Cyclohexanone

Repeated dose toxicity: other routes

Administrative data

Endpoint:

short-term repeated dose toxicity: other route

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1982

Materials and methods

Principles of method if other than guideline:

This study was performed to assess the toxicity of cyclohexanone administered iv for 28 consecutive days to normal Wistar and Gunn rats, and to ascertain the functional capacity of the homozygous Gunn rat to form an ether glucuronide of cyclohexanol.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexanone

EC Number:

203-631-1

EC Name:

Cyclohexanone

Cas Number:

108-94-1

Molecular formula:

C6H10O

IUPAC Name:

cyclohexanone

Specific details on test material used for the study:

99.8%, analytical reagent grade; McKesson Laboratories, Fairfield, Conn.

Test animals

Species:

rat

Strain:

other: Wistar und Gunn

Sex:

male

Details on test animals or test system and environmental conditions:

Wistar rats were 7-8 wk old and weighing 187-251 g, and Gunn rats weighing 193-259 g, were used. The rats were housed individually in suspended, stainless steel cages and fed Purina Laboratory Rodent Chow (certified, guaranteed ration) and deionized water ad libitum. Because several organic solvents have been reported to cause lenticular changes (Gordon and Klebeger, 1968; Rengstroff et al ., 1972), animals were evaluated before and after the study to obtain preliminary information about the potential of cyclohexanone to produce lenticular damage. The animals were screened for ocular lesions by using slit-lamp and indirect ophthalmoscopy. Rats with these types of lesions were excluded from the study.

Administration / exposure

Route of administration:

intravenous

Vehicle:

other: NaCl

Details on exposure:

A cyclohexanone solution was prepared daily in 0.9% sterile NaCl at a concentration of 0.5% (w/v) (5.0 mg/ml cyclohexanone). This solution was injected into normal Wistar and Gunn rats at a volume of 20 ml/kg to provide a dose of 100 mg/kg cyclohexanone. The doses were chosen to be multiple (approximately 100X and 200X) of the amount to which a patient could be potentially exposed in a clinical situation after the use of medical devices sealed with cyclohexanone. The 0.5% solution was further diluted 1 :2 with physiological saline to provide a concentration of 2.5 mg/ml cyclohexanone and administered to a second group of Wistar and Gunn rats at a volume of 20 ml/kg to provide a dose of 50 mg/kg. The rate of injection was approximately 2 ml/min. These solutions were assayed at the end of the study for cyclohexanone content. The physiological saline vehicle was administered to the animals in the control group at a volume of 20 ml/kg and an approximate rate of 2 ml/min. Twenty-four hours before the start of iv administration, 15 normal Wistar and 15 Gunn rats were placed in metabolic cages for 24-h urine collection. Urine samples were collected in containers protected from light with Al foil and bilirubin was assessed semiquantitatively by the Diazo tablet test. To assess whether all Gunn rats were hyperbilirubinemic, all rats were bled from the median orbital sinus for total serum bilirubin determinations (Gambino and Di Re, 1968). Twenty-five Gunn rats were markedly hyperbilirubinemic, with values ranging from 5.3 to 15.6 mg/dl. The other five Gunn rats had normal bilirubin values (<0 .5 mg/dl).

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Examinations

Observations and examinations performed and frequency:

Each rat was weighed before the daily injection, and the appropriate dose of cyclohexanone (50 or 100 mg/kg) or volume of saline was administered iv through the tail vein. Injections were given for 28 consecutive days. Immediately after the first injection and every 7 d thereafter, rats in groups III-VI, two normal Wistar, and two Gunn rats from the control groups were placed in metabolic cages for 24 h. Urine was collected and analyzed for free cyclohexanone, cyclohexanol, and the glucuronide conjugate of cyclohexanol [by enzymatic hydrolysis of the conjugate with ß-glucuronidase (Beer and Gallagher, 1955)] . In addition, sulfate conjugates of cyclohexanol were determined in selected urine samples.
After the last day of injection (d 28), all animals were placed in metabolic cages for 24 h and urine was collected for semiquantitative analysis of bilirubin and determinations of the parameters described above.
After the final urine collection (d 28), the surviving rats were weighed and their eyes were again examined for possible ocular changes.
Food was withheld from animals 24 h before the collection of blood and the necropsy procedure. Animals were then anesthetized and blood samples were collected from the abdominal aorta for hematologic and clinical chemistry analyses and for cyclohexanone and cyclohexanol determinations.

