Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST SYSTEM, ANIMAL RECEIPT, AND ACCLIMATION
Crl:CD(SD) rats were used as the test system for this study. This species and strain of animal is recognized as appropriate for repeated dose toxicity studies. The Sprague Dawley rat was selected because it is a widely used strain for which significant historical control data are available. The number of animals selected was based on regulatory guidelines (OECD Test Guideline 408 “Repeated Dose 90-Oral Toxicity in Rodents”). A minimum of 10 rats/sex/group are necessary for repeated-dose rodent studies in order to provide information on the possible health hazards likely to arise from prolonged exposure. These recommendations helped to ensure that the number of animals that survived to the end of the study was sufficient to permit a meaningful evaluation of toxicological effects.
Crl:CD(SD) rats (45 males and 45 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 02-Jul-2015. The animals were approximately 40 days old at receipt. Each animal was examined by a qualified technician on the day of receipt and weighed on the following day. Each animal was uniquely identified with a subcutaneous microchip (BMDS system) implanted in the dorsoscapular area. All animals were housed for an 11-day acclimation period. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior. Data collection during acclimation began on 03-Jul-2015. Individual body weights and cage food weights were recorded and detailed physical examinations were performed periodically during acclimation. Ophthalmic examination data were also recorded for animals prior to randomization.

ANIMAL HOUSING
Upon arrival, all animals were housed 2 to 3 per cage by sex in clean, solid bottom cages containing ground corncob bedding material (Bed O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). Following placement into treatment groups, animals were housed in groups of 2 of the same sex per cage. Assignments of individual animals to social groups during acclimation and following randomization are presented in Appendix D. The cages were cleaned and changed routinely at a frequency consistent with maintaining good animal health. The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the objectives of this study. The results of these analyses are maintained at WIL Research. The animals were temporarily separated as necessary to allow for the performance of protocol-specified activities. Animals whose cage mate were removed from the study (unscheduled death) were not re-paired and remained individually housed for the remainder of the study. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at WIL Research are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER, AND MAINTENANCE
The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to clinical pathology blood collection when food, but not water, was withheld. In addition, animals did not have access to diet during the conduct of Functional Observation Battery (FOB)
and Motor Activity (MA) assessments. Municipal water supplying the facility was analyzed for contaminants according to SOPs. The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
All animals were housed throughout acclimation and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71 ± 5°F (22 ± 3°C) and 50 ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. These data are summarized in Appendix E. Actual mean daily temperature ranged from 69.8°F to 71.1°F (21.0°C to 21.7°C) and mean daily relative humidity ranged from 35.2% to 60.2% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
On 06-Jul-2015 (7 days prior to the initiation of dose administration), all available rats were weighed and examined in detail for physical abnormalities. These data were collected using WTDMS™ and reviewed by the Study Director. The animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design. A printout containing the animal numbers and individual group assignments was generated, and the animals were then arranged into treatment groups and housed in social groups according to the printout. Individual body weights at randomization were within ± 20% of the mean for each sex. Animals not assigned to study were transferred to the WIL Research colony. Each group (Groups 1-4) consisted of 10 males and 10 females. These animals were then randomized into study replicates to allow for the reasonable conduct of the functional observational battery and motor activity assessments. Each dose group and sex were approximately equally represented within each study replicate. The animals were approximately 7 weeks old at the initiation of dose administration. Individual body weights ranged from 160 g to 199 g for males and from 120 g to 150 g for females at randomization.

ORGANIZATION OF TEST GROUPS, DOSAGE LEVELS, AND TREATMENT REGIMEN
The control and test substances were administered orally by gavage via an appropriately sized flexible Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily for 91 or 92 consecutive days, through the day prior to the scheduled necropsy. The dosing cannula was thoroughly wiped off prior to each dose administration to ensure that no test substance was deposited in the nasopharynx. The dose volume was 0.35, 0.09, 0.17, and 0.35 mL/kg for Groups 1, 2, 3, and 4, respectively. Individual doses were based on the most recently recorded body weights to provide the correct mg/kg/day dose. The first day of dosing was study day 0; the first week of dosing was study week 0.

