Reaction mass of 2-(cis-4-methylcyclohexyl)propan-2-ol and 2-(trans-4-methylcyclohexyl)propan-2-ol and cis-1-methyl-4-(propan-2-yl)cyclohexanol and trans-1-methyl-4-(propan-2-yl)cyclohexanol

Endpoint summary

Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422, no NOAEL for this six week repeat dose toxicity study with 14-day recovery period including a reproductive screen/ developmental toxicity could be identified as no adverse toxic effects were observed at the highest dose tested, i.e. 15000 ppm (representing mean achieved dose levels of 944 mg/kg bw/day for males and 971 mg/kg bw/day for non-mated females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 December 2013 to 17 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

GLP compliance:

yes (incl. QA statement)

Remarks:

November 2015

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: 340-397 g; Females: 224-273 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; . 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.
- 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 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 mixed together 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 after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For 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).

GROUPS
Groups 1, 2, 3 and 4: 0, 1500, 5000 and 15000 ppm, respectively

Details on mating procedure
- Toxicity phase and recovery phase males were paired with reproductive phase females. Toxicity and
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 re
ferred 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: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 100 and 20000 ppm were analysed to assess the stability and homogeneity of the test substance in the diet matrix.
Stability was confirmed at two days ambient temperature and 22 days when stored frozen at 100 ppm.
Stability was confirmed at eight days ambient temperature and 22 days when stored frozen at 20000 p
pm.
Diet in hoppers was changed daily.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test substance.

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 five consecutive weeks.
Toxicity phase females were treated daily for a minimum of five consecutive weeks up to necropsy in Week 6.
Five high dose 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.:

1 500 ppm

Remarks:

nominal in diet

Dose / conc.:

5 000 ppm

Remarks:

nominal in diet

Dose / conc.:

15 000 ppm

Remarks:

nominal in diet

No. of animals per sex per dose:

Toxicity phase : 5 animals/sex/dose for vehicle and high dose groups; 10 males/dose for 1500 and 5000 ppm dose groups; 5 females/dose for 1500 and 5000 ppm dose groups
Recovery phase: 5 for vehicle and high 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 OAD0015). Dose levels were selected as 1500, 5000 and 15000 ppm. In the dose range finding study the doses of 1500, 7500 or 15000 ppm were well tolerated, no adverse clinical signs or macroscopic changes in organ appearance were detected at any dose level. Effects of treatment at 15000 ppm included initial body weight loss and low food consumption for up to four days following the start of dosing; liver weights were also high in these animals.
- 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.

Positive control:

Not applicable

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
- 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. On each occasion, the examinations were performed at approximately the same time of day, by an observer unaware of the experimental group identities.
During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day.

NEUROBEHAVIOURAL EXAMINATION:
- Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal.
- Time schedule:
- Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed on all recovery animals in Groups 1 and 4 and the five lowest numbered surviving toxicity phase males and females in Groups 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 was measured.

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

FOOD CONSUMPTION:
- Time schedule for examinations: The weight of food supplied, that remaining and an estimate of any spilled was recorded daily for all animals. From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase. Food consumption was not recorded for males and females during the period when paired for mating (Day 22 to 28). Food consumption recordings recommenced on Day 29.

HAEMATOLOGY AND CLINICAL CHEMISTRY:
- Time schedule for collection of blood:
Haematology and clinical chemistry: Week 6; Five lowest numbered surviving toxicity phase males and females per group.
Clinical chemistry: Week 6; Five lowest numbered surviving toxicity phase males and females per group.
Recovery Week 2; All recovery phase animals
- 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).

