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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7-24 June 1981
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, conducted to follow regulatory guidelines of the day and main current guideline points, fully adequate for assessment.

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Principles of method if other than guideline:
The experimental methods used in this inhalation study are considered to be consistent with the main points of the OECD guideline No 475, for example, numbers of animals, cells scored, sampling times, cytogenetic parameters examined.
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Chemical structure
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:
Certified cyclohexane.
Clear colourless liquid
Lot 701739 Class 1B
99 Mol % pure

Test animals

Species:
rat
Strain:
other: CRL:COBS CD(SD)BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 weeks (males), 14 weeks (females)
- Housing: Housed individually in suspended, wire-mesh, stainless steel cages.
- Diet: Purina "Certified Laboratory Chow 5002 ®" available ad libitum, except during exposures.
- Water: Acidified water ad libitum, except during exposures.

ENVIRONMENTAL CONDITIONS
- Temperature controlled 
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
The concentrated cyclohexane vapour was diluted with room air in the chambers.
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body
10 male and 10 female rats per group were dosed by inhalation exposure to cyclohexane vapour at analysed concentrations of 0, 96.6, 307.2 and 1041.6 ppm, for 6 hours/day for 5 days.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Stainless steel and plexiglass chambers of 0.25 cubic meter volume. The chambers were constructed with a square cross-sectional area and pyramidal tops and bases. The chambers were operated in a dynamic mode using room air to dilute the test material. The cyclohexane vapour was generated by bubbling dry, oil-free, breathing quality air through liquid cyclohexane in 250 or 500 ml gas wash bottles fitted with fritted glass cylinders. The control rats were placed in a chamber to which no test material was added. Animals were housed individually throughout the exposure periods.
Chamber temperature range: 24 - 27°C
Total airflow for all chambers was 28.3 litres per minute.

TEST ATMOSPHERE
The concentration of cyclohexane in the chamber atmospheres was determined at least hourly.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
Daily for 5 days (Sunday - Thursday)
Post exposure period:
Bone marrow was sampled six hours after completion of the last exposure.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
0, 100, 300 and 1000 ppm
Remarks:
Doses / Concentrations:

Basis:
analytical conc.
0, 96.6, 307.2 and 1041.6 ppm
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent no treatment
Positive control(s):
Triethylenemelamine (TEM) at 10 mg/kg was the positive control substance and administered via a single intraperitoneal (IP) injection 24 hours prior to terminal kill.

Examinations

Tissues and cell types examined:
Bone marrow.
Routinely, 50 spreads were read for each animal. The locations of cells bearing aberrations were identified by the use of coordinates on the mechanical stage. A mitotic index based on at least 500 cells was also recorded. This index was calculated by scoring the number of cells in mitosis per 500 cells on each slide read.
Details of tissue and slide preparation:
Three hours prior to kill with CO2, the animals were injected IP with 4.0 mg/kg of colchicine. The adhering soft tissue and epiphyses of both tibiae were removed according to the method of Legator et al., (1969). The marrow was flushed from the bone and transferred to Hanks' balanced salt solution. The marrow button was collected by centrifugation and then resuspended in 0.075M KCL. The centrifugation was repeated and the pellet resuspended in fixative (methanol:acetic acid, 3:1) The fixative was changed once and the cells were left overnight at 4°C. Cells in fixative were dropped onto glass slides and air-dried. Spreads were stained with 5% Giemsa at pH 6.8. After drying, the slides were cover-slipped. Slides were coded for control of bias and then scored for chromosomal aberrations. Standard forms were used to score and record gaps, breaks, fragments and reunion figures.
Evaluation criteria:
The test substance was tested to establish any interaction with chromosomes to produce gross lesions or changes in chromosome numbers and whether these are of a type which can survive more than one mitotic cycle of the cell. All aberration figures detected by this assay resulted from breaks in the chromatin which either failed to repair or repaired in atypical combination. The type of aberration, its frequency and its correlation to dose in a given time period were considered in the evaluation of mutagenic potential.
Statistics:
Statistical analysis employed a Student t-test (Bancroft, 1957)

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There were no signs of toxicity in any rats during or after exposure.

Any other information on results incl. tables

Exposure conditions

 

Intended chamber concentration (ppm cyclohexane)

0

0

0

0

Mean chamber conc

0±0

0±0

0±0

0±0

Chamber temp

26.5±1.33

26.5±1.33

26.5±1.33

26.5±1.33

Number of samples

40

40

40

40

 

Summary of results

Treatment

Dose

% cells with structural aberrations

 

1 or more

2 or more

MI #

Negative control

N/A

1.0

0

1.2

Positive control

1.0 mg/kg

73.4**

66.9**

0.1

Cyclohexane

96.6 ppm

0.7

0.3

3.1

 

307.2 ppm

0.5

0.1

4.3

 

1041.6 ppm

0*

0

2.6

# Mitotic index based on at least 500 cells/animal

*significant at<0.05 by t-test

*significant at<0.01 by t-test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Cyclohexane was negative in inducing chromosomal aberrations in rat bone marrow cells under the conditions of this assay.
Executive summary:

Groups of 10 male and 10 female Sprague Dawley rats were exposed by inhalation (6 hours each day for 5 days) to nominal doses of cyclohexane (99%, certified) of 96.6, 307.2 or 1041.6 ppm. Bone marrow was taken 6 hours after the last dose and cytogenetic examination made. The structural aberration frequency did not differ significantly from the negative control for any tested dose. The percentages of cells showing one or more structural aberrations or two or more structural aberrations were likewise similar to the negative controls. The results were the same whether males or females or both sexes pooled were evaluated by the Student t-test. A concurrent positive control substance, triethylenemelamine, showed a significant increase in structural aberration frequency compared with controls. Cyclohexane was not clastogenic toward rat bone under the conditions of this test.