Sacrifice and pathology:

Animals were exsanguinated and necropsies performed, during which the weights of the heart, lung, liver, spleen, and kidneys were recorded. Specimens for histopathologic examination included cross sections of spleen, skeletal muscle (tissue surrounding the femur), right kidney, two laterally opposing portions of the lungs and liver, stomach, duodenum, pancreas, urinary bladder, brain, and eyes. The heart and left kidney were sectioned longitudinally. Specimens were fixed in 10% neutral buffered Formalin and stained with hematoxylin and eosin. Bone marrow smears were made from the femur and stained with Wright's stain for microscopic examination.

Statistics:

Three-way factorial analysis was applied to all data except the pretreatment values for total serum bilirubin and cyclohexanol conjugates in the urine. Two-way factorial analysis was applied to the pretreatment values of total serum bilirubin, as these were determined only in the Gunn rats. For the analysis of urinary cyclohexanol conjugates, the no-intercept linear model was used because control groups had zero values.

Results and discussion

Results of examinations

Description (incidence and severity):

No adverse effects were observed in the normal Wistar or Gunn rats treated with cyclohexanone at either dose.
Although the normal Wistar rats in all treatment groups initially showed signs of toxicity (e .g., piloerection and lethargy), they subsequently appeared normal and exhibited normal behavior.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One normal Wistar rat (group IV, 100 mg/kg cyclohexanone) died 5 d after the initiation of treatment.

Description (incidence and severity):

Daily weight changes of control and treated animals were also similar. Decreases in body weights in rats in all treatment groups coincided with the urine collection periods. This temporary weight loss may be attributed to the transfer of rats to metabolic cages. Besides this temporary loss, there were no observable differences in body weight changes when control and treated rats were compared.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Ophthalmologic observations revealed no lentricular changes that could be related to cyclohexanone treatment, although an increase in optical density of lenses was observed among Gunn rats (19 of 30) and normal Wistar rats (4 of 29) across all control and treatment groups.

Haematological findings:

no effects observed

Description (incidence and severity):

Likewise, no effects of cyclohexanone administration, as indicated by hemotology and clinical chemistry parameters, were noted in normal Wistar and Gunn rats.
At 24 h after iv administration of cyclohexanone, there were no detectable concentrations of cyclohexanone and/or its metabolite cyclohexanol in the plasma. The analytical method used had a lower limit of quantitation of 5 ug/ml and was linear over the range of concentrations studied, namely between 5 and 200 ug/ml cyclohexanone and cyclohexanol. These substances were thus cleared from the blood in less than 24 h . Under the dose schedule employed in the study, there was no accumulation of the compound in the plasma of normal Wistar or Gunn rats.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Likewise, no effects of cyclohexanone administration, as indicated by hemotology and clinical chemistry parameters, were noted in normal Wistar and Gunn rats. Only serum Ca concentrations in normal Wistar rats showed a dose-response relation to cyclohexanone. Serum Ca concentrations were significantly lower (p < 0.004) in rats treated with the high dose (100 mg/kg) of cyclohexanone. Serum Ca concentrations in both cyclohexanone-treated groups were significantly lower (p < 0.004) than those in the control group. However, Ca concentrations in all three groups were within the acceptable normal range for Wistar rats (Mitruka and Rawnsley, 1977).

