm)	Analysed concentration (ppm)			RME (%)	Difference from mean (%)	Procedural recoveries (%)
			Analysis 1	Analysis 2	Mean			At analysis	Mean
Week 1	1	0	ND	ND	-	-	-
	2	800	770	772	771	-3.6	±0.16	87.9	87.2
	3	2500	2400	2480	2390	-4.4	±0.43	86.0
	4	5000	4950	5220	5090	+1.8	±2.62	87.8
Week 9	1	0	ND	ND	-	-	-
	2	800	632	637	635	-20.6	±0.40	-	87.2^1
	3	2500	2220	2240	2230	-10.8	±0.53	-
	4	5000	4370	4450	4410	-11.8	±0.85	-

RME: Relative mean error, representing the deviation from nominal

ND: Not detected

¹ Week 9 results are corrected for the mean of Week 1 procedural recoveries

Conclusions:

The NOAEL for this screening study was 5000 ppm, equivalent to 298 mg/kg bw/day for males, 316 mg/kg bw/day for unmated females and 333 mg/kg bw/day for females during gestation. The dose level of 529 mg/kg bw/day ingested by females during lactation could be considered as adverse for those females due to low body weight gain/loss and low food consumption.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, groups of Crl:CD(SD) rats received REACTION MASS OF DL-LIMONENE, ALPHA‑ GAMMA- TERPINENES, TERPINOLENE orally, via the diet, at concentrations of 800, 2500 or 5000 ppm. Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of six consecutive weeks. Reproductive females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14-day period without treatment. Recovery phase males were used for pairing with reproductive phase females, but recovery females were not paired. A similarly constituted Control group was assigned to each phase, and received the vehicle, untreated diet, throughout the same relative treatment period.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology, blood chemistry, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic investigations were undertaken for adult animals. Microscopicpathology investigations were undertaken for the first five toxicity phase males and reproductive phase females in Group 1 (Control) and Group4 (5000 ppm) with examination of kidneys from males of Group 2 and 3. The clinical condition of offspring, litter size and survival, sex ratio and bodyweight were assessed, and macroscopic pathology investigations were undertaken. Offspring culled at Day 4 were examined for skeletal development and abnormalities. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.

Test item at dietary concentrations of 800, 2500 or 5000 ppm resulted in overall mean achieved doses of 50, 148 or 298 mg/kg bw/day for males, respectively. The overall mean achieved dose levels for unmated females were 53, 174 or 316 mg/kg bw/day, for mated females during gestation were 56, 172 or 333 mg/kg bw/day and during lactation were 97, 301 or 529 mg/kg bw/day at dietary concentrations of 800, 2500 or 5000 ppm, respectively.

On Day 8 of recovery one male that previously received 5000 ppm was found dead. The cause of death was undetermined but considered of unlikely relationship to treatment. Test item was well tolerated, with no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, blood chemistry, organ weights and macroscopic pathology. In addition the reproductive endpoints unaffected by treatment included oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, offspring clinical condition, litter size, survival, sex ratio or external or skeletal development.

Overall body weight gain and weekly mean food consumption data for the dosing period were generally low for animals receiving 2500 or 5000 ppm. During recovery, animals that had previously received 5000 ppm showed statistically significantly high body weight gain coupled with a slight increase in food consumption. During gestation body weight gain and mean food consumption for all treated females were generally similar to Control values. During lactation mean body weight gain and food consumption were significantly low for females receiving 5000 ppm, with body weight loss observed between Days 1-4.

Offspring body weight gain of litters at 5000 ppm was slightly low between Days 4-7 of age. Dams receiving 5000 ppm were also noted to have low food consumption and impaired body weight performance correlating with the offspring bodyweight effects; however within the context of this study the aetiology of these effects cannot be determined. As the offspring at 5000 ppm remained in good clinical conditions, body weights were not significantly impacted and survival was not affected, the effect on body weight change within the context of this study was not considered to be adverse.

Analysis of haematological parameters during Week 3 of study for females and Week 6 of study for males revealed low haematocrit and haemoglobin and large unstained cell counts, in males given 5000 ppm. Females receiving 5000 ppm had low reticulocytes (absolute count and percentage), mean cell haemoglobin concentration, and red cell distribution width and a slight increase in mean cell volume. There were no statistical differences to the Control 14 days after the cessation of dosing. 

Microscopically there were minimal hyaline droplets within the cortical tubules observed for all males examined (5/5) that received 2500 or 5000 ppm and the majority of males examined (4/5) at 800 ppm. This change was only observed in males and considered to be related to the administration of the test item. This change was not observed in the kidneys of any males that previously received 5000 ppm following a 2-week recovery period, indicating complete recovery.

Therefore, the NOAEL for this screening study was 5000 ppm, equivalent to 298 mg/kg bw/day for males, 316 mg/kg bw/day for unmated females and 333 mg/kg bw/day for females during gestation. The dose level of 529 mg/kg bw/day ingested by females during lactation could be considered as adverse for those females due to low body weight gain/loss and low food consumption.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

26 February to 25 March 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Preliminary study used as range-finder experiment for OECD 422 screening test, performed in GLP laboratory.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

Range-finder experiment for an OECD guideline 422 study, where clinical condition, bodyweight, food consumption, water consumption, oestrous cycles, blood chemistry, organ weight and macropathology investigations were undertaken in male and female rats exposed to test substance by dietary administration for 21 days.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Crl: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: Approximately 66 days
- Weight at study initiation: Males: 327-377 g; Females: 241-277 g
- Housing: Animals were housed in groups of 4/sex in polycarbonate cages
- Diet: SDS VRF1 Certified powdered diet; ad libitum
- Water: Potable water from public supply; ad libitum
- Acclimation period: 5 days

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

Route of administration:

oral: feed

Vehicle:

other: carrier diet, including corn oil

Details on oral exposure:

DIET PREPARATION
- Carrier diet: SDS VRF1 certified diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1)
- Method of preparation: 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.

- Frequency of preparation: Weekly
- Storage of preparation: Frozen (nominally -20 °C), until required for feeding

GROUPS
Groups 1, 2, 3 and 4: 0, 1500, 7500 and 15000 ppm, respectively

Stability and homogeneity
Homogeneity and stability of the test material in the vehicle from Study No. OAD0009. In that study the stability that was achieved over the range 500 to 20000 ppm was: One day ambient in open containers; Eight days ambient in closed containers; 22 days frozen.

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 (preparations replaced daily although stability was demonstrated up to 8 days).

Dose / conc.:

1 500 ppm

Remarks:

nominal in diet

Dose / conc.:

7 500 ppm

Remarks:

nominal in diet

Dose / conc.:

15 000 ppm

Remarks:

nominal in diet

No. of animals per sex per dose:

4

Control animals:

other: control group was provided with the carrier diet, including corn oil

Details on study design:

Dose selection rationale: Doses used in this study (0, 1500, 7500 and 15000 ppm) were selected in conjunction with the Sponsor.
In an OECD 422 study conducted with the related substance Terpinolene Multiconstituent (containing 30 % Terpinolene, 9.2 % d-Limonene and 9.2 % l Limonene), the NOAEL was set at 7500 ppm. The LD50 of the related substance was >2000 mg/kg bw in the rat. The dietary concentrations selected for this study were therefore 1500, 7500 and 15000 ppm.

Positive control:

Not applicable

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on 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 during the acclimatisation period, on the day that treatment commenced (Day 1), daily throughout the study and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 3 from all animals
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea and Creatinine.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Dry smears: Daily smears were taken using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the oestrous cycle.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; Animals were killed following 21 days of treatment by carbon dioxide asphyxiation and subjected to detailed necropsy.
ORGAN WEIGHTS: Yes; organ weights of adrenals, epididymides, heart, kidneys, liver, ovaries, prostate, spleen and testes were recorded. For bilateral organs, left and right organs were weighed together.
HISTOPATHOLOGY: No; but tissues with macroscopic abnormalities were preserved for microscopic examinations in 10% neutral buffered formalin for microscopic examination (except testes: In modified Davidson’s fluid).

Other examinations:

None

Statistics:

No data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs were limited to a single female receiving 15000 ppm observed as irritable with increased vocalisation and a female receiving 1500 ppm with hairloss from the forelimbs.
These signs are not attributed to the test material.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 15000 ppm males, and females at 7500 and 15 000 ppm, lossed weight during Day 1 to Day 4.
At 15000 and 7500 ppm body weight gain in males was low from Day 8 until necropsy.
At 1500 ppm body weight gain in males was low between Day 11 and 18.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

On Day 1 food consumption was markedly low for males and females receiving 15000 ppm.
From Day 2, food consumption for males improved, whilst for females food consumption improved from Day 4 of study, although mean consumption levels remained below Control values throughout treatment for both sexes, leading to overall food consumption of 76 and 79% of Control for males and females, respectively. Food consumption for animals receiving 7500 ppm was initially lower than the Control and slightly improved but mean consumption levels remained below Control values throughout treatment for both sexes leading to overall food consumption 87 and 84% of Control for males and females respectively.
At 1500 ppm, food consumption for males or females showed no adverse effects of treatment, although slightly low in males compared to Control (91% of Control).

