Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Several studies are available for this endpoint including both in vivo and in vitro studies: 2 bacterial mutagenicity assays; in in vitro UDS assay in rat hepatocytes; an in vitro Mouse Lymphoma assay; a mouse and rat in vivo micronucleus assay, and two in vivo comet assays (one performed in conjunction with the Rat micronucleus assay and one independant assay performed as part of compliance with US FDA food contact approval for use in water containers).
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:
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).
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
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
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
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
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
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.

None

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.

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

Additional information

Additional information from genetic toxicity in vivo:

Genotoxicity assessment:  

Although not all studies were conducted under current OECD and/or other regulatory guidelines or with the exact material under consideration for registration (the purity of the registered substance is broader than the test material used in some of the studies), their value for determination of genotoxic potential of the molecule is still practical. The studies were performed in accordance with respective standards at the time of the study and the data from genetic toxicity tests on this class of compounds are relevant and useful to support a genotoxicity evaluation of this chemical.

in vitro Studies

Bacterial mutation assay

Several relevant bacterial reverse mutation/mammalian-microsome (Ames) tests have been conducted and, importantly, the results are consistent across the studies.  The most recent and thorough evaluation was performed on 1,4-CHDM-DGE. The study was conducted using TA98, TA100, TA1535, and TA1537 strains of Salmonella typhimuriumand WP2uvrA (pKM 101) strain of Escherichia coli. In the first phase of this study, an initial toxicity-mutation test was performed, while the second phase was an independent confirmatory mutation test. The bacterial tester strains were exposed to the test substance in the presence and absence of a metabolic activation system (S-9 fraction prepared from Aroclor 1254-induced rat liver) using the pre-incubation procedure and DMSO as the vehicle.

In the initial toxicity-mutation assay, 1,4-CHDM DGE was exposed in duplicate at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate test doses along with the vehicle and appropriate positive controls. In the presence of metabolic activation for the initial toxicity-mutation assay, the test substance did not cause any precipitation on the basal agar plates, nor was toxicity observed up to 5000 μg/plate. Positive mutagenic responses were observed in the strains TA100 (from 500 to 5000 μg/plate) and TA1535 (from 150 to 5000 μg/plate). Tester strains TA98, TA1537 and WP2uvrA (pKM101) did not meet the fold-increase criteria for a positive mutagenic response; however, there was a dose-responsive increase up to 2.78-fold, 2.87-fold, and 1.97-fold, respectively, at 5000 μg/plate when compared to the vehicle control plates. In the absence of metabolic activation, no precipitation on the basal agar plates or toxicity was observed up to 5000 μg/plate, and positive mutagenic responses were observed in all the strains: TA98 (from 1500 to 5000 μg/plate), TA100 (from 500 to 5000 μg/plate), TA1535 (from 1500 to 5000 μg/plate), TA1537 (at 5000 μg/mL), and WP2uvrA (pKM101) (at 5000 μg/mL).

Based on these initial findings, the confirmatory mutation assay 1,4-CHDM DGE was exposed in triplicate at 150, 500, 1500, 2500 and 5000 μg/plate test doses along with the vehicle and appropriate positive controls. In the presence of metabolic activation, the test substance did not cause any precipitation or toxicity on the basal agar plates up to 5000 μg/plate, and there was a positive mutagenic response observed in the strains TA100 (from 500 to 5000 μg/plate), TA1535 (from 150 to 5000 μg/plate) and WP2uvrA (pKM101) (at 2500 μg/mL). Tester strains TA98 and TA1537 did not meet the 3-fold criteria for a positive mutagenic response, but there was an increase up to 2.97-fold and 2.83-fold, respectively, when compared to the vehicle control plates. In the absence of metabolic activation, there was a positive mutagenic response observed in the strains TA100 (from 500 to 5000 μg/plate), TA1535 (from 500 to 5000 μg/plate), and TA1537 (at 5000 μg/plate). Tester strains TA98 and WP2uvrA (pKM101) did not meet the fold-increase criteria for a positive mutagenic response; however, there was an increase up to 2.97-fold and 1.95-fold, respectively, when compared to the vehicle control plates.

In this study, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay. The results of concentration analysis of dose formulations from the initial as well as confirmatory mutation assays confirmed that the regulatory-required top dose level (5000 μg/plate) was achieved in both assays and the results support the validity of the study conclusion.

Similar mutagenic responses were noted in all other bacterial reverse mutation/mammalian-microsome (Ames) tests with other CHDM-DGE family members. These include Mendrala, 1983 (53% pure) and Arni, 1980 (unspecified purity).

In conclusion, it is determined that CHDM-DGE is mutagenic in the test systems utilized for detection of bacterial reverse mutations, based on direct experimental demonstration and supported by read-across to similar chemical compositions.

 

in vitro mammalian cell mutagenicity assay

A GLP-compliant guideline in vitro mammalian cell gene mutation assay (OECD 476;i.e., mouse lymphoma forward gene mutation assay; MLA) was performed in accordance with the global evaluation criteria for optimal assay performance with a mixture of 1,3- and 1,4-CHDM DGE (UNOXOL™)(Schisler and LeBaron, 2010). The mutagenic potential of the test material was assessed in an independent assay in the absence and presence of an externally supplied metabolic activation (S9) system at concentrations ranging from 2.5 to 80 μg/ml in the absence of S9 and from 40 to 140 μg/ml in the presence of S9. The highest concentration was based on the toxicity of the test material. The adequacy of the experimental conditions for detection of induced mutants was confirmed by employing positive control chemicals, methyl methane sulfonate for the assay without S9 and 20-methylcholanthrene for the assay with S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e. dimethyl sulfoxide). 

In the mutagenicity assay in the absence of metabolic activation, biologically significant increases in mutant frequencies of up to 6.9-fold over the current solvent controls were observed in cultures treated with the test material. In the presence of S9, mutant frequency increases of up to 11.9-fold over concurrent solvent controls were observed. Based on the results of this in vitro mouse lymphoma (L5178Y TK+/-) forward gene mutation assay, a mixture of 1,3- and 1,4-CHDM DGE was considered to be mutagenic in the absence and presence of an externally supplied metabolic activation (S9) system.

in vitro assessment of genotoxic potential

CHDM-DGE was assessed for the potential to induce repairable DNA damage (unscheduled DNA synthesis; UDS) in primary cultures of rat hepatocytes. The test material was administered at concentrations ranging from 1.3 x 10-7to 1.3 x 10-2M at half-log intervals. CHDM-DGE was clearly toxic to the hepatocyte cultures at 4 x 10-5M and greater concentrations and showed limited solubility at 1.3 x 10-2M. Hepatocyte toxicity was similar to control levels in cultures exposed to 4 x 10-7and 1.3 x 10-7M CHDM-DGE. CHDM-DGE did not induce significant UDS at any of the concentrations tested; however, the observed positive response with the positive control (2-acetylaminofluorene; 2-AAF) demonstrated that the hepatocyte cultures were capable of activating a known genotoxic agent and eliciting a DNA repair response.

In conclusion, under the conditions of this study, CHDM-DGE did not induce a DNA repair response in primary hepatocytesin vitro, which was inconsistent with the results of the bacterial mutation assays and the MLA mammalian cell mutagenicity assay. This discrepancy may be attributed to the inherent differences of thein vitrostudies including cellular metabolic competency, test material interaction and administration regimens of suspension vs adherent cells, and a difference in mammalian cell assay endpoints.

in vivo Studies

In vivo micronucleus test

An in vivo mouse peripheral blood micronucleus test performed under GLP and according to OECD 474 was conducted with 1,4-CHDM DGE (1,4-CHDM reaction products with epichlorohydrin; Schisler, 2013). In this study, 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 this study, 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. Indications of systemic bioavailability of the test material included mortality (3 of 6 animals) in the range finding portion of the study in animals given 2000 mg/kg bw/day (congestion of the lungs and kidneys were noted after gross examination), and hepatic hypertrophy was demonstrated in a separate orally-administered toxicity test in Crl:CD(SD) rats (Ellis-Hutchingset al., 2013). Therefore, 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin is considered negative in the mouse peripheral blood micronucleus test, under the experimental conditions used.

 

in vivo genotoxic assessment: combination comet and micronucleus study in the rat

The test substance, 1,4-CHDM DGE was evaluated for genotoxic activity by evaluating incidence of micronuclei in polychromatic erythrocytes in bone marrow and induction of primary DNA damage in liver, stomach, and duodenum in male Hsd:SD rats. The test substance was formulated in corn oil, which was also used as the vehicle control. The test substance and vehicle formulations were administered by oral gavage on 3 consecutive days (Study Days 1, 2, and 3). The first and second dose administrations were separated by approximately 24 hours while the second and third administrations were separated by ~ 21 hours. Ethyl methanesulfonate (EMS, 200 mg/kg/day) was used as the positive control substance in the study and was administered twice; ~ 24 hours and 3 – 4 hours prior to organ collection (Study Days 2 and 3). All formulations were administered at a dose volume of 10 ml/kg/treatment. 

