Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

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
Study period:
2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

- Pre-existing data:
There are no pre-existing data available that would address the pre-natal developmental toxicity study endpoint (GLP or non-GLP). A rat pre-natal developmental toxicity study is not adequate according to the regulation. There are no historical human data that could be used in place of this study.

- (Q)SAR:
There are no accepted QSAR approaches for predicting the outcome of this endpoint. However, there were no Reproductive Toxicity alerts for MiAK.

- In vitro methods
There are no accepted in vitro approaches for predicting the outcome of this endpoint

- Weight of evidence
WoE is not possible as there are not similar substances that could be used for such a comparison.

- Grouping and read-across
The Registrant has not identified any suitable analogues that could be used to provide data for this endpoint. Other similar substances are quite data poor.

- Substance-tailored exposure driven testing
Based on the use patterns of the registered substance in coatings, exposure-based waiving is not possible.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Test Substance Identification : 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate (TMPD-MIB)
Alternate Identification : Eastman Texanol™ ester alcohol
Batch/Lot No. : TD19034470 TD21007237
Expiration/Retest Date : 11 Dec 2022 19 Apr 2023
Purity : 99.3% 99.3%
Correction Factor None
Density 0.9464 g/mL
Storage Conditions 18°C to 24°C


Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity testing by regulatory agencies. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test substance. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Crl:CD(SD) rats were used as the test system for this study. This species and strain of animal is recognized as appropriate for subchronic 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-Day Oral Toxicity in Rodents”). A minimum of 10 animals/sex/group was 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 survive until the end of the study will be sufficient to permit a meaningful evaluation of toxicological effects. In addition to the main study groups, an interim dose group consisting of 5 animals/sex/group of control and high dose was treated for 30 days, sacrificed and the only parameters measured at necropsy were urinalysis and kidney weights and histology/immunohistochemistry.

Crl:CD(SD) rats were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 03 Feb 2021. The animals were approximately 6 weeks 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 implanted in the dorsoscapular area. All animals were housed for a 7-day acclimation. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior. Individual body weights and cage food weights were recorded, and detailed physical examinations were performed periodically during acclimation. Ophthalmic examinations were performed on all animals during acclimation prior to randomization.

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 2-3 of the same sex per cage. 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. The animals were temporarily separated as necessary to allow for the performance of protocol-specified activities. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at Charles River are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment.

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 Charles River. Reverse osmosis and ultraviolet irradiation 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 and during the conduct of FOBs and motor activity assessments when food, but not water, was withheld. Municipal water supplying the facility was analyzed for contaminants according to SOPs. The results of the diet and water analyses are maintained at Charles River. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. 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 68°F to 78°F (20°C to 26°C) and 30% to 70% respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. 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.

Route of administration:
oral: gavage
Details on route of administration:
Test substance formulations (TMPB-MIB 300,700 and 1000mg/kg/day and vehicle control were administered once daily, for 29 consecutive days (Interim Study animals) or for 90 consecutive days (Main Study animals) via oral gavage
Vehicle:
other: 0.5% methylcellulose (400 cps) and 0.1% Cremophor EL in deionized water
Details on oral exposure:
Vehicle control and test substance formulations 300, 700 and 1000 mg/kg/day were administered once daily, for 29 consecutive days (Interim Study animals- control & high dose group alone) or for 90 consecutive days (Main Study animals) via oral gavage.
Dose Volume : 5 mL/kg and Dose concentration : 60, 140 and 200 mg/mL for 300, 700 and 1000 mg/kg/day doses respectively
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by a gas chromatography (GC) method with flame ionization detection (FID) using a validated analytical procedure. Dose analysis results were verified prior to dose administration at each sampling interval.
Duration of treatment / exposure:
once daily for 29 consecutive days (Interim Study animals) or for 90 consecutive days (Main Study animals) via oral gavage.
Frequency of treatment:
Once Daily
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
60mg/mL
Dose / conc.:
700 mg/kg bw/day (actual dose received)
Remarks:
140 mg/mL
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
200 mg/mL
No. of animals per sex per dose:
Interim study : Vehilce & 1000 mg/kg/day TMPD-MIB group: 5 rats per sex per group
Main study : Vehicle, 300c, 700 and 1000 mg/kg/day TMPD-MIB group : 10 rats per sex per group
Control animals:
yes, concurrent vehicle
Details on study design:
Group No. Test Material Dose Level (mg/kg/day) Dose Volumea (mL/kg) Dose Concentration (mg/mL) Number of Animals
Interim Studyb Main Studyc
Males Females Males Females
1 Vehicle 0 5 0 5 5 10 10
2 TMPD-MIB 300 5 60 0 0 10 10
3 TMPD-MIB 700 5 140 0 0 10 10
4 TMPD-MIB 1000 5 200 5 5 10 10
a Based on the most recent body weight measurement. The first day of dosing was based on Day 1 body weights.
b Animals assigned to the Interim Study were euthanized on Day 30.
c Animals assigned to the Main Study were euthanized on Day 91.
Positive control:
No
Observations and examinations performed and frequency:
Animals were observed at least twice daily (morning and afternoon) for any mortality, beginning upon arrival through termination. 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 3 days of receipt, on the day of randomization, weekly (± 2 days) during the study period, and on the days of the scheduled necropsy. Due to social housing, some observations could not be attributed to a single animal.

Individual animal body weights were recorded for all the animals within 3 days of receipt, on the day of randomization, weekly (± 2 days) during the study period, and on the days of the scheduled necropsy. Food Consumption was recorded once weekly (± 2 days) throughout the study period, beginning on Day 1.

