Exo-1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptan-2-ol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-10-19 to 1987-11-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(1S,2S)-1-methyl-4-(propan-2-yl)-7-oxabicyclo[2.2.1]heptan-2-ol

Cas Number:

87172-89-2

IUPAC Name:

(1S,2S)-1-methyl-4-(propan-2-yl)-7-oxabicyclo[2.2.1]heptan-2-ol

Test material form:

solid

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rats were supplied by Charles River U.K. Ltd., Manston, Kent. Approximately 60 weanling rats of each sex were delivered under Specific Pathogen Free (SPE) conditions in wire lined filter boxed to the SPF Experimental Unit, Sittingbourne Research Centre.
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: males: 153.5 - 162.0 g; females: 116.2 - 119.2 g
- Housing: The animals were singly housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Approximately one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 °C
- Humidity (%): 45-70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The animals were dosed by oral intubation at 3 mL per kilogram body weight (based on the most recent body weight measurement) once each day until scheduled necropsy.

Vehicle:

other: polyethylene glycol:water (1:1 v/v)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was formulated in polyethylene glycol: water (1:1 v/v). Solutions were prepared to provide the equivalent of 333 mg/mL, 166.6 mg/mL, 33.3 mg/mL, 3.33 mg/mL, 0.33 mg/mL and 0 mg/mL. The formulations were prepared weekly throughout the study and stored at approximately 30 °C to keep the test substance in solution.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sub-samples of the test solutions were taken by volume and added to ethanol. The total volume was adjusted to a known value, and further dilutions, if required, were made using more ethanol. The solutions thus produced were analysed without further treatment using gas chromatography.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once each day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Doses / concentrations 5

Dose / conc.:

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

No. of animals per sex per dose:

Test group: 7/ sex
Control group: 14/sey

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily on weekdays and once daily on weekends
- Each animals was examined carefully for evidence of changes in skin, fur, eyes and visible mucous membranes, for abdominal patterns of behaviour, disturbance of major body systems (e.g. respiratory, alimentary, urinary, nervous system) and for goss deviations in food and water consumption.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly intervals

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food hoppers were weighted at weekly filling and topped up later in the week; weekly food intakes during the study wer calculated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken from each animal at necropsy by cardiac puncture into tubes containing EDTA (ethylenediamine tetra-acetic acid)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy by cardiac puncture into tubes containing heparin.
- Animals fasted: No
- How many animals: All animals

For details refer to section 'Any other information on materials and methods incl. tables'

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (see table) / No / Not specified

Statistics:

A one-way analysis of variance with treatment and block factors was used for all variates. Following analysis of variance, differences between the control and treated group means were assessed for significance using the Williams' t test (Williams, 1971; Williams 1975). On occasions where a monotonic dose response could not be assumed, Dunnett's test was used (Dunnett, 1964).
Body and organ weights are reported as analysed with initial body weight as a covariate provided a significant covariance relationship was observed.
Organ weights were further analysed using terminal body weight as a covariate. Although not a true covariance analysis, the analysis does provide an aid to the interpretation of organ weights when there are differences in terminal body weights. The analysis attempts to estimate what the organ weights would have been if all the animals had had the same terminal body weight.
Wilcoxon's two sample rank sum test (Hill and Peto, 1971; Lehman, 1961) was used for BANDS (non-segmented polymorphic neutrophils) and IGT. Food intakes, and other clinical chemistry and haematology variates were examined using the analysis of variance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights were increased at 500 and 1000 mg/kg in males and at 500 and 1000 mg/kg in females, with histological evidence of enzyme induction and a macrovesicular vacuolation at 1000 mg/kg in both sexes.
Kidney weights were increased at 500 and 1000 mg/kg in males and at 1000 mg/kg in females.

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL OBSERVATIONS AND MORTALITY:
All top dose animals were comatose within a few hours of their first dose, except one male which became ataxic and then died and two males which were ataxic for about 5 hours. The coma lasted several hours, most animals recovered overnight but some died in coma or were humanely killed the next day; two others died or were killed after being comatose subsequent to their second dose. Few toxic signs were observed among the surviving animals, although coma did occur sporadically after approximately one dose in 13. One female died after being comatose in the last week of dosing.
Several of the animals with ataxia repeatedly scrached their heads.
Three animals appeared to have blood in their urine, two of these subsequently died.
At 500 mg/kg one male and four females had ataxia or abasia on the first day of dosing only, one other female had ataxia and abasia on the third day of dosing only; they had no other signs of toxicity.
Animals in the other dose groups survived to the end of the study with no significant clinical signs.