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Visual assessment indicated low water consumption for the animals receiving either 7500 or 15000 ppm

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma levels of urea and creatinine were slightly elevated at 7500 and 15000 ppm, with evidence of a dose response for creatinine but not urea.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Consistent effects on organ weight included high absolute, body weight adjusted and body weight relative liver weights in treated groups, with an apparent dose response for males but not for females. High body weight adjusted and body weight relative kidney weights in treated groups which demonstrated a dose response, were observed in both sexes.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- Clinical signs were limited to a single female receiving 15000 ppm observed as irritable with increased vocalisation and a female receiving 1500 ppm with hairloss from the forelimbs. These signs are not attributed to the test item.

BODY WEIGHT AND WEIGHT GAIN
- Males receiving 15000 ppm showed mean body weight loss during Days 1 to 4, there was no effect during the following four day period but low gains were apparent from Day 8 until necropsy. At 7500 ppm male body weight gain was low from Day 8 until necropsy. At 1500 ppm body weight gain in males was low between Day 11 and 18. These changes resulted in overall body weight gain 74, 57 and 32 % of Control at 1500, 5000 and 15000 ppm, respectively.
- Females receiving 7500 or 15000 ppm showed mean body weight loss between Day 1 and 4 of study. From Day 4 the body weight performance at 7500 or 15000 ppm stabilised but mean body weights remained below Day 1 values up to termination; a dose response was apparent. Body weight performance of females receiving 1500 ppm was unaffected by treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- On Day 1 food consumption was markedly low for males and females receiving 15000 ppm. From Day 2, food consumption for males improved, whilst for females food consumption improved from Day 4 of study, although mean consumption levels remained below Control values throughout treatment for both sexes, leading to overall food consumption 75 and 79 % of Control for males and females, respectively. Food consumption for animals receiving 7500 ppm was initially lower than the Control and slightly improved but mean consumption levels remained below Control values throughout treatment for both sexes, leading to overall food consumption 87 and 84 % of Control for males and females, respectively.
- At 1500 ppm, food consumption for males or females showed no adverse effects of treatment, although slightly low in males compared to Control (91 % of Control).

WATER CONSUMPTION
- Visual assessment indicated low water consumption for the animals receiving either 7500 or 15000 ppm.

CLINICAL CHEMISTRY
- There were no adverse effects of treatment on blood plasma liver parameters.
- Plasma levels of urea and creatinine were slightly elevated at 7500 and 15000 ppm, with evidence of a dose response for creatinine but not urea.

ORGAN WEIGHTS
- Terminal body weight was low for males at all dose levels and females that received 7500 ppm or higher. Due to the differences in terminal body weight adjusted mean organ weights and body weight relative organ weights were evaluated in addition to absolute organ weights. Consistent effects on organ weight included high absolute, body weight adjusted and body weight relative liver weights in treated groups, with an apparent dose response for males but not for females. High body weight adjusted and body weight relative kidney weights in treated groups which demonstrated a dose response, were observed in both sexes.
- Other differences were restricted to a single sex and/or showed inconsistency between adjusted and body weight relative organ weight and therefore may be an artefact of the differences in terminal body weight rather than a true effect of treatment.

GROSS PATHOLOGY
- There were no macroscopic findings that could be attributed to treatment.

OTHER FINDINGS
- Oestrus cycles were unaffected by treatment with Multiconstituent Dipentene.

Critical effects observed:

not specified

Achieved dose:

At dietary concentrations of 1500, 7500 and 15000 ppm overall mean achieved dose levels (Days 1-21) were 104, 484 and 899 mg/kg bw/day and 102, 476 and 930 mg/kg bw/day for males and females, respectively.

Achieved dose (mg/kg bw/day) at 7500 and 15000 ppm was initially low, reflecting the food consumption of these groups. In general achieved dose reflected variability in the daily food consumption values.

