Registration Dossier

Administrative data

Description of key information

This was GLP study based on the OECD 422 guideline (Reproduction/Developmental toxicity screening Test).
The purpose of this study was to evaluate the potential effects of 1,4-CHDM DGE, on general toxicity, neurological and reproductive function, and
prenatal/early neonatal growth and survival of the offspring, following repeated gavage administration.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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 422 and EPA OPPTS 870.3650 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Portage, Michigan)
- Age at study initiation: Approximately eight weeks of age at initiation of treatment.
- Housing: After assignment to study, animals were housed singly in solid bottom stainless steel cages, except during breeding and during the gestation/littering phase of the study. The solid bottom cages contained ground corn cob nesting material with laboratory grade wood shavings for enrichment purposes. During breeding, one male and one female were placed in stainless steel cages with wire mesh floors that were suspended above catch pans in order to better visualize copulation and plugs. After mating, dams (and their litters) were housed in plastic cages provided with ground corn cob nesting material with some heat-treated laboratory grade wood shavings until completion of lactation. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
- Diet (ad libitum): Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form
- Water (ad libitum): Municipal water provided
- 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): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: All dosing solutions were prepared by mixing the test material in corn oil at concentrations of 0, 2.5, 15, or 75 mg/ml and administered at a dose volume of 4 ml/kg body weight to achieve the targeted dose levels. Dose solutions were not corrected for purity and were prepared periodically throughout the study based upon stability data. Dose volumes were adjusted using the most current body weight. The control rats were dosed with corn oil at 4 ml/kg body weight.

Analysis to determine concentration of the test material of all dosing solutions from the first mix of the study was initiated prior to the start of dosing. The low- and high-dose solutions from the first mix of the study were analyzed to confirm homogenous distribution of the test material concurrent with dose confirmation. The method used for analyzing the test material in corn oil was liquid chromatography-mass spectrometry (LC/MS).

A previously conducted study showed 1,4-CHDM DGE to be stable for at least 23 days in corn oil at concentrations ranging from 0.25 to 250 mg/ml. The established concentration range spanned those used in this study and dose solutions were used within the established stability duration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): recommended by various regulatory agencies
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in corn oil from the control and all dose levels was determined pre-exposure and ranged from 98.5 to 110% of targeted values. Homogeneity analyses conducted on the low-dose and high-dose solutions indicated that the preparations were homogeneously mixed based on relative standard deviations of ≤ 1.3%.

Dose confirmation and homogeneity assessment was conducted to determine the concentrations of 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin in corn oil prepared on 4/24/13 by the TERC pharmacy. The dose solutions were vortex-mixed before they were sampled. For control and 2.5 mg/mL samples, duplicate 1-mL aliquots were collected from random locations directly from the sample jars. For 15 and 75 mg/mL samples, 1-mL aliquots were collected from top, middle, and bottom locations from the sample jars. These sample aliquots were diluted with 25 mL of isopropanol and then, using the Hamilton Microlab 600 Diluter, the solutions were diluted with isopropanol to achieve the final concentration of 0.953 – 1.17 μg/mL. Analyses were performed by high performance liquid chromatography with mass spectral detection, with electrospray ionization (positive ion mode) operating in multiple reaction monitoring mode (HPLC/ESI-MS/MS). The concentration of 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin was quantified (as measured by 1,4-CHDM DGE) by using external standard calibration. Stability has been demonstrated by monitoring 1,4-CHDM DGE (1,4-Cyclohexanedimethanol diglycidyl ether) and 1,4-CHDM MGE (1,4-Cyclohexanedimethanol monoglycidyl ether) in corn oil over 23 days. As both components were shown to be stable over this time course, and 1,4-CHDM DGE is the primary component of the test material, 1,4-CHDM DGE was monitored for this study.

Low spike samples were fortified at ~ 2.50 mg/mL by the addition of ~2500 μg of 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin using 250 μL of a stock solution (~10000 μg/mL) into a 10 dram vial containing approximately 1-mL of corn oil (with weight recorded). Mid spike samples were fortified at ~ 15 mg/mL by the addition of ~0.015 g of 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin into a 10 dram vial followed by an addition of approximately 1-mL of corn oil (with weight recorded). High spike samples were fortified at ~ 75 mg/mL by the addition of ~0.075 g of 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin into a 10 dram vial followed by an addition of approximately 1 mL of corn oil (with weight recorded). The spike samples were diluted using the same techniques stated for the samples. Average spike recovery was applied to the samples final calculated concentration.

A stock solution was prepared at 9646 μg 1,4-cyclohexanedimethanol, reaction products with epichlorohydrin per mL of isopropanol. The stock was diluted with isopropanol to concentrations of 0.0193 – 4.82 μg/mL. Aliquots of 1-mL of the final dilutions were transferred to glass autosampler vials. All standards were analyzed by HPLC/ESI-MS/MS.

Analysis conditions -
HPLC System: Agilent 1200 Series
Binary Pump: Model: G1312B
Autosampler: Model: G1367C
Column manager: Model: G1316B
Mass Spectrometer: Model: Applied Biosystems API-4000 QQQ
HPLC Column: Agilent XDB-C18, 4.6 x 50 mm, 1.8μm
Guard Cartridge: None
Column Temperature: 20 °C
Autosampler Temperature: Not controlled
Gradient Conditions:
Eluent A: Water + 0.1% formic acid
Eluent B: Acetonitrile + 0.1% formic acid
Time (min) Flow (μL/min) %B
0 800 10
0.50 800 10
3.00 800 100
5.00 800 100
5.01 1600 10
6.50 1600 10
6.51 800 10