Ophthalmic examination (including Interim Study and unused alternate animals), neurobehavioral and motor activity assessments of all main study animals were performed once during the pretreatment period following randomization and near the end of the dosing period (Day 86-87). On the day of the scheduled necropsy, Vaginal lavages were performed from all main study female animals to determine the stage of estrous.
Sacrifice and pathology:
Samples for clinical Pathology analysis were collected on Day 30 from groups 1 and 4 (Interim Study animals) for urinalysis and on Day 91 from groups 1 through 4 for hematology, coagulation, clinical chemistry and urinalysis. On Day 91, blood samples were collected from group 1 through 4 and obtained serum was analyzed for throid hormones (T3, T4 and TSH).

A complete necropsy was conducted on all (interim and main study 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): Protocol specified organswere weighed at necropsy for all scheduled euthanasia animals.

Tissue Collection and Preservation
Animal identificationa Large intestine, cecum
Artery, aorta Large intestine, colon
Bone marrow, sternum Large intestine, rectum
Bone marrow, smear (from femur)b Larynx
Bone, sternum Liver
Brain Lung
Epididymis (2)c Lymph node, mandibular (2)f
Esophagus Lymph node, mesenteric
Eyes (2)d Macroscopic abnormalities (gross lesions; when possible)
Ganglion, dorsal root, lumbara,e Muscle, skeletal (2)f
Gland, adrenal (2) Nerve, optic (2)d
Gland, clitoral (2)a Nerve, sciatic (2)f
Gland, lacrimal (extra-orbital [2])a Nerve, tibial (2)a
Gland, Harderian (2)a Ovary (2)
Gland, mammary Oviduct (2)a
Gland, parathyroid (2) Pancreas
Gland, pituitary Skin
Gland, preputial (2)a Small intestine, duodenum
Gland, prostate with seminal vesicle and coagulating glands Small intestine, ileum
Gland, salivary, submandibular (2)f Small intestine, jejunum
Gland, salivary, sublingual (2)a Spinal cord
Gland, salivary, parotid (2)a Spleen
Gland, seminal vesicle (2) Stomach
Gland, thyroid (2) Testis (2)c
Gland, Zymbal’s (2)a Thymus
Gut-associated lymphoid tissueg Tongue
Heart Trachea
Kidney (2)h Ureter (2)a, Urinary bladder
Uterus/Cervix and Vagina
a Not processed for histology or examined microscopically.
b Bone marrow smears were obtained at scheduled and unscheduled necropsies, but not placed in formalin; slides were not processed for histology or examined microscopically.
c Preserved in modified Davidson’s fixative.
d Preserved in Davidson’s fixative.
e Collected, but not examined macroscopically.
f One of the pair was processed for histology and examined microscopically.
g From small intestine: Peyer’s patch or solitary lymphoid follicle.
h The left kidney was maintained in 10% neutral-buffered formalin for preparation of hematoxylin eosin stained paraffin sections, while the right
kidney was transferred to 70% ethanol after 48–72 hours for immunohistochemistry. At the interim necropsy, only kidneys were collected.


Organs Weighed at Necropsy:
Brain Kidneyb
Epididymis Liver
Gland, adrenal Ovary with oviduct
Gland, pituitary Spleen
Gland, prostate with gland, seminal- Testis
-vesicle and coagulating glands Thymus
Gland, thyroid with gland, parathyroida Uterus with cervix
Heart
a Weighed following fixation.
b At the interim necropsy, only kidneys were weighed.

Also refer to tables on hematology, coagulation, urinalysis parameters and thyroid hormone/ sample collection schedule in "any other information" on materials and methods session.
Statistics:
Refer to Statistical methods " any other information" on materials and methods session due to character limits in this field.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related clinical observations were noted in the ≥ 300 mg/kg/day group males and females.
Test substance-related clinical observations noted in the 300 mg/kg/day group males and females was limited to red fur staining around the mouth noted at 1–2 hours postdosing.
Test substance-related clinical observations noted in the 700 mg/kg/day group males and females included wet fur and/or red fur staining (mouth, lower jaw, muzzle, nose, ventral cervical, abdominal, forepaws, and/or periorbital areas) noted at 1–2 hours postdosing.
Test substance-related clinical observations noted in the 1000 mg/kg/day group males and females included wet fur and/or red fur staining (mouth, lower jaw, muzzle, nose, ventral cervical, abdominal, forepaws, and/or periorbital areas) noted at 1–2 hours postdosing. Observations of red fur staining around the mouth were noted in all test substance treated groups, with the highest incidence noted in the 1000 mg/kg/day group
Female No. 4512 (1000 mg/kg/day group) was noted with wet fur around the muzzle and sustained convulsions at approximately 1 hour postdosing on Day 86. Unlike Female No. 4509, Female No. 4512 recovered from the convulsions (within approximately 10 minutes) and was euthanized at the Day 91 terminal euthanasia. There were also no apparent test substances-related changes in neurobehavioral assessments and motor activity in Female No. 4512 on Day 85, nor in any other animal on study. Finally, there were no test substance related microscopic findings in the neurological tissues (brain, spinal cord, nerves) of any animal on study. Therefore, the convulsions noted in Female Nos. 4509 and 4512 were not considered related to the test substance
All other clinical observations in the test material-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.

Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no test substance-related effects on survival.
Female No. 4509 (1000 mg/kg group) was noted with wet fur around the mouth and sustained convulsions at approximately 1 hour postdosing on Day 80. On examination by the veterinary staff, the rat was noted to be actively convulsing and occasionally would not have full convulsions but head tremors. Due to the continued convulsions the animal was euthanized on Day 80. Dark red discoloration of the glandular stomach was observed macroscopically with no microscopic correlate. No test substance-related microscopic findings were noted in the nervous system or in remaining tissues evaluated; the cause of the convulsions was undetermined but not considered to be related to administration of the test substance. All other animals survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Test substance-related higher body weights were noted in the 300 and 700 mg/kg/day group males.
Test substance related higher mean body weight gains (statistically significant during Week 8 [Days 50–57] and cumulatively throughout the study period [Days 1-90]) were noted in the 300 mg/kg/day group males throughout the study period compared to the control group. This resulted in higher mean body weights at all scheduled intervals during the study period with mean body weights 9.7% higher compared to the control group at the end of the study period on Day 90.
Test substance related higher mean body weight gains (statistically significant from Week 2 [Days 8–15] to Week 4 [Days 22–29], during Week 8 [Days 50–57], and cumulatively throughout the Interim and Main Study periods [Days 1–29 and Days 1–90]) were noted in the 700 mg/kg/day group males throughout the study period compared to the control group. This resulted in higher mean body weights at all scheduled intervals (statistically significant from Day 22 to 90) with mean body weights 13.8% higher compared to the control group on Day 90.
There were no other test substance-related effects on body weight. Any other differences in body weights and/or body weight gains, regardless of statistical significance, were considered not to be toxicologically relevant and were attributed to biological variation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Test substance-related higher food consumption was noted in the 300 and 700 mg/kg/day group males.
Test substance related higher mean food consumption (up to 17.0% higher, occasionally statistically significant) was noted in the 300 mg/kg/day group males throughout the study period (except during Week 5 [Days 29–36]) compared to the control group. This correlated to the higher mean body weights and body weight gains noted throughout the study period.
Test substance related higher mean food consumption (up to 35.4% higher, frequently statistically significant) was noted in the 700 mg/kg/day group males throughout the study period compared to the control group. This correlated to the higher mean body weights and body weight gains noted throughout the study period.
Higher mean food consumption (up to 40.3% higher, intermittently statistically significant) was noted in the 300 mg/kg/day group females beginning Week 2 (Days 8–15) and continuing throughout the dosing period compared to the control group. However, given the lack of a dose response and correlation to body weights, the relationship to the test substance was uncertain.
There were no other test substance-related effects on food consumption. Any other differences in food consumption, regardless of statistical significance, were considered not to be toxicologically relevant and were attributed to biological variation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
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.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related alterations in hematology parameters were noted in the ≥ 700 mg/kg/day group males and 1000 mg/kg/day group females.
Test substance-related alterations in hematology parameters were limited to lower red blood cell counts (RBC), hemoglobin (HGB), and hematocrit (HCT) and higher reticulocytes (RETIC) and red cell distribution width (RDW) noted in the ≥ 700 mg/kg/day group males and 1000 mg/kg/day group females at the terminal euthanasia (Day 91). Although mean values in these groups were not always statistically significant from the control group nor dose-responsive, a sufficient number of individual values within each test substance-treated group were outside the range of individual values in the respective control groups and these changes were part of trend seen in both sexes at higher dosage levels that may indicate that the animals had recovered from anemia. These mild changes were considered nonadverse. Any other differences in hematology values, regardless of statistical significance, were attributed to biologic variation and were thus considered unrelated to test substance administration.
Coagulation: Test substance-related alterations in coagulation parameters were noted in the ≥ 700 mg/kg/day group females. Test substance-related alterations in coagulation parameters were limited to higher mean fibrinogen (FIB) noted in the females at ≥ 700 mg/kg/day at the terminal euthanasia (Day 91) when compared to the control group. There was no biological relevance of this nonadverse change. There were no other test substance-related effects on coagulation parameters. There were no other statistically significant differences when the control and test substance treated groups were compared.


Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related alterations in clinical chemistry parameters were noted in the ≥ 300 mg/kg/day group males and females. Higher cholesterol (CHOL) was noted in the ≥ 300 mg/kg/day group males and females at the terminal euthanasia (Day 91) when compared to the control group, with correlative higher high density lipoprotein cholesterol (HDL) and low density lipoprotein cholesterol (LDL) in the ≥ 300 mg/kg/day group males and in the ≥ 700 mg/kg/day group females. In addition, test substance-related lower mean total bilirubin (TBIL) was noted in the 1000 mg/kg/day group females (Day 91). All of these changes were considered adaptive and nonadverse and may be related to microsomal enzyme induction. There were no other test substance-related effects on clinical chemistry parameters. Any other differences in clinical chemistry values, regardless of statistical significance, were attributed to biologic variation and were thus considered unrelated to test substance administration.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis: There were no clear test substance-related effects on thyroid hormones.
Higher mean T4 was noted in the 700 and 1000 mg/kg/day group males and 1000 mg/kg/day group females on Day 91. Higher mean TSH was noted in the 1000 mg/kg/day group males and females on Day 91. However, not all of these mean changes were statistically significant. Changes were driven by a small number of individual animals with large outlying values and most values in the 1000 mg/kg/day group were within the range of values of the respective control group. Therefore, the relationship of these changes to test substance administration in and of themselves was uncertain. However, given the changes noted in the liver and thyroid gland weights, correlating histological changes, and that the downstream effects of hepatocellular enzyme induction often leads to similar changes in serum thyroid hormones, these changes were considered likely test substance-related, but nonadverse.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related alterations in urinalysis parameters were noted in the 1000 mg/kg/day group males and females. Test substance related higher mean urine volume (males only) and lower mean urine pH were noted in the 700 mg/kg/day group males and females at the terminal euthanasia (Day 91) when compared to the control group.
Test substance related higher mean urine volume and lower mean urine pH were noted in the 1000 mg/kg/day group males and females at the interim euthanasia (Day 30) and higher mean urine volume (males only) and lower mean urine pH were noted in the 1000 mg/kg/day group males and females at the terminal euthanasia (Day 91) when compared to the control group. Changes in urinalysis data were considered to be nonadverse.
There were no other test substance-related effects on urinalysis parameters. There were no other statistically significant differences when the control and test substance treated groups were compared.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Neurobehavioral Assessments
Activity observations, autonomic observations, exicitability observations, neuromuscular observations, physiological and sensorimotor observationsActivity observations were unaffected by test substance administration. There were no differences between the control and test substance-treated groups (by sex) at the Day 86 evaluation and/or the values were similar to those noted during the pretreatment evaluation.
Motor activity patterns (total and ambulatory activity counts) were unaffected by test substance administration. 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 Week 13. The only statistically significant effect was a mean lower male ambulatory activity at 700 mg/kg/day when all intervals are combined. However, there was no statistical significance when male total activity was evaluated and no similar effect on activity in the 1000 mg/kg/day group males and was thus considered unrelated to administration of the test substance. Any other differences noted, regardless of statistical significance, were attributed to biological variation, and were thus considered unrelated to administration of the test substance.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Interim Euthanasia Animals (Day 30): Test substance-related statistically significantly higher mean kidney weights (absolute and relative to terminal body weight) were noted in the 1000 mg/kg/day group males.

