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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclohex-1,4-ylenedimethanol
EC Number:
203-268-9
EC Name:
Cyclohex-1,4-ylenedimethanol
Cas Number:
105-08-8
Molecular formula:
C8H16O2
IUPAC Name:
cyclohexane-1,4-diyldimethanol
Constituent 2
Reference substance name:
[4-(hydroxymethyl)cyclohexyl]methanol
IUPAC Name:
[4-(hydroxymethyl)cyclohexyl]methanol
Constituent 3
Reference substance name:
Cyclohexane-1,4-dimethanol; 1,4-Bis(hydroxymethyl)cyclohexane; CHDM
IUPAC Name:
Cyclohexane-1,4-dimethanol; 1,4-Bis(hydroxymethyl)cyclohexane; CHDM
Details on test material:
- Test article (as cited in report): EC99-0207, CHDM-D90
- Composition: CHDM-D90 is a 90/10 weight percent solution of 1,4-cyclohexanedimethanol in water
- Source: Eastman Chemical Company
- Lot number: 42490
- Physical Description: Transparent, colorless, viscous liquid
- Storage Temperature: Ambient temperature
- Date received: September 2, 1999 (Dose Rangefinding Study)
September 23, 1999 (Definitive Study)
Specific details on test material used for the study:
Test Article
Sponsor's Identification: EC99-0207, CHDM-D90,
Lot No. 42490
Date Received: September 2, 1999 (Dose Rangefinding Study)
September 23, 1999 (Definitive Study)
Physical Description: Transparent, colorless, viscous liquid
Storage Conditions: Ambient temperature

Test animals

Species:
rat
Strain:
other: Crl:CD (SD) IGS BR
Details on species / strain selection:
Chromosome mutations and numerical changes are the cause of many human genetic diseases and there is substantial evidence to suggest that they are also implicated in the development and progression of cancer. The mammalian in vivo chromosomal aberrations test is routinely used for the detection of these chromosomal events in the bone marrow of animals after treatment with a test article. Structural chromosomal aberrations are the result of breaks (clastogenicity) or breaks and rearrangements of the DNA strand within the chromosome and are visible with the light microscope in cells that are in metaphase of the cell cycle. They can be present in a single chromatid (termed "chromatid damage") or involve an identical locus in both chromatids (termed "chromosome damage"). After test article administration, the bone marrow cells were sampled based on the rate of the cell cycle. The cell cycling time for rodents is estimated to range from 12-18 hours. The two harvest times (18 and 42 hours after test article administration) were chosen to detect the induction of chromosomal aberrations by test articles in both the presence and absence of test article effects on the cell cycle time of the bone marrow cells. The early 18-hour time was expected, in the absence of delay or in the presence of slight delay (e.g.,0-6 hr), to assess metaphase cells that had completed at least one full cell cycle. The 42-hour harvest time was expected to detect metaphase cells which under conditions of cell cycle delay would have progressed through at least one cell cycle (or more than one cycle in the absence of delay).
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 8-9 weeks
- Weight at study initiation for rangefinding study: 229-263 g (males) and 167-195 g (females)
- Weight at study initiation for main study: 280-375 g (males) and 186-237 g (females)
- Housing: Housed individually in clean, suspended, stainless steel wire-mesh cages for at least 7 days following receipt in an environmentally controlled room.
- Diet: PMI® Feeds, Inc. Certified Rodent Lab Diet® 5002 (chow) ad libitum
- Water: Tap water ad libitum
- Acclimation period: 7 days
- Animal Identification: ear tag
- Randomization: by computer randomization program into study groups

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-79
- Humidity (%): 30-70
- Air handling: 10 complete air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

IN-LIFE DATES:
Study Initiation Date: September 3, 1999
Experimental Start Date: September 13, 1999
Experimental Completion Date: October 8, 1999

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Sterile deionized water
Details on exposure:
Dose Preparation: The highest stock solution of the test article, CHDM-D90, was prepared by adding the appropriate volume of deionized water to a measured quantity of the test article and mixing until a solution was obtained. The lower dose levels were prepared as dilutions of the highest stock solution.

Animals were weighed prior to dosing and doses were based upon individual animal weights. The weight variation of the animals did not exceed ±20% of the mean weight of each sex. All dose calculations were based on a specific gravity of 1.02 for the test substance.

Rangefinding study: Three animals/sex/group received a single dose of test article by oral gavage at dose levels of 200, 500, 800, 1500, and 2000 mg/kg/bw (dosing volume = 10 mL/kg) and were observed for signs of toxicity for 2 days. At the conclusion of the study, animals were euthanized by CO2 inhalation followed by incision of the diaphragm.