Urinalysis findings:

no effects observed

Description (incidence and severity):

Measurements of free cyclohexanone and free cyclohexanol in the urine showed a positive correlation between dose administered and excretion of these substances. However, urinary excretion of free cyclohexanone and cyclohexanol is a minor pathway in the overall elimination of cyclohexanone, as it accounted for less than 1% of the administered dose.
Cyclohexanol conjugates in urine were determined as total conjugates, glucuronide conjugates, and sulfate conjugates. The Gunn rats excreted the administered cyclohexanone as glucuronide conjugates of cyclohexanol. Urinary concentrations of the total conjugates and the glucuronide conjugates of cyclohexanol, whether expressed as milligrams per 24 h or as milligrams per milliliter of urine, were similar in normal Wistar and Gunn rats at the two doses. Statistical analysis showed that the linear dose responses are significant (p<0.001) for all glucuronide and total conjugates (expressed as milligrams per 24 h or per milliliter of urine). Glucuronide conjugate excretions were different for Wistar and Gunn rats (p <0.001) on d 7 and d 14. However, the only statistically significant difference between total and glucuronide conjugates (expressed as milligrams per 24 h or per milliliter of urine, and before or after adjusting for dose administered per kilogram of body weight) was found on d 7 of urine collection. This difference was observed in cyclohexanone-treated Wistar and Gunn rats at the two doses.
Total conjugates were higher than glucuronide conjugates; this difference increased as dose increased (p <0.001) and was not considered to have biological significance.
The percent of the dose excreted in the urine as glucuronides of cyclohexanol in Wistar rats ranged from 15.1 to 20.2% for the 50 mg/kg group and from 18.8 to 28.5% for the 100 mg/kg group. Urinary excretion of glucuronides of cyclohexanol in Gunn rats accounted for 16.9-25.0% and 24.2-34.1% of the administered dose for the 50 and 100 mg/kg groups, respectively .
Urine samples collected on d 1 and 7 of treatment were analyzed for the presence of sulfate conjugates of cyclohexanol. With the procedure employed, no traces of sulfate conjugates of cyclohexanol were detected. No pathological lesions were noted that could be attributed to cyclohexanone treatment. Two cases of pulmonary foreign body emboli were found and these caused a giant cell foreign body reaction. This was observed in one normal Wistar rat receiving 100 mg/kg and one Gunn rat receiving 50 mg/kg cyclohexanone. This is a common finding after multiple tail injections and could have been due to skin, keratin, hair, and so on.

Description (incidence and severity):

Behavioral patterns of all animals were similar, regardless of the treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One normal Wistar rat (group IV, 100 mg/kg cyclohexanone) died 5 d after the initiation of treatment. Necropsy of this rat did not reveal any gross abnormalities except that the lungs appeared congested throughout.
At necropsy, no gross pathological lesions were observed that were treatment-related.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

One normal Wistar rat (group IV, 100 mg/kg cyclohexanone) died 5 d after the initiation of treatment. The histopathologic assessment showed diffuse acute pulmonary edema and hemorrhage as well as hemorrhage around the aorta. None of the other animals had a similar pathological sign. Therefore, the death was probably not treatment-related.
Histopathologically, Wistar rats were essentially normal: one control rat (group I) had focal epicardial necrosis; one rat (group III) had mild focal pericarditis ( the same rat also had focal chronic pneumonia and acute pleuritis); four rats (one in group I, one in group III, and two in group IV) had mild chronic interstitial pneumonia; two rats (group III and group IV) had mild chronic bronchitis. The liver, spleen, and kidneys were generally normal.
The Gunn rats, however, had a high incidence of chronic pneumonitis (26 of 29 rats), interstitial nephritis (11 of 29), and tubular atrophy (17 of 29). These changes were found across all treatment groups and thus were not treatment-related.
Bone marrow smears indicated normal cell morphology and maturation. There did not appear to be any tendency toward proliferation of erythrocytes, granulocytes, or platelets, or any indication of hypocellularity.
However, bone marrow smears from 10 Gunn rats (5 controls, 4 in group V, and 1 in group VI) appeared to have more than the normal amount of mast cells. Because of the incidence in all treatment groups, this change is not considered to be treatment-related.

Effect levels

Dose descriptor:

NOAEL

Remarks:

both strains

Effect level:

100 mg/kg bw/day

Sex:

male

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The Gunn rat, despite its inability to form glucuronide conjugates of bilirubin, is capable of forming glucuronide conjugates of cyclohexanol after administration of cyclohexanone. With the dose schedule employed in this study, there was neither accumulation nor evidence of potential toxicity in normal Wistar and Gunn rats when cyclohexanone was administered iv for 28 consecutive days.