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Toxicological information

Genetic toxicity: in vivo

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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:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 474, EPA 870.5395, EC B.12 guidelines and in accordance with the Principles of Good Laboratory Practices (GLP).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
Minor exceptions such as concentration check and stability for the positive control substance (cyclophosphamide monohydrate) used in this study were not performed, GLP characterization was conducted concurrently, however these had no impact on the study
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,4 Cyclohexanedimethanol Reaction Products with Epichlorohydrin
- Analytical purity: The purity of the test material was determined to be 9.5 area % 1,4-Cyclohexanedimethanol monoglycidiyl ether, 45.0 area %, 1,4-Cyclohexanedimethanol diglycidyl ether, and 25.0 area % of the reaction product of 1,4-cyclohexanedimethanol with 3 equivalents of epichlorohydrin by gas chromatography/flame ionization detector with tentative identification of the major components by nuclear magnetic resonance and gas chromatography mass spectrometry
- Impurities (identity and concentrations): no information available
- Lot number : Lot # 1H1050NZP1

Test animals

Species:
mouse
Strain:
other: Crl:CD1(ICR) mice
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Kingston, New York)
- Age at study initiation: Approximately 8 weeks
- Assigned to test groups randomly: yes
- Fasting period before study: not applicable
- Housing: After assignment, animals were housed one per cage in stainless steel cages. Cages had wire mesh floors and were suspended above absorbent paper. Non-woven gauze was placed in the cages to provide a cushion from the flooring for the rodents’ feet. The gauze also provided environmental enrichment. Cages contained a hanging feeder and a pressure activated lixit valve-type watering system.
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: at least 1 week

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 cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: recommended vehiclle by various regulatory agencies
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions of the test material were prepared once and used on each of the days of administration based on known stability of the test material in the vehicle. 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) served as the negative control. The concentrations of the test material in the dosing solutions used for the micronucleus test were verified using high performance liquid chromatography and mass spectrometry (HPLC/MS) with external standard quantitation.

Duration of treatment / exposure:
Groups of male mice were administered 0 (negative control), 375, 750, or 1500 mg/kg bw/day of the test material on two consecutive days. Cyclophosphamide (CP) was administered only once at a dose level of 40 mg/kg bw.
Frequency of treatment:
Groups of male mice were administered 0 (negative control), 375, 750, or 1500 mg/kg bw/day of the test material on two consecutive days. Cyclophosphamide (CP) was administered only once at a dose level of 40 mg/kg bw.
Post exposure period:
not applicable
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
375 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
750 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1500 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
There were six mice/dose except in the 1500 mg/kg bw/day group, where seven mice were treated in the event of deaths occurring among the treated animals of this group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP)
- Justification for choice of positive control(s): recommended positive control by various regulatory agencies
- Route of administration: oral
- Doses / concentrations: 40 mg/kg bw/day

Examinations

Tissues and cell types examined:
Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry with traditional blood smears prepared as a backup
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the results of a dose range finding study - Animals (one/sex/dose) were dosed with 1000 or 2000 mg/kg of the test material on two consecutive days and observed for any signs of toxicity for at least 72 hours after the initial dosing. Based upon the results of the initial dosing, an additional two mice/sex/dose were dosed with 1000 or 2000 mg/kg bw/day. These animals were also observed for at least 72 hours after the initial dose for any signs of toxicity. Due to the mortality observed at the high dose (i.e., 2000 mg/kg bw/day), a lower dose level of 1500 mg/kg bw/day was administered (three/sex/dose). These animals were also observed for at least 72 hours after the initial dose for any signs of toxicity. All mortalities and/or moribund animals were necropsied to ascertain whether the condition was related to a dosing mishap