A dose range finding (DRF) assay was conducted exposing three rats/sex to the dose levels of 125, 250, 500, 1000 or 2000 mg/kg/treatment. Based on the mortality observed in DRF assay, a dose of 750 mg/kg/treatment was determined to be the maximum tolerated dose (MTD) and was tested as high dose in the definitive assay. Two lower doses at 187.5 and 375 mg/kg/treatment were also tested. Since no differences in the clinical signs of toxicity between the sexes were observed in DRF study, only male rats were used in the definitive assay. The vehicle and positive control substances were run concurrently and all animals were observed for clinical signs of toxicity. Approximately 3.5 ± 0.5 hours after last dose administration, animals in each group were euthanized and organs (liver, gladular stomach, dueodunum) were collected and processed for comet slide preparation. Tissues from the left liver lobe and duodenum were also processed for histopathological evaluation. Tissues from the gladular stomach were stored in formalin. Bone marrow was microscopically evaluated for the presence of micronucleated polychromatic erythrocytes (MPCEs; 2000 PCEs/animal) and the data was statistically analyzed using binomial distribution and Kastenbaum-Bowman Tables for significant level of p ≤ 0.05. DNA damage in liver, stomach, and duodenal cells was detected using a microgel electrophoretic technique, automated scoring system, and by measuring % tail DNA, tail moment and tail migration per 100 cells per animal. A statistical analysis of data was performed using mean group values for % tail DNA only (ANOVA, Dunnett’s test, for significant level of p ≤ 0.05).

In the DRF study, all rats at 125, 250, 500 mg/kg/treatment appeared normal by clinical observation during the study period. Mortality was observed in 2/3 male and 2/3 female rats at 1000 mg/kg and in all male and female rats at 2000 mg/kg as well as lethargy and piloerection; however, no appreciable changes in the mean group body weights were observed in any of the treatment groups. In the main study, all animals in the vehicle control substance group and all animals at 187.5 and 375 mg/kg/treatment group appeared normal by clinical observation during the study period. One of the rats in the 750 mg/kg/treatment group was found dead prior to euthanasia. Lethargy and/or piloerection were among the clinical signs observed in this dose group. Diarrhea was noted in the animals dosed with EMS on Study Day 3. No appreciable changes in the group mean body weights were observed in any of the treatment groups. In the bone marrow analysis, dose dependent reductions up to 20% in the PCEs/EC ratio in the test substance groups compared to the vehicle control group was observed indicating the test substance induced cytotoxicity. No statistically significant increase in the incidence of MPCEs in the test substance groups was observed relative to the concurrent negative control group (binomial distribution, p ≥ 0.05, Kastenbaum-Bowman Tables). The positive control (EMS) induced a statistically significant increase in the incidence of MPCEs relative to the concurrent negative control (p ≤ 0.05, binomial distribution, Kastenbaum-Bowman Tables).

In the comet assay, the mean percent of clouds in the liver cells in the test substance treated groups was comparable % of clouds seen in the vehicle control group. The mean percent of clouds in the stomach and duodenal cells were below the % of clouds seen in the vehicle control group. No statistically significant increase in % tail DNA (DNA damage) in stomach cells was observed in any of the test substance treatment groups relative to the concurrent vehicle control group (p >0.05, ANOVA, Dunnett’s test). No statistically significant increase in % tail DNA (DNA damage) in liver, or duodenal cells was observed in the 187.5 or 375 mg/kg/day treatment groups relative to the concurrent vehicle control group (p >0.05, ANOVA, Dunnett’s test). However, a statistically significant (~4.1 fold increase) was observed in the liver and duodenal cells of the 750 mg/kg/day treated groups. The positive control, EMS, induced a statistically significant increase in the % tail DNA in liver cells (t-test, p < 0.05). The % tail DNA and the incidence of MPCEs in the vehicle control group did not exceed the respective historical negative (vehicle) control range. These results indicate that all criteria for valid micronucleus assay and comet assay, as specified in the protocol, were met.