Conclusions:

It is concluded that a high dose level between 1500 ppm and 7500 ppm would be suitable for use on a subsequent OECD 422 combined repeated dose and reproductive screening study and a dose level less than 1500 ppm should be considered for the low dose level.

Executive summary:

In a repeated dose toxicity range-finding study, groups of Crl:CD(SD) rats (4/sex/dose) received REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE orally, via the diet at concentrations of 1500, 7500 or 15000 ppm. A similarly constituted control group received untreated diet throughout the same treatment period. During the study, clinical condition, body weight, food consumption, oestrous cycles, blood chemistry, organ weight and macropathology investigations were undertaken.

Administration of the test item at dietary concentrations of 1500, 7500 and 15000 ppm resulted in overall mean achieved dose levels (Days 1-21) of 104, 484 and 899 mg/kg bw/day and 102, 476 and 930 mg/kg bw/day for males and females, respectively.

There were no premature deaths and no signs at routine physical examination that could be attributed to treatment.

Overall (Days 1 to 21) bodyweight gain, food and water consumption for males receiving 7500 or 15000 ppm was low when compared with Controls; a dose response was apparent. At 1500 ppm bodyweight gain for males was low between Days 11 and 18 of treatment, whilst food and water consumption was similar to Control values. Females receiving 7500 or 15000 ppm showed an overall loss in bodyweight during the treatment period correlating with the low food and water consumption over the same period. Body weight performance, food and water consumption were unaffected by treatment for females at 1500 ppm.

Oestrous cycles were unaffected by treatment.

Plasma levels of urea and creatinine were slightly elevated at 7500 and 15000 ppm for both sexes.

Consistent effects on organ weight included high liver weights in treated groups and high kidney weights in treated groups for both sexes. 

A dose response was apparent for males but not females.

There were no macroscopic observations that could be attributed to treatment.

It is concluded that a high dose level between 1500 ppm and 7500 ppm would be suitable for use on a subsequent OECD 422 combined repeated dose and reproductive screening study and a dose level less than 1500 ppm should be considered for the low dose level.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

November 2015

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

227-813-5

EC Name:

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

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-isopropenyl-1-methylcyclohexene

Constituent 2

Reference substance name:

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

EC Number:

227-815-6

EC Name:

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

Cas Number:

5989-54-8

Molecular formula:

C10H16

IUPAC Name:

(4S)-isopropenyl-1-methylcyclohexene

Constituent 3

Reference substance name:

p-mentha-1,4-diene

EC Number:

202-794-6

EC Name:

p-mentha-1,4-diene

Cas Number:

99-85-4

Molecular formula:

C10H16

IUPAC Name:

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

Constituent 4

Reference substance name:

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

EC Number:

209-578-0

EC Name:

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

Cas Number:

586-62-9

Molecular formula:

C10H16

IUPAC Name:

4-isopropylidene-1-methylcyclohexene

impurity 1

Reference substance name:

(3R)-isopropylidene-6-methylcyclohexene

Cas Number:

43124-56-7

Molecular formula:

C10H16

IUPAC Name:

(3R)-isopropylidene-6-methylcyclohexene

impurity 2

Reference substance name:

(3S)-isopropylidene-6-methylcyclohexene

Cas Number:

62504-16-9

Molecular formula:

C10H16

IUPAC Name:

(3S)-isopropylidene-6-methylcyclohexene

impurity 3

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

impurity 4

Reference substance name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-336-2

EC Name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-03-6

Molecular formula:

C10H16

IUPAC Name:

(1R,4S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 5

Reference substance name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-337-8

EC Name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-04-7

Molecular formula:

C10H16

IUPAC Name:

(1S,4R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 6

Reference substance name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

2243-33-6

Molecular formula:

C10H16

IUPAC Name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

impurity 7

Reference substance name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

EC Number:

224-167-6

EC Name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

4221-98-1

Molecular formula:

C10H16

IUPAC Name:

(5R)-isopropyl-2-methylcyclohexa-1,3-diene

Test material form:

liquid

Details on test material:

Batch No.: 146750
Purity: 84.8% (sum of the five main constituents)
Name of test material (as cited in study report): Reaction mass of dl-limonene, alpha- gamma-terpinenes, terpinolene
Physical state: colourless - slightly yellow liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 22 October 2013