TREATMENT AND SAMPLING TIMES : Approximately 48 hours after the last dosing, peripheral blood samples were collected from all animals. Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry with traditional blood smears prepared as a backup. Samples were prepared and analyzed per instructions in the Mouse MicroFlow Micronucleus Analysis Kit Manual (Litron Laboratories, Rochester, New York). At the end of the specified interval following treatment, a peripheral blood sample was collected from the orbital sinus of all surviving animals into anticoagulant solution following anesthesia with isoflurane. Briefly, the blood samples from the first six animals of each treatment group were fixed in ultracold (-70 to -80°C) methanol within five hours of collection. All fixed blood samples were stored at -80°C. Fixed blood samples were washed with a cold, buffered salt solution and isolated by centrifugation. The resulting cell pellets were stored at 4°C until staining. Blood samples were ultimately incubated with RNAse to degrade the high levels of RNA present in the reticulocytes (RET) and a fluorescently labeled antibody to the transferrin receptor (anti-CD71-FITC) to specifically identify the RET. A propidium iodide solution was added to each sample immediately before flow cytometry (FCM) (Beckman Coulter Gallios flow cytometer) analysis to stain the DNA, including that of micronuclei. Blood samples were analyzed by high-speed FCM. In this system, the sample was moved at a high velocity past a laser set to provide 488 nm excitation. The fluorescent wavelengths emitted by each cell were collected by photomultiplier tubes. Using the previously described staining procedure, the propidium iodide-stained DNA of the micronuclei emitted a red fluorescence and the anti-CD71-FITC antibody emitted a high green fluorescent signal permitting differentiation between cells with and without micronuclei. In addition to obtaining fluorescent profiles, FCM simultaneously provided
cell size information by determining the light scatter properties of each cell or combination of cells.

DETAILS OF 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.

METHOD OF ANALYSIS: 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, MN-NCE, and MN-RET observed. The analyses were conducted utilizing a flow cytometer (Beckman Coulter Gallios).

OTHER: The relative body temperatures of the treated animals were monitored during the renge finding test (RFT) using programmable transponders (BioMedic Data Systems, Seaford, Delaware). The temperatures were generally collected by scanning the microchip prior to each dosing, and approximately 2, 5, and 24 hours after dosing as well as prior to sacrifice. Temperatures were monitored during the main study as a body temperature increase of 1C or a decrease of at least 3C for more than 5 hours was observed during the RFT.
Evaluation criteria:
A test was considered valid if all of the following conditions are 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:
Standard staistical methods were employed

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose Range-Finding Test (RFT)
Phase I
Targeted dose levels of 1000 or 2000 mg/kg bw/day were used in the initial phase of the RFT using male and female mice (one/sex/dose). The male and female mice dosed at 1000 mg/kg bw/day had no remarkable changes in body weight, body temperature, or clinical observations
In mice dosed at 2000 mg/kg bw/day, the male mouse had no clinical observations while the female mouse spontaneously died after dosing on the second day with no prior clinical observations including no significant body temperature changes Examination of this female mouse dosed at 2000 mg/kg bw/day revealed congestion of the lungs and kidneys, although no evidence of a gavage error was noted.
Based on this information, an additional two mice/sex were dosed at 1000 or 2000 mg/kg bw/day. In the male mice dosed at 1000 mg/kg bw/day, one had no significant clinical observations for the entire observation period and the second mouse had fecal soiling and soft feces on the second day of dosing. By the third day the clinical signs that were observed in the mouse dosed at 1000 mg/kg bw/day had largely recovered with no remarkable clinical observations for the remainder of the in-life including no significant changes in body weight or body temperature. In the female mice dosed at 1000 mg/kg bw/day, one displayed no remarkable observations for the entire in-life portion of the test including no significant changes in body weight or body temperature. The remaining female mouse dosed at 1000 mg/kg bw/day had a body temperature decrease of 4.0ºC five hours after dosing on the initial day with no other remarkable clinical signs on that day. On the second day of dosing this mouse had slow and labored respiration, decreased activity, partially closed eye lids, a head tilt, and a body temperature decrease of 10.7°C five hours post dosing and was declared moribund. Pathological evaluation revealed that the esophagus of this animal was punctured due to a probable gavage error.
In male mice dosed at 2000 mg/kg bw/day, one had a decreased quantity of feces on the third day of observation with no prior clinical signs, however, this mouse largely recovered by the fourth day of observation with minimal changes in body temperature and body weight. The remaining male mouse dosed at 2000 mg/kg bw/day spontaneously died after the initial dose of the test material. Examination of this mouse revealed congestion in the lungs, although no evidence of a gavage error was noted. A female mouse dosed at 2000 mg/kg bw/day spontaneously died on the second day of dosing with no prior clinical observations including no significant changes in body temperature. Examination of this
mouse revealed friable liver tissue with no evidence of a gavage error. The remaining female mouse dosed at 2000 mg/kg bw/day showed no remarkable observations on the two days of dosing, however, had a decrease in feces on the third day of observation. This female mouse largely recovered by the end of the in life with no significant changes in body temperature or body weight.

