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Diss Factsheets

Administrative data

Description of key information

Repeat dose (Oral):
US EPA OPPTS 870.3100 90 day oral toxicity study in rodents was conducted with p-tert-amylphenol (Craig, 2012): NOAEL (based on effects to the stomach) = 200 mg/kg/d

OECD guideline 422, 13 week study on P-tert butyl phenol (Japanese Ministry of Health 1996): Combined repeat dose and reproductive toxicity = NOAEL 60 mg.kg/day
2- generation study on P-tert butyl phenol (Clubb & Jardine 2006): NOAEL for systemic effects = 70 mg/kg/day
Teratology study on p-tert amylphenol (Siglin 1991): NOAEL for systemic maternal effects = 50 mg/kg/day
Repeat dose (inhalation): No specific study.
Repeat dose (dermal): 90-day repeat dose dermal study (Siglin 1992): NOAEL = 25 mg/kg/day (systemic effects); 2.5. mg/kg/day (local effects).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
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
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
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
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
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
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
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
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.
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
Key result
Critical effects observed:
no

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 108

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 108

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

 

 

 

 

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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Further information is included in the attachment entitled “Read-across between p-tert-amylphenol (CAS 80-46-6) and Sodium p-tertiary amylphenol (CAS 31366-95-7) and p-tert-butylphenol (CAS 98-54-4).

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target and source chemicals display a high structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target chemical is p-tert-amylphenol and the two source chemicals are Sodium p-tertiary amylphenol and p-tert-butylphenol.

3. ANALOGUE APPROACH JUSTIFICATION
All substances consist of a branched tertiary alkyl chain attached to a phenolic ring in the 4-position (para) to the hydroxyl substituent and are close structural analogues. In addition, the target and source chemicals display similar physico-chemical properties. Based on similar structure and function, it can be assumed that Phase 1 and Phrase 2 metabolism will be similar.

4. DATA MATRIX
A detailed data matrix can be found in the attached read-across document.
Reason / purpose for cross-reference:
read-across source
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight gains
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
reduced food intakes
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
no effects observed at necropsy
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
70 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduced body weight gain at intakes of 200 mg/kg/day and above
Key result
Critical effects observed:
not specified

0, 800, 2500, 7500 ppm corresponds to approximate intakes of 0, 70, 200, 600 mg/kg bw/day

Conclusions:
There were signs of maternal toxicity in the form of reduced body weight gains at doses >/= 200 mg/kg/day p-tert butyl phenol adminstered in diet. Apart from this observation, there were no specific treatment related clinical signs observed at any dose. Therefore a precautionary NOAEL is  800  ppm (70  mg/kg bw/day in females) based upon maternal reduced body weights. 
Executive summary:

In a two-generation reproduction study, p-tert butyl phenol was administered orally to Sprague Dawley rats (28 males and females F0 generation; 24 males and females F1 generation) at 0, 800, 2500 and 7500 ppm indiet yielding approximate intakelevels of 0,70, 200 and 600mg/kg bw/day.

There were no treatment related clinical signs.

There were no abnormalities noted in the pups associated with treatment.

At 200 mg/kg/day and 600 mg/kg/day there was a decrease in weight gain seen in F0 animals, and in both generations during gestation. In the F1 generation, overall weights of animals at these levels were lower, reflecting the lower weaning weights of these animals.

At 200 mg/kg and 600 mg/kg, food consumption was notably reduced in F0 animals, and in F1 animals at 600 mg/kg.

The values for achieved intake were generally proportional to the dietary concentrations.

The NOAEL is 800 ppm (70 mg/kg bw/day in females).

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Further information is included in the attachment entitled “Read-across between p-tert-amylphenol (CAS 80-46-6) and Sodium p-tertiary amylphenol (CAS 31366-95-7) and p-tert-butylphenol (CAS 98-54-4).

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target and source chemicals display a high structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target chemical is p-tert-amylphenol and the two source chemicals are Sodium p-tertiary amylphenol and p-tert-butylphenol.

3. ANALOGUE APPROACH JUSTIFICATION
All substances consist of a branched tertiary alkyl chain attached to a phenolic ring in the 4-position (para) to the hydroxyl substituent and are close structural analogues. In addition, the target and source chemicals display similar physico-chemical properties. Based on similar structure and function, it can be assumed that Phase 1 and Phrase 2 metabolism will be similar.