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited
- Age of F0 animals at study initiation: Approximately 10 weeks
- Weight at study initiation: Males: 338-385 g; Females: 225-274 g
- Housing: No. of animals per cage - Males: pre pairing and post pairing - up to 5 animals; Females: pre
pairing - up to 5 animals; During pairing - one male and one female; Gestation - one female; Lactation:
one female + litter. Toxicity phase and recovery phase - up to five animals of one sex. Cages comprised
of a polycarbonate body with a stainless steel mesh lid. Solid (polycarbonate) bottom cages were used
during the acclimatisation, gestation, littering and lactation and maturation periods. Grid bottomed cages
were used during pairing.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: carrier diet, including corn oil

Details on oral exposure:

- Carrier diet: SDS VRF1 certified diet.
- Stabiliser: Corn oil (test material to corn oil ratio 5:1)
- Method of preparation: 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.
- Frequency of preparation: Weekly
- Storage of preparation: Frozen (nominally -20 °C), until required for feeding
GROUPS
Groups 1, 2, 3 and 4: 0, 800, 2500 and 5000 ppm, respectively

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Homogeneity and stability of the test material in the carrier diet was determined from Study No. OAD0009. In that study the stability that was achieved over the range 500 to
20000 ppm was: One day ambient in open containers; Eight days ambient in closed containers; 22 days frozen
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 9 of formulation were analysed for achieved concentration of the test substance.
Samples that were taken in Week 6 of treatment were discarded without analysis due to incorrect storage.

Duration of treatment / exposure:

Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of six consecutive weeks.
Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14-day period without treatment

Frequency of treatment:

Continuously (fresh diet given daily). During the recovery period, all animals were given untreated diet (without corn oil).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

800 ppm

Remarks:

nominal in diet

Doses / concentrations 2

Dose / conc.:

2 500 ppm

Remarks:

nominal in diet

Doses / concentrations 3

Dose / conc.:

5 000 ppm

Remarks:

nominal in diet

No. of animals per sex per dose:

Toxicity phase : 5 males/dose for vehicle and 5000 ppm dose groups; 10 males/dose for 800 and 2500 ppm dose groups
Reproductive phase: 10 females/dose
Recovery phase: 5 animals/sex/dose for vehicle and 5000 ppm dose groups

Control animals:

other: control group was provided with the carrier diet, including corn oil

Details on study design:

- Dose selection rationale: Dose levels were selected in conjunction with the Sponsor following the completion of the dose range finder study (Study number OAD0019). In the dose range finding study the
doses of 1500, 7500 or 15000 ppm were well tolerated, there were no mortalities and no adverse clinical signs or macroscopic changes detected. The body weght effect observed in males and females receiving 15000 ppm was considered too marked for use in an OECD 422 study. The magnitude of body weight
effect at 7500 ppm for females was also considered to preclude this dose for use during gestation, parturition and early lactation.
- Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each adult animal. During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery phase animals in Group 1 and 4 and the five lowest numbered toxicity males in Group 2 and 3 during Week 6 of treatment.
- Motor activity: During Week 6 of treatment, the motor activity of all recovery animals in Groups 1 and 4 and the five lowest numbered surviving toxicity phase males and females in Groups 2 and 3.

BODY WEIGHT: Yes
- Time schedule for examinations:
Toxicity phase males and recovery phase males and females: Before feeding on the day that treatment commenced (Day 1), weekly thereafter and before necropsy.

- Time schedule for examinations:
Toxicity phase males, recovery phase males and females and reproductive phase females before pairing: Daily. From these records the mean daily consumption per animal (g/rat/day) was calculated for each cage; Food consumption was not recorded for males during the period when paired for mating (Day 22 to
26). Food consumption recordings recommenced on Day 27.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematology: Week 3 (prior to pairing) - Five lowest numbered reproductive phase females per group and recovery phase females; Week 6 - Five lowest numbered toxicity phase males per group; Recovery Week 2 - All surviving recovery phase animals
Clinical chemistry: Week 3 (prior to pairing) - Five lowest numbered reproductive phase females per group and recovery phase females; Week 6 - Five lowest numbered toxicity phase males per group
- Animals fasted: Yes, Blood samples were collected after overnight withdrawal of food.
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Percentage reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte co
unt: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count,
Morphology: Anisocytosis, Macrocytosis, Microcytosis, Hypochromasia, Hyperchromasia, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (gGT), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

Sacrifice and pathology:

SACRIFICE
- Toxicity phase animals: Following completion of Week 6 investigations.
- F0 females: Scheduled kill - Day 7 of lactation; Failing to produce viable litter - Day 25 after mating.