Definitive study: Six animals/sex/group received a single dose of test article by oral gavage at dose levels of 0 (vehicle control), 500, 1000, and 2000 mg/kg/bw (dosing volume = 10 mL/kg) and were observed for signs of toxicity. Eighteen hours after test article administration, animals were euthanized and bone marrow was extracted. An additional 6 animals/sex/group dosed with 0 and 2000 mg/kg/bw of the test article were observed for toxicity for 42 hours after test article administration, then animals were euthanized and bone marrow extracted. Cyclophosphamide 60 was dissolved in sterile deionized water (stock solution was 6.0 mg/mL) and was the positive control for the study with scheduled euthanasia at 18 hours after test article administration.
Duration of treatment / exposure:
once
Frequency of treatment:
once
Post exposure period:
Dose rangefinding study: 2 days
Definitive study: 18-42 hours
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
500 and 1000 mg/kg/bw : 6
0 and 2000 mg/kg/bw : 12
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide (Sigma lot number 108H0568, CAS No. 6055-19-2) was dissolved in sterile deionized water (Covance batch number 32) to make a stock concentration of 6.0 mg/mL.
- Administered to 6 animals/sex/group

Examinations

Tissues and cell types examined:
Approximately 1.5-2.5 hours prior to euthanasia, experimental animals were injected intraperitoneally with 2.0 mg/kg of colchicine (10 mL/kg). Following euthanasia, the tibias and femurs were removed from the first five surviving animals (in each treatment and control group) for marrow extraction. The marrow was flushed from the bone and transferred to Hank’s balanced salt solution. The marrow pellet was collected by centrifugation and then resuspended in 0.075M potassium chloride. The centrifugation was repeated and the pellet was resuspended in fixative (methanol: acetic acid, 3:1). Any additional animals in excess of the first five survivors were euthanized and discarded with no marrow extracted. Bone marrow metaphase cells were examined for chromosomal aberrations and polyploidy.
Details of tissue and slide preparation:
The fixative was then changed and the fixed cells were dropped onto glass slides and air dried. The metaphase cells were stained with 5% Giemsa and air dried; the slides were coverslipped. Slides were coded for control of scoring bias.
Evaluation criteria:
Cells were selected for scoring on the basis of good chromosome morphology, and only cells with the number of centromeres equal to the rat model number 42 ± 2 were analyzed. A sample of 100 metaphase cells from each animal was scored for aberrations. For the positive control animals, 25 metaphase cells were analyzed per animal because of the high frequency of aberrations (25% or more of the cells having one or more aberrations). For each cell bearing an aberration, the microscope stage location was recorded so that the cell could be relocated, if necessary. Percent polyploidy and endoreduplication were also analyzed and tabulated by evaluating 100 metaphases per animal. A mitotic index was calculated by scoring the number of cells in mitosis per 1000 cells observed and converting the number to a percent.

Gaps were not counted as significant aberrations. Open breaks were considered as indicators of genetic damage, as were configurations resulting from the repair of breaks. The latter included translocations, multiradials, rings, multicentrics, etc. Cells with more than one aberration were considered to indicate more genetic damage than those containing evidence of single events.

Comparison with a concurrent vehicle control which happens to show an unusually low frequency of aberrations may suggest statistical significance which is not biologically relevant. In these instances, the treatment data were considered against the historical control database.

The type of aberration, its frequency, the statistical significance of any increase and its correlation to dose in a given time period were all considered in evaluating the clastogenic potential for the test article. The usual criteria for a positive response are a statistically significant increase in the number of structural aberrations for at least one dose level and a dose-related response.
Statistics:
Analyses were performed on a per animal basis for the following variables: number of cells with at least one structural aberration and the number of cells with two or more structural aberrations.

Analysis of variance (ANOVA) was used to compare positive control to the vehicle control group. Specifically, Levene’s test was performed to test for variance homogeneity. In the case of variance heterogeneity, the data were ranked. Dunnett’s t-test was performed to compare test article-treated group means to the vehicle control. In this case, additional tests such as linear regression and Terpstra-Jonckheere test for monotone trend were also performed to evaluate any possible dose-response.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE RANGEFINDING STUDY:
All animals at all dose levels appeared normal after dosing and remained healthy throughout the observation intervals. Based on these results the dose levels for the chromosomal aberrations assay were selected to be 500, 1000, and 2000 mg/kg.

DEFINITIVE CHROMOSOMAL ABERRATIONS ASSAY:
The test article induced no mortality or signs of clinical toxicity at any of the dose levels selected. There were no statistically significant increases in either structural chromosomal aberrations or numerical chromosome changes. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest time points. Under the conditions of the present assay, the test article was judged to be negative for induction of structural or numerical chromosome damage.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
1,4-Cyclohexanedimethanol showed no evidence of cytotoxicity or clastogenic activity in an in vivo chromosomal aberrations assay when tested in male and female Crl:CD (SD) IGS BR rats exposed up to the limit dose of 2000 mg/kg bw as given in OECD Guideline 475. Based on an absence of genotoxic/mutagenic effects in this study, 1,4-cyclohexanedimethanol is not classifiable for Germ Cell Mutagenicity according to GHS.
Executive summary:

In a chromosomal aberrations assay, CHDM-D90 was administered to groups of 6 Crl:CD (SD) IGS BR rats/sex/group by oral gavage at dose levels of 0, 500, 1000, and 2000 mg/kg bw. Vehicle and positive controls were also prepared. Mitotic index was used to assess cytotoxicity and select concentrations for metaphase analysis. Metaphase cells were examined for structural chromosome damage, chromosome rearrangements, and a polyploid number of chromosomes. Positive controls induced the appropriate response. There was no evidence of cytotoxicity or chromosomal aberrations induced over background in 1,4-cyclohexanedimethanol-exposed rats at any test concentration up to the limit dose of 2000 mg/kg bw. There were no statistically significant increases in either structural chromosomal aberrations or numerical chromosome changes when compared to the solvent control. It was concluded that 1,4-cyclohexanedimethanol showed no evidence of clastogenic activity under the conditions of exposure in this study.