4. DATA MATRIX
A detailed data matrix can be found in the attached read-across document.
Reason / purpose for cross-reference:
read-across source
Vehicle:
other: 0.5% aqueous methyl cellulose
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
stridor associated with dyspnea
Mortality:
mortality observed, treatment-related
Description (incidence):
stridor associated with dyspnea
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
males only
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
males only
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS were observed in females belonging to the high-dose group 200 mg/kg bw/day and consisted of stridor associated with dyspnea,
ie causing respiratory distress.

HAEMATOLOGY: Significant changes in haematology parameters in high-dose males compared to control males included a decrease in red blood cell counts, which was not dose-dependent, and a significant increase in white blood cells counts, which appeared to be dose-dependent. In contrast, no changes in haematology were observed in females.

CLINICAL CHEMISTRY: Significant changes in clinical chemistry parameters in males included significant decreases in plasma albumin levels in
mid- and high-dose males, which appeared to be dose-dependent and were accompanied by a significant reduction in protein levels at the
high-dose level. High-dose males also presented with a significant decrease in plasma potassium concentrations and a significant increase in
plasma inorganic phosphate concentrations; however, these changes were not dose-dependent. In contrast, no changes in clinical chemistry were
observed in females.

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Respiratory distress observed in females and the changes in blood parameters (albumin, potassium & inorganic phosphate) observed males at the high-dose level of 200 mg/kg body weight/day
Key result
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Respiratory distress observed in females and the changes in blood parameters (albumin, potassium & inorganic phosphate) observed in males
Key result
Critical effects observed:
not specified

Table: Haematological and blood chemical findings of male rats treated orally with p-tert-butylphenol in the combined repeat dose and reproductive/developmental toxicity screening test

a) haematology

Male (13)

 Dose (mg/kg)  0  20  60  200
 Red blood cells (count)  844 ± 37  821 ± 29

833 ± 20

 805 ± 27 **

 Hemaglobin (g/dl)

 15.5 ± 0.9

 15.4 ± 0.4

15.3 ± 0.4 

 15.0 ± 0.4

 Hematocrit (%)

 44.9 ± 2.4

 44.7 ± 1.2

 44.6 ± 1.2

 43.8 ± 1.4

 MCV (ml)

 53.2 ± 1.8

54.4 ± 1.3 

 53.5 ± 1.3

 54.5 ± 2.1

 MCHC (pg)

 18.4 ± 0.6

18.7 ± 0.5 

 18.4 ± 0.4

 18.7 ± 0.7

 MCHC (%)

 34.6 ± 0.4

34.4 ± 0.4 

 34.4 ± 0.4

 34.3 ± 0.5

 White blood cells (count)  57 ± 12  59 ± 20  63 ± 11  79 ± 14 **
 Band neutrophil (%)  0 ± 0  0 ± 0  0 ± 0  0 ± 0
 Segement neutrophil (%)  13 ± 4  10 ± 5  16 ± 3  11 ± 4
 Eosinophil (%)  1 ± 1  1 ± 1  1 ± 1  0 ± 1
 Basophil (%)  0 ± 0  0 ± 0  0 ± 0  0 ± 0
 Monocyte (%)  4 ± 2  3 ± 1  3 ± 1  2 ± 2
 Lymphocyte (%)  82 ± 6  85 ± 6  80 ± 4  86 ± 4
 Platelet (counts)  105.4 ± 8.7  107.3 ± 7.0  110.4 ± 7.4  110.4 ± 7.4

 

Values are expressed as Mean ± S.D. Parenthesis indicates number of animals.