GROSS NECROPSY
- All adult animals were subject to a detailed necropsy. 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together.
Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
The five lowest numbered surviving toxicity phase males and the five lowest numbered reproductive phase females with live litter in Groups 1 and 4 at scheduled termination
Abnormalities only: All animals
Kidneys: All males from the toxicity (Group 2 and 3) and recovery (Group 1 and 4) phases which had tissues retained.
- Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid Schiff (PAS) method.
- Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of Testes in modified Davidson’s fluid; Eyes In Davidson’s fluid

Statistics:

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

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence (7/15 compared to 0/15 in Control) of vocalisation was apparent for females receiving 5000 ppm; this is not an uncommon finding in this species but may indicate that females at
this dietary concentration were more irritable. Brown staining affected the head or upper dorsal thorax of animals during the study, males and females were still affected during the recovery phase. These
observations lacked a dose relationship indicating it is unlikely to be related to the administration of Multiconstituent Dipentene

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male that previously received 5000 ppm (Group 4 Male animal number 40) was found dead on Day 8 of recovery. None of the macroscopic or microscopic findings were considered to be related to treatment or have contributed to the death of this animal; the cause of death was undetermined. In the absence of any other deaths in this dose group an association to treatment is considered unlikely

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain up to pairing (Days 1 to 22) and for the complete treatment period was low among animals receiving 2500 or 5000 ppm although statistical significance (p<0.05) was only attained for recovery phase females receiving 5000 ppm during Days 1 to 43; a dose dependant trend was not apparent for females. During recovery animals that had previously received 5000 ppm displayed statistically higher body weight gain when compared with Controls (p<0.01 for males and p<0.05 for females).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Weekly mean food consumption data indicated males receiving 2500 or 5000 ppm throughout the dosing period and all females receiving 5000 ppm before pairing was marginally lower than Controls this difference was not considered to be adverse at the degree observed. The food consumption during recovery was noted to be slightly higher than Control for animals that previously received 5000 ppm.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

During Week 6 of study, males receiving 5000 ppm were recorded to be slightly but statistically significantly low haematocrit, haemoglobin concentration and large unstained cell count. However these data
were within historical control data. During recovery these parameters for males which had previously received 5000 ppm were similar to Control.

During Week 3 of study, females receiving 5000 ppm indicated slightly low reticulocytes, mean cell haemoglobin concentration, red cell distribution width and slightly increased mean cell volume all differences attaining statistical significance. However these data are within historical control data. After 14 days recovery there were no statistical differences to the Control.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings considered to be related to treatment with Multiconstituent Dipentene were observed in the male kidney. Minimal hyaline droplets within the cortical tubules were observed in all kidneys examined microscopically from males that received 5000 or 2500 ppm and the majority of males that received 800 ppm.
A similar change was not observed in the kidneys of any females.
Microscopically there was minimal hyaline droplet accumulation in the cortical tubules of the kidneys of males only at all dose levels and this is consistent with the accumulation of alpha 2u globulin. This common pathological change has been associated with the exposure to a variety of hydrocarbons-containing chemicals, and is restricted to male rats. The minimal levels of droplet accumulation observed were considered to be non-adverse. In addition, following a 2-week recovery period hyaline droplets were not observed in the kidneys of any males previously receiving 5000 ppm, indicating complete recovery. No pathological changes observed in the reproductive tissues examined were considered to be related to the administration of Multiconstituent Dipentene. This observation is not relevant to humans.
A similar change was not observed in the kidneys of any females.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
- No adverse signs were observed in association with dose administration and there were no signs at routine physical examination that could be related to treatment.
- An increased incidence (7/15 compared to 0/15 in Control) of vocalisation was apparent for females receiving 5000 ppm; this is not an uncommon finding in this species but may indicate that females at this dietary concentration were more irritable. Brown staining affected the head or upper dorsal thorax of
animals during the study, males and females were still affected during the recovery phase. These observations lacked a dose relationship indicating it is unlikely to be related to the administration of test item.
- On Day 8 of recovery one male that previously received 5000 ppm was found dead. The cause of death was undetermined but considered of unlikely relationship to treatment.