Divert Valve:
0 – 1.5 minutes: Waste
1.5 – 5.0 minutes: MS
5.0 – 7.0 minutes: Waste
Injection Volume: 6 μL
Needle Wash: 30:30:30:10:0.1 Methanol: Acetonitrile: 2-Propanol: Water: Formic Acid
Wash Time: 15 seconds at flush port
Ionization: Electrospray
Ion Polarity: Positive
Mode: Multiple Reaction Ion Monitoring (MRM)
DGE: Precursor = 274.3 amu; Product =109.2 amu, collision energy= 17 V
Declustering Potential = 30 V
Dwell Time = 150 ms
Ion Source Voltage: 5000 V
Curtain Gas: 20 psi
CAD: 4
Source Temperature: 400 °C
Gas 1: 20 psi
Gas 2: 20 psi
Interface Heater: On
Entrance Potential: 5 V
Collision Cell Exit Potential: 5 V
Duration of treatment / exposure:
approximately 7-8 weeks. Females were dosed once daily for approximately two weeks prior to breeding, continuing through breeding (two weeks), gestation (three weeks), and through post-partum day (lactation day, LD) 4. Males were dosed beginning approximately two weeks prior to breeding and continuing through breeding (two weeks) for a treatment period of at least 33 days.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
10 mg 1,4-CHDM DGE per kilogram body weight per day (mg/kg/day)
Basis:
other: nominal concentration
Remarks:
Doses / Concentrations:
60 mg 1,4-CHDM DGE per kilogram body weight per day (mg/kg/day)
Basis:
other: nominal concentration
Remarks:
Doses / Concentrations:
300 mg 1,4-CHDM DGE per kilogram body weight per day (mg/kg/day)
Basis:
other: nominal concentration
No. of animals per sex per dose:
12 male + 12 female per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a recent range-finding study, three male and three female Crl:CD(SD) rats per group were administered 0, 500, 750, or 1000 1,4-CHDM DGE mg/kg/day in corn oil via oral gavage for up to 21 days (0 or 500 mg/kg/day for 21 days; 750 or 1000 mg/kg/day for 14 days). Gavage administration of 1000 mg/kg/day 1,4-CHDM DGE to males exceeded the maximum tolerated dose (MTD) as evidenced by considerable decreases in feed consumption and body weight losses; therefore, the rats were humanely euthanized after five days of treatment. Gross necropsy of these animals revealed increased size of the liver, congestion of the liver and kidney, and point of contact irritation in the stomach represented by multifocal congestion in the nonglandular mucosa.
In the remaining groups, treatment-related in-life findings included clinical observations, decreases in feed consumption, and/or body weight gains at all dose levels, with the exception of females given 500 mg/kg/day. Clinical observations were limited to clear perioral soiling in one male and one female given 500 mg/kg/day, all males and females given 750 mg/kg/day, and all females given 1000 mg/kg/day. Body weight gains were decreased throughout the treatment period in males given 500 and 750 mg/kg/day, while decreases in females given 750 or 1000 mg/kg/day were limited to the first seven or nine days of the study, respectively, compared to controls. Decreases in feed consumption were observed in males given 750 mg/kg/day and females given 1000 mg/kg/day during the first week of the study compared to controls. Feed consumption of males and females given 500 mg/kg/day, females given 750 mg/kg/day, and body weight gain of females given 500 mg/kg/day were similar to controls.
Treatment-related gross pathological findings included thickened nonglandular mucosa of the stomach in all males and females given 750 mg/kg/day and all females given 1000 mg/kg/day. Dose-dependent, treatment-related increases in absolute and relative liver weights were observed in all treatment groups (males and females), when compared to those of the controls. All females given 750 or 1000 mg/kg/day had higher absolute and relative kidney weights than controls. A histopathological examination was conducted on the livers, kidneys, and stomachs of males and females in the control, 500, and 750 mg/kg/day dose groups to clarify the observed toxicity.
Histopathological examination of the livers revealed treatment-related very slight (females, 500 mg/kg/day), slight (males, 500mg/kg/day; females, 750 mg/kg/day) and moderate (males, 750 mg/kg/day) centrilobular/midzonal hepatocellular hypertrophy. All males and females given 500 or 750 mg/kg/day had treatment-related slight-to-moderate diffuse hyperkeratinosis and hyperplasia of the non-glandular mucosa of the stomach. In addition, diffuse inflammation of the non-glandular mucosa and submucosa was noted in all males (very slight) and two females (slight) in the 750 mg/kg/group. One male in the 500 mg/kg/day group had very slight multifocal inflammation of the non-glandular mucosa. One male in the 750 mg/kg/day group had a focal ulcer of the nonglandular mucosa. There were no treatment-related histopathological effects indentified in the kidneys of animals given 500 and 750 mg/kg/day, thus the observation of higher kidney weights in females was interpreted to be of no toxicological significance. Under the conditions of this range-finding study, due to the stomach histopathological findings in all animals from the 500 and 750 mg/kg/day groups, dose levels of 0, 10, 60, or 300 mg/kg/day were selected for this definitive screening study.
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were conducted on all rats pre-exposure and weekly throughout the study. Mated females received DCO examinations on GD 0, 7, 14, and 20, and lactation day (LD) 3.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed at least once during the pre-exposure period and on the first day of dosing. Male body weights continued to be recorded weekly throughout the study. Females were weighed weekly during the pre-mating and mating periods. During gestation, females were weighed on GD 0, 7, 14, 17, and 20. Females that delivered litters were weighed on LD 1 and 4. Body weight analyses were conducted for the following days: GD 0, 7, 14, and 20. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4.

FOOD CONSUMPTION : Yes
- Time schedule: Feed consumed was determined weekly during the two-week pre-breeding period for males and females by weighing feed crocks at the start and end of a measurement cycle. During breeding, feed consumption was not measured for males or females due to cohousing. Following breeding, feed consumption was not measured for males. For females during gestation, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on LD 1 and 4.

