p-(1,1-dimethylpropyl)phenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-01 to 2012-01-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

(1998)

Deviations:

yes

Remarks:

Minor protocol deviations not affecting the outcome of the study were noted

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: U.S. Food and Drug Administration, Office of Food Additive Safety. Toxicological principles for the safety assessment of food ingredients.

Version / remarks:

Redbook 2000: 2000 July 7; updated October 2001, November 2003, and April 2004.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

p-(1,1-dimethylpropyl)phenol

EC Number:

201-280-9

EC Name:

p-(1,1-dimethylpropyl)phenol

Cas Number:

80-46-6

Molecular formula:

C11H16O

IUPAC Name:

4-(2-methylbutan-2-yl)phenol

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): phenol, 4-(1,1-dimethylpropyl)-
- Lot/batch No.: 20409018

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 53 days
- Weight at study initiation: 80 male and 80 female animals (weighing 216 to 247 g and 167 to 203 g
- Fasting period before study: Not Stated
- Housing: individually housed in suspended, stainless steel, wire-mesh type cages in an environmentally controlled room
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

DETAILS OF FOOD AND WATER QUALITY: Block Lab Diet® (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum, except during designated periods. The lot number from each diet lot used for this study was recorded. Certification analysis of each diet lot was performed by the manufacturer. Tap water was available ad libitum via an automatic watering system.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2011-11-01 To: 2012-01-31

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

The vehicle and test article were administered once daily for 90 or 91 consecutive days during the study via oral gavage; depending on the animals’ scheduled day of termination. The dose levels of 50, 200, and 600 mg/kg/day were administered at a dose volume of 10 mL/kg. The control group received the vehicle in the same manner as the treated groups. The vehicle and test article were withdrawn from stirred formulations throughout administration. Individual doses were based on the most recent body weights.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations prepared for the study were evaluated for homogeneity and concentration. While the preparations were stirring, appropriate samples were collected using a positive displacement pipette and placed into 20 mL amber glass scintillation vials.

Samples were stored refrigerated at 2 to 8 °C until analyzed. All analytical work was conducted by MPI Research, Inc., Mattawan, Michigan, using an analytical method developed and validated under MPI Research Study Number 1038-017.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral LD50 has been demonstrated to be 1830 mg/kg in rats in previous toxicity studies. In a two week range-finding toxicity study (MPI Research Study Number 1038-014) body weight gain was progressively lower at 1000 mg/kg/day. Evaluation of the results indicated that animals would not likely tolerate 1000 mg/kg/day (limit dose) for 90 days, so 600 mg/kg/day was chosen as the high dose for this study. The mid (50 mg/kg/day) and low (200 mg/kg/day) doses are approximate log intervals of the high dose level.

- Rationale for animal assignment (if not random): Random

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily. A cage-side clinical observation of each animal was performed once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed clinical examination of each animal was performed prior to the initiation of test article administration and weekly during the study.
- Parameters checked: The observations included, but were not limited to changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture, and reactivity to handling, as well as the presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights for all animals were measured and recorded at receipt, prior to randomization, and weekly during the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmoscopic examinations were conducted on all animals pretest to test article administration and prior to terminal necropsy
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Hematology and clinical chemistry blood samples (approximately 1.5 mL) were collected from the jugular vein on Days 7 and 45.
- Anaesthetic used for blood collection: Yes - carbon dioxide inhalation
- Animals fasted: Yes
- Parameters checked: Leukocytes, Erythrocytes, Haemoglobin, Haematocrit, Mean cell volume, Mean cell haemoglobin, Mean cell haemoglobin concentration, Platelets, Absolute Reticulocytes, Neutrophils, Band Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Other cells, Activated Partial Thromboplastin Time, Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinical pathology evaluations were conducted on Days 7 and 45 and prior to terminal necropsy.
- Animals fasted: Yes for Days 7 and 45, but not prior to terminal necropsy.
- How many animals: 10 animals/sex/group
- Parameters checked: Sodium, Potassium, Chloride, Calcium, Phosphorus, Alkaline phosphatase, Total bilirubin, Gamma Glutamyltransferase, Aspartate Aminotransferase, Alanine Aminotransferase, Urea Nitrogen, Creatinine, Total protein, Albumin, Globulin, Albumin/globulin ratio, Triglyceride, Cholesterol, Glucose