**: significant difference from control, p < 0.01

b) blood chemistry

 Dose (mg/kg)  0  20  60  200
 Total protein (g/dl)  5.4 ± 0.2  5.4 ± 0.2  5.5 ± 0.3  5.1 ± 0.4
 Albumin (mg/dl)  3.1 ± 0.2  3.0 ± 0.2  2.0 ± 0.2  2.7 ± 0.2
 A!G  1.31 ± 0.16  1.21 ± 0.14  1.10 ± 0.16  1.18 ± 0.20
 BUN (mg/dl)  15 ± 3  16 ± 2  15 ± 3  16 ± 3
 Creatinine (mg/dl)  0.7 ± 0.1  0.7 ± 0.1  0.7 ± 0.2  0.6 ± 0.1
 Glucose (mg/dl)  127 ± 15  123 ± 11  128 ± 12  127 ± 11
 Total cholesterol (mg/dl)  29 ± 4  30 ± 6  27 ± 9  28 ± 6
 Total bilirubin (mg/l)  0.09 ± 0.03  0.07 ± 0.05  0.07 ± 0.02  0.07 ± 0.02
 Na (mEq/l)  143.4 ± 1.0  144.1 ± 0.9  143.3 ± 1.0  143.4 ± 0.8
 K (mEq/l)  3.95 ± 0.22  3.80 ± 0.16  3.85 ± 0.15  3.72 ± 0.14 *
 Cl (mEq/l)  108.7 ± 1.3  108.8 ± 1.2  107.8 ± 1.4  107.9 ± 1.8
 Ca (mg/dl)  8.3 ± 0.2  8.5 ± 0.4  8.3 ± 0.3  8.4 ± 0.3
 Inorg phos. (mg/dl)  5.5 ± 0.4  5.7 ± 0.5  5.7 ± 0.4  6.2 ± 0.4
 ALP (U/l)  301 ± 52  280 ± 35  269 ± 55  291 ± 38
 GPT (U/l)  32 ± 4  32 ± 9  32 ± 5  28 ± 4
 GOT (U/l)  66 ± 9  68 ± 12  72 ± 10  69 ± 6
 Y GTP (U/l)  0 ± 0  0 ± 0  0 ± 1  0 ± 1

 Values are expressed as Mean ± S.D. Parenthesis indicates number of animals.

*: significant difference from control, p < 0.05

**: significant difference from control, p < 0.01

Conclusions:
The NOEL for repeat dose toxicity is 60 mg/kg bw/day in males and females.
No effects on reproductive endpoints were observed at any dose.

Executive summary:

In a 13 week combined repeat dose and reproductive toxicity study (according to OECD guideline 422) p-tert butyl phenol was administered to13 female and 13 male Sprague Dawley rats by oral gavage at dose levels of 0, 20, 60, 200 mg/kg bw/day).

Respiratory distress was observed in females at the top dose, and changes to clinical chemistry and haematology parameters were observed at the top dose in males. The NOEL for repeat dose oral toxicity is 60 mg/kg bw/day in males and females.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
70 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1. GLP study performed to OECD guideline