Sacrifice and pathology:

SACRIFICE:
Toxicity phase animals: After at least Week 6 investigations completed.
- 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 males and females 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.
Light microscopy
- Terminal sacrifice:
Groups 1 and 4: Five lowest numbered F0 males and five Toxicity phase F0 females - All specified in table 7.8.1/1
All adult animals from all groups/phases - Abnormalities

Statistics:

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

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one decedent in the study, a recovery female given 15000 ppm (animal No. 72), attributed to an accidental death. Significant macroscopic findings were seen in the left side of the head comprising of trauma to the bone of the cranium with clotted blood in the cranial cavity and haemorrhagic areas in the subcutis.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was no clear indication of an effect of the test material on the unmated females at any dose level or males receiving 5000 or 1500 ppm. Changes detected at 15000 ppm in males though attributed to treatment, were considered not to be adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Short lasting reduction in food consumption was observed between day 1 and 4, in animals receiving 15000 ppm but was attributed to the low palatability of the preparation and is not considered to be adverse.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood urea concentration was higher, attaining statistical significance, compared with Control with an indication of a dose trend. However, urea concentration was within historical control data (historical control 90 percentile range : 4.72-6.60 mmol/L, n=20, year 2014-2015).
Chloride concentration was significantly lower than control in males receiving 15000 or 5000 ppm but stayed in historical control data (historical control 90 percentile range : 98-103 mmol/L, n=20, year 2014-2015).
Other changes detected at the week 6 sampling occasion included bile acid concentration in females which was significantly lower than Control at all dose levels with a dose response apparent, lower levels were noted in males at the low and intermediate dose levels only however the highest concentration group was similar to control. It was not accompagnied by histopathological findings.
Cholesterol concentration for both males and females receiving 15000 ppm were higher than Control, with an indication of a dose trend in males only. After 14 days without the test material a complete recovery was observed and it was not associated with any pathological change and did not affect the clinical condition of the animal therefore, cholesterol higher concentration is not considered to be adverse.

Calcium was significantly higher than Control in males and females at 15000 ppm, also attaining significance at 5000 ppm in males only. No other mineral concentration was modificated. Females receiving 15000 ppm also had slight but significantly higher total protein levels at 15000 ppm but neither albumin nor albumin globulin ratios were significantly affected.After 14 days without the test material there were no significant differences to Control in all previously affected parameters after 14 days off dose. It was noted that calcium in males was marginally but significantly lower than Control but this was considered to have arisen by chance.

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):

Adjusted liver weight for toxicity phase males and females receiving 15000 ppm were statistically higher than Control, this was also apparent in males only at 5000 ppm. However it was mainly based in slight body weight decrease in this group.
After 14 days off dose, none of the parameters affected above were significantly different to the Control.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with Dihydroterpineol multiconstituent were seen in kidneys of male animals.
Hyaline droplets in the cortical tubules were seen in all male treated groups with corticomedullary granular casts seen in males given 5000 and 15000 ppm of Dihydroterpineol multiconstituent. An increase incidence and severity of cortical tubular basophilia was seen in males given 5000 and 15000 ppm of Dihydroterpineol multiconstituent.
This is not relevant in humans

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

An adenocarcinoma was recorded in the mammary gland of a female given 1500 ppm of Dihydroterpineol multiconstituent. This was considered to be incidental.

Details on results:

BODY WEIGHT:
The bodyweight change of males receiving 15000 ppm was significantly lower than Control for the first week of study. During week 1 of study females receiving 15000 ppm showed bodyweight stasis. This was accompanied by a period of low food consumption which was attributed to the animals becoming accustomed to the diet. Thereafter there was no notable difference in body weight gain to the Control although for the 3 week pre-pairing period and the 6 week treatment period, the overall weight gain of the males receiving 15000 ppm remained significantly lower than Control.

FOOD CONSUMPTION:
Food consumption of animals receiving 15000 ppm on Day 1 of study was markedly lower than Control. Intake increased each day and by Day 4 of dosing there was no notable effect on food consumption at any dose level. This short lasting reduction in food consumption is attributed to the low palatability of the preparation and is not considered to be adverse.

SENSORY REACTIVITY AND GRIP STRENGHT:
There was no effect of Dihydroterpineol multiconstituent on sensory reactivity or grip strength.

MOTOR ACTIVITY:
There was considered to be no effect of treatment on motor activity in males or females at dose levels up to 15000 ppm.
There were two statistically significant periods of high low beam activity (at 54 minutes and 60 minutes) above the Control for females at 15000 ppm. This difference was noted for females only and group mean values were within the historical Control range

HAEMATOLOGY:
There were no effects of Dihydroterpineol multiconstituent on any of the haematology parameters assessed.