URINALYSIS: Yes
- Time schedule for collection of urine: prior to terminal necropsy
- Metabolism cages used for collection of urine: No
- Animals fasted: Not specified
- Parameters checked: Volume, Specific gravity, pH, Glucose, Bilirubin, Ketones, Occult Blood, Protein, Urobilinogen, Appearance, Color, Casts, Crystals, Amorphous, Epithelial Cells, Leukocytes, Erythrocytes, Sperm, Yeast, Bacteria

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during Week 12
- Dose groups that were examined: All animals
- Battery of functions tested: The observations included, but were not limited to, evaluation of activity and arousal, posture, rearing, bizarre behavior, clonic and tonic movements, gait, mobility, stereotypy, righting reflex, response to stimulus (approach, click, tail pinch, and touch), palpebral closure, pupil response, piloerection, exophthalmus, lacrimation, salivation, and respiration. The amount, qualitative and/or quantitative measures, of defecation and urination were also recorded. Forelimb and hindlimb grip strength was measured using the procedure described by Meyer, et al., and hindlimb splay was quantitatively measured as described by Edwards and Parker. Pain perception was assessed by measuring the latency of response to a nociceptive (thermal) stimulus when each animal was placed on a hot plate apparatus set to 52 °C, as described by Ankier. Body
weight and temperature were also measured. Motor activity was measured by placing animal into the assigned Hamilton-Kinder enclosure for monitoring. The duration of monitoring was 60 minutes with the data summarized into 10 minute segments. A range of different activities were assessed in a three dimensional array and were recorded. Only basic movement, fine movement, rearing, and distance (cm) were used in comparisons
between treated and control animals as the most representative activity parameters.

THYROID FUNCTION
- Time schedule for examinations: once during Week 4 and prior to terminal necropsy
- Dose groups that were examined: 10 animals/sex/group
- Animals fasted: Yes
- Anaesthetic used for blood collection: Yes - carbon dioxide inhalation
- Parameters checked: thyroid stimulating hormone (TSH), triiodothyronine (T3), and thyroxine (T4)

SPERM ANALYSIS
- Time schedule for examinations: once at terminal necropsy
- Dose groups that were examined: 10 males/group
- Parameters checked: sperm motility, sperm concentration, % abnormal

ESTROUS CYCLE DETERMINATIONS
- Time schedule for examinations: daily beginning Week 9
- Dose groups that were examined: all females
- Parameters checked: determination of estrous cycle

BONE MARROW ANALYSIS
- Time schedule for examinations: once at terminal necropsy
- Dose groups that were examined: control and high dose
- Parameters checked: Myeloblast, Promyelocyte, Myelocyte Neutrophil, Metamyelocyte Neutrophil, Band Neutrophil, Segmented Neutrophil, Eosinophil, Basophil, Mitotic Figure - Myeloid, Rubriblast, Prorubricyte, Rubricyte - Basophilic, Rubricyte - Polychromatophilic, Metarubricyte, Mitotic Figure - Erythroid

Sacrifice and pathology:

Necropsy examinations were performed under procedures approved by a veterinary pathologist on animals that died after blood collection and all surviving animals euthanized at the scheduled necropsy. The animals were euthanized by carbon dioxide inhalation. Euthanasia was confirmed by exsanguination via the abdominal vena cava. The animals were examined carefully for external abnormalities including palpable masses. The skin was reflected from a ventral midline incision and any subcutaneous masses were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities. The organs were removed, examined, and, where required, placed in fixative. The pituitary was fixed in situ. All designated tissues were fixed in neutral buffered formalin, except for the eye (including the optic nerve) and testes, which were fixed using a modified Davidson’s fixative. Formalin was infused into the lung via the trachea and into the urinary bladder. A full complement of tissues and organs was collected from all animals.

Body weights and protocol-designated organ weights were recorded for all surviving animals at the scheduled necropsy and appropriate organ weight ratios were calculated (relative to body and brain weights). Paired organs were weighed together. The thyroid/parathyroid gland and pituitary gland were weighed following fixation. A combined weight for the thyroid and parathyroid glands was obtained.

Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues. The slides were examined by a board-certified veterinary pathologist. A four-step grading system was utilized to define gradable lesions for comparison between dose groups. The stomach, glandular and stomach, nonglandular were determined to be potential target organs and were examined for all animals.

The following organs and tissues were examined (* indicates if weighed):
- Adrenal (2)*
- Aorta
- Bone with marrow [femur]
- Bone with marrow [sternum]
- Bone marrow smear [2 collected]
- Brain [cerebrum, midbrain, cerebellum, medulla/pons]*
- Epididymis (2)*
- Eye including optic nerve (2)
- Gastrointestinal tract:
esophagus
stomach [glandular and nonglandular]
duodenum
jejunum
ileum
cecum
colon
rectum
- Gonads:
ovary (2)*
testis (2)*
- Gross lesions
- Heart*
- Joint, tibiofemoral
- Kidney (2)*
- Liver [3 sections collected; 2 examined]*
- Lung [collected whole; 2 sections examined]
- Lymph nodes: mandibular [2 collected; 1 examined] and mesenteric
- Mammary gland [process females only]
- Pancreas
- Pituitary*
- Prostate and seminal vesicle (2)
- Salivary gland, mandibular [2 collected; 1
examined]
- Sciatic nerve
- Skeletal muscle, biceps femoris
- Skin
- Spinal cord [cervical, thoracic, and lumbar]
- Spleen*
- Thymus*
- Thyroid/parathyroid (2)*
- Tongue
- Trachea
- Urinary bladder
- Uterus [both horns]/Cervix*
- Vagina

Statistics:

The control group was compared to the 3 treatment groups for the statistical analyses described below.

The raw data were tabulated within each time interval, and the mean and standard deviation and/or incident counts (categorical variables) were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared to the control group using the analysis outlined below. Data for some endpoints, as indicated, were transformed by either a log or rank transformation prior to conducting the specified analysis.

Group Pair-wise Comparisons (Levene’s/ANOVA-Dunnett’s/Welch’s) were used for the following endpoints:
-Body Weights
-Food Consumption
-Hematology (except leukocyte counts)
-Coagulation
-Clinical Chemistry
-Organ Weights
-Absolute Weights
-Relative to Body and Brain Weights
-FOB (Continuous Endpoints)
--Body Weight
--Body Temperature
--Defecation
--Urination
--Rearing
--Thermal Response
--Forelimb Grip Strength
--Hindlimb Grip Strength
--Hindlimb Splay
Motor Activity
Estrous Cycle (mean cycle time)
Estrous Cycle (number of cycles/period)
Sperm Analysis
--Concentration

Arcsin-Square-Root Transformation was used for the following endpoints:
-Sperm Analysis
--% Abnormal
--% Motility

Cochran Mantel Haenszel Test was used for the following endpoints:
-FOB (Categorical Endpoints)

Log Transformation/Group Pair-wise Comparisons were used for the following endpoints:
-Leukocyte Counts
--Total Leukocyte Counts
--Differential Leukocyte Counts

Rank Transformation with Dunnett’s Test was used for the following endpoints:
-Urinalysis
--Urine Volume
--Specific Gravity
--pH

