Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
-sensory activity, grip strength, and motor activity assessments not performed
Qualifier:
according to
Guideline:
other: OPPTS Guideline 870.3050
Deviations:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Butanediol diglycidylether, epoxy resins
- Physical state: Liquid
- Analytical purity: 100%
- Lot/batch No.: 91-13719/06
- Expiration date of the lot/batch: 29 September 2011
- Stability under test conditions: Documentation regarding the purity and stability of the test substance is on file with the Sponsor and WIL.
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 38 days old at receipt; 7 weeks old at the initiation of dose administration
- Weight at study initiation: 180 g to 220 g for males and from 154 g to 177 g for females at the time of randomization.
- Fasting period before study: Not reported
- Housing: housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board
- Diet (e.g. ad libitum): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, ad libitum
- Water (e.g. ad libitum): Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, ad libitum
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 to 22.2
- Humidity (%): 38.7 to 46.0
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethylcellulose in deionized water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The vehicle suspension was prepared approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored refrigerated. The vehicle was mixed throughout preparation, sampling, and dose administration procedures.
The test substance formulations were weight/volume (test substance/vehicle) mixtures. The test substance formulations were prepared daily as single formulations for each dosage level and stored at room temperature. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

- Dosing volume: 10 mL/kg

VEHICLE
- Justification for use and choice of vehicle (if other than water): carboxymethylcellulose sodium, medium viscosity (No justification for use and choice of vehicle provided)
- Concentration in vehicle: 0, 2.5, 10.0, 20.0, and 40.0 mg/mL (test substance concentration)
- Lot/batch no. (if required): YM0461
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of dose administration (study day 0), quadruplicate samples (approximately 1 mL each) for homogeneity determination were collected from the top, middle, and bottom strata of the 2.5 and 40 mg/mL dosing formulations. Two of the quadruplicate samples collected from the middle strata for homogeneity determination were used for concentration determination. In addition, quadruplicate samples 10.0, and 20.0 mg/mL dosing formulations for concentration determination. On study days 7, 14, and 21, quadruplicate samples (approximately 1 mL each) for concentration determinations were collected from the middle strata of all dosing suspensions, including the control group. One duplicate set was analyzed and the remaining duplicate set was frozen (approximately -10ºC to -30ºC) and retained as backup samples. All analyses were conducted by the WIL Analytical Chemistry Department using a validated GC/MS method.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 100, 200, 400 mg/kg body weight/day
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Test substance dosing concentrations were selected by the Sponsor based on results from acute toxicity testing and were meant to cover a broad range of doses to evaluate toxicity.

The selected route of administration for this study was oral (gavage) because this is the route preferred for subchronic testing.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, once in the morning and once in the afternoon (for mortality and moribundity); twice daily, at the time of dose administration and approximately 1 to 2 hours following dose administration (clinical examination)
- Cage side observations checked: mortality and moribundity


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, beginning at least 1 week prior to test substance administration, at the time of randomization, and prior to the scheduled necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, beginning at least 1 week prior to randomization.


FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the scheduled necropsy (Week 4)
- Anaesthetic used for blood collection: Yes (isoflurane and carbon dioxide)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Total leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLATELET), prothrombin time (PT), activated partial thromboplastin time (APTT), reticulocyte count [percent (RETIC) and absolute (RETIC ABSOLUTE)], differential leukocyte count [percent and absolute, neutrophil (NEU), lymphocyte (LYMPH), monocyte (MONO), eosinophil (EOS), basophil (BASO), large unstained cell (LUC)], red cell distribution width (RDW), hemoglobin distribution width (HDW), platelet estimate, and red cell morphology (RBC Morphology)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the scheduled necropsy (Week 4)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Albumin, total protein, globulin [by calculation], albumin/globulin ratio (A/G Ratio) [by calculation], total bilirubin (Total Bili), urea nitrogen, creatinine, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), glucose, total cholesterol (Cholesterol), calcium, chloride, phosphorus, potassium, sodium, triglycerides (Triglyceride), and sorbitol dehydrogenase (SDH)


URINALYSIS: Yes
- Time schedule for collection of urine: at the scheduled necropsy (Week 4)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Specific gravity (SG), pH, urobilinogen (URO), total volume (TVOL), color (COL), clarity (CLA), protein (PRO), glucose (GLU), ketones (KET), bilirubin (BIL), occult blood (BLD), leukocytes (LEU), nitrites (NIT), and microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
HISTOPATHOLOGY: Yes; Adrenals (2), aorta, bone with marrow, femur, sternum, bone marrow smear (femur), brain, cerebrum level 1, cerebrum level 2, cerebellum with medulla/pons, cervix, epididymides (2), exorbital lacrimal glands (2), eyes with optic nerve (2), gastrointestinal tract, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, kidneys (2), liver (sections of 2 lobes), lungs (including bronchi, fixed by inflation with fixative), lymph nodes, axillary (2), mandibular (2), mesenteric, nasal cavity, ovaries with oviducts (2), pancreas, peripheral nerve (sciatic), peyer’s patches, pharynx, pituitary, prostate, salivary glands (mandibular [2]), seminal vesicles with coagulating gland (2), skeletal muscle (rectus femoris), skin (with mammary gland), spinal cord (cervical, thoracic, lumbar), spleen, testes (2), thymus, thyroid (with parathyroids, if present [2]), trachea, Urinary bladder, uterus, vagina, gross lesions (when possible)

- The following organs were weighed from all animals at the scheduled necropsy: Adrenals, brain, epididymides, heart, kidneys, liver, ovaries with oviducts, prostate with seminal vesicles and coagulating glands, spleen, testes, thymus, thyroid with parathyroids, and uterus with cervix
Other examinations:
None
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ by ±1 in the last significant figure.

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.

Body weight, body weight change, food consumption, clinical pathology (except gamma glutamyltransferase), and organ weight data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Gamma glutamyltransferase values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. Gamma glutamyltransferase data were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
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:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
Test substance-related wet clear material around the mouth was noted in the 200 and 400 mg/kg/day group males and females.

In the 400 mg/kg/day group males, wet clear material around the mouth was noted in 5/5 males at the time of dosing as early as study day 6 and in 5/5 males 1 to 2 hours following dose administration as early as study day 6. In the 400 mg/kg/day group females, wet clear material around the mouth was noted in 2/5 females at the time of the detailed physical examination on study day 14, in 5/5 females at the time of dosing as early as study day 5, and in 5/5 females 1 to 2 hours following dose administration as early as study day 8. This observation was noted throughout the dosing period in males and females, but to a lesser extent in females.

In the 200 mg/kg/day group males, wet clear material around the mouth was noted in 2/5 males at the time of dosing as early as study day 8 and in 3/5 males 1 to 2 hours following dose administration as early as study day 12. In general, the wet clear material around the mouth was observed throughout the dosing period. This clinical observation was also noted in the 200 mg/kg/day group females, but with fewer occurrences.

There were no other test substance-related clinical observations. All other clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.


BODY WEIGHT AND WEIGHT GAIN
Test substance-related effects on body weights were noted in the 400 mg/kg/day group males. Lower mean body weights, body weight gains, and cumulative body weight gains (occasionally significant compared to the control group) were noted throughout the dosing period.

Body weights were not affected in the 25, 100, and 200 mg/kg/day group males or in the 25, 100, 200, and 400 mg/kg/day group females.


FOOD CONSUMPTION
Food consumption was unaffected by test substance administration. There were no statistically significant differences when the control and test substance-treated groups were compared.


HAEMATOLOGY
Test substance-related higher mean absolute neutrophil, lymphocyte, monocyte, basophil, large unstained cell (LUC), and white blood cell counts were noted in the 400 mg/kg/day group males and/or females.

Statistically significantly higher mean absolute basophil counts were noted in the 400 mg/kg/day group males. Statistically significantly higher mean absolute neutrophil, lymphocyte, monocyte, and LUC counts, and consequently higher mean white blood cell counts were noted in the 400 mg/kg/day group males and females. These effects were considered to be test substance-related; however, all mean values were well within WIL historical control reference ranges (version 2.9), reference ranges are wide due to variability in these parameters, and individual values in this study were often variable as well.

There were no other test substance-related effects on hematology and coagulation parameters. Statistically significantly lower mean percent red cell distribution widths were noted in the 100 mg/kg/day group males and higher mean absolute reticulocyte counts were noted in the 400 mg/kg/day group females when the control and test substance-treated groups were compared; however, these differences were not considered test substance-related due to lack of a dose-response relationship, no correlating effects in red cell parameters, and mean values within WIL historical control reference range (version 2.9).