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991-03-07 to 1992-08-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: EPA guideline method 82-3 adopted with no deviations
Qualifier:
according to guideline
Guideline:
EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
All animals were acclimated to the laboratory environment for a period of 11 days. Individual body
weights were determined on the day following receipt and on days -7 and -1.
During the week preceding study initiation (days -7 to 4, all rats were acclimated
to binders. The binders were placed on the animals for 2 hours on day -7, 4 hours on
day -6, and 6 hours on days -5 to -3.
Animals were housed throughout the study in an environment-controlled room with
a 12-hour light/l2-hour dark cycle. The controls were set to maintain a room temperature
of 65-78OF and a relative humidity of 40-70%. The room temperature and relative
humidity were determined and recorded a minimum of once daily.
Purina Certified Rodent Meal@ #5002 and municipal tap water were provided to the
animals ad libitum.
Type of coverage:
semiocclusive
Vehicle:
ethanol
Details on exposure:
The fur was clipped from the dorsal trunk area on the day prior to the first dose and when necessary thereafter (a minimum of once weekly).
The clipped and treated area on each animal was approximately 10% of the body surface area.
The test article and the vehicle control were administered by dermal application five days per week for 13 weeks.
Each rat’s dose was held in contact with the skin using a porous 2 x 3 inch 12-ply gauze dressing which was secured in place using an ovewrap of Coban self-adherent wrap. The edges of the Coban wrap were further secured using 1 inch wide athletic tape.
During the study, plastic collars and/or harnesses (made using athletic tape) were applied to the rats when necessary to prevent excessive
chewing on the binding materials and potential oral exposure to the test article/vehicle. After a daily exposure period of approximately six hours, the wrapping materials were removed and the treated skin of each animal was wiped with clean gauze moistened with distilled water.
A volume of 6ml/kg test substance was used over an area of 10 cm^2.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prestudy analytical chemistry evaluations indicated that PTAP was homogeneous and stable in the vehicle for up to 8 days when stored at room temperature. Analysis of dosing solutions resulted in average test article recoveries ranging from 93.8 to 105.9% indicating that the solutions were
accurately prepared.
Duration of treatment / exposure:
6 hours; treated skin of each animal was wiped with clean gauze moistened with distilled water
Frequency of treatment:
Once daily for 5 days per week, for a period of 13 weeks.
Remarks:
Doses / Concentrations:
0, 2.5, 10, 25 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10 animals per group
Control animals:
yes, concurrent vehicle
Details on study design:
Study design
No of animals
Group Male Female material Level (mg/kg/day) Conc. (mg/ml) Volume (ml/kg)
1 10 10 Vehicle 0 0 6
2 10 10 PTAP 2.5 0.42 6
3 10 10 PTAP 10 1.67 6
4 10 10 PTAP 25 4.17 6
Positive control:
None
Observations and examinations performed and frequency:
Clinical Observations: The animals were observed daily for overt signs of toxicity. Mortality and moribundity checks were performed twice daily, in
the morning and afternoon.
Dermal Observations: The application site on each animal was examined daily prior to dosing for signs of erythema, edema, desquamation and other adverse skin reactions. A final dermal observation was performed on all surviving animals on the day of scheduled sacrifice.
Bodv Weights and Food Consumption: Individual body weights and food consumption were measured weekly. Terminal body weights were
determined on the day of scheduled sacrifice.
Opthalmological Examinations: Ophthalmological examinations were performed by a board certified veterinary ophthalmologist prior to initiation
(day -4) and near the conclusion of the study (day 85). Eyes were dilated using 0.5% MydriacyP ophthalmic solution prior to biomicroscopic and
indirect ophthalmoscopic examination.
Sacrifice and pathology:
Clinical pathology
Blood was collected from all surviving rats on the day of scheduled sacrifice (day 92 or 93) for evaluation of selected hernatology and clinical
chemistry parameters. The animals were fasted overnight prior to blood collection. Blood samples were obtained via the orbital plexus while the
animals were under light halothane anesthesia. The following parameters were evaluated.
Haematology: Erythrocyte count, Hematocrit, Hemoglobin concentration, Mean corpuscular hernoglobin (MCH), Mean corpuscular hernoglobin
concentration (MCHC), Mean corpuscular volume (MCV), Platelet count, Reticulocyte count, Total and differential leukocyte counts
Clinical Chemistry: Alanine aminotransferase, Albumin, Albumin/globulin ratio, Alkaline phosphatase, Aspartate aminotransferase, Calcium
Chloride, Creatinine, Fasting glucose, Globulin (calculated), Phosphorus, Potassium, Sodium, Total bilirubin, Total serum protein, Urea nitrogen

All animals were subjected to a complete gross necropsy at the time of death or sacrifice. The gross necropsy included examination of the
external surfaces of the body and all internal viscera. Surviving animals were sacrificed on study day 92 or 93 by exsanguination following carbon dioxide asphyxiation. Five anirnals/sex/group were sacrificed on each day, when possible. Fresh organ weights were obtained at scheduled
sacrifice for the liver, kidneys, testes, adrenals, brain and heart of surviving animals. Paired organs were weighed together. Photographic slides of the exposure sites were taken at scheduled necropsy. The following organs and tissues from surviving animals were preserved in 10% neutral
buffered formalin.
- Accessory genital organs (epididymides, seminal vesicles, prostate or uterus and vagina)
- Adrenals
- All gross lesions
- Aorta
- Brain (including medulla/pons, cerebellar cortex and cerebral cortex)
- Cecum
- Colon
- Duodenum
- Esophagus
- Exorbital lachrymal glands
- Eyes
- Femur (including articular surface)
- Heart with aorta
- Ileum
- Jejunum
- Kidneys
- Liver
- Lungs
- Mammary gland
- Mesenteric lymph node
- Ovaries
- Pancreas
- Peripheral nerve (sciatic)
- Pituitary
- Rectum
- Skeletal muscle (thigh)
- Skin (treated) - dorsal back
- Skin (untreated) - hip region
- Spinal cord (cervical, midthoracic and lumbar)
- Spleen
- Sternum with bone marrow
- Stomach
- Submaxillary salivary glands
- Testes
- Thymus
- Thyroid/parathyroid
- Trachea
- Urinary bladder