BODY WEIGHT AND FOOD INTAKE
The top dose animals had reduced food intakes and only gained about half as much body weight as the controls. The 500 mg/kg animals and the 100 mg/kg females had reduced body weights and food intakes throughout the study, although this was not always statistically significant.

ORGAN WEIGHTS
Liver weights were increased at 100, 500 and 1000 mg/kg in males, and at 500 and 1000 mg/kg in females. These increases may be related to a functional hypertrophy caused by exposure to xenobiotics resulting in a stimulation of the normal detoxification system.
Kidney weights were increased in both sexes at 1000 mg/kg, and at 500 mg/kg in males. this may be related to the blood seen in the urine of the top dose animals and the histopathological finfings.
Adrenal weights were increased in all top dose animals. The top doses males showed a small statistically significant increase in heart weight, of no toxicological importance.

PATHOLOGY
Treatment related macroscopic and microscopic observations were only found in the top dose group.
The livers of the top dose group had a range of minor tinctorial changes visible macroscopically that were inconsistent and of uncertain significance. Several animals had histological changes consistent with enzym induction, and a macrovesicular vacuolation which is considered related to treatment. the bladders of premature decedents had a range of lesions from mild submucosal haemorrhage to severe haemorrhagic cystitis. This typ of lesion is normally due to the presence of an irritant material in the bladder, i.e. the test substance or metabolite in the urine. No other treatment related histopathological changes were identified.

HAEMATOLOGY AND CLINICAL CHEMISTRY
Female rats at 500 and 1000 mg/kg had mild macrocytic hypochromic anaemia i.e. reduced haemoglobin and mean corpuscular haemoglobin concentration, and an increased erythrocyte volume. Females at 500 mg/kg also had a reduced erythrocyte count. At 100 mg/kg females gat a very mild normochromic anaemia i.e. reduced haemoglobin.
Male rats had no significant signs of anaemia as such, but both males and females had evidence of compensation for anaemia. Females at 10 to 100 mg/kg and males at 100 to 1000 mg/kg had increased reticulocytes and all animals at 100 mg/kg had increased normoblasts (females hat a non statistically significant increase).
Males had increased platelet counts at 100 to 1000 mg/kg and increased plateletcrit at 500 and 1000 mg/kg; females had an increased platelet count at 1000 mg/kg only. Mean platelet volumes were decreased at 500 and 1000 mg/kg in males and 100 to 1000 mg/kg in females. Females at 500 and 1000 mg/kg had increased polymorphic neutrophils and decreased in lymphocytes at 1000 mg/kg. This may reflect a mild inflammatory reaction. other small statistically sifnificant haematological changes are of no toxicological importance.
The haematological findings in the higher dose level my be related to the haemorrhagic effects seen in the bladders of the decedents i.e. the anaemia and increased platelets could be due to haemorrhage earlier in the study.
Although no macroscopic changes were observed at necropsy in the bladders of the survivors, it is likely that some haemorrhage did occur early in the study in some survivors e.g. blood was seen in the urine of one of the males in the top dose group on day 1.
All top dose animals had increased serum bilirubin, probably related to the increase in erythrocyte turnover.
Urea nitrogen and creatinine were increased at 500 mg/kg in males and decreased at 1000 mg/kg in females. There were no treatment related histopathological changes in the kidney, but kidney weights were increased in these groups as well as in the 1000 mg/kg males.
Cholesterol levels were increased in the 100 and 500 mg/kg males and triglycerides were decreased in females at 100 to 1000 mg/kg.
Alkaline phosphate levels were markedly decreased at 500 and 1000 mg/kg in both sexes. A decrease of this magnitude was probably due to a reduction in the intestinal alkaline phosphatase isoenzyme, brought about by either the reduced food intakes, or a direct effect of the compound on the intestine.
Other statistically significant clinical chemical differences are not toxicologically important.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

haematology

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

other: hematopoietic system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified