2-hydroxyethyl [(1R,2S,5R)-2-isopropyl-5-methyl-cyclohexyl] carbonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-11-24 to 1994-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981-05-12

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Remarks:

1994-03-16

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[({[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]oxy}carbonyl)oxy]ethan-1-ol

EC Number:

701-039-2

Cas Number:

156324-78-6

Molecular formula:

C13H24O4

IUPAC Name:

2-[({[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]oxy}carbonyl)oxy]ethan-1-ol

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (U.K.) Limited, Manston, Kent
- Age at start of treatment: approx. five to six weeks old
- Weight at start of treatment: males: 129 g to 160 g; females: 120 g to 159 g
- Housing: housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper.
- Diet (ad libitum): pelleted diet (Rat and Mouse SQC Expanded Diet No. 1)
- Water (ad libitum): mains water
- Acclimation period: six days

DETAILS OF FOOD AND WATER QUALITY:
The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 22 °C
- Relative humidity: 37 - 59 %.
- Air changes: at least fifteen air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected by the study Sponsor as a possible route of human exposure and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man.

Vehicle:

polyethylene glycol

Remarks:

400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a solution in the vehicle. Formulations were prepared weekly and stored at 4°C in the dark.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability, homogeneity, and concentration of the test material formulations were analysed. The test item concentration in the test samples was determined by gas chromatography using an external standard technique.
For homogeneity determination, the test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container shaking in between sampling. The sampling was performed in triplicate.
For stability determination, the test material formulations were sampled and analysed initially and then after stroage at approximately 4 °C in the dark for ten days.
For the verification of the test material formulation concentration, the test material formulations were sampled and analysed within three days of preparation.
Results:
The formulation showed to be stable for at least ten days. In addition the results indicated that the prepared formulations were within ± 10 % of the nominal concentration.

Duration of treatment / exposure:

28 consecutive days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Main groups: 5 males / 5 females
Recovery groups (vehicle control recovery and 1000 mg/kg/day recovery group): 5 males / 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels for the 28-day repeated dose toxicity study were chosen based on the results of a range-finding study. In the range finding study groups of 3 male and 3 female Sprague-Dawley CD strain rats were dosed with either 150, 400, or 1000 mg/kg/day of the test item in polyethylene glycol 400 by gavage for a duration of 13 consecutive days. A vehicle control group was run concurrently. The following parameters were examined/recorded: clinical signs, mortality, body weight, and gross pathology.
Results:
Mortality:
- 400 mg/kg/day: two females were found dead prior to dosing on Day 6 (toxicological significane of these deaths was unclear)

Clinical signs:
- 1000 mg/kg/day: clinical signs of toxicity were observed for all animals on Days 6 and 7 and included hunched posture, piloerection, lethargy and noisy respiration. Most of these animals appeared to recover from any adverse effect of treatment thereafter although sporadic changes in appearance were still evident for two of the females treated at this dose level until Day 10.
Animals showed increased salivation immediately after dosing from Day 2 onwards together with isolated incidents of associated fur wetting and red/brown staining around the mouth.
- 150 or 400 mg/kg/day: animals treated at 400 or 150 mg/kg/day also showed increased salivation immediately after dosing from Days 6 and 7 all respectively. Such findings involving short-lived salivation are common when the test material formulation has an unpleasant taste or is slightly irritant and may not be indicative of systemic toxicity.

Body weight:
No treatment-related bodyweight effects were detected during the study.

Gross pathology:
400 mg/kg/day: one male dosed had a small white nodule on the limiting ridge of the stomach at necropsy. No further macroscopic abnormalities were detected at terminal kill.
Two female decedents showed several macroscopic changes at necropsy including an haemorrhagic and/or thickened glandular gastric region, a congested small intestine, a darkened liver, dark areas on the thymus, patchy pallor of the kidneys, darkened adrenals and dark red lungs. Such findings represent normally expected post mortem changes and are unlikely to be indicative of systemic toxicity.

The dose levels for the main twenty-eight day study were chosen, following consultation with the sponsor, as: 15, 150, and 1000 mg/kg/day plus a control group treated with vehicle only.

- Post-exposure recovery period in satellite groups: 14 days

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule (treatment period): immediately before dosing and one and five hours after dosing during the working week or immediately before dosing and one hour after dosing at weekends.
- Time schedule (treatment-free period): twice daily, morning and afternoon (once daily at weekends and on public holidays).
- Cage side observations checked: overt signs of toxicity, ill-health or behavioural change

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations (treatment period): Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28
- Time schedule for examinations (treatment-free period): Days 35 and 42

Body weights were also recorded at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each group determined and mean weekly diet consumption calculated as g food/kg body weight/week: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Change in mean bodyweight (g)/ mean food consumption (g/rat/week): Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily from Day 8 onwards

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: animals from the treatment groups at the end of the treatment period (Day 28) and satellite group animals at the end of the treatment-free period (Day 42).
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals from the treatment groups and recovery groups
- Parameters checked: haematocrit, haemoglobin, erythrocyte count, total leucocyte count, differential leucocyte count, platelet count, mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration, and clotting time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: animals from the treatment groups at the end of the treatment period (Day 28) and satellite group animals at the end of the treatment-free period (Day 42).
- Animals fasted: No
- How many animals: all animals from the treatment groups and recovery groups
- Parameters checked: blood urea, total protein, albumin, albumin/globulin ratio (by calculation), sodium, potassium, chloride, calcium, inorganic phosphorus, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, glucose, total bilirubin, and creatinine

URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

On completion of the dosing period or, in the case of satellite group animals, at the end of the treatment-free period; all animals were killed.

Gross pathology:
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ weights:
The following organs from animals that were killed at the end of the study, dissected free from fat, were weighed before fixation: adrenals, brain, gonads, heart, kidneys, liver, pituitary, and spleen.

Histopathology:
Samples of the following tissues were removed from all animals and preserved: adrenals, aorta (thoracic), bone & bone marrow (femur), bone & bone marrow (sternum), brain, caecum, colon, duodenum, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical and mesenteric), muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, skin (hind limb), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, and uterus.
The following preserved tissues from all animals (treatment groups, recovery groups, and control groups) were prepared as paraffin blocks, sectioned (5 µm) and stained for microscopic examination:
adrenals, spleen, heart, kidneys, liver, and testes. Macroscopically observed lesions were also processed.
Initially, microscopic examination was confined to the vehicle control group and 1000 mg/kg bw dose group of the treatment period only but since there were indications of treatment-related hepatic changes, examination was subsequently extended to include sections of liver from all animals in the remaining dose groups.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
Absolute and relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating 'F-max' test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal Wallis non-parametric analysis of variance and Mann Whitney U-Test.

Results and discussion

Results of examinations

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:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg/day: animals of either sex showed a statistically significant increase in liver weight, relative to bodyweight, compared with controls. Male relative kidney weight was also slightly elevated at this dose level (statistically significant). Treatment-related organ weight changes had regressed amongst satellite 1000 mg/kg/day dose group animals following a further fourteen days without treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day: speckled kidneys were observed for several males at terminal kill.

1000 mg/kg/day: speckled kidneys were observed for all males at terminal kill. One male also had pale kidneys at necropsy. Treatment-related macroscopic renal changes had regressed amongst recovery group males following a further fourteen days without treatment. No histopathologic correlate could be observed.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day: treatment-related hepatic changes were observed. Centrilobular hepatocyte enlargement (minimal: 1/5 males) and a reduced severity of periportal hepatocyte vacuolation (minimal: 4/5 males; slight: 1/5 males) were observed in relation to treatment for male rats. Female rats were unaffected.
1000 mg/kg/day: treatment-related hepatic changes were observed. Centrilobular hepatocyte enlargement (minimal: 2/5 males; slight: 1/5 males) and a reduced severity of periportal hepatocyte vacuolation (minimal: 2/5 males; slight: 1/5 males) were observed in relation to treatment for male rats. Female rats were unaffected.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS.
15 and 150 mg/kg/day: animals showed no clinically observable signs of toxicity during the study.

1000 mg/kg/day (satellite group): one female was hunched, ataxic and showed tiptoe gait following dosing on Day 17 whilst one male from the same group showed pilo-erection on Day 7 of treatment. Clinical signs of toxicity were no longer evident several hours later and, in the absence of any further changes in appearance at this dose level, the toxicological significance of these findings is unclear.

1000 mg/kg/day (main group): animals of either sex also showed increased salivation from Day 5 of treatment onwards. This was mostly short-lived and visible only immediately after dosing although more persistent salivation was seen on isolated occasions. Associated findings of fur wetting, red/brown staining around the mouth and noisy respiration were also detected although the latter finding only occurred sporadically. Increased salivation and any associated findings were no longer evident in satellite 1000 mg/kg/day dose animals following cessation of treatment. Observations of this nature often represent a localised response to the oral administration of an unpleasant tasting or slightly irritant test material formulation and in the absence of any evidence of gastric irritation, may not be of any toxicological importance.