Terminal Euthanasia Animals (Day 91): Test substance-related higher mean kidney weights noted at the interim euthanasia were observed at the end of the terminal period (Day 91). In addition, higher mean liver and thyroid gland weights were noted at the terminal euthanasia. Higher mean kidney weights noted in the 700 and 1000 mg/kg/day group males had a weak dose response relationship, but correlated microscopically with alpha2u-globulin nephropathy. Higher mean liver weights correlated with hepatocellular hypertrophy. Higher mean thyroid/parathyroid weights correlated with follicular cell hypertrophy; higher mean thyroid gland weights lacked a clear dose response relationship in the test substance treated group females but correlated with follicular cell hypertrophy and were thus
considered to be test substance related.
No other test substance-related organ weight changes were noted. There were other isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, other organ weight differences observed were considered incidental and unrelated to administration of the test substance.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Interim Euthanasia Animals (Day 30): No test substance-related macroscopic findings were noted. The macroscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test substance. Dark red foci in the renal cortex were observed in the control and with a higher incidence in the 1000 mg/kg/day group males when compared with the control. There were no microscopic correlates in the control and 1000 mg/kg/day groups, thus the observation was considered a perimortem change unrelated to administration of the test substance.

Terminal Euthanasia Animals (Day 91): No test substance-related macroscopic findings were noted. The macroscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Interim Euthanasia Animals (Day 30): Minimal alpha2u-globulin nephropathy was characterized by increased concentration, size, and area of distribution of brightly eosinophilic renal epithelial cytoplasmic droplets (hyaline droplets) concentrated in the S2 segment of proximal convoluted tubules in hematoxylin and eosin stained kidney sections when compared to the control group males. Mild alpha2u-globulin nephropathy was characterized by similarly increased intracytoplasmic droplets with one or more of the following tubular changes: rare basophilic tubules, rare epithelial cell necrosis/exfoliation, and/or 1 to 2 granular casts at the junction between outer and inner stripes of the outer medulla per kidney section. Droplets stained positive with both the Mallory Heidenhain and alpha2u globulin immunohistochemistry (IHC) stains in the control and 1000 mg/kg/day group males with greater staining distribution and intensity at 1000 mg/kg/day, confirming the intracytoplasmic accumulation of protein and alpha2u globulin, respectively. Some alpha2u globulin staining in control male sections is normal, as alpha2u-globulin is a constitutive protein in the male rat kidney.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Terminal Euthanasia Animals (Day 91): The microscopic characteristics of minimal and mild alpha2u-globulin nephropathy. Moderate alpha2u-globulin nephropathy was characterized by increased concentration, size, and area of distribution of hyaline droplets and frequent tubular changes including basophilic tubules, rare epithelial cell necrosis/exfoliation, and greater than 3 granular casts per section of kidney; karyomegaly was not a microscopic feature. Droplets stained positive with both the Mallory Heidenhain and alpha2u-globulin IHC stains in the control and test substance administered group males with greater staining distribution and intensity in the test substance administered groups.
Increased incidence and severity grade of chronic progressive nephropathy was noted in the 700 and 1000 mg/kg/day group males. This common spontaneous change in male and females rats was characterized by increased incidence and severity grade of a spectrum of renal tubular changes including increased basophilia of renal tubules, thickened tubular basement membranes, and infiltration by variable numbers of mononuclear cells. The microscopic features of chronic progressive nephropathy were thus distinct from those of alpha2u-globulin nephropathy. The increased incidence and severity grade of chronic progressive nephropathy was considered consistent with alpha2u-globulin-related accentuation in rats (Dill et al., 2003).
Hepatocellular hypertrophy was characterized by increased homogeneous eosinophilic hepatocellular cytoplasm in a centrilobular distribution.
Within the thyroid gland, follicular cell hypertrophy was characterized by increased follicular cell height often associated with lower follicular colloid content.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test substance.

Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
no

Test Substance-Related Hematology Changes

Group

2

3

4

Dose (mg/kg/day)

300

700

1000

Sex

M

F

M

F

M

F

RBC

 

 

 

 

 

 

   Day 91

0.95x

0.96x

0.97x

HGB

 

 

 

 

 

 

   Day 91

0.95x*

0.96x*

0.98x

HCT

 

 

 

 

 

 

   Day 91

0.96x*

0.96x

0.98x

RDW

 

 

 

 

 

 

   Day 91

1.06x**

1.02x

1.05x*

RETIC

 

 

 

 

 

 

   Day 91

1.21x

1.07x

1.27x*

M = males; F = females.

* = Statistically significantly different from the control group at ≤ 0.05 using Dunnett’s test.

** = Statistically significantly different from the control group at ≤ 0.01 using Dunnett’s test.

A dash (—) indicates absence of change. Numerical values indicate fold change of the treated group mean value relative to the control group mean value.