The following table presents the study group assignment:

Group Treatment Dosage Level Dosage volume Number of Animals
Number (mg/kg/day) (mL/kg) Males Females
1 control substance 0 0.35 10 10
2 m-DIPB 75 0.09 10 10
3 m-DIPB 150 0.17 10 10
4 m-DIPB 300 0.35 10 10

Dosage levels were selected in consultation with the Sponsor based on the results of a previous 28-day oral toxicity study in Sprague-Dawley rats conducted by the Sponsor (O’Donoghue, 1986). Rats in this study were given 21 doses of either 0, 100, 300, or 1000 mg/kg/day over 28 days (dosing did not occur on weekends or holidays). There were no effects on body weight or food consumption; however, mild irritation to the stomach and increased liver weights were observed in the 1000 mg/kg/day group. The selected route of administration for this study was oral (by gavage) because the oral route is the possible route of exposure for the general population.




Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Approximately weekly, aliquots of the control substance were prepared for daily dispensation to the control group (Group 1) and stored at room temperature (18°C to 24°C). The pH of the first dispensation of the control substance was 6.34. Approximately weekly, the neat test substance was divided into aliquots for daily dispensation and stored at room temperature (18°C to 24°C).
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSES
Analyses to demonstrate the homogeneity, stability, and concentration of the test substance were not conducted as part of this study. The test substance was expected to be stable for the duration of testing if stored properly as determined by the Sponsor. Characterization of the test substance has previously been conducted and the test substance was administered in pure form.
Duration of treatment / exposure:
Approximately 90 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
300 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
75 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
Positive control:
none
Observations and examinations performed and frequency:
SURVIVAL
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. Animals found dead were examined macroscopically as soon as possible to ensure that tissues were not lost due to autolysis.

CLINICAL OBSERVATIONS
Clinical examinations were performed at the time of dose administration and 1-2 hours following dose administration. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals within 4 days of receipt, on the day of randomization, weekly (± 2 days) during the study period, and on the day of the scheduled necrops y. Due to social housing, some treatments could not be offered to a single animal. In these instances, the treatment was recorded in a separate computer protocol for the social group and these observations are presented in Appendix D. Any observations noted outside of the above-specified intervals were also recorded.

BODY WEIGHTS
Individual body weights were recorded within 4 days of receipt, on the day of randomization, study day 0, weekly (± 2 days) during the study period, and on the day prior to the scheduled necropsy (nonfasted). The last nonfasted body weights were collected on the final day of study week 12, which was the day prior to the first day of the scheduled necropsy. These second weekly body weights during study week 12 are identified in the text and on the report tables as study week 13. Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION
Cage food weights were recorded once weekly (± 2 days) beginning following randomization and throughout the study period. Food consumption was calculated as g/animal/day. The last food weights were collected on the final day of study week 12, which was the day prior to the first day of the scheduled necropsy. These second weekly food consumptions during study week 12 are identified in the text and on the report tables as study week 13. When food consumption could not be measured for a given interval.

FOB ASSESSMENTS
FOB assessments were recorded for all animals during study week 12. The FOB utilized at WIL Research is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988; Moser et al., 1991, and O’Donoghue, 1989). Testing was performed by the same biologists, whenever possible, without knowledge of the animal’s group assignment. The FOB was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. All animals were observed for the following parameters as described below:

HOME CAGE OBSERVATIONS
Posture
Convulsions/tremors
Feces consistency

HANDLING OBSERVATIONS
Ease of removal from cage
Lacrimation/chromodacryorrhea
Piloerection
Palpebral closure
Eye prominence
Red/crusty deposits
Ease of handling animal in hand
Salivation
Fur appearance
Respiratory rate/character
Mucous membranes/eye/skin color
Muscle tone

OPEN FIELD OBSERVATIONS
Mobility
Rearing
Convulsions/tremors
Grooming
Bizarre/stereotypic behavior
Time to first step (seconds)
Gait
Arousal
Urination/defecation
Gait score
Backing
Note: Open field observations were evaluated over a 2-minute observation period.

SENSORY OBSERVATIONS
Approach response
Startle response
Pupil response
Forelimb extension
Air righting reflex
Touch response
Tail pinch response
Eyeblink response
Hindlimb extension
Olfactory orientation

NEUROMUSCULAR OBSERVATIONS
Hindlimb extensor strength
Hindlimb foot splay
Grip strength-hind and forelimb
Rotarod performance

Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer et al. (1979). The animal was allowed to grip a T-shaped grip bar with its forepaws and was pulled back gently along a platform until its grip was broken. As the backward locomotion continues, the animal’s hindpaws reach a T-shaped rearlimb grip bar, which it is allowed to grasp and then forced to release by continued pulling. Mark-10 series EG digital force gauges (Mark-10 Corporation, Copiague, NY) were used to record the maximum strain required to break forelimb and hindlimb grip. The average of 3 valid measurements was taken as the animal’s score for each grip strength measure.