MORTALITY:
15, 150, and 1000 mg/kg/day: no deaths

BODY WEIGHT:
15, 150, and 1000 mg/kg/day: all animals showed normal bodyweight development during the study. Animals treated with the test material showed similar bodyweight gains to controls over the treatment period.

FOOD CONSUMPTION:
15, 150, and 1000 mg/kg/day: no adverse effect on food consumption during the study.

FOOD EFFICIENCY:
15, 150, and 1000 mg/kg/day: animals treated with the test material showed similar weekly food efficiency to controls.

WATER CONSUMPTION:
A treatment-related effect on water consumption was suspected following daily visual inspection of the water bottles during the first week of treatment. Measurement of water consumption was, therefore, initiated on Day 8. The visual findings were not confirmed by measurement and there was no appreciable quantitative difference in water intake between test and control group animals.

1000 mg/kg/day (recovery group): females showed a slightly greater water intake than the corresponding control group during the dosing period (not observed in the corresponding main group females; toxicological significance of this finding was considered to be highly dubious).

HAEMATOLOGY:
15, 150, and 1000 mg/kg/day: no treatment-related changes in the haematological parameters measured.

15, 150, and 1000 mg/kg/day: animals of either sex showed a statistically significant reduction in neutrophil counts compared with controls although none of the individual values were abnormally low for rats of the strain and age used in this study. The intergroup differences showed no dose-related relationship and were considered to be entirely due to a higher than expected neutrophil count in the controls.

1000 mg/kg/day (recovery group): lymphocyte count was reduced in males compared with controls at the end of the fourteen day recovery period but, in the absence of a similar finding in the corresponding main group males at the end of the twenty-eight day dosing period, this intergroup difference was considered to be of no toxicological importance.

The remaining statistically significant intergroup differences involving a reduction in leucocyte count or an increase in monocytes were confined to low, intermediate or low and high dose animals (dose-relate; not to be treatment-related).

CLINICAL CHEMISTRY:
15, 150, and 1000 mg/kg/day: no treatment-related changes in the blood chemical parameters measured.

150 mg/kg/day and 1000 mg/kg/day: females showed a slight but statistically significant reduction in plasma alanine aminotransferase concentration compared with controls. None of the individual values were abnormally low for rats of the strain and age used in the study. Finding was considered not to be toxicologically significant.

15, 150, and 1000 mg/kg/day: a slight but statistically significant reduction in female plasma inorganic phosphorus concentration was detected compared with controls with several of the individual values outside the normally expected range for rats of the strain and age used. There was no further evidence of nephropathy or changes in the concentration of other plasma electrolytes. A convincing dose-relationship could not be demonstrated and the intergroup differences were considered to have arisen fortuitously.

1000 mg/kg/day (recovery group): males showed a statistically significant increase in total plasma protein and alanine aminotransferase levels compared with controls at the end of the fourteen day treatment-free period but, in the absence of any similar findings for corresponding main group males at the end of the twenty-eight day dosing period, these slight increases were considered not to be toxicologically significant.

15 mg/kg/day: the remaining statistically significant intergroup differences involved a reduction in plasma urea, an increase in the albumin/globulin ratio and an increase in alkaline phosphatase (not dose-related finding; not treatment-related).

ORGAN WEIGHTS:
15 and 150 mg/kg/day: no treatment-related organ weight changes

1000 mg/kg/day: males showed a slight but statistically significant reduction in testicular weight compared with controls. No such intergroup difference was evident when testicular weight was expressed relative to bodyweight. Finding was not considered to be toxicologically significant.
1000 mg/kg/day: satellite females showed a statistically significant increase in relative spleen weight compared with controls at the end of the fourteen day recovery period but in the absence of a similar finding at the end of the twenty-eight day dosing period, this was considered of no toxicological importance.

GROSS PATHOLOGY:
15 mg/kg/say: males treated with the test material showed no toxicologically significant macroscopic abnormalities at terminal kill.

15, 150, and 1000 mg/kg/day: all females treated with the test material showed no toxicologically significant macroscopic abnormalities at terminal kill.

1000 mg/kg/day (recovery group): one male had an enlarged and darkened spieen at the end of the fourteen day recovery period. The weight of this spieen was abnormally high for rats of the strain and age used in the study but there were no treatment-related splenic abnormalities, either macroscopic or microscopic, at the end of the twenty-eight day dosing period and this isolated finding was considered to be of no toxicological importance.