Test Substance-Related Coagulation Changes

Group

2

3

4

Dose (mg/kg/day)

300

700

1000

Sex

M

F

M

F

M

F

FIB

 

 

 

 

 

 

   Day 91

1.12x*

1.15x**

M = males; F = females.

* = Statistically significantly different from the control group at ≤ 0.05 using Dunnett’s test.

** = Statistically significantly different from the control group at ≤ 0.01 using Dunnett’s test.

A dash (—) indicates absence of change. Numerical values indicate fold change of the treated group mean value relative to the control group mean value.

Test Substance-Related Clinical Chemistry Changes

Group

2

3

4

Dose (mg/kg/day)

300

700

1000

Sex

M

F

M

F

M

F

TBIL

 

 

 

 

 

 

   Day 91

0.31x**

CHOL

 

 

 

 

 

 

   Day 91

1.36x

1.16x

1.26x

1.18x

1.30x

1.27x*

HDL

 

 

 

 

 

 

   Day 91

1.31x**

1.19x

1.22x*

1.37x*

1.26x**

LDL

 

 

 

 

 

 

   Day 91

1.47x

1.45x

1.25x

1.40x

1.50x*

M = males; F = females.

* = Statistically significantly different from the control group at ≤ 0.05 using Dunnett’s test.

** = Statistically significantly different from the control group at ≤ 0.01 using Dunnett’s/Dunn’s test.

A dash (—) indicates absence of change. Numerical values indicate fold change of the treated group mean value relative to the control group mean value.

Test Substance-Related Urinalysis Changes

Group

2

3

4

Dose (mg/kg/day)

300

700

1000

Sex

M

F

M

F

M

F

Urine volume

 

 

 

 

 

 

   Day 30

N/A

N/A

N/A

N/A

1.85x

3.00x

   Day 91

1.88x*

3.32x*

Urine pH

 

 

 

 

 

 

   Day 30

N/A

N/A

N/A

N/A

0.82x**

0.90x

   Day 91

0.88x**

0.85x*

0.89x**

0.81x**

M = males; F = females; N/A = not applicable.

* = Statistically significantly different from the control group at ≤ 0.05 using Dunnett’s/Dunn’s test.

** = Statistically significantly different from the control group at ≤ 0.01 using Dunnett’s/Dunn’s test.

A dash (—) indicates absence of change. Numerical values indicate fold change of the treated group mean value relative to the control group mean value.

Summary of Organ Weight Data – Interim Euthanasia (Day 30)a

Sex

Males

Females

Group

1

4

1

4

Dose (mg/kg/day)

0

1000

0

1000

No. Animals per Group

5

5

5

5

Kidney (No. Weighed)

(5)

(5)

(5)

(5)

Absolute value

3.27

3.82**

1.73

1.83

    Standard deviation

0.16

0.30

0.20

0.18

    % difference

N/A

16.79

N/A

5.84

% of body weight

0.788

0.94**

0.734

0.78

    Standard deviation

0.04

0.04

0.07

0.06

    % difference

N/A

19.90

N/A

5.88

N/A = not applicable.

a  Organ weight values rounded to the appropriate decimal place to account for the size of the organ (values presented to all decimal places in Provantis tables).

** = Statistically significantly different from the control group at ≤ 0.01 using Dunnett’s test.

Bold= Values considered to be test substance-related.

Summary of Organ Weight Data – Terminal Euthanasia (Day 91)a

Sex

Males

Females

Group

1

2

3

4

1

2

3

4

Dose (mg/kg/day)

0

300

700

1000

0

300

700

1000

No. Animals per Group

10

10

10

10

10

10

10

10

Kidney (No. Weighed)

(10)

(10)

(9)

(10)

(10)

(10)

(10)

(9)

Absolute value

3.65

4.01

4.78**

4.06

1.89

2.05

2.09

2.04

    Standard deviation

0.40

0.40

0.53

0.23

0.19

0.21

0.20

0.21

    % difference

N//A

9.68

30.98

11.26

N/A

8.15

10.28

7.82

% of body weight

0.704

0.705

0.805**

0.763

0.668

0.682

0.714

0.690

    Standard deviation

0.06

0.05

0.09

0.07

0.05

0.05

0.04

0.04

    % difference

N/A

0.21

14.38

8.47

N/A

2.01

6.83

3.31

% of brain weight

166.91

180.46

207.12**

185.60*

94.70

100.43

99.35

101.19

    Standard deviation

18.82

11.01

22.40

13.37

9.73

10.22

7.92

10.72

    % difference

N/A

8.12

24.09

11.20

N/A

6.05

4.91

6.86

Liver (No. Weighed)

(10)

(10)

(10)

(10)

(10)

(10)

(10)

(9)

Absolute value

14.77

17.87**

20.34**

20.89**

8.26

9.92**

10.85**

11.52**

    Standard deviation

1.70

1.84

1.81

2.66

0.86

0.94

1.05

1.54

    % difference

N/A

20.99

37.75

41.47

N/A

20.06

31.34

39.48

% of body weight

2.84

3.14**

3.44**

3.90**

2.91

3.30**

3.71**

3.88**

    Standard deviation

0.17

0.14

0.16

0.26

0.21

0.17

0.20

0.27

    % difference

N/A

10.46

20.91

37.15

N/A

13.30

27.32

33.35

% of brain weight

675.22

805.05**

882.85**

953.25**

412.52

486.18*

515.96**

571.33**

    Standard deviation

81.49

55.89

70.36

122.08

41.13

46.40

44.34

82.62

    % difference

N/A

19.23

30.75

41.18

N/A

17.85

25.07

38.50**

Thyroid/parathyroid (No. Weighed)

(10)

(10)

(10)

(10)

(10)

(10)

(9)

(9)