MOTOR ACTIVITY
Motor activity was assessed for all animals during study week 12. Motor activity, recorded after the completion of the FOB, assessment was conducted using a personal computer-controlled system that utilizes a series of infrared photobeams surrounding an amber, plastic rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. Each animal was tested separately. Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor acti vity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY
Blood and urine samples for clinical pathology evaluations (hematology, coagulation, serum chemistry, and urinalysis) were collected from all surviving animals on the day of the scheduled necropsy (study week 13). The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected for hematology and serum chemistry evaluation via the jugular vein. Blood was collected for coagulation parameters at the time of euthanasia via the vena cava of animals euthanized by inhalation of carbon dioxide. Blood was collected into tubes containing potassium (K2) EDTA (hematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

The following parameters were evaluated:

HEMATOLOGY AND COAGULATION
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)
Absolute (RETIC Absolute)
Mean platelet volume (MPV)
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
Red cell morphology
(RBC Morphology)

SERUM CHEMISTRY
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
Sorbitol dehydrogenase (SDH)
Triglycerides (Triglyceride)
Appearance

URINALYSIS
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)
Microscopy of sediment
[Tabular abbreviations appear
on individual tables]

OPHTHALMIC EXAMINATIONS
Ocular examinations were conducted on all animals prior to randomization (06-Jul-2015; study week -1) and all surviving animals in Groups 1 and 4 during the last week of the dosing period (10-Oct-2015; study week 12). All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.

Sacrifice and pathology:
MACROSCOPIC EXAMINATION
A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. 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. The following tissues and organs were collected and placed in 10% neutral-buffered formalin (except as noted):

Adrenals (2)
Aorta
Bone with marrow
Femur
Sternum
Bone marrow smear
(from femur)
Brain
Cervix
Epididymides (2)
Eyes with optic nerve (2)
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys (2)
Larynx
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed
by inflation with fixative)
Lymph nodes
Axillary (2)
Mandibular (2)
Mesenteric
Ovaries (2) with oviducts
Pancreas
Peripheral nerve (sciatic)
Peyer’s patches
Pharynx
Pituitary
Prostate
Salivary glands (mandibular [2])
Seminal vesicles (2)
Skeletal muscle (rectus femoris)
Skin with mammary gland
Spinal cord (cervical, thoracic, lumbar)
Spleen
Testes (2)
Thymus
Thyroid (with parathyroids [2])
Tongue
Trachea
Urinary bladder
Uterus
Vagina
Gross lesions (when possible)

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled necropsy:

Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary
Prostate with seminal vesicles
Spleen
Testes
Thymus
Thyroid

HISTOLOGY AND MICROSCOPIC EXAMINATION
After fixation, protocol-specified tissues were trimmed according to WIL Research SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin. Microscopic examination was performed on all tissues listed in Section 5.9.1. from all animals found dead and in the control and 300 mg/kg/day groups at the scheduled necropsy. Gross lesions were examined from all animals and correlated to microscopic findings if possible. In addition, the stomach (nonglandular and glandular) and liver were identified as potential target tissues and were examined from all animals. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.
Statistics:
STATISTICAL ANALYSIS
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and/or the number of animals or cages (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables.

STATISTICS CONDUCTED BY WIL RESEARCH
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, continuous FOB, body temperature, clinical pathology, 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. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test (Steel and Torrie, 1980).