The remaining macroscopic abnormalities seen at terminal kill, multiple raised white areas on the spieen and a misshapen right kidney with hydronephrosis, represent normally expected low incidence findings in laboratory maintained rats of the strain and age used in the study and were considered not to be treatmentrelated.

HISTOPATHOLOGY:
Besides the findings concerning the liver, all remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

After the oral administration of 15, 150, or 1000 mg/kg/day of the test item no deaths occurred during the study. Furthermore, no treatment-releated effects were observed for clinical signs, body weight and weight changes, food consumption, food efficiency, water consumption, haematology, and clinical chemistry.

During necropsy, male animals of the 150 mg/kg/day dose group showed, speckled kidneys at terminal kill. Furthermore, at this dose level treatment-related hepatic changes were observed, such as centrilobular hepatocyte enlargement (minimal: 1/5 males) and a reduced severity of periportal hepatocyte vacuolation (minimal: 4/5 males; slight: 1/5 males).

At the 1000 mg/kg/day dose level, speckled kidneys were observed for all males at terminal kill. One male also had pale kidneys at necropsy. Treatment-related macroscopic renal changes had regressed amongst recovery group males following a further fourteen days without treatment.

Relative organ weight changes were observed in animals at the 1000 mg/kg/day dose level in liver (statistical significant increase) and kidney (male only, statistical significant increase). Treatment-related organ weight changes had regressed amongst satellite 1000 mg/kg/day dose group animals following a further fourteen days without treatment.

Treatment-related hepatic changes were observed during histopathology. Centrilobular hepatocyte enlargement (minimal: 2/5 males; slight: 1/5 males) and a reduced severity of periportal hepatocyte vacuolation (minimal: 2/5 males; slight: 1/5 males) were observed in relation to treatment for male rats.

Based on the the organ weights findings in the liver of males at the 1000 mg/kg/day dose level and the histopathological findings in the liver of males at the 150 and 1000 mg/kg/day dose levels, a No Observed Adverse Effect Level (NOAEL) for test item of 150 mg/kg/day and 1000 mg/kg/day was concluded for males and females, respectively.

Executive summary:

A repeated dose oral toxicity study was performed with the test item according to the OECD guideline 407 (1981). The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley CD strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Polyethylene glycol 400). Two satellite groups, each of five males and five females, were treated with the high dose (1000 mg/kg/day) or the vehicle alone throughout the twenty-eight day study period and then maintained without treatment for a further fourteen days. The following parameters were evaluated: clinical signs, body weight, food consumption, food efficiency, water consumption, haematology, clinical chemistry, organ weights, macroscopic examination, and histopathology of selected tissues.

After the oral administration of 15, 150, or 1000 mg/kg/day of the test item no deaths occurred during the study. Furthermore, no treatment-releated effects were observed for clinical signs, body weight and weight changes, food consumption, food efficiency, water consumption, haematology, and clinical chemistry.

During necropsy, male animals of the 150 mg/kg/day dose group showed, speckled kidneys at terminal kill. Furthermore, at this dose level treatment-related hepatic changes were observed, such as centrilobular hepatocyte enlargement (minimal: 1/5 males) and a reduced severity of periportal hepatocyte vacuolation (minimal: 4/5 males; slight: 1/5 males).

At the 1000 mg/kg/day dose level, speckled kidneys were observed for all males at terminal kill. One male also had pale kidneys at necropsy. Treatment-related macroscopic renal changes had regressed amongst recovery group males following a further fourteen days without treatment.

Relative organ weight changes were observed in animals at the 1000 mg/kg/day dose level in liver (statistical significant increase) and kidney (male only, statistical significant increase). Treatment-related organ weight changes had regressed amongst satellite 1000 mg/kg/day dose group animals following a further fourteen days without treatment.

Treatment-related hepatic changes were observed during histopathology. Centrilobular hepatocyte enlargement (minimal: 2/5 males; slight: 1/5 males) and a reduced severity of periportal hepatocyte vacuolation (minimal: 2/5 males; slight: 1/5 males) were observed in relation to treatment for male rats.

Based on the organ weights findings in the liver of males at the 1000 mg/kg/day dose level and the histopathological findings in the liver of males at the 150 and 1000 mg/kg/day dose levels, a No Observed Adverse Effect Level (NOAEL) for test item of 150 mg/kg/day and 1000 mg/kg/day was concluded for males and females, respectively.