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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Three genotoxicity assays are available for this substance. A Bacterial mutagenicity assay, an in vitro micronucleus assay and an in vivo micronucleus assay.
Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Outbred Crl:CD1(ICR)
Sex:
male
Details on test animals 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.)
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
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
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.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2000 mg/kd/day
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro data:

Bacterial mutagenicity

The mixture of 1,3- and 1,4-cyclohexandicarboxaldehyde was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). In the dose range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. Cytotoxicity was evidenced by a complete lack of any microcolony background lawn at the dose levels of 3330 and 5000 μg/plate in both tester strains. Based on the results of the dose range finding test, the first mutation assay used a concentration range of 33 to 3330 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Cytotoxicity was evidenced by a complete lack of any microcolony background lawn in all three tester strains.

In an independent repeat of the assay with additional parameters, a concentration range of 10 to 2000 μg/plate in the absence S9-mix and at 10 to 3330 μg/plate in the presence of 10% (v/v) S9-mix was used in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity was observed in all tester strains, except in tester strain WP2uvrA in the absence of S9-mix.

The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the mixture of 1,3- and 1,4-cyclohexandicarboxaldehyde is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Mammalian Chromosome aberration

1,3- and 1,4-cyclohexanedicarboxaldehyde was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (solvent control) to 1410 μg 1,3- and 1,4-cyclohexanedicarboxaldehyde per ml of culture medium. The duration of treatment was 4 or 24 hours without S9 and 4 hours with S9. The highest concentration was based on the limit dose of 10 mM in this assay system. Based upon the mitotic indices, cultures treated for 4 hours with targeted concentrations of 0 (solvent control), 44.1, 88.1, and 176.3 μg/ml in the absence and presence of S9 activation were selected for determining the incidence of chromosomal aberrations in the initial assay (Assay A1). A significant increase in the incidence of aberrant cells relative to the solvent control was observed at the highest concentration (176.3 μg/ml) in the absence of metabolic activation with a 4 hour treatment. There were no significant increases in the frequencies of cells with aberrations in the presence of S9 activation. A confirmatory assay was conducted in the absence of metabolic activation for 4 and 24 hours with concentrations ranging from 0 (solvent control) to 250 μg/ml to confirm the response observed in the initial assay. Based on the mitotic indices, cultures treated for 4 hours with targeted concentrations of 100, 150, and 175 μg/ml were selected for determining the incidence of chromosomal aberrations. A significant increase in aberrant cell frequencies was observed at the 150 and 175 μg/ml treatment levels. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of abnormal cells in all assays. Based upon the dose related and reproducible increase in aberrant cell frequencies, 1,3- and 1,4-cyclohexanedicarboxaldehyde was considered to be genotoxic in the absence of metabolic activation in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Mammalian cell mutagenicity:

Due to the positive in vitro chromosome aberration assay and in accordance with Column 1 of annex VIII of the REACH legal text, an in vitro mamallian cell mutagenicity assay has been waived.

In vivo studies

Mouse peripheral blood micronucleus assay

Due to the positive result (in the absence of metabolic activation) in the in vitro assay, an in vivo assay was performed to further assess the genotoxicity in the whole organism. 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. 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.

Rat liver UDS assay:

Due to the positive finding in vitro it was necessary to conduct a second in vivo assay in order to satisfy the requirements of the Chinese New Substance Regulation. Therefore a Rat liver UDS assay was conducted. The assay was perfomed using male sprague dawley rats. Dose levels were 450, 900 and 1800 mg/kg bw (based on a range finding study). A vehicle control (corn oil) was used as well as a positve control (dimethylnitrosamine, 35 mg/kg bw). The test materials were given as a single oral gavage dose and the livers collected at 2 -4 hours post dose and 12 -16 hours post dose. 3 liver samples were analysed per dose, per timepoint.

In this assay, the reaction mass of 1,3- and 1,4-Cyclohexanedicarboxaldehyde did not cause a significant increase in average mean mean net nuclear grain count (MNNGC) at any dose level or harvest time. At both harvest times, the proportion of cells in repair in the vehicle control group was less than 15%, the average mean MNNGC of the vehicle control group was less than 1, and the average mean MNNGC of the positive control group was at least 5 counts over that of the vehicle control group.

Based on these results, all criteria for a valid study were met, and 1,3- and 1,4-Cyclohexanedicarboxaldehyde was negative for genotoxic potential in hepatocytes from male Sprague-Dawley rats given a single oral dose of up to 1800 mg/kg in the Unscheduled DNA synthesis (UDS) Assay with Mammalian CellsIn Vivo.

Summary:

Based on the absence of genotoxicity in the in vivo studies using the highest attainable concentrations and the lack of genotoxicity in vitro when metabolic activation was present, the weight of evidence supports the conclusion that the reaction mass of 1,3 and 1,4-cyclohexanecarboxaldehyde is not genotoxic in vivo.


Justification for selection of genetic toxicity endpoint
Two in vivo genetic toxicity test results are available indicating that the test material does not damage DNA or cause chromosome aberration as evaluated in the mouse micronucleus assay and rat liver UDS assay

Justification for classification or non-classification

The data do not support the need to classify for mutagenicity