Cyclohexane

Repeated dose toxicity: other routes

Administrative data

Endpoint:

repeated dose toxicity: other route

Remarks:

other: I.P. injection 5 times/week for 2 weeks and time-course experiment for 10 consecutive days of i.p. injection

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Non-guideline study, GLP status unknown, published in peer reviewed literature but route of exposure (intra-peritoneal) not relevant for human exposures.

Data source

Reference

Reference Type:

publication

Title:

Evaluation of the subacute nephrotoxicity of cyclohexane and other industrial solvents in the female Sprague-Dawley rat

Author:

Bernard AM, de Russis R, Normand J, Lauwerys R

Year:

1989

Bibliographic source:

Toxicology Letters 45 (1989) 271-280

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

Female Sprague-Dawley rats were injected i.p. with 1.5 or 1 g/kg cyclohexane, 5 times per week for 2 weeks and urinary excretion of ß2-microglobulin,albumin and N-acetyl-ß-glucosaminidase measured. In a time-course experiment, rats were injected for10 consecutive days with 0.375, 0.75 or 1.5 g/kg cyclohexane or 0.4 g/kg cyclohexanol, controls were injected with olive oil only (2.5 ml/kg). The urinary excretion of ß2-microglobulin, and the effects on effects on glomerular filtration rate, renal plasma flow, relative kidney weights and renal concentrating ability were assessed.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexane

EC Number:

203-806-2

EC Name:

Cyclohexane

Cas Number:

110-82-7

Molecular formula:

C6H12

IUPAC Name:

cyclohexane

Details on test material:

analytical grade used

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 2-4 months
- Weight at study initiation: 150-250 g
- Fasting period before study:
- Diet: A.03 pellets (Usine d'alimentation ratiooelle, Epinay-sur-Orge, France) ad libitum (except during urine collection)
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature : 22°C
- Humidity : 65%
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

olive oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

14 days

Frequency of treatment:

Injected i.p. 5 times per week for 2 weeks. In a time course experiment, injected daily for 10 consecutive days

Doses / concentrations

Remarks:

Doses / Concentrations:
Nominal 1.5 and 1.0 g/kg cyclohexane in olive oil (5 times per week for 2 weeks). In time course experiment, nominal 0.375, 0.75, 1.5 g/kg cyclohexane or 0.4 g/kg cyclohexanol in olive oil (daily for 10 consecutive days).

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Urine collection: After each series of 10 injections, a 24-hr urine collection was carried out by placing rats in individual stainless-steel metabolism cages equipped with urine/faeces separators. No food was given during urine collection but water was provided ad libitum. In the time-course experiment, a 24-h urine collection was carried out before the first injection, then every 2 days for 10 days. The collection flasks contained 10 mg of NaN3 as preservative.

Examinations

Observations and examinations performed and frequency:

The concentration of albumin and ß2-microglobulin in urine was determined using an automated immunoassay based on latex particle agglutination. The activity of N-acetyl-ß-glucosaminidase in was assayed using 4-methylumbelliferyl-N-acetyl-ß-D-glucosaminide as substrate. The renal plasma flow and the glomerular filtration rate were estimated simultaneously from the clearance of 125I-iodohippurate and 51Cr-EDTA respectively. Clearance was determined from a single timed blood sample collected 60 min after intravenous injection of the radioisotopes. By measuring the osmolality of urine collected over 8 h following a 24-h water deprivation period the kidney concentrating ability could be determined. The concentrations of cyclohexanol and cyclohexanone in rat urine were determined by gas chromatography.

Statistics:

Differences between groups assessed by one-way analysis of variance followed by Dunett's multiple comparison test (P < 0.05 statistically significant). If parameters not normally distributed , statistical analysis performed on log-transformed data and geometric means used to present the results.

Results and discussion

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

glomerular filtration rate, renal plasma flow or relative kidney weights

Effect level:

1 500 mg/kg bw/day (nominal)

2

Dose descriptor:

other: excretion of ß2-microglobulin

Effect level:

1 500 mg/kg bw/day (nominal)

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Following an i.p injection of 1.5 g/kg cyclohexane, the only significant effect observed was an increased urinary excretion of ß2-microglobulin in rats. There were no effects in glomerular filtration rate, renal plasma flow or relative kidney weights. The renal concentrating ability was significantly depressed in rats treated with 1.5 g/kg cyclohexane.

There were no significant effects at doses of 1 g/kg cyclohexane and lower.

 

Applicant's summary and conclusion

Conclusions:

Cyclohexane, administered to female Sprague-Dawley rats, caused tubular injury as shown by a statistically significant increase in ß2-microglobulinuria, which was both time and dose-dependent. It was not accompanied by changes in the glomerular filtration rate and the renal plasma flow, but at the highest dose (1.5 g/kg) the renal concentrating ability was depressed. These renal tubular effects can most likely be ascribed to cyclohexanol, the main metabolite of cyclohexane.

Executive summary:

The subacute nephrotoxicity of cyclohexane was assessed following repeated i.p. injections to female Sprague-Dawley rats. Dose levels of 1.5 and 1 g/kg (administered 5 times per week for 2 weeks) were used in a sub-acute investigation, with 0.375, 0.75 and 1.5 g/kg (daily for 10 consecutive days) given in a time course study. There was a statistically significant increase in ß2-microglobulinuria, which was both time and dose-dependent. It was not accompanied by changes in the glomerular filtration rate and the renal plasma flow, but at the highest dose (1.5 g/kg) the renal concentrating ability was depressed. The authors believe that these renal tubular effects can most likely be ascribed to cyclohexanol, the main metabolite of cyclohexane.

This study used the i.p route and very high doses and the results, therefore, have limited relevance to real life exposures to man.