FUNCTIONAL TESTS: Yes
The functional tests (sensory evaluation, rectal temperature, grip performance, and motor activity) were conducted pre-exposure and during the last week of the treatment period. For the females, this took place on LD 4.
- Sensory Evaluation: The sensory evaluation included a test for nociception (responsiveness to tail pinch) and for startle response (responsiveness to sharp noise). The evaluation was conducted in a clear plastic box.
- Rectal temperature was measured by carefully placing a rectal thermistor (Physitemp, Clifton, New Jersey) approximately 4 cm into the rectum for approximately 10 seconds. Temperature was then recorded. The thermistor was validated at 37 °C once prior to the start and upon completion of the study. The instrument was re-calibrated if the temperature recordings differ from the reference thermometer by more than ± 0.5 °C.
- Hindlimb grip performance was tested according to the procedure described by Mattsson et al. (1986). Briefly, the observer places the rat’s forelegs on a plastic bench and the hindfeet were set on a horizontal screen attached to an electronic strain gauge (Chatillon, Greensboro, North Carolina). The observer then smoothly but firmly pulled backward on the tail until the rat’s grip on the screen was broken. An electronic strain gauge was used to record the rat’s resistance to the pull in grams. The average of three trials was used for statistical analysis. Forelimb grip performance was similarly tested. In this application, a bench was not used, and the rats were placed so that the forefeet were on the screen and the hindfeet were suspended approximately 10 cm above the plastic platform.
- Motor Activity: An automated system was used for motor activity (MA) data collection. No entry into the MA test room was allowed during the testing period. Each test session consisted of eight 8-minute epochs, totaling 64 minutes of testing per animal per test session. This duration was chosen based on the results of a validation study indicating that performance of control animals approached asymptote in 50-60 minutes in Crl:CD(SD) rats (Marty and Andrus, 2007). Activity counts for each epoch were recorded.
- Motor Activity Cage Calibration: Cages used for testing were calibrated prior to testing each day. Calibration was performed with a rod attached to a rotary motor that broke the infrared beam at a constant speed. The duration of each beam break was calculated to ensure equivalence across chambers.
- Motor Activity Cage Allocation: Rats were allocated to the motor activity cages in such a way that the counterbalancing of treatment groups and sexes across cages and test times was maximized.

CLINICAL PATHOLOGY: Yes
Adult animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia with CO2/O2 at the scheduled necropsy.
- HEMATOLOGY : Blood samples for a complete blood count were mixed with ethylenediaminetetraacetic acid (EDTA). Blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped and archived for potential future evaluation if warranted. Standard hematologic parameters were assayed using the Advia 120 Hematology Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York).
- COAGULATION : Blood samples were collected in sodium citrate tubes, centrifuged, the plasma collected, and assayed using the ACL9000 Analyzer (Instrumentation Laboratory, Bedford Massachusetts).
- CLINICAL CHEMISTRY : Blood samples were collected and serum was separated from cells as soon as possible. Serum parameters were measured using a cobas c311 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana).
- URINE ANALYSES : Urine samples were obtained from all males the week prior to the scheduled necropsy per the protocol, urine samples were not collected from females. Males were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this procedure. Microscopic evaluation of the microsediments from pooled urine samples from the male rats of each group was evaluated.
Sacrifice and pathology:
The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.

Representative samples of tissues were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides that were fixed in Bouin’s or another appropriate fixative. Transponders were removed and placed in jars with the tissues.

The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain based on Kopf et al. (1964) and were examined for the presence and
number of implantation sites. The uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.

Weights of the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, and thyroid with parathyroids (weighed after fixation) were recorded, and organ:body weight ratios calculated.

Histopathologic examination of the tissues was conducted on all control and high-dose adult rats. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope. The following tissues from the remaining groups were processed, sectioned, and stained: liver, kidneys, stomach, thyroid and relevant gross lesions. Relevant gross lesions were microscopically examined from all animals. All target tissues (liver, stomach, and thyroid) were microscopically examined from the low- and intermediate-dose group animals to define a NOEL. Kidney tissue from the low- and intermediate dose group animals were not microscopically examined because there were no treatment-related histopathological changes in the kidneys of the high-dose group.

The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at 5 μm and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis were qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
Other examinations:
not applicable
Statistics:
Standard statistical methods were employed
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related clinical observations in animals given 300 mg/kg/day were limited to transient, clear perioral soiling in one male during the last week of treatment, three females during the gestation period, and one female during the lactation period.
Mortality:
mortality observed, treatment-related
Description (incidence):
Treatment-related clinical observations in animals given 300 mg/kg/day were limited to transient, clear perioral soiling in one male during the last week of treatment, three females during the gestation period, and one female during the lactation period.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males and females given 300 mg/kg/day had statistically-significant, treatmentrelated increases in absolute (26.1 and 21.5%, respectively) and relative (26.5 and 19.3%, respectively) liver weights when compared to those of the controls
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related histopathological observations were present in the stomach, liver, and thyroid gland, considered to adaptive (non-adverse) changes
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS) - All animals survived the full duration of the study. Treatment-related clinical observations in animals given 300 mg/kg/day were limited to transient, clear perioral soiling in one male during the last week of treatment, three females during the gestation period, and one female during the lactation period. No treatment-related effects on behavior or demeanor were observed in animals given 60 or 10 mg/kg/day during the study. There were no additional notable observations made during the clinical or cage-side observations. Detailed clinical examinations performed on all rats revealed no treatment related findings. Likewise, there were no treatment related findings on the functional tests - sensory evaluation, grip strength, rectal temperature and motor activity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS) - There were no treatment-related effects on body weights or body weight gains for males at any dose level tested throughout the duration of the study. Similarly, there were no significant differences or effects related to treatment on body weights or body weight gains of females at any dose level tested during the pre-mating, gestation, or lactation periods. There were no effects related to treatment on the amount of feed consumed by any of the dose groups when compared to their respective controls throughout the study.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS) - There were no treatment-related effects at any dose level on any reproductive parameters or pup survival indices.

ORGAN WEIGHTS (PARENTAL ANIMALS) - Males and females given 300 mg/kg/day had statistically-significant, treatment related increases in absolute (26.1 and 21.5%, respectively) and relative (26.5 and 19.3%, respectively) liver weights when compared to those of the controls. The increased absolute and relative liver weights corresponded with hypertrophy of the hepatocytes at these dose levels. Males given 10 mg/kg/day had statistically significant decreases in relative thyroid weights when compared to those of the controls. These decreases were interpreted to be unrelated to treatment due to the lack of a dose-response relationship. All other organ weights of males and females in all treatment groups were similar to those of the controls.

GROSS PATHOLOGY (PARENTAL ANIMALS) - There were no treatment-related gross pathologic observations. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure of rats to 1,4-CHDM DGE.

HISTOPATHOLOGY (PARENTAL ANIMALS) - Treatment-related histopathological observations were present in the stomach, liver, and thyroid gland.
Stomach - Males and females given 60 or 300 mg/kg/day of 1,4-CHDM DGE had very slight or slight hyperkeratosis and hyperplasia of the nonglandular mucosa. The nonglandular effects were diffuse, but most prominent in the limiting ridge of the stomach. The limiting ridge is a raised fold of the stomach mucosa lined by stratified squamous epithelium and represents the border between the forestomach and glandular stomach. The histopathological effects in the stomach were interpreted to be a result of localized contact irritation from repeated oral gavage of the test material. There were no treatment-related histopathologic changes in stomachs of males or females given 10 mg/kg/day.
Liver - Males and females given 300 mg/kg/day had very slight panlobular or centrilobular-to-midzonal hepatocellular hypertrophy with increased cytoplasmic eosinophilia. The cytoplasm of the hypertrophied hepatocytes contained a finely granular-to-homogeneous cytoplasm with very slightly increased eosinophilia as compared to that of the controls. The hepatocellular hypertrophy with increased cytoplasmic eosinophilia corresponded to the higher liver weights noted in males and females given 300 mg/kg/day. Additionally, males at this dose level also had a treatment-related decrease in vacuolization of the hepatocytes, both on a periportal and individual cell basis that was interpreted to be a non-adverse finding of no toxicological significance. Males given 60 mg/kg/day had very slight centrilobular-to-midzonal hepatocellular hypertrophy with increased cytoplasmic eosinophilia. The hepatocellular hypertrophy (in males and females given 300 mg/kg/day and males given 60 mg/kg/day), along with the modest increases in liver weights of males and females given 300 mg/kg/day, both relative to controls, were likely due to increased hepatic microsomal enzyme activity. There were no treatment-related histopathologic changes in livers of males given 10 mg/kg/day or females given 10 or 60 mg/kg/day.
Thyroid Gland: Females given 300 mg/kg/day had slight treatment-related hypertrophy of thyroid gland follicular cells and a concomitant decrease in the amount of colloid in the follicular lumens. The thyroid hypertrophy was considered to be secondary to induction of liver microsomal enzymes (resulting in hypertrophy of hepatocytes) and subsequent increased biliary. excretion of thyroid hormones, with resultant chronic stimulation of thyroid stimulating hormone production. It is well known that pregnancy affects all aspects of thyroid management (Larsen and Ingbar, 1992), further supporting the adaptive aspect of this finding in the dams.
There were no treatment related histopathologic changes in the thyroid glands of females given 10 or 60 mg/kg/day or in males at any dose level.

OTHER FINDINGS (PARENTAL ANIMALS) -

Hematology - Males given 10 mg/kg/day had statistically-significant lower red blood cell counts and females given 10 mg/kg/day had statistically-significant higher white blood cell counts when compared to those of the controls. These alterations were interpreted to be spurious and unrelated to treatment due to the lack of a dose-response relationship. There were no other statistically-significant or treatment-related alterations in any of the hematology parameters for males or females at any dose level when compared to those of the controls.

Coagulation - There were no treatment-related differences in the prothrombin times of males and females at any dose level when compared to controls.

Clinical chemistry - Males given 300 mg/kg/day had a statistically-significant, treatment-related increase in serum albumin levels when compared to those of the controls. Additionally, although not statistically-significant, there was a decrease in serum globulin levels and an increase in albumin/globulin ratio noted at this dose level. Males given 300 mg/kg/day also had a slight decrease in triglycerides when compared to those of the controls. While this decrease was not statistically significant, a relationship to treatment could not be discounted as the individual animal values were consistently lower than the concurrent controls. These treatment-related increases and/or decreases were interpreted to be associated with the liver effects identified at this dose level. A statistically-significant increase in blood urea nitrogen was present in males given 300 mg/kg/day; however, this was interpreted to be unrelated to treatment as 1) there was no histopathologic evidence of kidney disease and/or any indication of renal dysfunction and 2) the difference from controls was marginal. Total bile acids were also increased (not statistically identified) in the male high dose group, but this was driven by a single animal and thus interpreted to be unrelated to treatment.
There were no other statistically-significant or treatment-related alterations in any of the clinical chemistry parameters for males or females at any dose level when compared to those of the controls.

Urinalysis - There were no treatment-related effects on urinalysis parameters for males at any dose level.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the limited to adaptive (non-adverse) changes in clinical chemistry, organ weight and histopathological responses
Dose descriptor:
NOEL
Remarks:
neurological and reproductive toxicity, effects on prenatal/neonatal growth and survival
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no neurological or reproductive toxicity, no effects on prenatal/neonatal growth and survival
Critical effects observed:
no

None

Conclusions:
Based on the limited to adaptive (non-adverse) changes in clinical chemistry, organ weight and histopathological responses and under the conditions of the study, the systemic no observed adverse effect level (NOAEL) for both the sexes is 300 mg/kg/day and the NOEL for neurological and reproductive toxicity or for effects on prenatal/neonatal growth and survival was 300 mg/kg/day, the highest dose tested.
Executive summary:

The purpose of this study was to evaluate the potential effects of 1,4- cyclohexanedimethanol, reaction products with epichlorohydrin, hereafter referred to as 1,4- CHDM DGE, on general toxicity, neurological and reproductive function, and prenatal/early neonatal growth and survival of the offspring, following repeated oral gavage administration. Groups of 12 male and 12 female Crl:CD(SD) rats were administered 0, 10, 60, or 300 milligrams 1,4-CHDM DGE per kilogram body weight per day (mg/kg/day) in corn oil via oral gavage. Females were dosed once daily for approximately two weeks prior to breeding, continuing through breeding (two weeks), gestation (three weeks), and through post-partum day (lactation day, LD) 4. Females were necropsied on LD 5. Males were dosed beginning approximately two weeks prior to breeding and continuing through breeding (two weeks) for a treatment period of at least 33 days. Males were necropsied on test day 34 or 35. Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, postmortem examinations included a gross necropsy of the adults with collection of organ weights and histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed.

Gavage administration of 1,4-CHDM DGE resulted in no treatment-related effects on body weight, body weight gain, or feed consumption in males or females in any treated groups compared to controls. The only treatment-related clinical observation was transient, clear perioral soiling in one male and three females given 300 mg/kg/day. Treatment-related effects on clinical chemistry parameters were limited to increased serum albumin, decreased serum globulin, and a slight decrease in triglyceride levels in males given 300 mg/kg/day. These findings were consistent with liver effects in males at this dose level. Males and females given 60 or 300 mg/kg/day had treatment-related very slight or slight hyperkeratosis and hyperplasia of the nonglandular mucosa of the stomach. The histopathological effects in the stomach were interpreted to be a result of localized, point of contact irritation from repeated oral gavage of the test material. There were no treatmentrelated histopathological changes in stomachs of males or females given 10 mg/kg/day. Males and females given 300 mg/kg/day had treatment-related very slight panlobular or centrilobular-to-midzonal hepatocellular hypertrophy with increased cytoplasmic eosinophilia that corresponded to treatment-related increases in absolute (26.1 and 21.5%, respectively) and relative (26.5 and 19.3%, respectively) liver weights when compared to those of the controls. Additionally, males at this dose level also had a treatment-related decrease in vacuolization of the hepatocytes, both on a periportal and individual cell basis that was interpreted to be a non-adverse finding of no toxicological significance. Males given 60 mg/kg/day had treatment-related very slight centrilobular-to-midzonal hepatocellular hypertrophy with increased cytoplasmic eosinophilia. Taken together, the liver effects in both sexes given 300 mg/kg/day and males given 60 mg/kg/day were interpreted to be an adaptive (non-adverse) response to accommodate increased metabolism by the hepatocytes following exposure to the test material. All other organ weights of males and females in all treatment groups were similar to those of the controls. There were no treatment-related histopathologic changes in livers of males given 10 mg/kg/day or females given 10 or 60 mg/kg/day. Secondary to the liver effects described above, females given 300 mg/kg/day had slight treatment-related hypertrophy of thyroid gland follicular cells and a concomitant decrease in the amount of colloid in the follicular lumens. There were no treatment-related histopathological changes in thyroid glands of females given 10 or 60 mg/kg/day or males at any dose level.

There were no treatment-related effects of 1,4-CHDM DGE on neurological or reproductive function, or prenatal neonatal growth or survival of the offspring.