ORGANS WEIGHTS:
Liver weight for toxicity phase males and females receiving 15000 ppm were statistically higher than Control, this was also apparent in males only at 5000 ppm. Toxicity phase males, females receiving 15000 ppm had high adrenal weights in comparison with Control; the difference did not attain statistical significance for toxicity phase females.
Low thymus weight recorded for toxicity phase females at all levels did not demonstrate a dose response and was considered not to be an adverse effect of treatment.
After 14 days off dose, none of the parameters affected above were significantly different to the Control. Males which had previously received 15000 ppm had high spleen weights in comparison to recovery Control but these Control animals had lower spleen weights than were recorded in animals killed at the end of the treatment period. Females at this dose level had slightly but significantly lower kidney weights

MACROPATHOLOGY:
There was no indication of any reaction to the test material in the profile of macroscopic changes observed in toxicity phase males and females, reproductive phase females or recovery phase males and females.

HISTOPATHOLOGY:
Animals killed after 5 weeks of treatment
Changes related to treatment with Dihydroterpineol multiconstituent were seen in kidneys of male animals.
Hyaline droplets in the cortical tubules were seen in all male treated groups with corticomedullary granular casts seen in males given 5000 and 15000 ppm of Dihydroterpineol multiconstituent. An increase incidence and severity of cortical tubular basophilia was seen in males given
5000 and 15000 ppm of Dihydroterpineol multiconstituent.

Animals killed after 14 days of recovery
Changes related to treatment with Dihydroterpineol multiconstituent were seen in kidneys of male recovery animals.
The findings of cortical tubular basophilia and corticomedullary granular casts, seen in male terminal animals, were also present in the recovery animals previously treated with 15000 ppm of Dihydroterpineol multiconstituent. Hyaline droplets in the cortical tubules, seen in terminal animals, were not seen in recovery animals. Cortical tubular basophilia, although present in all previously treated animals, was present at a minimal grade only which may indicate partial recovery.
The granular casts were seen at a slightly reduced incidence in the recovery animals previously treated with 15000 ppm compared to terminal animals which may indicate partial recovery.

For males the target organ was the kidney, detected in blood chemistry by low chloride and high urea concentrations and at microscopic examination, principally as hyaline droplet formation which was observed at all dose levels. The corticomedullary granular cysts and cortical tubular basophilia, observed at the 5000 or 15000 ppm dose levels, were most likely secondary to the accumulation of hyaline droplets in the cortical tubules. The pathology seen in the kidneys of male animals probably accounts for the increase in the blood urea that was observed. After 14 days recovery, hyaline droplets were not seen in the kidneys of the recovery animals. There was an indication of partial recovery of the corticomedullary granular cysts and cortical tubular basophilia. As the changes were mostly minimal or slight in severity and as recovery was in progress and the root cause of the change had resolved, this
change is not considered to be adverse.

Hyaline droplets in the renal tubules of the kidney of the rat are considered to represent the accumulation of α2u globulin. Glomerular filtration is an important pathway for removing proteins from circulation with the amount of protein reaching the urinary space depending on the glomerular filtration rate, its plasma concentration and the characteristics of the protein. The majority of protein within the tubular fluid is taken up by the proximal tubule cells and transported to lysosomes where fusion and hydrolysis takes place with the amino acids returned to circulation. Compounds that affect the level of filtered proteins or their catabolism can result in their accumulation in the proximal tubular cells forming droplets of variable size which appear eosinophilic or hyaline in haematoxylin stained sections.
Hyaline droplets are particularly prominent in the proximal renal tubules of untreated male rats which are thought to be due mainly to the presence of a specific protein, α2u globulin (Alden, 1986). This is a normal urinary protein in rats and as outlined above is filtered by the glomerulus and reabsorbed in the proximal tubules. Accumulation of this protein can be increased after treatment with a wide range of compounds, an example being hydrocarbons which are thought to bind with α2u globulin within the lysosomes producing a complex that is poorly catabolised.
Hyaline droplets that form after administration of xenobiotics may attain much larger size and contain more prominent angular crystalloid structures. If the tubular cells become overloaded with droplets and crystalloid structures, loss of cytoplasmic integrity may occur leading to cell death, regeneration and repair. Cellular debris may form granular appearing casts within tubular lumens of the outer medulla. Hyaline droplet formation is not seen in female rats, which is thought to be due the higher rate of catabolism of α2u globulin. Although humans excrete proteins of a similar nature to α2u globulin they are only found in trace amounts so the finding and related pathology is considered to have limited relevance to man.

Key result

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Higest dose level tested corresponding to 944 and 971 mg/kg bw/day for males and females, respectively

Critical effects observed:

yes

Lowest effective dose / conc.:

5 000 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Conclusions:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422, no NOAEL for this six week repeat dose toxicity study with 14-day recovery period including a reproductive screen/ developmental toxicity could be identified as no adverse toxic effects were observed at the highest dose tested, i.e. 15000 ppm (representing mean achieved dose levels of 944 mg/kg bw/day for males and 971 mg/kg bw/day for non-mated 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 dihydroterpineol multiconstituent orally, via the diet, at concentrations of 1500, 5000 or 15000 ppm.Toxicity phase males were treated daily for three weeks before pairing, throughout pairing and up to necropsy after a minimum of five consecutive weeks. Toxicity phase females were treated daily for a minimum of five consecutive weeks up to necropsy in Week 6. Five high dose 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.

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 all adult animals. Microscopic pathology investigations were undertaken in the first five Toxicity phase males and Toxicity phase females in Group 1 (Control) and Group4 (15000 ppm). 

The mean concentrations of dihydroterpineol multiconstituent in formulations were within applied limits of +10%/-15%, confirming the accuracy of formulation. The difference from mean values was <3%, confirming precise analysis. With the exception of one recovery in Week 1, procedural recovery values remained within 96.7 and 103.0%, confirming the continued accuracy of the method.

There were no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, haematology and macroscopic pathology.

Analysis of serum after six weeks of exposure indicated high urea and low chloride concentrations in animals receiving 15000 ppm. Bile acid was significantly lower than control in females at all dose levels and males at the intermediate and low dose. Cholesterol was higher than Control for males and females receiving 15000 ppm with an indication of a dose trend at 5000 ppm in males. Calcium was significantly higher than Control in males and females at 15000, at 5000 ppm in males only. Females receiving 15000 ppm had slight but significantly higher total protein at 15000 ppm neither albumin or albumin globulin ratios were significantly affected. At analysis during recovery week two, none of the above changes were apparent. 

At necropsy the liver and adrenal weights for males and females at 15000 ppm and males at 5000 ppm (liver only) were higher than Control. After 14 days recovery these differences were not apparent.

Findings seen in the kidneys of male animals comprised of hyaline droplets in the cortical tubules, cortical tubular basophilia and corticomedullary granular casts, all at generally minimal or slight severity. Hyaline droplet formation was seen in all treated male groups after six weeks of treatment but not following 14 days of recovery. Cortical tubular basophilia and corticomedullary granular casts were seen in males given 5000 and 15000 ppm of dihydroterpineol multiconstituent. After cessation of treatment for 14 days in males given 15000 ppm there was a slight reduction in the incidence or severity of these findings, which may indicate partial recovery.

Therefore, no NOAEL for this six week repeat dose toxicity study with 14-day recovery period including a reproductive screen/ developmental toxicity could be identified as no adverse toxic effects were observed at the highest dose tested, i.e. 15000 ppm (representing mean achieved dose levels of 944 mg/kg bw/day for males and 971 mg/kg bw/day for non-mated females.

Reference 2

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

5 February to 4 March 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Preliminary study used as range-finder experiment for a 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: 347-379 g; Females: 238-261 g
- Housing: Animals were housed in groups of 4/sex in polycarbonate cages
- Diet: Standard rodent diet (SDS VRF1 Certified Diet); ad libitum
- Water: Potable water from public supply; ad libitum
- Acclimation period: 6 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 mixed together 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 after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet.
Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For 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.