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No adverse cage-side clinical observations were noted. An increased incidence of salivation was noted among males and females at ≥200 mg/kg/day. This finding was considered incidental as there was not similar findings noted in either the weekly detailed observations or FOB evaluations.
No adverse clinical observations were noted in the weekly observations. Test article-related increased incidence of brown, yellow, or red discolored anogenital areas were noted in both sexes at ≥200 mg/kg/day. However, these findings were not considered adverse.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A single male and female at 600 mg/kg/day died following blood collections on Day 22. These were not considered test article-related as no overt signs of toxicity were noted at this level and no other deaths occurred.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test article-related body weight changes were noted at ≤200 mg/kg/day. In males at 600 mg/kg/day, mean body weight was lower (6-13%) after 1 week of treatment through the duration of treatment. Mean female body weight at this level was only slightly affected (5-6%) during Weeks 11 to 13.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was transiently lower (8-14%) in both sexes at 600 mg/kg/day during Week 1 only. No other test article-related effects were noted. Food consumption was statistically significantly higher among females at 200 and 600 mg/kg/day during Week 5. However, these were not considered test article-related due to the direction of the change.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At termination in both sexes and in all groups, there were mild increases in lymphocytes (up to 1.5-fold) relative to Day 7 that correspond to an increase in total leukocytes (up to 1.5-fold), consistent with age-related changes or epinephrine-mediated response. These changes were generally progressive over time and tended to be more pronounced in males than females.
There were several other changes in hematologic parameters that were associated with age and were not test article-related.
At termination in both sexes receiving 600 mg/kg/day, there were minimal prolongations in prothrombin time (up to 1.1-fold) relative to vehicle controls that were likely test article-related, but of minor biologic relevance based on their small magnitude.
No test article-related findings among bone marrow parameters were observed in either sex. All fluctuations among individual and mean values were considered within an acceptable range for biologic variability. Megakaryocytes were generally observed in adequate numbers. Both myeloid and erythroid cell lines were well represented and matured appropriately to completion. Cells from all lines appeared morphologically unremarkable.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At Days 7 and 45, males receiving 600 mg/kg/day had mild reductions in glucose (up to 19%) relative to vehicle controls that were considered test article-related. These changes were possibly secondary to altered glucose absorption and/or metabolism.
At all intervals in both sexes receiving 600 mg/kg/day, there were mild decreases in albumin (up to 7%) relative to vehicle controls that were reflected in a mild reduction in the albumin/globulin ratio (up to 10%). These changes were likely test-article related but of limited biologic relevance based on their small magnitude.
At Days 7 and 45, there were sporadic statistically significant decreases in urea nitrogen (up to 19%) relative to vehicle controls in both sexes and in all treatment groups that were unlikely to be biologically relevant based on their sporadic nature, small magnitude and lack of correlative findings.
At Day 7, males receiving ≥ 200 mg/kg/day had minor reductions in alkaline phosphatase (ALP, up to 32%) relative to vehicle controls that were likely secondary to decreased food consumption and indirectly test article-related.
There were several other statistically significant alterations among clinical chemistry analytes that were not considered test article-related due to their sporadic nature, small magnitude, direction of the change, and association with age.
There were minor fluctuations among thyroid assays (T4, T3, and TSH) that were consistent with normal biologic variation and not test article-related. The Clinical Pathologist from MPI (Laura Cregar, DVM, DACVP) has stated that she did not consider there to be a meaningful effect on TSH, as most individual values overlapped with the range of controls. Any apparent differences in group means were considered secondary to biologic variation. The range of individual values in controls is an indicator of the expected biologic variation in rats, and concurrent controls serve as the best reference for expected values. Also, she has stated that there were no effects on indirect tests of thyroid function (red cell mass, cholesterol), direct tests of thyroid function (T3, T4 or TSH) or histopathology to indicate there is an effect on the thyroid. As there were no direct, especially histopathology, indirect or other toxicological changes to indicate a thyroiditis or other auto immune thyroid condition, there was no evidence to provide a hypothesis to test for thyroid autoantibodies and it is therefore not considered to be a request than can be experimentally justified on the data generated. As a follow-up, the Clinical Pathologist from MPI reviewed the thyroid hormone tables with the addition of statistical analysis. There was a statistical decrease in T3 in males at the Week 4 collection only. This decrease was not considered meaningful as it was small (16%), transient and only occurred in a single sex. Given the variability of individual animal data, the pathologist felt the statistical change was the result of biologic variation and did not represent a meaningful test article-related effect.