1,3- AND 1,4-CYCLOHEXANDICARBOXYALDEHYDE

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

482-020-3

EC Name:

-

Molecular formula:

C8H12O2

IUPAC Name:

cyclohexane-1,3-dicarbaldehyde; cyclohexane-1,4-dicarbaldehyde

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): 1,3 and 1,4-Cyclohexanecarboxaldehyde
- Composition of test material, percentage of components: A mixture of 1,3-cyclohexanedicarboxaldehyde and 1,4-cyclohexanedicarboxaldehyde
- Lot/batch No.: Lot# 201001644 2nd pass Bottle # 2
- Storage condition of test material: ambient

Test animals

Species:

mouse

Strain:

other: Outbred Crl:CD1(ICR)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Kingston, New York)
- Age at study initiation: 8 weeks
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ] animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study.
- Housing: animals were housed one per cage in stainless steel cages
- Diet (e.g. ad libitum): LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form ad libitum
- Water (e.g. ad libitum): municipal water ad libitum
- Acclimation period: at least one week prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil, CAS number 8001-30-7, Sigma

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions of the test material were prepared and used fresh on each of the two
consecutive days of administration. A frozen stock solution of CP dissolved in distilled
water (thawed and brought to room temperature prior to use) served as the positive
control. The vehicle used to mix the test material (corn oil, CAS number 8001-30-7,
Sigma) served as the negative control.

Duration of treatment / exposure:

Groups of male mice were administered test material on two consecutive days. Animals (six mice/dose) were
dosed on March 20-21, 2012. To the positive control group, CP was administered only
once.

Frequency of treatment:

daily

Post exposure period:

48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 1000, or 2000 mg/kg bw/day
Basis:
other: based on results of RFT

No. of animals per sex per dose:

6

Positive control(s):

Cyclophosphamide monohydrate (CP) was administered only
once at a dose level of 40 mg/kg bw

Examinations

Tissues and cell types examined:

Approximately 5,000 RETs were analyzed per blood sample. The number of
normochromatic erythrocytes (NCE), MN-NCE, RET, and MN-RET were recorded for
each sample and the frequency of MN-RET was determined to provide an indication of
genotoxic potential. The frequency of RETs relative to total erythrocytes was determined
to provide an indication of perturbations in hematopoietic activity indicative of cell
toxicity. For each of the treatment groups, a mean and standard deviation was calculated
to describe the frequency of RET and MN-RET observed. The analyses were conducted
utilizing a Beckman Coulter Gallios™ flow cytometer.

Details of tissue and slide preparation:

Duplicate cell smears were prepared and stored to serve as backups in the event that the
flow cytometric analysis was not possible. Blood was collected into a microtainer tube
coated with EDTA (Becton Dickinson, Franklin Lakes, New Jersey). Wedge smears
were prepared, fixed in methanol, and stored at room temperature.

Evaluation criteria:

A test was considered valid if all of the following conditions were met:
• The range of MN-RET values in the negative controls were within reasonable
limits of the recent laboratory background range.
• There was a significant increase in the incidence of MN-RET in the positive
control treatment as compared to the concurrent negative controls.
• The mean for percent RET value in one or more of the test material treated
groups was ≥ 20% of the control value indicating no undue effect on
erythropoiesis (toxicity).
A test material was considered positive in this assay if the following criterion was met:
• Statistically significant increase in MN-RET frequency at one or more dose
levels accompanied by a dose response.
A test material was considered negative in this assay if the following criterion was met:
• No statistically significant dose-related increase in MN-RET when compared to
the negative control.
A test result not meeting the criteria for either the positive or the negative response was
considered to be equivocal.

Statistics:

MN-RET and percent RET was tested for equality of variance using Bartlett's test (alpha
= 0.01). If the results from Bartlett's test were significant, then the data for
the parameter may have been subjected to a transformation to obtain equality of the
variances. The transformations examined were the common log, the inverse, and the
square root in that order. The data was reviewed and an appropriate form of the data was
selected and subjected to the following analysis.
Based on the similarity of the response between sexes in the RFT, the MN-RET data and
the data on percent RET were analyzed by a one-way analysis of variance (where only
males were used). The alpha level at which tests were conducted was
0.05.
The MN-NCE was not analyzed statistically and was only used as an adjunct end point to
evaluate the biological significance of the MN-RET results.
The final interpretation of biological significance of the responses was based on both
statistical outcome and scientific judgment.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2000 mg/kd/day

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Treatment-related toxicity including a slight decrease in body temperature, a slight decrease in the percent RET value in the test material-treated animals, and clinical signs including decreased feces and perineal soiling were observed in male mice administered two consecutive daily doses of 1,3 and 1,4-cyclohexanecarboxaldehyde up to the limit dose of 2000 mg/kg bw/day. Based upon the results of the study reported herein, it was concluded that the test material, 1,3 and 1,4-cyclohexanecarboxaldehyde, did not induce a significant increase in the frequencies of micronucleated reticulocytes in the peripheral blood when given as a single oral dose on two consecutive days to male Crl:CD1(ICR) mice. Hence, 1,3 and 1,4-cyclohexanecarboxaldehyde is considered negative in this test system under the experimental conditions used.

Executive summary:

The in vivo genotoxic potential of 1,3 and 1,4-cyclohexanecarboxaldehyde was evaluated by

examining the incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood.

The test material was administered to male Crl:CD1(ICR) mice by single oral gavage on two

consecutive days at dose levels of 0 (negative control), 500, 1000, and 2000 (limit dose)

mg/kg body weight (bw). The highest dose level of 2000 mg/kg bw/day was based upon the

results of a range-finding test where at this dose, no treatment-related deaths were observed

in male or female mice. The analytically determined concentrations of the test material in the

dosing solutions used for the first day of dosing in the micronucleus ranged from 86.3% to

93.8% of the targeted concentrations. All animals were observed for clinical signs prior to

dosing and at 2, 5, and 24 hours following each dosing. Groups of animals were euthanized

48 hours after the second treatment for the collection of peripheral blood and evaluation of

RET (approximately 5,000/animal) for MN by flow cytometry. The proportion of RET was

also determined based upon 5,000 RET per animal and the results expressed as a percentage.

Mice treated with 40 mg/kg bw cyclophosphamide monohydrate by a single gavage dose and

euthanized 48 hours later served as positive controls.

All animals survived to the end of the observation period. Treatment-related clinical signs

including decreased feces and perineal soiling were noted in the 2000 mg/kg bw/day

treatment group. There were no statistically significant increases in the frequencies of MNRET

or statistically significant effects on the percent RET in groups treated with the test

material as compared to the negative controls. There was a significant increase in the

frequency of MN-RET and a decrease in the percentage of RET in the positive control

chemical group as compared to the negative control group. Based upon the results of the study reported herein, it was concluded that the test material, 1,3 and 1,4-

cyclohexanecarboxaldehyde, did not induce a significant increase in the frequencies of

micronucleated reticulocytes in the peripheral blood when given by daily gavage on two

consecutive days to male Crl:CD1(ICR) mice. Therefore, 1,3 and 1,4-

cyclohexanecarboxaldehyde is considered negative in this test system under the experimental

conditions used.