Study Conclusion

Under the conditions of this study, 1,4-CHDM DGE at oral dose levels up to and including 750 mg/kg/day, did not induce a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. No statistically significant increase in the DNA damage in the stomach cells relative to the concurrent negative control in male Hsd:SD rats was noted. However, a statistically significant increase (~4.1 fold) in the liver and duodenal cells compared to the vehicle control was observed at a dose of 750 mg/kg/day was observed. Histopathological evaluation of these tissues indicated primary signs of toxicity in the duodenum tissue as evidenced by necrosis of crypt epithelial cells in a dose dependent manner. Minimal centrilobular hypertrophy noted in the high dose group was considered as an adaptive response.

 

In conclusion, the data from two independent micronucleus tests (i.e., mouse peripheral blood and rat bone marrow) indicate that 1,4-CHDM DGE does not have in vivo clastogenic or aneugenic potential, as evaluated under the conditions of these studies. The in vivo comet data for the high dose CHDM DGE group indicated an increase in % tail DNA in the duodenal cells (point-of-contact) and in the liver. Although the high dose liver comet data were statistically identified, several important considerations are relevant in the interpretation of these data. First, the maximal treatment-related induction of % tail DNA by CHDM DGE was within the historical control range of the vehicle for the laboratory for comet analysis of the liver, and the induction of % tail DNA was approximately 1/5thof that for the positive control. Second, the MTD may have been exceeded in the main study. A thorough range finding study was undertaken and 4 of 6 rats died with administration of 1000 mg/kg bw/day and all rats died at the limit dose of 2000 mg/kg bw/day, while no clinical observations or other signs of toxicity were noted in animals administered 500 mg/kg bw/day or below. In the main study a dose of 750 mg/kg bw/day was chosen as the MTD, but resulted in the death of one of the animals on the study along with the clinical observations of lethargy and piloerection in the remaining animals at this dose. Third, while a complete histopathological evaluation was undertaken in the target tissues to investigate potential histopathological alterations and only minimal observations were noted; recent experience has demonstrated a delayed necrotic/apoptotic response such that comet alterations may precede the onset of frank histopathologic cytotoxicity by days. Forth, the comet study was performed consistent with standard practice at the time of study conduct; however, currently an OECD guideline has not yet been finalized to clearly establish standard study conduct and interpretation. Importantly, the endpoint (i.e., % Tail DNA) as a measurement of DNA damage is less clear when compared to standard genotoxic endpoints of mutagenicity, clastogenicity, and aneugenicity.

In light of these points, and the equivocal nature (at best) of the comet response in the liver, a repeat comet assay is warranted to further characterize any potential genotoxic or DNA damage response to CHDM-DGE.

 

Review Summary

Based on the above genotoxicity data CHDM-DGE was determined to have in vitro mutagenic potential, as indicated in the bacterial and mammalian cell mutation assays, with and without metabolic activation. The in vivo clastogenic/aneugenic data were clearly negative when evaluated in both the rat and mouse micronucleus tests. These results are consistent with epoxy-containing compounds, which demonstrate in vitro genotoxicity while in vivo responses are negative for systemic measures of genotoxicity. The liver comet data are suggestive of potential DNA damage, although comparisons to other epoxy-containing compounds are not readily available as common use of the assay is relatively recent. Development and finalization of an OECD guideline will standardize conduct and acceptance of data from this assay, especially with regard to what a positive comet response means biologically. When evaluated in a weight of evidence approach from the battery of genetic toxicity test data available, all collected under GLP- and guideline-compliant conditions, the data indicate that CHDM-DGE is reactive in vitro but not genotoxic in traditional in vivo endpoints of micronucleus and UDS.

Potential further testing

In support of Food Contact applications in the USA, the in vivo rat comet assay is being repeated in a study that confirms to the current draft OECD guideline in order to confirm whether the results from the existing study are equivocal or indicate positive activity for this compound in vivo.

Once available, the data from this study will be added into the REACH dossier in an update.


Justification for selection of genetic toxicity endpoint
GLP and OECD 474 guideline study.

Justification for classification or non-classification

The weight of evidecnce supports the conclusion that although CHDM DGE appears to be genotoxic in in vitro test systems, in vivo it is not clastogenic or genotoxic. As such, there is no requirement for classification at this time, nor is there a need to perform further studies to assess potential Germ cell mutagenicity.