Tissues collected at necropsy from control animals, high-dose animals and all animals found dead or sacrificed during the study, as well as the
treated skin, untreated skin, lungs, liver, kidneys and gross lesions from all low and mid-dose animals, were processed for histopathological
examination. The tissues were trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Histology was performed by
Histo Techniques, Powell, OH, and the tissues were examined microscopically by Dr. Robert G. Geil, a board certified veterinary pathologist.
Statistics:
Continuous data, including body weights, weight gain, food consumption, organ weights and clinical pathology, were analyzed by One Way
Analysis of Variance (ANOVA). When significance was observed with ANOVA, control to treatment group comparisons were performed using
Dunnett's Test or a modified version of Dunnett's Test. All tests were two-tailed with a minimum significance level of 5%.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Dose-related dermal irritation was observed during the study for males and females in the 10.0 and 25.0 mg/kg/day groups. The dermal findings in these groups included erythema and desquamation which progressed to eschar formation with subsequent eschar exfoliation. The incidence
and severity of the dermal irritation were increased in the 25.0 mg/kg/day group where the eschar formation progressed to cover >50% but
<75% of the application site in two males and five females. Ulcerations were also observed within the exposure site of three males and five females in the 25.0 mg/kg/day group. Eschar formation in the 10.0 mg/kg/day group progressed to cover >10% but <25% of the application site in one male and three females. No signs of dermal irritation were observed for males or females in the control or 2.5 mg/kg/day group.
Key result
Dose descriptor:
NOEL
Remarks:
(systemic)
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOEL
Remarks:
(local)
Effect level:
2.5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
not specified

For the local skin irritation effect, a NOAEL is given as 2.5 mg/kg/day. For the purposes of DNEL derivation or risk characterisation to convert this into a mg/cm^2 can be done as follows:

Conc of PTAP in dosing solution = 0.42 mg/ml

Total amount of dosing solution applied/kg = 6 ml/kg

Estimated average weight of rat = 220g

Total amount of dosing solution applied per rat = (6/1000) x 220 = approx 1.3 ml/rat

Total in mg per rat = 1.3ml x 0.42mg = 0.5 mg/rat

Surface area applied = Dorsal region = 10% of total surface area of a 200g rat = approx 25cm^2 = approx 20 microg/cm^2

(This value is used in the endpoint summary for local effects)

Conclusions:
PTAP did not produce systemic toxicity by the dermal route up to a top dose tested of 25 mg/kg/day (dosed in 6 mg/ml over an area of 10 cm^2).
PTAP did cause dermal irritation at a dose of 10 mg/kg/day and 25 mg/kg/day.
Executive summary:

This study was performed to evaluate the potential toxicity of Nipacide PTAP when administered dermally to Sprague Dawley rats over the course of 91 days. The study consisted of a control group and three treatment groups with ten animals per sex per group. The test article was diluted with the vehicle (50% v/v ethanol in distilled water) and administered five days/week, for thirteen weeks, at levels of 2.5, 10.0 and 25.0 mg/kg/day. All doses were given at a constant volume of 6.0 ml/kg. Control animals were administered with the vehicle under the same experimental conditions and at an equivalent dose volume. The rats were observed daily for clinical signs of toxicity and application sites were examined prior to dosing for adverse skin reactions.

No treatment-related mortality or clinical signs of toxicity were observed during the study. Substantial, dose-dependent dermal irritation was produced by the test article in the 10.0 and 25.0 mg/kg/day groups. The dermal irritation was characterized by

erythema, desquamation, eschar formation and subsequent eschar exfoliation. There were no apparent test article-related changes noted among the groups with respect to body weight, body weight gain, food consumption, ophthalmology, clinical pathology, necropsy or organ weight data. Similarly, no treatmentrelated histopathological changes were noted in any of the study animals beyond the dermal changes noted above.

Based on the above results, a dosage level of 25.0 mg/kg/day was considered a no observed-effect level (NOEL) for systemic toxicity.

A level of 2.5 mg/kg/day was considered a NOEL for dermal effects of PTAP in rats.

For the local skin irritation effect, a NOAEL is given as 2.5 mg/kg/day. For the purposes of DNEL derivation or risk characterisation to convert this into a microgram/cm^2 can be done as follows:

Conc of PTAP in dosing solution = 0.42 mg/ml

Total amount of dosing solution applied/kg = 6 ml/kg

Estimated average weight of rat = 220g

Total amount of dosing solution applied per rat = (6/1000) x 220 = approx 1.3 ml/rat

Total in mg per rat = 1.3ml x 0.42mg = 0.5 mg/rat

Surface area applied = Dorsal region = 10% of total surface area of a 200g rat = approx 25cm^2 = approx 20 microg/cm^2