STATISTICS CONDUCTED BY BIOSTAT CONSULTANTS, INC.
All repeated measures analysis of variance (RANOVA) statistical analyses for total and ambulatory motor activity counts recorded prior to the initiation of dose administration and after dosing were conducted by BioSTAT Consultants, Inc., Portage, MI, using SAS® version 9.2 software (SAS Institute, Inc., 2002-2008). The results of these analyses are presented in Appendix K. Each analysis endpoint was analyzed, by sex and session, with a RANOVA. Factors in the model included treatment group (TRT),
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were 7 animals found dead prior to the scheduled necropsy. One male and 1 female in the 75 mg/kg/day group were found dead on study days 25 and 31, respectively. One 150 mg/kg/day group male was found dead on study day 73. Two males in the 300 mg/kg/day were found dead on study days 19 and 68, and 2 females in the 300 mg/kg/day group were found dead on study days 67 and 85. The cause of death for these animals were either unknown and/or resultant from aspiration of the test substance and were not considered test substance-related.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were 7 animals found dead prior to the scheduled necropsy. One male and 1 female in the 75 mg/kg/day group were found dead on study days 25 and 31, respectively. One 150 mg/kg/day group male was found dead on study day 73. Two males in the 300 mg/kg/day were found dead on study days 19 and 68, and 2 females in the 300 mg/kg/day group were found dead on study days 67 and 85. The cause of death for these animals were either unknown and/or resultant from aspiration of the test substance and were not considered test substance-related.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
lower glucose concentrations that were noted in 150 and 300 mg/kg groups were considered to be non-adverse findings
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):
Test substance-related effects on organ weights were noted in all treatment groups and included higher liver weights in the 75, 150, and 300 mg/kg groups, which also correlated with hepatocellular hypertrophy microscopically in two 300 mg/kg/day females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A single test substance-related gross observation of dark red area in the glandular portion of the stomach was noted in a 300 mg/kg/day group female.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal degeneration of the superficial epithelium accompanied by minimal to mild accumulation of cellular debris (necrotic debris) in the stratum corneum and minimal neutrophilic inflammation in the underlying submucosa of the non-glandular stomach
Histopathological findings: neoplastic:
not examined
Details on results:
SURVIVAL
A total of 7 animals were found dead prior to the scheduled necropsy. Male no. 4733 from the 150 mg/kg/day group and male no. 4747 from the 300 mg/kg/day group were found dead after dosing on study days 73 and 19, respectively. There were no test substance-related clinical observations noted for these animals. The cause of death for these animals was unknown. Male no. 4733 had test substance-related minimal erosion in the nonglandular stomach that was considered insufficient to result in death. Female no. 4772 from the 75 mg/kg/day group and female nos. 4795 and 4781 from the 300 mg/kg/day group were found dead on study days 31, 67, and 85, respectively. There were no test substance-related clinical observations noted for these animals. The cause of death in these animals was unknown. Female no. 4772 was found cannibalized by her cagemate, which most likely occurred post-mortem because there were no signs of trauma or injury. Female no. 4795 was found dead following dosing and had necropsy findings of dark red contents surrounding the heart and dark red areas in the lungs that correlated microscopically with hemorrhages. The animal also had 8.0 ml of red fluid in the thoracic cavity. It was unclear if these findings were caused by gavage accident as no tears in the esophagus were noted at necropsy. In addition, no microscopic findings to justify gavage accident were noted in sections of esophagus, trachea, and lung. Female no. 4781 had test substance-related minimal cellular debris in the stratum corneum and neutrophilic inflammation in the nonglandular stomach that were considered insufficient to result in death.

Male no. 4763 from the 75 mg/kg/day group was found dead following dosing on study day 25. A clinical observation of cool body was noted for this animal prior to death and a body weight loss of 8% was observed from study week 2 to 3. Microscopic findings in this animal that likely resulted in death included necrosis of bronchiolar epithelium with secondary regenerative hyperplasia of the epithelium and peribronchiolar chronic active inflammation, in addition to pyogranulomatous inflammation in the alveoli. The pyogranulomatous inflammation correlated with the necropsy finding of a nodule on the right intermediate lobe of the lung. The findings were centered in the airways and were therefore considered likely caused by aspiration of the test substance. Male no. 4754 from the 300 mg/kg/day group was found dead prior to dosing on study day 68. A clinical observation of yellow material around the mouth was noted for this animal prior to death on study day 67. Microscopic findings in this animal were similar to male no. 4763, with findings centered in the airways, likely secondary to aspiration of the test substance. This animal also had evidence of secondary systemic inflammation, characterized by myeloid hyperplasia in the sternal and femoral bone marrow, and evidence of secondary stress, characterized by lymphoid depletion in the thymus and spleen. Both inflammation and stress were attributed to the findings in the lungs.

CLINICAL OBSERVATIONS
Test substance-related clinical observations of clear material around the mouth and/or salivation were periodically noted in the 150 and 300 mg/kg/day group males and females, but at a low incidence. 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 WEIGHTS
Body weights were unaffected by test substance administration. By the end of the dosing period, the mean body weights in the 300 mg/kg/day group were slightly lower than the control group but these changes were attributed to individual animal variation.

FOOD CONSUMPTION
Food consumption was unaffected by test substance administration.

FOB ASSESSMENTS (TOXICOLOGY GROUPS)
HOME CAGE OBSERVATIONS
Home cage observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 12 evaluation, with the exception of the posture parameter (a higher number of animals were alert and oriented toward the observer and a corresponding lower number of animals were sitting or standing normally) in the 150 mg/kg/day group females. This was not considered test substance-related because the observation was not statistically significant different than controls in the high-dose group and there were no correlating observations in the remaining neurobehavioral assessments.