BODY WEIGHT
- Body weight gain up to pairing (Days 1 to 22) and for the complete treatment period was low among animals receiving 2500 or 5000 ppm although statistical significance (p<0.05) was only attained for recovery phase females receiving 5000 ppm during Days 1 to 43; a dose dependant trend was not apparent
for females. During recovery animals that had previously received 5000 ppm displayed statistically higher body weight gain when compared with Controls (p<0.01 for males and p<0.05 for females).

FOOD CONSUMPTION
- Weekly mean food consumption data indicated males receiving 2500 or 5000 ppm throughout the dosing period and all females receiving 5000 ppm before pairing was marginally lower than Controls this difference was not considered to be adverse at the degree observed. The food consumption during
recovery was noted to be generally slightly higher than Control for animals that previously received 5000 ppm.

TEST SUBSTANCE INTAKE:
- Overall mean achieved doses at 800, 2500 or 5000 ppm were 50, 148 or 298 mg/kg bw/day for males, respectively. Overall mean achieved dose levels for recovery females throughout receiving 800, 2500 or 5000 ppm were 53, 174 or 316 mg/kg bw/day, respectively.

ORGAN WEIGHTS
- Organ weights for toxicity phase males killed after 6 weeks of treatment, and recovery males and females killed following 2 weeks of recovery indicated no adverse effects of dosing.
- Males at 5000 ppm terminated after Week 6 of study had slightly low adjusted mean prostate and seminal vesicle weights (0.85X and 0.86X Control, respectively) but these differences did not attain statistical significance. At the end of the 2-week recovery period prostate and seminal vesicle weights were similar
to Controls for males which had previously received 5000 ppm.

GROSS PATHOLOGY:
- Examination of males after 6 weeks of dosing and males and females following a 2 week recovery period did not reveal any findings that could be attributed to treatment.

HISTOPATHOLOGY:
Males killed after 6 weeks of dosing: Treatment related findings: Minimal hyaline droplets within the cortical tubules were observed in all kidneys examined microscopically from males that received 5000 or 2500 ppm and the majority of males that received 800 ppm. A similar change was not observed in the kidneys of any females.
Incidental findings: All other findings were considered to be incidental and unrelated to treatment and were consistent with common background microscopic changes previously seen in control Sprague Dawley rats.
Animals killed after 2 weeks of recovery
Treatment related findings: The kidneys from all recovery males and any abnormalities identified at the macroscopic examination were examined microscopically. No findings were considered to be related to treatment.
The incidence and distribution of all findings were consistent with common background changes previously seen in control Sprague Dawley rats.
OTHER FINDINGS:
OTHER FINDINGS (PARENTAL ANIMALS)
- Sensory reactivity and grip strength: Sensory reactivity and grip strength examined during Week 6 of dosing and Days 4 to 6 of lactation (females only) were considered unaffected by the administration of test item.
- Motor activity: There was no effect on either rearing (high beam) or ambulatory (low beam) motor activity.
- Haematology: Analysis of haematological parameters during Week 3 of study for females and Week 6 of study for males revealed low haematocrit and haemoglobin and large unstained cell counts, in males given 5000 ppm. Females receiving 5000 ppm had low reticulocytes (absolute count and percentage), mean cell haemoglobin concentration, and red cell distribution width and a slight increase in mean cell volume.
There were no statistical differences to the Control 14 days after the cessation of dosing.
- Biochemistry: Analysis of biochemical parameters during Week 6 of study for males and Week 3 of study for males indicated there were no adverse effects of the administration of test item. In males, a statistical significance was attained for low alanine amino transferase activity at all dose levels (0.81X to 0.84X Control), a dose response was not apparent and a change of this degree was considered not to be adverse. Females receiving 2500 or 5000 ppm showed slightly low alanine amino transferase activity (0.78X Control for both), with 5000 ppm attaining statistical significance and females at 5000
ppm showed significantly low cholesterol levels (0.90X Control). The degree of change recorded was considered not to be adverse.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed at the highest concentration tested

2

Dose descriptor:

NOAEL

Effect level:

298 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effect observed at the highest concentration tested

3

Dose descriptor:

NOAEL

Effect level:

316 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effect observed at the highest concentration tested

Target system / organ toxicity

Critical effects observed:

no

Lowest effective dose / conc.:

2 500 ppm

System:

urinary

Organ:

kidney

Any other information on results incl. tables

Formulation analysis:

The mean concentrations of test item in test formulations analysed for the Week 1 were within applied limits of +10 %/-15 % of nominal concentrations, confirming accurate formulation. The investigation into Week 9 showed that the concentration of Alpha‑terpinene in both the formulations and procedural recoveries had

reduced by approximately 50 % due to the oxidization of the sub-sample of Multiconstituent Dipentene used for the analyses over the course of the study. When the mean Week 1 procedural recovery is used to correct the Week 9 formulations, Group 2 is outside acceptable limits with an RME of -20.6 % from the nominal concentration, Groups 3 and 4 are both within applied limits.