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At termination in both sexes receiving 600 mg/kg/day, there were moderate increases in urine volume (up to 4.4-fold) with a corresponding decrease in specific gravity relative to vehicle controls that were likely test article-related. However this is considered to represent a functional difference in the absence of any corresponding treatment related renal pathology and may have been associated with the renal excretion of high concentrations of conjugates following the metabolism and detoxification of large bolus doses of the test article (600 mg/kg). There were some variations between treatment groups among physical (appearance), biochemical (occult blood, protein, etc) and microscopic (leukocytes, erythrocytes, etc) urinary components; however, these findings were considered within an acceptable range for biological and/or procedure-related variability.
The pathologist at MPI (Daniel Patrick, DVM, DACVP, Associate Director, Principal Pathologist) has confirmed that there were no hyaline droplets present above a recording threshold, and no apparent test article-related increase.
As there was an absence of any apparent renal pathology indicative of nephropathy and evidence of any increase above background in hyaline droplets, there was no case or indication to conduct immunohistochemistry for the presence of α 2u-globulin.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test article-related effects on any FOB parameter examined. Several statistical significant changes were noted and included: lower forelimb grip strength (both sexes, 600 mg/kg/day); higher hindlimb grip strength (males at all treated levels); higher body temperature (males at all treated levels); lower body weight (males, 600 mg/kg/day); and an increase in thermal response time (females, 200 mg/kg/day). These changes were not considered test article-related due to the direction of the changes and/or due to lack of a dose response.
There were no test article-related effects on motor activity. Statistical significant changes were noted in some parameters and included: increased rearing (males, 600 mg/kg/day and females, 50 and 200 mg/kg/day); and decreases in basic, fine movement and/or total distance (females, 50 and 200 mg/kg/day). These changes were not considered test article-related as they represented normal exploration (rearing) or due to a lack of a dose response.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test article-related organ weight changes were limited to decreased thymus weights in males at 200 and 600 mg/kg/day. The thymus weight decreased by 15.31% and 30.38% in the 200 and 600 mg/kg/day doses, respectively. There were no microscopic correlates for these weight changes.
All other organ weight changes lacked microscopic correlates and were considered to be incidental. In addition, there were no treatment related thyroid weight increases in males or females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test article-related macroscopic findings were limited to the nonglandular stomach and included swelling/thickening, nodule, and red to tan foci. These observations correlated with epithelial hyperplasia of the nonglandular stomach.
All other macroscopic observations (mammary gland enlarged, jejunum deformity/malformation, skin hair sparse, uterus enlargement, small epididymides, liver hepatodiaphragmatic nodule, and small testes) were non dose-related and of low incidence, and were considered incidental.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test article-related microscopic findings were limited to epithelial hyperplasia of the nonglandular stomach in males at ≥ 50 mg/kg/day and females at ≥ 200 mg/kg/day. This finding was infrequently associated with erosion/ulceration of the nonglandular stomach, and mucosal hypertrophy of the glandular stomach.
The hyperplasia of the nonglandular stomach was characterized by thickening of the epithelium with hyperkeratosis. The thickening diffusely affected the nonglandular stomach, but was more pronounced adjacent to and within the limiting ridge. The nonglandular hyperplasia was associated with minimal to mild nonglandular erosion/ulceration within the limiting ridge in a male and female at 600 mg/kg/day. Within the adjacent glandular mucosa of the glandular stomach, the parietal cells were minimally enlarged in two males and a female at 600 mg/kg/day.
All other microscopic observations were considered to be incidental.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No test article-related effects were noted in estrous cycling and in the sperm evaluation.