Absolute value

0.0204

0.0222

0.0253*

0.0246*

0.0169

0.0211

0.0210

0.0194

    Standard deviation

0.0029

0.0037

0.0037

0.0038

0.0024

0.0037

0.0052

0.0043

    % difference

N/A

9.09

24.13

20.98

N/A

24.57

23.65

14.33

% of body weight

0.0039

0.0039

0.0043

0.0047

0.0060

0.0070

0.0073

0.0065

    Standard deviation

0.0006

0.0005

0.0006

0.0011

0.0009

0.0012

0.0017

0.0014

    % difference

N/A

-0.98

8.42

18.79

N/A

17.47

21.14

9.21

% of brain weight

0.9279

1.0000

1.0955*

1.1222*

0.8450

1.0326

0.9871

0.9600

    Standard deviation

0.1163

0.1485

0.1497

0.1649

0.1131

0.1713

0.2204

0.2192

    % difference

N/A

7.77

18.07

20.93

N/A

22.20

16.82

13.58

N/A = not applicable.

a  Organ weight values rounded to the appropriate decimal place to account for the size of the organ (values presented to all decimal places in Provantis tables).

* = Statistically significantly different from the control group at ≤ 0.05 using Dunnett’s test.

** = Statistically significantly different from the control group at ≤ 0.01 using Dunnett’s test.

Bold= Values considered to be test substance-related.

Summary of Microscopic Findings – Interim Euthanasia (Day 30)

Sex

Males

Females

Group

1

4

1

4

Dose (mg/kg/day)

0

1000

0

1000

No. Animals per Group

5

5

5

5

Kidney (No. Examined)

(5)

(5)

(5)

(5)

Alpha2u-globulin nephropathy

0

5

0

0

     Minimal

-

1

-

-

     Mild

-

4

-

-

Alpha2u-globulin IHC positive

5

5

0

0

     Minimal

5

0

-

-

     Mild

0

5

-

-

Mallory Heidenhain positive

5

5

0

1

     Minimal

5

0

-

1

     Moderate

0

5

-

0

- - no noteworthy findings.

Summary of Microscopic Findings – Terminal Euthanasia (Day 91)

Sex

Males

Females

Group

1

2

3

4

1

2

3

4

Dose (mg/kg/day)

0

300

700

1000

0

300

700

1000

No. Animals per Group

10

10

10

10

10

10

10

10

Kidney (No. Examined)

(10)

(10)

(10)

(10)

(10)

(10)

(10)

(9)

Alpha2u-globulin nephropathy

0

2

7

10

0

0

0

0

     Minimal

-

2

3

2

-

-

-

-

     Mild

-

0

2

2

-

-

-

-

     Moderate

-

0

2

6

-

-

-

-

Alpha2u-globulin IHC positive

10

10

10

10

0

0

0

0

     Minimal

7

1

0

0

-

-

-

-

     Mild

3

8

7

5

-

-

-

-

     Moderate

0

1

3

5

-

-

-

-

Mallory Heidenhain positive

10

10

10

10

0

0

0

0

     Minimal

10

5

0

0

-

-

-

-

     Mild

0

5

6

7

-

-

-

-

     Moderate

0

0

4

3

-

-

-

-

Chronic progressive nephropathy

1

1

6

9

1

0

0

0

     Minimal

1

1

4

8

1

-

-

-

     Mild

0

0

2

1

0

-

-

-

Liver (No. Examined)

(10)

(10)

(10)

(10)

(10)

(10)

(10)

(9)

Hypertrophy; hepatocellular

0

9

9

9

0

5

8

7

     Minimal

-

8

8

9

-

5

7

6

     Mild

-

1

1

0

-

0

1

1

Gland, thyroid (No. Examined)

(10)

(10)

(10)

(10)

(10)

(10)

(10)

(9)

Hypertrophy; follicular cell

0

3

8

7

0

1

3

3

     Minimal

-

3

8

7

-

1

3

3

- = no noteworthy findings.

Estrous Cyclicity: Vaginal lavages were collected from the primary study females on the day of necropsy to assist in histological evaluation of estrogen-sensitive tissues. The study pathologist did not identify any test substance-related microscopic findings in female estrogen-sensitive tissues.

Conclusions:
In conclusion, administration of TMPD-MIB by once daily oral gavage to Crl:CD(SD) rats at dose levels of 300, 700, and 1000 mg/kg/day for 90 consecutive days or at 1000 mg/kg/day for 29 consecutive days resulted in adverse renal changes at 1000 mg/kg/day in males at the interim euthanasia, and adverse renal changes at 700 and 1000 mg/kg/day in males at the terminal euthanasia. The findings were characterized by alpha2u‑globulin nephropathy with correlating chronic progressive nephropathy and higher kidney weights. All other test substance-related findings were considered nonadverse. Based on these results, the no-observed-adverse-effect level (NOAEL) following 90 consecutive days of dosing was considered to be 300 mg/kg/day for male rats and 1000 mg/kg/day for female rats.
Executive summary:

The objective of this study was to determine the potential toxicity of 2,2,4‑trimethyl‑1,3‑pentanediol monoisobutyrate (TMPD-MIB), when given by oral gavage for at least 90 consecutive days to Sprague Dawley rats. In addition, this study included an interim necropsy examination to evaluate the potential toxicity of the test substance on the kidneys.

Interim Study animals were dosed once daily for 29 consecutive days via oral gavage. Main Study animals were dosed once daily for 90 consecutive days via oral gavage.

The following parameters and end points were evaluated in the Interim Study: mortality, clinical signs, body weights, body weight gains, food consumption, and urinalysis. Also, the following parameters were examined in the kidney: macroscopic examination, organ weight, and microscopic examination with hematoxylin and eosin (H&E) staining, Mallory Heidenhain staining, and alpha2u‑globulin staining.