CLINICAL CHEMISTRY
Test substance-related alterations in serum chemistry parameters in the 400 mg/kg/day group males included lower mean globulin levels and subsequent higher A/G ratios, higher mean urea nitrogen values, higher mean alanine aminotransferase (ALT) values, and lower mean total protein values. Test substance-related higher triglyceride values were noted in the 400 mg/kg/day group females.

A statistically significantly higher mean A/G ratio was noted in the 400 mg/kg/day group males, and this value was above WIL historical control reference range (version 2.9). As the mean albumin value was similar to the control group, this ratio difference was associated with lower mean globulin levels. This difference from the control group value, while not statistically significant, was below the WIL historical control reference range (version 2.9). Mean total protein was also slightly lower in the 400 mg/kg/day group males when compared to the control group, although the difference lacked statistical significance. Statistically significant higher mean urea nitrogen and slightly higher mean alanine aminotransferase (ALT) values were also present in the 400 mg/kg/day group males. These effects were considered related to test substance administration.

Statistically significant higher triglycerides were noted in the 400 mg/kg/day group females. Though this effect was considered test substance-related, the mean value was within the WIL historical control reference ranges (version 2.9).

There were no other test substance-related effects on serum chemistry parameters. Statistically significantly lower total bilirubin values were noted in the 100 and 400 mg/kg/day group males; however, these differences were not considered to be test substance-related due to the lack of a dose-response trend, a high control group value (greater than the WIL historical control reference range [version 2.9]) being used for comparison, a toxicologically insignificant direction of change, and the mean values being within WIL historical control reference range (version 2.9). Statistically significantly higher chloride values were noted in the 400 mg/kg/day group males; however, this difference was not considered to be test substance-related due to the low magnitude of change, no effects noted on other electrolyte parameters, and the mean value being within WIL historical control reference range (version 2.9).

URINALYSIS
There were no test substance-related effects on urinalysis parameters. However, one statistically significant difference was observed when the control and test substance-treated groups were compared. Higher total urine volume in the 400 mg/kg/day group females was not considered to be test substance-related as there was no dose response, the magnitude of the difference was small, expected variability for this parameter is large, and the mean value was within WIL historical control reference range (version 2.9).


ORGAN WEIGHTS
Test substance-related lower final body weights were noted in the 400 mg/kg/day group males. Higher liver weights relative to body weights were noted in the 400 mg/kg/day group males and females and were considered related to test substance administration.

Statistically significantly lower final body weights were noted in the 400 mg/kg/day group males and were considered test substance-related. Mean liver weight relative to body weight differences were statistically significant in the 400 mg/kg/day group males and females when compared to the control group mean; due to final body weight differences in the males, only relative (to final body weight) values were employed as these values were considered to be more accurate in assessing a test substance-related effect. All mean values were within WIL historical control reference range (version 2.9), and there were no histologic correlates.
Statistically significantly higher brain weights relative to final body weights were noted in the 400 mg/kg/day group males; however, this difference was considered to be a result of the test substance-related effect on final body weight and, therefore, not directly related to test substance administration. There were no other test substance-related effects on organ weights. There were no test substance-related alterations in mean final body weight in the females.

GROSS PATHOLOGY
Thickened stomach was noted in the 400 mg/kg/day group males and females and was considered to be test substance-related due to correlation with histologic changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test substance-related histologic alterations were observed in the stomach (nonglandular portion), exorbital lacrimal glands, and pancreas.

Hyperplasia of the stratified squamous epithelium of the nonglandular portion of the stomach was identified in 0/5, 1/5, 1/5, 3/5, and 5/5 males and 0/5, 0/5, 1/5, 4/5, and 4/5 females in the control, 25, 100, 200 and 400 mg/kg/day groups, respectively. This lesion correlated to gross observations of “thickened” stomach identified in both males and females in the 400 mg/kg/day group. At minimal to mild levels, a slight increase in keratin production was observed with only minimal hyperplasia of the underlying epithelial layers. At moderate to severe levels, rete peg formation, mucosal ridges, and remarkably increased keratin production were prominent. In 1 animal of each sex, associated submucosal edema and inflammation were also observed. The glandular stomach was unaffected.