HANDLING OBSERVATIONS
Handling observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 12 evaluation.

OPEN FIELD OBSERVATIONS
Open field observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 12 evaluation.

SENSORY OBSERVATIONS
Sensory observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 12 evaluation.

NEUROMUSCULAR OBSERVATIONS
Neuromuscular observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 12 evaluation.

PHYSIOLOGICAL OBSERVATIONS
Physiological observations were unaffected by test substance administration. There were no statistically significant differences when the test substance-treated males and females were compared to the control group at the study week 12 evaluation.

MOTOR ACTIVITY
Motor activity patterns (total and ambulatory activity counts) were unaffected by test substance administration. There were no statistically significant changes for the test substance-treated males and females when compared to the control group at the study week 12 evaluation. Values obtained from the 6 epochs evaluated (0-10 minutes, 11-20 minutes, 21-30 minutes, 31-40 minutes, 41-50 minutes, and 51-60 minutes) and the overall 60-minute test session were comparable to the concurrent control values. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the animals were evaluated on study week 12.

CLINICAL PATHOLOGY
For the presentation of clinical pathology results in this report, the terms “higher” or “lower” refer to comparisons between test substance-treated group means versus the concurrent control group mean.

HEMATOLOGY AND COAGULATION
Hematology and coagulation parameters were unaffected by test substance administration. There were no statistically significant differences when the control and test substance-treated groups were compared.

SERUM CHEMISTRY
Lower glucose values were noted in the 300 mg/kg/day group males (15.2%) and females (11.2%); the group mean value was not statistically significant in females. The mean values for males and females were within the WIL Research historical cont rol data ranges, with the exception of a few individual animals in both sexes that showed values lower than the WIL Research historical control data ranges. In the absence of contributing factors commonly observed in treated laboratory rats, such as decreased food consumption, this finding was considered likely test substance-related, although
nonadverse, due to the low magnitude and lack of correlating findings. Lower chloride values in the 75 and 300 mg/kg/day group males (1.9% and 0.9%, respectively) were of minimal magnitude, showed no dose response, and were within the WIL Research historical control data ranges; therefore, they were considered normal biological variation.

URINALYSIS
Urinalysis parameters were unaffected by test substance administration. There were no statistically significant differences when the control and test substance-treated groups were compared.

OPHTHALMIC EXAMINATIONS
No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

ANATOMIC PATHOLOGY
MACROSCOPIC EXAMINATION
A single test substance-related gross observation of dark red area in the glandular portion of the stomach was noted in a 300 mg/kg/day group female (no. 4809). This change had a microscopic correlate of erosion.

ORGAN WEIGHTS
Test substance-related higher liver weights (relative to final body weights) were noted in males at all dosage levels (8.3%, 11.2%, and 14.5% in the 75, 150, and 300 mg/kg/day, respectively), in addition to the 150 and 300 mg/kg/day group females (13.0% and 17.3%, respectively). Corresponding absolute and relative (to brain weight) liver weight values were also higher in males and females, albeit statistically significant only in the 150 mg/kg/day group males (absolute and relative to brain weight) and for the relative to brain weight in the 150 and 300 mg/kg/day group females. Statistically significant higher kidney weight (relative to final body weight) in the 300 mg/kg/day group females was caused by a slightly lower final body weight in this
group and considered unrelated to test substance administration. There were no other test substance-related effects on organ weights. However, some statistically significant differences were observed when the control and test substance treated groups were compared. These included lower adrenal gland weights (absolute and relative to brain weight) in the 75 and 300 mg/kg/day group males, lower pituitary weights (absolute and relative to final body and brain weights) in the 150 mg/kg/day group females, and higher thyroid/parathyroid weights (absolute and relative to brain weight) in the 150 mg/kg/day group females. These changes had no microscopic correlates, showed no dose responses, and were within the WIL Research historical control data ranges; therefore, they were considered normal biological variation.