Stability and homogeneity
Homogeneity and stability of the test material in the vehicle from Study No. OAD0014. In that study stability at ambient conditions for two days at 100 ppm and 20000 ppm was confirmed, and for eight days at 20000 ppm only. Frozen stability for 22 days has been confirmed at both 100 and 20000 ppm.

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 due to stability at ambient temperature).

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.
An OECD 422 study was conducted with a related substance, Menthanyl Acetate Multiconstituent (containing 36% of cis-1-methyl-1-(4-methylcyclohexyl) ethyl acetate, 27% of trans-1-methyl-1-(4-methylcyclohexyl) ethyl acetate, 19% of cis-4-isopropyl-1-methylcyclohexyl acetate and 17% of trans-4-isopropyl-1-methylcyclohexyl acetate), the NOAEL was set at 5000 ppm. The LD50 of a related substance was > 2000 mg/kg bw in the rat.
The dose levels in this study were therefore set at 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 and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyser.
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea and Creatinine.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Oestrous cycles
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 of for each animal killed after completion of the study. 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.

Other examinations:

None

Statistics:

No data

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

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:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- No clinical signs or mortality was observed during the study.

BODY WEIGHT AND WEIGHT CHANGE
Among males, all groups showed some mean body weight loss following the start of treatment but males receiving 15000 ppm showed greater mean weight loss than in Controls and other treated groups between Days 1 and 2 of treatment.
Thereafter, a period of good weight gain was recorded and body weight change was generally similar to Controls from Day 4 to Day 22. There was no difference among males receiving 7500 ppm or 1500 ppm and Controls.

Among females receiving 15000 or 7500 ppm, mean body weight loss of 12 g and 8 g respectively was recorded between Days 1 and 2 of treatment compared with mean weight stasis in Controls. Thereafter, mean body weight gain was variable in both groups but overall body weight change was 100 and 118% of Controls, respectively. There was no difference among females receiving 1500 ppm and Controls

FOOD CONSUMPTION
Food consumption for the first 3 days at 15000 ppm for males and females or 7500 ppm for males only was lower than control. Females at 7500 ppm had low food consumption for the first day only. Thereafter differences to the control were small and /or lacking dose related trends.

GROSS PATHOLOGY
- No macroscopic abnormalities were observed.

CLINICAL BIOCHEMISTRY
High blood serum urea concentrations with a dose trend were observed in females; there was an indication of a similar effect in males but to a smaller degree and without a dose trend.
High blood serum creatinine concentrations were observed with a dose response in females only.
The liver markers of alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase did not indicate any adverse effects of treatment.

ORGAN WEIGHT
Liver weights for males and females were higher than Controls for animals receiving 15000 ppm; there was also a suggestion of slightly high liver weights at 7500 ppm.
There was also a marginal increase in adrenal weights at 15000 ppm (males and females) however this is considered not to be adverse at the degree observed.

OTHER FINDINGS
OESTROUS CYCLES:
- There was no effect of treatment on oestrus cycle activity of females receiving Dihydroterpineol multiconstituent at dietary levels up to 15000 ppm.

Critical effects observed:

no

None

Conclusions:

In a 21-day repeated dose toxicity range-finding study, the NOAEL in rats was considered to be 1500 ppm equivalent to 102 and 107 mg/kg bw/day in males and females, respectively.

Executive summary:

In a repeated dose toxicity range-finding study, groups of Crl:CD(SD) rats (4/sex/dose) received dihydroterpineol multiconstituent orally, via the diet at concentrations of 1500, 7500 or 15000 ppm during 21 days. 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.

 

Overall mean achieved dose levels for animals receiving 1500, 7500 or 15000 ppm were 102, 468 or 986 mg/kg bw/day for males and 107, 496 or 987 mg/kg bw/day for females, respectively. 

There were no premature deaths and no changes in clinical condition were detected during the study. There was no effect of treatment on oestrus cycle activity. 

At 15000 ppm, males and females showed greater mean body weight loss between Days 1 and 2 of treatment compared with Controls, and low food consumption during the first 3 days of treatment. At 7500 ppm, food consumption of males was slightly low during the first 3 days of treatment, and females showed mean body weight loss between Days 1 and 2 of treatment and slightly low food consumption on the first day of treatment.

High blood serum urea concentrations were observed in females with a dose trend apparent. High blood serum creatinine concentrations were observed; the females displayed a more classical dose response than males. 

Liver weights for males and females were higher than Control for animals receiving 15000 ppm; there was also a suggestion of slightly high liver weights at 7500 ppm. There were no macroscopic abnormalities detected in any animals receiving dihydroterpineol multiconstituent.

 

None of the effects observed in this study preclude the use of 15000 ppm as the highest dose level in a subsequent OECD 422 screening study. Dose levels of 1500 and 5000 ppm would be suitable as low and intermediate levels to establish dose response relationships for any treatment-related effects observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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) ratsreceived dihydroterpineol multiconstituent orally, via the diet, at concentrations of 1500, 5000 or15000 ppm.Toxicity phase males weretreated dailyfor three weeks before pairing, throughout pairing and up to necropsy after a minimumof five consecutive weeks. Toxicity phase femaleswere treateddaily for a minimumof five consecutive weeksup to necropsy in Week 6. Five high dose Recovery phase male and female rats were treated daily for a minimumofsixconsecutive 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.

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 investigationswere undertaken for all adult animals. Microscopicpathology investigations were undertaken in thefirst five Toxicity phase males and Toxicity phase females in Group 1 (Control) and Group4 (15000 ppm). 

The mean concentrations of dihydroterpineol multiconstituent in formulations were within applied limits of +10%/-15%, confirming the accuracy of formulation. The difference from mean values was <3%, confirming precise analysis. With the exception of one recovery in Week 1, procedural recovery values remained within 96.7 and 103.0%, confirming the continued accuracy of the method.

There were no adverse effects attributed to treatment on clinical condition, sensory reaction, grip strength, motor activity, haematology and macroscopic pathology.

Analysis of serum after six weeks of exposure indicated high urea and low chloride concentrations in animals receiving 15000 ppm. Bile acid was significantly lower than control in females at all dose levels and males at the intermediate and low dose. Cholesterol was higher than Control for males and females receiving 15000 ppm with an indication of a dose trend at 5000 ppm in males. Calcium was significantly higher than Control in males and females at 15000, at 5000 ppm in males only. Females receiving 15000 ppm had slight but significantly higher total protein at 15000 ppm neither albumin or albumin globulin ratios were significantly affected. At analysis during recovery week two, none of the above changes were apparent. 

At necropsy the liver and adrenal weights for males and females at 15000 ppm and males at 5000 ppm (liver only) were higher than Control. After 14 days recovery these differences were not apparent.

Findings seen in the kidneys of male animals comprised of hyaline droplets in the cortical tubules, cortical tubular basophilia and corticomedullary granular casts, all at generally minimal or slight severity. Hyaline droplet formation was seen in all treated male groups after six weeks of treatment but not following 14 days of recovery. Cortical tubular basophilia and corticomedullary granular casts were seen in males given 5000 and 15000 ppm of dihydroterpineol multiconstituent. After cessation of treatment for 14 days in males given 15000 ppm there was a slight reduction in the incidence or severity of these findings, which may indicate partial recovery.

Therefore, no NOAEL for this six week repeat dose toxicity study with 14-day recovery period including a reproductive screen/ developmental toxicity could be identified as no adverse toxic effects were observed at the highest dose tested, i.e. 15000 ppm (representing mean achieved dose levels of 944 mg/kg bw/day for males and 971 mg/kg bw/day for non-mated females).

Justification for classification or non-classification

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted in rats according to OECD Guideline 422 and in compliance with GLP, no NOAEL could be identified as no adverse toxic effects were observed at the highest dose tested, i.e. 15000 ppm (representing mean achieved dose levels of 944 mg/kg bw/day for males and 1065 and 1874 mg/kg bw/day for reproductive phase females during gestation and lactation, respectively).

Therefore, dihydroterpineol multiconstituent does not need to be classified according to CLP Regulation (EC) n° 1272/2008.