Secretory depletion within the exorbital lacrimal glands was noted in 1/5, 1/5, 1/5, 2/5, and 3/5 males in the control, 25, 100, 200, and 400 mg/kg/day groups, respectively; this effect was absent in females. Given the similar incidence in the control, 25, and 100 mg/kg/day groups, this finding was considered an incidental background lesion in these animals, while secretory depletion was considered to be test substance-related in the 200 and 400 mg/kg/day groups based on a dose-responsive higher incidence and/or severity.

Zymogen depletion within the pancreas was observed in 1/5, 0/5, 1/5, 0/5, and 5/5 males and 1/5, 2/5, 2/5, 3/5, and 2/5 females in the control, 25, 100, 200, and 400 mg/kg/day groups, respectively. Based on incidence and severity, this finding was considered to be test substance-related in the 400 mg/kg/day group males. Although normal biological variability cannot be entirely excluded, based on the results of this study, zymogen depletion within the pancreas was considered to be test substance-related in the 25, 100, 200, and 400 mg/kg/day group females. The low number of animals per group complicated interpretation, particularly in the 25 mg/kg/dose group females. The relationship of this finding to the test substance was less apparent in the females. Although a single animal in the control group was minimally affected, a higher incidence was noted throughout the test substance-treated groups, with an increased severity in individuals in the 100 and 400 mg/kg/day dose groups. No dose response was observed.

Test substance-related zymogen depletion (mostly minimal to mild) noted in the pancreas of all test substance-treated females and 400 mg/kg/day group males, and secretory depletion of the exorbital lacrimal glands (minimal to mild) in the 200 and 400 mg/kg/day group males and females were of unknown toxicological significance and may represent a stress response.

There were no other test substance-related histopathologic changes. Histopathologic findings were considered to be incidental, manifestations of spontaneous diseases or related to some aspect of experimental manipulation other than exposure to the test substance. There was no test substance related alteration in the prevalence, severity, or histologic character of these incidental tissue alterations.

Effect levels

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Based on lower body weights and body weight gains (males), as well as changes to hematological and clinical chemistry parameters and higher liver weights (both sexes).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Test substance-related clinical findings were limited to wet clear material around the mouth noted in the 200 and 400 mg/kg/day males and females.

Test substance-related lower body weights, body weight gains, and cumulative body weight gains were noted in the 400 mg/kg/day group males throughout the dosing period. This effect was evident in subsequent test substance-related lower final body weights noted at the scheduled necropsy.

Test substance-related higher absolute neutrophil, lymphocyte, monocyte, basophil, large unstained cell (LUC), and consequent white blood cell counts were noted in the 400 mg/kg/day group males and/or females.

Test substance-related alterations in serum chemistry parameters in the 400 mg/kg/day group males included lower globulin levels and subsequent higher A/G ratios, higher urea nitrogen values, higher alanine aminotransferase (ALT) values, and lower total protein values. Test substance-related higher triglyceride values were noted in the 400 mg/kg/day group females.

Test substance-related higher liver weights (relative to body weight) were noted in the 400 mg/kg/day group males and females.

Test substance-related histologic changes included hyperplasia of the stratified squamous epithelium of the nonglandular stomach in the 25, 100, 200, and 400 mg/kg/day group males and the 100, 200, and 400 mg/kg/day females correlating to gross observations of “thickened” stomach in the 400 mg/kg/day group animals, secretory depletion in the exorbital lacrimal glands of the 200 and 400 mg/kg/day group males and females, and secretory depletion of zymogen granules in the pancreas of the 400 mg/kg/day group males and the 25, 100, 200, and 400 mg/kg/day group females. Findings of epithelial hyperplasia within the nonglandular stomach in this study are consistent with a local irritant effect of the test substance (GRILONIT RV 1806, MSDS No. 231053, 2010). Although this portion of the stomach (cardia) is comprised of keratinized stratified squamous epithelim in the rodent (Brown et al., 1990), the human stomach bears only glandular mucosa throughout (Burkitt et al., 1993). There were no histologic alterations of the glandular portion of the stomach of rats in this study.

Applicant's summary and conclusion