The following parameters and end points were evaluated in the Main Study: mortality, clinical signs, bodyweights, body weight gains, food consumption, estrous cyclicity, ophthalmology, functional observational battery, motor activity, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), thyroid hormone analysis, organ weights, and macroscopic and microscopic examinations.

In the Main Study, there were no test substance-related effects on mortality, estrous cyclicity, functional observational battery, or motor activity. There were no test substance-related ophthalmic or macroscopic findings.

The analyzed dosing formulations were within the protocol-specified range of target concentrations for suspensions and were homogeneous.

Test substance-related clinical observations noted in the 300 mg/kg/day group males and females was limited to red fur staining around the mouth. Test substance-related clinical observations noted in the 700 and 1000 mg/kg/day group males and females included wet fur and/or red fur staining (mouth, lower jaw, muzzle, nose, ventral cervical, abdominal, forepaws, and/or periorbital areas).

Test substance-related higher body weights were noted in the 300 and 700 mg/kg/day group males throughout the study period.

Test substance-related higher food consumption was noted in the 300 and 700 mg/kg/day group males throughout the study period and correlated to the higher body weights and body weight gains.

Non-adverse, test substance-related alterations in hematology parameters were limited to lower red blood cells, hemoglobin, and hematocrit and higher reticulocytes and red cell distribution width (males only) noted in the ≥ 700 mg/kg/day group males and 1000 mg/kg/day group females at the terminal euthanasia (Day 91).

Non-adverse, test substance-related alterations in coagulation parameters were limited to higher fibrinogen noted in the females at ≥ 700 mg/kg/day at the terminal euthanasia (Day 91).

Non-adverse, test substance‑related alterations in clinical chemistry parameters included higher cholesterol in the ≥ 300 mg/kg/day group males and females (with correlative higherhigh density lipoprotein cholesterol [HDL] and lowdensity lipoprotein cholesterol [LDL] in the ≥ 300 mg/kg/day group males and in the ≥ 700 mg/kg/day group females), and lower total bilirubin in the 1000 mg/kg/day group female at the terminal euthanasia (Day 91).

Non-adverse, test substance‑related alterations in urinalysis parameters at the interim euthanasia (Day 30) included higher urine volume and lower urine pH in the 1000 mg/kg/day group males and females. In addition, test substance-related higher urine volume (males only) and lower urine pH were noted in the 700 and 1000 mg/kg/day group males and females at the terminal euthanasia (Day 91).

Test substance-related alterations in organ weights included higher kidney weights in the 1000 mg/kg/day group males at the interim euthanasia (Day 30) and in the ≥ 700 mg/kg/day group males at the terminal euthanasia (Day 91). In addition, test substance-related higher liver weights were noted in the ≥ 300 mg/kg/day group males and females and higher thyroid gland weights were noted in the ≥ 700 mg/kg/day group males and females at the terminal euthanasia (Day 91).

Test substance-related microscopic findings included accumulation of alpha2u-globulin, which was confirmed by positive alpha2u-globulin IHC stains. The protein composition of droplets was confirmed by Mallory Heidenhain stains. Minimal to mild alpha2u‑globulin nephropathy was noted in the 1000 mg/kg/day group males at the interim euthanasia (Day 30) and minimal to moderate alpha2u-globulin nephropathy in the ≥ 300 mg/kg/day group males at the terminal euthanasia (Day 91). Chronic progressive nephropathy was additionally noted with increased incidence and severity in the 700 and 1000 mg/kg/day group males, correlated with higher kidney weights, and was considered adverse at ≥ 700 mg/kg/day.

Additionally, test substance-related minimal to mild hepatocellular hypertrophy in the liver and minimal follicular cell hypertrophy within the thyroid gland were noted in the ≥ 300 mg/kg/day group males and females at the terminal euthanasia (Day 91), which correlated with higher liver and thyroid/parathyroid weights. Liver and thyroid gland changes were considered adaptive and nonadverse.

Higher mean T4 was noted in the 700 and 1000 mg/kg/day group males and 1000 mg/kg/day group females on Day 91. Higher mean TSH was noted in the 1000 mg/kg/day group males and females on Day 91. Although the relationship of these serum thyroid changes to the test substance was not clear in and of themselves, given the changes noted in the liver and thyroid gland weights and correlating histological changes and that the downstream effects of hepatocellular enzyme induction often leads to similar changes in serum thyroid hormones, these changes were considered likely test substance-related, but nonadverse.

In conclusion, administration of TMPD-MIB by once daily oral gavage to Crl:CD(SD) rats at dose levels of 300, 700, and 1000 mg/kg/day for 90 consecutive days or at 1000 mg/kg/day for 29 consecutive days resulted in adverse renal changes at 1000 mg/kg/day in males at the interim euthanasia, and adverse renal changes at 700 and 1000 mg/kg/day in males at the terminal euthanasia. The findings were characterized by alpha2u‑globulin nephropathy with correlating chronic progressive nephropathy and higher kidney weights. All other test substance-related findings were considered nonadverse. Based on these results, the no-observed-adverse-effect level (NOAEL) following 90 consecutive days of dosing was considered to be 300 mg/kg/day for male rats and 1000 mg/kg/day for female rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
System:
other: Increased various organ weights in males only
Organ:
kidney
liver
thyroid gland

Additional information

The objective of this study was to determine the potential toxicity of 2,2,4‑trimethyl‑1,3‑pentanediol monoisobutyrate (TMPD-MIB), when given by oral gavage for at least 90 consecutive days to Sprague Dawley rats. In addition, this study included an interim necropsy examination to evaluate the potential toxicity of the test substance on the kidneys.

Interim Study animals were dosed once daily for 29 consecutive days via oral gavage. Main Study animals were dosed once daily for 90 consecutive days via oral gavage.

The following parameters and end points were evaluated in the Interim Study: mortality, clinical signs, body weights, body weight gains, food consumption, and urinalysis. Also, the following parameters were examined in the kidney: macroscopic examination, organ weight, and microscopic examination with hematoxylin and eosin (H&E) staining, Mallory Heidenhain staining, and alpha2u‑globulin staining.

The following parameters and end points were evaluated in the Main Study: mortality, clinical signs, bodyweights, body weight gains, food consumption, estrous cyclicity, ophthalmology, functional observational battery, motor activity, clinical pathology parameters (hematology, coagulation, clinical chemistry, and urinalysis), thyroid hormone analysis, organ weights, and macroscopic and microscopic examinations.

In the Main Study, there were no test substance-related effects on mortality, estrous cyclicity, functional observational battery, or motor activity. There were no test substance-related ophthalmic or macroscopic findings.

The analyzed dosing formulations were within the protocol-specified range of target concentrations for suspensions and were homogeneous.

Test substance-related clinical observations noted in the 300 mg/kg/day group males and females was limited to red fur staining around the mouth. Test substance-related clinical observations noted in the 700 and 1000 mg/kg/day group males and females included wet fur and/or red fur staining (mouth, lower jaw, muzzle, nose, ventral cervical, abdominal, forepaws, and/or periorbital areas).

Test substance-related higher body weights were noted in the 300 and 700 mg/kg/day group males throughout the study period.

Test substance-related higher food consumption was noted in the 300 and 700 mg/kg/day group males throughout the study period and correlated to the higher body weights and body weight gains.

Non-adverse, test substance-related alterations in hematology parameters were limited to lower red blood cells, hemoglobin, and hematocrit and higher reticulocytes and red cell distribution width (males only) noted in the ≥ 700 mg/kg/day group males and 1000 mg/kg/day group females at the terminal euthanasia (Day 91).

Non-adverse, test substance-related alterations in coagulation parameters were limited to higher fibrinogen noted in the females at ≥ 700 mg/kg/day at the terminal euthanasia (Day 91).

Non-adverse, test substance‑related alterations in clinical chemistry parameters included higher cholesterol in the ≥ 300 mg/kg/day group males and females (with correlative higherhigh density lipoprotein cholesterol [HDL] and lowdensity lipoprotein cholesterol [LDL] in the ≥ 300 mg/kg/day group males and in the ≥ 700 mg/kg/day group females), and lower total bilirubin in the 1000 mg/kg/day group female at the terminal euthanasia (Day 91).

Non-adverse, test substance‑related alterations in urinalysis parameters at the interim euthanasia (Day 30) included higher urine volume and lower urine pH in the 1000 mg/kg/day group males and females. In addition, test substance-related higher urine volume (males only) and lower urine pH were noted in the 700 and 1000 mg/kg/day group males and females at the terminal euthanasia (Day 91).

Test substance-related alterations in organ weights included higher kidney weights in the 1000 mg/kg/day group males at the interim euthanasia (Day 30) and in the ≥ 700 mg/kg/day group males at the terminal euthanasia (Day 91). In addition, test substance-related higher liver weights were noted in the ≥ 300 mg/kg/day group males and females and higher thyroid gland weights were noted in the ≥ 700 mg/kg/day group males and females at the terminal euthanasia (Day 91).

Test substance-related microscopic findings included accumulation of alpha2u-globulin, which was confirmed by positive alpha2u-globulin IHC stains. The protein composition of droplets was confirmed by Mallory Heidenhain stains. Minimal to mild alpha2u‑globulin nephropathy was noted in the 1000 mg/kg/day group males at the interim euthanasia (Day 30) and minimal to moderate alpha2u-globulin nephropathy in the ≥ 300 mg/kg/day group males at the terminal euthanasia (Day 91). Chronic progressive nephropathy was additionally noted with increased incidence and severity in the 700 and 1000 mg/kg/day group males, correlated with higher kidney weights, and was considered adverse at ≥ 700 mg/kg/day.

Additionally, test substance-related minimal to mild hepatocellular hypertrophy in the liver and minimal follicular cell hypertrophy within the thyroid gland were noted in the ≥ 300 mg/kg/day group males and females at the terminal euthanasia (Day 91), which correlated with higher liver and thyroid/parathyroid weights. Liver and thyroid gland changes were considered adaptive and nonadverse.

Higher mean T4 was noted in the 700 and 1000 mg/kg/day group males and 1000 mg/kg/day group females on Day 91. Higher mean TSH was noted in the 1000 mg/kg/day group males and females on Day 91. Although the relationship of these serum thyroid changes to the test substance was not clear in and of themselves, given the changes noted in the liver and thyroid gland weights and correlating histological changes and that the downstream effects of hepatocellular enzyme induction often leads to similar changes in serum thyroid hormones, these changes were considered likely test substance-related, but nonadverse.

In conclusion, administration of TMPD-MIB by once daily oral gavage to Crl:CD(SD) rats at dose levels of 300, 700, and 1000 mg/kg/day for 90 consecutive days or at 1000 mg/kg/day for 29 consecutive days resulted in adverse renal changes at 1000 mg/kg/day in males at the interim euthanasia, and adverse renal changes at 700 and 1000 mg/kg/day in males at the terminal euthanasia. The findings were characterized by alpha2u‑globulin nephropathy with correlating chronic progressive nephropathy and higher kidney weights. All other test substance-related findings were considered nonadverse. Based on these results, the no-observed-adverse-effect level (NOAEL) following 90 consecutive days of dosing was considered to be 300 mg/kg/day for male rats and 1000 mg/kg/day for female rats.

Justification for classification or non-classification

The primary systemic effects found in the key 90 -day repeat was small changes in the organ weights from the liver, thyroid and kidney. The effects were small, but significant and were not corrorborated by other findings such as histopathology or clinical chemistry. Thus, neither STOT classification is warranted and the test article is not classified based on repeat dose studies.