Table 7.8.1/4: Analysed concentrations for Multiconstituent Dipentene in SDS VRF1 certified diet:

Occasion	Group	Nominal inclusion (ppm)	Analysed concentration (ppm)			RME (%)	Difference from mean (%)	Procedural recoveries (%)
			Analysis 1	Analysis 2	Mean			At analysis	Mean
Week 1	1	0	ND	ND	-	-	-
	2	800	770	772	771	-3.6	±0.16	87.9	87.2
	3	2500	2400	2480	2390	-4.4	±0.43	86.0
	4	5000	4950	5220	5090	+1.8	±2.62	87.8
Week 9	1	0	ND	ND	-	-	-
	2	800	632	637	635	-20.6	±0.40	-	87.2^1
	3	2500	2220	2240	2230	-10.8	±0.53	-
	4	5000	4370	4450	4410	-11.8	±0.85	-

RME: Relative mean error, representing the deviation from nominal

ND: Not detected

¹ Week 9 results are corrected for the mean of Week 1 procedural recoveries

Applicant's summary and conclusion

Conclusions:

The NOAEL for this screening study was 5000 ppm, equivalent to 298 mg/kg bw/day for males, 316 mg/kg bw/day for unmated females.

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, groups of Crl:CD(SD) rats received REACTION MASS OF DL-LIMONENE, ALPHA‑ GAMMA- TERPINENES, TERPINOLENE orally, via the diet, at concentrations of 800, 2500 or 5000 ppm.

Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of six consecutive weeks. Recovery phase male and female rats were treated daily for a minimum of six consecutive weeks, followed by a 14-day period without treatment. Recovery phase males were used for pairing with reproductive phase females, but recovery females were not paired. A similarly constituted Control group was assigned to each phase, and received the vehicle, untreated diet, throughout the same relative treatment period.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology, blood chemistry, oestrous cycles, organ weight and macroscopic investigations were undertaken for adult animals. Microscopicpathology investigations were undertaken for the first five toxicity phase males in Group 1 (Control) and Group 4 (5000 ppm) with examination of kidneys from males of Group 2 and 3.

Test item at dietary concentrations of 800, 2500 or 5000 ppm resulted in overall mean achieved doses of 50, 148 or 298 mg/kg bw/day for males, respectively. The overall mean achieved dose levels for unmated females were 53, 174 or 316 mg/kg bw/day.

On Day 8 of recovery one male that previously received 5000 ppm was found dead. The cause of death was undetermined but considered of unlikely relationship to treatment. Test item was well tolerated, with no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, blood chemistry, organ weights and macroscopic pathology.

Overall body weight gain and weekly mean food consumption data for the dosing period were generally low for animals receiving 2500 or 5000 ppm. During recovery, animals that had previously received 5000 ppm showed statistically significantly high body weight gain coupled with a slight increase in food consumption.

During gestation body weight gain and mean food consumption for all treated females were generally similar to Control values.

Analysis of haematological parameters during Week 3 of study for females and Week 6 of study for males revealed low haematocrit and haemoglobin and large unstained cell counts, in males given 5000 ppm. Females receiving 5000 ppm had low reticulocytes (absolute count and percentage), mean cell haemoglobin concentration, and red cell distribution width and a slight increase in mean cell volume. There were no statistical differences to the Control 14 days after the cessation of dosing.

Microscopically there were minimal hyaline droplets within the cortical tubules observed for all males examined (5/5) that received 2500 or 5000 ppm and the majority of males examined (4/5) at 800 ppm. This change was only observed in males and considered to be related to the administration of test item. This change was not observed in the kidneys of any males that previously received 5000 ppm following a 2-week recovery period, indicating complete recovery.

Therefore, the NOAEL for this screening study was 5000 ppm, equivalent to 298 mg/kg bw/ day for males, 316 mg/kg bw/day for unmated females.