MICROSCOPIC EXAMINATION
Test substance-related microscopic findings were noted in the stomach of animals from all test substance-treated groups and the liver of the 300 mg/kg/day group females. Test substance-related findings in the nonglandular stomach occurred in the region adjacent to the transition with the glandular portion (limiting ridge). Changes in males occurred at all dose groups (≥75 mg/kg/day groups) and were characterized by minimal degeneration of the superficial epithelium accompanied by minimal to mild accumulation of cellular debris (necrotic debris) in the stratum corneum and minimal neutrophilic inflammation in the underlying submucosa. Lesions in a few individual males progressed to erosion (necrosis and loss of epithelial surface) or ulcer (necrosis and loss extending to the muscularis mucosa). Findings were generally dose responsive in males. The erosion and ulcer were graded mild or minimal because they occurred as single lesions confined to the limiting ridge. In addition, they were not associated with clinical pathology findings. In females, changes in the nonglandular stomach occurred at all dose groups (≥75 mg/kg/day) and were characterized by minimal degeneration of the superficial epithelium (except in the 300 mg/kg/day group females) accompanied by minimal neutrophilic inflammation in the submucosa and minimal or mild cellular debris in the stratum corneum, also in the limiting ridge. A single animal in the 300 mg/kg/day dose group showed edema of the submucosa in this region, but erosion or ulcer were not observed in females. A dose response was not noted in females. The glandular stomach of a single female in the 300 mg/kg/day group had a single erosion in the mucosa. Despite the low occurrence, this finding was considered likely test substance-related due to lesions of similar nature in the nonglandular portion of the stomach. Test substance-related minimal hepatocellular hypertrophy occurred in 2 females from the 300 mg/kg/day group and correlated with higher liver weights in these animals. Hypertrophy was characterized by increased cytoplasmic size of centrilobular hepatocytes. There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Two 300 mg/kg/day group females had findings of necrosis of the bronchiolar epithelium and peribronchiolar chronic active inflammation. One of these females also had pyogranulomatous inflammation in alveolar spaces adjacent to the affected bronchioles. These findings were likely caused by aspiration of the test substance , but were not sufficient to result in death, as occurred for animal nos. 4763 and 4754.

The objective of the study was to evaluate the potential toxicity of m-DIPB when administered daily by oral gavage to Sprague Dawley rats for a minimum of 90 consecutive days. This included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity (MA) assessment.

Deaths occurred in all test substance-treated groups (except females in the 150 mg/kg/day group) but were not considered test substance-related because there were no clinical or microscopic findings attributable to the test substance that would be sufficient to result in death. Nonadverse test substance-related findings included clinical observations of clear material around the mouth/salivation in the 150 and 300 mg/kg/day groups, lower
glucose values in the 300 mg/kg/day group, and higher liver weights in the 75, 150, and 300 mg/kg/day group males and the 150 and 300 mg/kg/day group females. Findings in the nonglandular stomach occurred in males and females at all dose levels (75, 150, and 300 mg/kg/day). In males, findings progressed to erosions in 1 male at each dose level and to an ulcer in 1 male at 300 mg/kg/day. The erosion in the glandular stomach of the single female in the 300 mg/kg/day group was presumed to be caused by direct test substance-related effects on the mucosa. Although stress has been associated with similar degenerative/necrotic lesions in the gastric mucosa that can lead to erosions/ulcers (Greaves 2012), the gastric findings were considered test substancerelated due to the occurrence at all dose levels, and were suggestive of chemical irritation. No evidence of stress was observed in the parameters evaluated in animals affected by the gastric findings. The gastric findings in the context of this study were considered adverse when there was progression to erosions and ulcers. The gastric lesions in the female animals were milder and therefore appear to be better tolerated by treatment in this sex. Increased liver weights with microscopic correlate of hepatic hypertrophy in two 300 mg/kg/day group females was considered likely an adaptive change, secondary to enzyme induction, caused by test substance metabolism in the liver. There was a low incidence of the microscopic correlates hepatocellular hypertrophy for the widespread higher mean liver weights. This is generally accepted in weight incre ases below 20% compared to controls, as in this study (Hall et al., 2012). Adaptive liver hypertrophy of low magnitude, without evidence of hepatotoxicity and functional changes in liver, as i n this study, is considered nonadverse (Hall et al., 2012).
Dose descriptor:
LOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: oral administration of m-DIPB to male Crl:CD(SD) rats at dosage levels of 75, 150, and 300 mg/kg/day for up to 91 or 92 days resulted in adverse microscopic changes in the stomach (leading to erosion and/or ulcer).
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, oral administration of 1,3-diisopropylbenzene (m-DIPB) to Crl:CD(SD) rats at dosage levels of 75, 150, and 300 mg/kg/day for up to 91 or 92 days resulted in adverse microscopic changes in the stomach (leading to erosion and/or ulcer) at all dosage levels in males and at 300 mg/kg/day in females. Nonadverse test substance-related effects included clinical observations of clear material around the mouth/salivation in the 150 and 300 mg/kg/day groups, lower glucose values in the 300 mg/kg/day group, and higher liver weights at all dosage levels in males and in the 150 and 300 mg/kg/day group females that correlated with hepatocellular hypertrophy microscopically in two 300 mg/kg/day females. As a result of the a dverse gastric findings, the no-observed-adverse-effect level (NOAEL) was 150 mg/kg/day in the females and was unable to be determined in the males.
Executive summary:

The objective of the study was to evaluate the potential toxicity of 1,3-diisopropylbenzene (m-DIPB) when administered daily by oral gavage to Sprague Dawley rats for a minimum of 90 consecutive days. This included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity (MA) assessment.

m-DIPB was administered orally by gavage once daily for 91 or 92 consecutive days to 3 groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 75, 150, and 300 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received the control substance (deionized water) on a comparable regimen. The dose volume was 0.35, 0.09, 0.17, and 0.35 mL/kg for Groups 1, 2, 3, and 4, respectively. Each group consisted of 10 animals/sex. Following 91 or 92 consecutive days of dose administration, all surviving animals were euthanized. For toxicology assessment, all animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body weights were recorded weekly (± 2 days). Cage food weights were recorded once weekly (± 2 days) beginning following randomization. Functional observational battery (FOB) and motor activity (MA) data were recorded for all animals during study week 12. Ophthalmic examinations were performed on all animals during study week -1 and for all surviving animals in Groups 1 and 4 during study week 12. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals assigned to the scheduled (study week 13) necropsy. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from the animals found dead and all animals in the control and high-dose groups at the scheduled necropsy. In addition, the stomach (nonglandular and glandular) and liver were identified as potential target tissues and were examined microscopically from all animals. Gross lesions were examined microscopically from all animals.

There were no test substance-related effects on survival. There were 7 animals found dead prior to the scheduled necropsy. One male and 1 female in the 75 mg/kg/day group were found dead on study days 25 and 31, respectively. One 150 mg/kg/day group male was found dead on study day 73. Two males in the 300 mg/kg/day were found dead on study days 19 and 68, and 2 females in the 300 mg/kg/day group were found dead on study days 67 and 85. The cause of death for these animals were either unknown and/or resultant from aspiration of the test substance and were not considered test substance-related. All other animals survived to the scheduled necropsy. There were no test substance-related effects on body weights, food consumption, hematology and coagulation, or urinalysis parameters. There were no test substance-related ophthalmic findings. FOB and MA assessments were unaffected by test substance administration. Test substance-related clinical observations noted for the 150 and 300 mg/kg/day group males and females at low incidences included clear material around the mouth/salivation. Test substance-related lower glucose values were noted in the 300 mg/kg/day group males and females and was considered nonadverse. A single test substance-related gross observation of dark red area in the glandular portion of the stomach was noted in a 300 mg/kg/day group female. Test substance-related effects on organ weights were noted in all treatment groups and included higher liver weights in the 75, 150, and 300 mg/kg/day group males and the 150 and 300 mg/kg/day group females.

Microscopic changes in the nonglandular stomach occurred in the region adjacent to the transition with the glandular portion (limiting ridge) in all dose groups. These findings were characterized by minimal degeneration of the superficial epithelium accompanied by minimal to mild accumulation of cellular debris (necrotic debris) in the stratum corneum and minimal neutrophilic inflammation in the underlying submucosa. Lesions in a few individual males progressed to erosion or ulceration. A single female in the 300 mg/kg/day group showed edema of the submucosa in this region, but no erosion or ulceration was observed. Also, the glandular stomach of a single female in the 300 mg/kg/day group had a single erosion in the mucosa. These gastric findings exhibited a dose response in males and were considered adverse for both males and females. Test substance-related nonadverse minimal hepatocellular hypertrophy was also noted in 2 females from the 300 mg/kg/day group and correlated with higher liver weights in these animals.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
75 mg/kg bw/day
Study duration:
subchronic
Species:
rat

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

The potential toxicity of m-diisopropylbenzene (m-DIPB) was evaluated following repeated oral doses for 21 days over a 29-day period. The test material was administered by oral gavage to groups of rats at doses of 0, 100, 300, or 1000 mg/kg. During this time, body weights, food consumption, and clinical observations were recorded at weekly intervals. At the conclusion of dosing, rats were sacrificed and subjected to routine hematology, clinical chemistry, and histopathological evaluations. m-DIPB was well tolerated when administered by gavage. The exposure resulted in mild irritation to the stomach and increased liver weights. The increase in liver weights was probably an adaptive response since there was no indication of degenerative effects on microscopic examination and no evidence of increased serum enzyme levels. The significance, if any, of the changes in glucose and creatinine levels in the high-dose males is unclear. The changes in glucose and creatinine were within levels commonly encountered in this laboratory and would not be considered clinically significant. The significance of increased renal hyalin droplets in males is also not clear. Based on previous experience, it appears that the effect is probably due to exposure to the test substance but its importance for human health evaluation is not known. Target organs include the stomach due to direct contact, possibly the liver based on increased organ weight, and possibly the kidneys based on the presence of hyalin droplets. The no-effect level was close to but below 100 mg/kg.

In a subsequent study, the effects of m-DIPB were observed following daily oral administration via gavage to Sprague Dawley rats for a minimum of 90 consecutive days. This included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity (MA) assessment. Dosage levels were 75, 150, and 300 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received the control substance (deionized water) on a comparable regimen. The dose volume was 0.35, 0.09, 0.17, and 0.35 mL/kg for Groups 1, 2, 3, and 4, respectively. Each group consisted of 10 animals/sex. Following 91 or 92 consecutive days of dose administration, all surviving animals were euthanized. For toxicology assessment, all animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body weights were recorded weekly (± 2 days). Cage food weights were recorded once weekly (± 2 days) beginning following randomization. Functional observational battery (FOB) and motor activity (MA) data were recorded for all animals during study week 12. Ophthalmic examinations were performed on all animals during study week -1 and for all surviving animals in Groups 1 and 4 during study week 12. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals assigned to the scheduled (study week 13) necropsy. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from the animals found dead and all animals in the control and high-dose groups at the scheduled necropsy. In addition, the stomach (nonglandular and glandular) and liver were identified as potential target tissues and were examined microscopically from all animals. Gross lesions were examined microscopically from all animals.

There were no test substance-related effects on survival. There were 7 animals found dead prior to the scheduled necropsy. One male and 1 female in the 75 mg/kg/day group were found dead on study days 25 and 31, respectively. One 150 mg/kg/day group male was found dead on study day 73. Two males in the 300 mg/kg/day were found dead on study days 19 and 68, and 2 females in the 300 mg/kg/day group were found dead on study days 67 and 85. The cause of death for these animals were either unknown and/or resultant from aspiration of the test substance and were not considered test substance-related. All other animals survived to the scheduled necropsy. There were no test substance-related effects on body weights, food consumption, hematology and coagulation, or urinalysis parameters. There were no test substance-related ophthalmic findings. FOB and MA assessments were unaffected by test substance administration. Test substance-related clinical observations noted for the 150 and 300 mg/kg/day group males and females at low incidences included clear material around the mouth/salivation. Test substance-related lower glucose values were noted in the 300 mg/kg/day group males and females and was considered nonadverse. A single test substance-related gross observation of dark red area in the glandular portion of the stomach was noted in a 300 mg/kg/day group female. Test substance-related effects on organ weights were noted in all treatment groups and included higher liver weights in the 75, 150, and 300 mg/kg/day group males and the 150 and 300 mg/kg/day group females.

Microscopic changes in the nonglandular stomach occurred in the region adjacent to the transition with the glandular portion (limiting ridge) in all dose groups. These findings were characterized by minimal degeneration of the superficial epithelium accompanied by minimal to mild accumulation of cellular debris (necrotic debris) in the stratum corneum and minimal neutrophilic inflammation in the underlying submucosa. Lesions in a few individual males progressed to erosion or ulceration. A single female in the 300 mg/kg/day group showed edema of the submucosa in this region, but no erosion or ulceration was observed. Also, the glandular stomach of a single female in the 300 mg/kg/day group had a single erosion in the mucosa. These gastric findings exhibited a dose response in males and were considered adverse for both males and females. Test substance-related nonadverse minimal hepatocellular hypertrophy was also noted in 2 females from the 300 mg/kg/day group and correlated with higher liver weights in these animals.


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
inhalation is not considered to be a relevant route of potential human exposure

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
inhalation is not considered to be a relevant route of potential human exposure

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Dermal is not considered to be a relevant route of potential human exposure

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Dermal is not considered to be a relevant route of potential human exposure

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: stomach

Justification for classification or non-classification

Following exposure of rats to m-DIPB by oral gavage for 4 weeks, the data indicate that test article-related effects were observed in the stomach, liver and kidneys. However the potential mechanism for these effects are related to adaptive responses due to local irritation (stomach/liver) and hyalin droplets (kidney) that are not relevant to human health.  Based upon these findings, m-DIPB does not satisfy the criteria for classification as an ocular irritant according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).