Based on the limited to adaptive (non-adverse) changes in clinical chemistry, organ weight and histopathological responses and under the conditions of the study, the systemic no observed adverse effect level (NOAEL) for both the sexes is 300 mg/kg/day and the NOEL for neurological and reproductive toxicity or for effects on prenatal/neonatal growth and survival was 300 mg/kg/day, the highest dose tested.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
good

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral

The repeated dose oral toxicity potential of CHDM DGE was assessed in an OECD 422 “Combined gavage repeated dose toxicity study with the reproductive/developmental toxicity screening test” compliant study in Crl:CD(SD) rats (Ellis-Hutchings et al., 2013). This study assessedgeneral toxicity, neurological and reproductive function, and prenatal/early neonatal growth and survival of the offspring, following repeated oral gavage administration. Groups of 12 male and 12 female Crl:CD(SD) rats were administered 0, 10, 60, or 300 milligrams CHDM DGE per kilogram body weight per day (mg/kg/day) in corn oil via oral gavage. Females were dosed once daily for approximately two weeks prior to breeding, continuing through breeding (two weeks), gestation (three weeks), and through post-partum day (lactation day, LD) 4. Females were necropsied on LD 5. Males were dosed beginning approximately two weeks prior to breeding and continuing through breeding (two weeks) for a treatment period of at least 33 days. Males were necropsied on test day 34 or 35. Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, postmortem examinations included a gross necropsy of the adults with collection of organ weights and histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed.

Gavage administration of 1,4-CHDM DGE resulted in no treatment-related effects on body weight, body weight gain, or feed consumption in males or females in any treated groups compared to controls. The only treatment-related clinical observation was transient, clear perioral soiling in one male and three females given 300 mg/kg/day. Treatment-related effects on clinical chemistry parameters were limited to increased serum albumin, decreased serum globulin, and a slight decrease in triglyceride levels in males given 300 mg/kg/day. These findings were consistent with liver effects in males at this dose level. Males and females given 60 or 300 mg/kg/day had treatment-related very slight or slight hyperkeratosis and hyperplasia of the nonglandular mucosa of the stomach. The histopathological effects in the stomach were interpreted to be a result of localized, point of contact irritation from repeated oral gavage of the test material. There were no treatment-related histopathological changes in stomachs of males or females given 10 mg/kg/day. Males and females given 300 mg/kg/day had treatment-related very slight panlobular or centrilobular-to-midzonal hepatocellular hypertrophy with increased cytoplasmic eosinophilia that corresponded to treatment-related increases in absolute and relative liver weights when compared to those of the controls. Additionally, males at this dose level also had a treatment-related decrease in vacuolization of the hepatocytes, both on a periportal and individual cell basis that was interpreted to be a non-adverse finding of no toxicological significance. Males given 60 mg/kg/day had treatment-related very slight centrilobular-to-midzonal hepatocellular hypertrophy with increased cytoplasmic eosinophilia. Taken together, the liver effects in both sexes given 300 mg/kg/day and males given 60 mg/kg/day were interpreted to be an adaptive (non-adverse) response to accommodate increased metabolism by the hepatocytes following exposure to the test material. All other organ weights of males and females in all treatment groups were similar to those of the controls. There were no treatment-related histopathologic changes in livers of males given 10 mg/kg/day or females given 10 or 60 mg/kg/day. Secondary to the liver effects described above, females given 300 mg/kg/day had slight treatment-related hypertrophy of thyroid gland follicular cells and a concomitant decrease in the amount of colloid in the follicular lumens. There were no treatment-related histopathological changes in thyroid glands of females given 10 or 60 mg/kg/day or males at any dose level.

There were no treatment-related effects of 1,4-CHDM DGE on neurological or reproductive function, or prenatal neonatal growth or survival of the offspring.

Based on the limited to adaptive (non-adverse) changes in clinical chemistry, organ weight and histopathological responses and under the conditions of the study, the systemic no observed adverse effect level (NOAEL) for both the sexes is 300 mg/kg/day and the NOEL for neurological and reproductive toxicity or for effects on prenatal/neonatal growth and survival was 300 mg/kg/day, the highest dose tested.

      

 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP and OECD 422 Study

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
no data available

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
no data available

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
no data available

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
no data available

Justification for classification or non-classification

The Key effects observed in this study were limited to adaptive effects in the liver and local point of contact irritation at the site of administration (stomach). As the effects were not severe in nature and did not occurr at doses falling within the classification criteria for STOT RE. As such no classification for repeated dose toxicity is required.