Details on results:

Following 13 weeks of oral (gavage) dosing of the substance once daily at 0, 50, 200, and 600 mg/kg/day, the No-Observed-Adverse-Effect Level (NOAEL) was determined to be 200 mg/kg/day based on progressively lower body weight among males throughout the study and the adverse microscopic findings in the stomach in both sexes at 600 mg/kg/day. There was no evidence of neurotoxicity or a reproductive effect in any of the parameters examined.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

The detailed test article-related changes in thymus weights (without microscopic changes) are shown in the table below:

Test Article-related Organ Weight Changes - Terminal Male (Percent change relative to control)
Dose level: mg/kg/day	200	600
Number Examined	20	19
Thymus (g)	↓15.31a	↓30.38b
Thymus/BWt%	↓11.35	↓18.49a
Thymus/BrWt ratio	↓14.03	↓30.10b
aSignificantly different from control; (p<0.05) bSignificantly different from control; (p<0.01) ↓ - Decreased BWt - Body Weight BrWt - Brain Weight

 

The test article-related microscopic findings of the stomachs are shown in the table below:

Test Article-related Microscopic Observations – Terminal
Dose level: mg/kg/day	Vehicle Control		50		200		600
Sex	M	F	M	F	M	F	M	F
Number Examined	20	20	20	20	20	20	20*	20*
Stomach,nonglandular
erosion/ulcer	0	0	0	0	0	0	1	1
- minimal	0	0	0	0	0	0	1	0
- mild	0	0	0	0	0	0	0	1
hyperplasia, epithelial cell	0	0	5	0	18	10	20	20
- minimal	0	0	5	0	5	8	0	0
- mild	0	0	0	0	13	2	0	1
- moderate	0	0	0	0	0	0	20	19
Stomach, glandular
hypertrophy	0	0	0	0	0	0	2	1
- minimal	0	0	0	0	0	0	2	1
M – Male F – Female * – includes animal that died during study

 

The test article had no effect on estrous cycling:

Summary of Estrous Cycling
	Vehicle Control			50 mg/kg/day			200 mg/kg/day			600 mg/kg/day
Endpoint	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Mean Cycle Length (Days)	5.1	1.03	20	4.9	0.94	20	4.8	0.91	20	4.6	0.46	19
No. of Cycles (Count)	6.0	1.36	20	6.5	1.05	20	6.6	1.15	20	6.2	0.85	19
N - Number of measures used to calculate mean SD - Standard Deviation No. - Number

 

There were no test article related effects on sperm:

Summary of Sperm Evaluation
	Vehicle Control			50 mg/kg/day			200 mg/kg/day			600 mg/kg/day
Endpoint	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
Sperm Motility Percent Motility	76.0	10.03	10	66.7	25.87	10	77.5	11.97	10	83.9	9.02	9
Total Sperm Concentration per Cauda Epididymis x 10^8	2.678	0.7992	10	2.964	0.4733	10	2.972	0.5361	10	2.775	0.7919	9
Sperm Concentration per gram Cauda Epididymis x 10^8	7.992	2.3190	10	8.798	0.9921	10	8.927	1.5730	10	8.365	1.9751	9
Percent Abnormal	3.00	1.333	10	2.70	1.085	10	3.20	1.798	10	2.33	1.479	9
N - Number of measures used to calculate mean SD - Standard Deviation

 

There were no test article related effects on the various thyroid assays:

Summary of Special Chemistry Values - MALE
	Vehicle Control			50 mg/kg/day			200 mg/kg/day			600 mg/kg/day
Endpoint / Study Interval	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T4 μg/dL
-Week 4	4.685	1.0340	10	4.150	1.2008	10	4.494	1.3585	10	4.090	1.1076	10
- Terminal	4.238	0.8311	10	4.596	0.8753	10	4.698	1.0146	10	4.151	0.6095	9
T3 ng/mL
-Week 4	1.393	0.2257	10	1.307	0.2227	10	1.292	0.1301	10	1.176	0.1828	10
- Terminal	1.090	0.1299	9	1.080	0.2733	10	1.139	0.2041	9	0.992	0.1010	9
TSH ng/mL
-Week 4	0.847	0.2099	10	0.785	0.3988	10	0.884	0.4065	10	0.948	0.3989	10
- Terminal	0.549	0.2321	10	0.470	0.1823	9	0.609	0.2976	9	0.554	0.2449	8
N - Number of measures used to calculate mean SD - Standard Deviation

 

Summary of Special Chemistry Values - FEMALES
	Vehicle Control			50 mg/kg/day			200 mg/kg/day			600 mg/kg/day
Endpoint / Study Interval	Mean	SD	N	Mean	SD	N	Mean	SD	N	Mean	SD	N
T4 μg/dL
-Week 4	2.931	1.1304	10	3.044	0.5539	10	3.061	0.6200	10	3.910	1.5005	10
- Terminal	3.207	0.4381	10	3.769	0.7910	10	3.667	0.5473	10	3.817	0.4838	9
T3 ng/mL
-Week 4	1.508	0.1757	10	1.435	0.2283	10	1.389	0.1576	10	1.452	0.2310	10
- Terminal	1.428	0.2814	10	1.284	0.1724	10	1.405	0.2125	10	1.194	0.1872	9
TSH ng/mL
-Week 4	0.543	0.2601	10	0.596	0.1816	10	0.551	0.2816	10	0.554	0.1830	9
- Terminal	0.544	0.3234	9	0.663	0.3566	10	0.689	0.2367	10	0.612	0.2813	9
N - Number of measures used to calculate mean SD - Standard Deviation

 

 

 

 

Applicant's summary and conclusion

Conclusions:

Following 13 weeks of oral (gavage) dosing of p-tert-Amylphenol once daily at 0, 50, 200, and 600 mg/kg/day, the No-Observed-Adverse-Effect Level (NOAEL) was determined to be 200 mg/kg/day based on progressively lower body weight among males throughout the study and the adverse microscopic findings in the stomach in both sexes at 600 mg/kg/day. There was no evidence of neurotoxicity or a reproductive effect in any of the parameters examined.

Executive summary:

Three treatment groups of 20 male and 20 female CD® [Crl:CD®(SD)] rats were administered p-tert-Amylphenol  by oral gavage using a vehicle at respective dose levels of 50, 200, and 600 mg/kg/day for 13 weeks. One additional group of 20 animals/sex served as the control and received the vehicle, 0.5% methylcellulose in deionized water. The vehicle or test article was administered to the appropriate groups once daily at a dose volume of 10 mL/kg.

Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals. Cage-side clinical observations were conducted once daily. Detailed clinical observations were conducted prior to initial test article administration and weekly thereafter. Functional observational battery (FOB) and motor activity evaluations were conducted once during Week 12. Body weights and food consumption were measured and recorded weekly. Ophthalmoscopic examinations were conducted pretest to test article administration and prior to terminal necropsy. Estrous cycles were determined daily beginning Week 9 through termination. Blood and urine samples for clinical pathology evaluations were collected from designated animals on Days 7 and 45 (blood only) and prior to terminal necropsy. Special chemistry evaluations were collected from designated animals once during Week 4 and prior to terminal necropsy. At study termination, necropsy examinations were performed and organ weights were recorded. Tissues were microscopically examined for animals at 0 and 600 mg/kg/day. Target organs were microscopically examined for all animals. Sperm analysis was conducted on designated males at the terminal necropsy.

There were no test article-related mortalities, adverse clinical findings, or ophthalmologic findings. No test article-related effects were noted in the FOB or motor activity evaluations. Male body weight at 600 mg/kg/day was lower for the entire treatment period and females at this level were affected during Weeks 11 to 13. Food consumption was only transiently lower at this level during Week 1.

There were no test article-related effects noted among thyroid assays (T4, T3, and TSH) or in bone marrow parameters. No test article-related effects were noted in estrous cycling or sperm evaluations.

Minor test article-related clinical pathology changes in males and/or females at 600 mg/kg/day included: minimal prolongations in prothrombin time at termination; mild reductions in glucose on Days 7 and 45; mild decreases in albumin at all intervals; and moderate increases in urine volume with a corresponding decrease in specific gravity. These changes were not considered adverse as they were not biologically relevant based on their sporadic nature, small magnitude and lack of correlative findings.

Test article-related organ weight changes were limited to decreased thymus weights in males at 200 and 600 mg/kg/day. However, these findings were not considered adverse as there were no microscopic correlates for these weight changes.

Test article-related macroscopic findings were limited to the nonglandular stomach and included swelling/thickening, nodule, and red to tan foci. These observations correlated with epithelial hyperplasia of the nonglandular stomach in males at ≥ 50 mg/kg/day and females at ≥ 200 mg/kg/day. This finding was infrequently associated with erosion/ulceration of the nonglandular stomach, and mucosal hypertrophy of the glandular stomach at 600 mg/kg/day. The findings at 600 mg/kg/day were considered adverse.