Phase II
Based on the mortality observed at 2000 mg/kg bw/day in male and female mice, a lower dose level of 1500 mg/kg bw/day was given to additional mice (three/sex/dose). In the male mice dosed at 1500 mg/kg bw/day, all three animals had decreased feces on the third day of observation, however, largely recovered for the remainder of the observation period with no significant changes in body temperature or body weight. In one female mouse dosed at 1500 mg/kg bw/day, decreased feces was observed on the third day of observation with minimal changes in body temperature or body weight and this mouse recovered from the decreased feces by the end of the observation period. A second female mouse dosed at 1500 mg/kg bw/day displayed fecal soiling on the initial day of dosing and decreased feces on the third day of observation with no other remarkable clinical observations including no significant body temperature or body weight changes. The final female mouse dosed at 1500 mg/kg bw/day displayed no remarkable clinical observations throughout the in-life including no changes in body temperature or body weight.
Based on the results of the RFT, the main micronucleus test was dosed at 0 (negative control), 375, 750, or 1500 mg/kg bw/day to male mice, due to the similarity in toxicity between the sexes. Body temperatures were monitored during the micronucleus test as body temperature changes were observed during the RFT.

Main Test - Micronucleus Assay (MNT)
The highest dose tested in the MNT was 1500 mg/kg bw/day. The middle- and low doses were 750 mg/kg bw/day and 375 mg/kg bw/day, respectively. The analytically determined concentrations of the test material in the dosing solutions used for dosing of the MNT ranged from 100.8 to 103.4% of the targeted concentrations.
The treatments did not have a remarkable effect on the body weight of the animals. There were no significant treatment-related indications of toxicity upon daily observation during the in-life portion of the MNT at any dose level. Minimal body temperature changes were observed at all dose levels.
There were no significant differences in MN-RET frequency between the groups treated with the test material and the negative controls. The overall adequacy of the experimental conditions used for the detection of induced micronuclei was ascertained based on the observation of a significant increase in the frequencies of micronucleated RET in the positive control group. The percent RET values observed in the test material-treated animals were not significantly different from the negative control values. The percent RET values of the positive control animals were found to be significantly lower than those of the negative control animals.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In the micronucleus test, no treatment-related toxicity was observed in male mice administered two consecutive daily doses of 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin up to 1500 mg/kg bw/day. In the range finding portion of this study, mortality was observed in animals given 2000 mg/kg bw/day (three of six) and upon gross examination congestion of the lungs and kidneys was noted. Based upon the results of the study reported herein, it was concluded that the test material, 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin, did not induce a significant increase in the frequency 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, the test material 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin, is considered negative for micronucleus induction in this test system under the experimental conditions used.
Executive summary:

The in vivo genotoxic potential of 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin (reaction product containing 2,2’-(1,4 -cyclohexanediylbis(methyleneoxymethylene))bisoxirane) was evaluated by examining the incidence of micronucleated reticulocytes (MN-RET) in the peripheral blood in mice. The test material was administered to male Crl:CD1(ICR) mice by a single oral gavage dose on two consecutive days at dose levels of 0 (negative control), 375, 750, or 1500 mg/kg body weight (bw). The highest dose level of 1500 mg/kg bw/day was based upon the results of a range-finding test where at a higher dose, treatment-related deaths, clinical observations, and changes in body temperatures were observed in male and 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 100.8 to 103.4% 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. In the micronucleus test, all animals survived to the end of the observation period. No treatment-related clinical signs were noted in any treatment group. There were no statistically significant increases in the frequencies of MN-RET 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, 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin, 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, 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin is considered negative in this test system under the experimental conditions used.