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Good quality OECD guideline

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991-03-07 to 1992-08-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: EPA guideline method 82-3 adopted with no deviations
Qualifier:
according to guideline
Guideline:
EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
All animals were acclimated to the laboratory environment for a period of 11 days. Individual body
weights were determined on the day following receipt and on days -7 and -1.
During the week preceding study initiation (days -7 to 4, all rats were acclimated
to binders. The binders were placed on the animals for 2 hours on day -7, 4 hours on
day -6, and 6 hours on days -5 to -3.
Animals were housed throughout the study in an environment-controlled room with
a 12-hour light/l2-hour dark cycle. The controls were set to maintain a room temperature
of 65-78OF and a relative humidity of 40-70%. The room temperature and relative
humidity were determined and recorded a minimum of once daily.
Purina Certified Rodent Meal@ #5002 and municipal tap water were provided to the
animals ad libitum.
Type of coverage:
semiocclusive
Vehicle:
ethanol
Details on exposure:
The fur was clipped from the dorsal trunk area on the day prior to the first dose and when necessary thereafter (a minimum of once weekly).
The clipped and treated area on each animal was approximately 10% of the body surface area.
The test article and the vehicle control were administered by dermal application five days per week for 13 weeks.
Each rat’s dose was held in contact with the skin using a porous 2 x 3 inch 12-ply gauze dressing which was secured in place using an ovewrap of Coban self-adherent wrap. The edges of the Coban wrap were further secured using 1 inch wide athletic tape.
During the study, plastic collars and/or harnesses (made using athletic tape) were applied to the rats when necessary to prevent excessive
chewing on the binding materials and potential oral exposure to the test article/vehicle. After a daily exposure period of approximately six hours, the wrapping materials were removed and the treated skin of each animal was wiped with clean gauze moistened with distilled water.
A volume of 6ml/kg test substance was used over an area of 10 cm^2.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prestudy analytical chemistry evaluations indicated that PTAP was homogeneous and stable in the vehicle for up to 8 days when stored at room temperature. Analysis of dosing solutions resulted in average test article recoveries ranging from 93.8 to 105.9% indicating that the solutions were
accurately prepared.
Duration of treatment / exposure:
6 hours; treated skin of each animal was wiped with clean gauze moistened with distilled water
Frequency of treatment:
Once daily for 5 days per week, for a period of 13 weeks.
Remarks:
Doses / Concentrations:
0, 2.5, 10, 25 mg/kg/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10 animals per group
Control animals:
yes, concurrent vehicle
Details on study design:
Study design
No of animals
Group Male Female material Level (mg/kg/day) Conc. (mg/ml) Volume (ml/kg)
1 10 10 Vehicle 0 0 6
2 10 10 PTAP 2.5 0.42 6
3 10 10 PTAP 10 1.67 6
4 10 10 PTAP 25 4.17 6
Positive control:
None
Observations and examinations performed and frequency:
Clinical Observations: The animals were observed daily for overt signs of toxicity. Mortality and moribundity checks were performed twice daily, in
the morning and afternoon.
Dermal Observations: The application site on each animal was examined daily prior to dosing for signs of erythema, edema, desquamation and other adverse skin reactions. A final dermal observation was performed on all surviving animals on the day of scheduled sacrifice.
Bodv Weights and Food Consumption: Individual body weights and food consumption were measured weekly. Terminal body weights were
determined on the day of scheduled sacrifice.
Opthalmological Examinations: Ophthalmological examinations were performed by a board certified veterinary ophthalmologist prior to initiation
(day -4) and near the conclusion of the study (day 85). Eyes were dilated using 0.5% MydriacyP ophthalmic solution prior to biomicroscopic and
indirect ophthalmoscopic examination.
Sacrifice and pathology:
Clinical pathology
Blood was collected from all surviving rats on the day of scheduled sacrifice (day 92 or 93) for evaluation of selected hernatology and clinical
chemistry parameters. The animals were fasted overnight prior to blood collection. Blood samples were obtained via the orbital plexus while the
animals were under light halothane anesthesia. The following parameters were evaluated.
Haematology: Erythrocyte count, Hematocrit, Hemoglobin concentration, Mean corpuscular hernoglobin (MCH), Mean corpuscular hernoglobin
concentration (MCHC), Mean corpuscular volume (MCV), Platelet count, Reticulocyte count, Total and differential leukocyte counts
Clinical Chemistry: Alanine aminotransferase, Albumin, Albumin/globulin ratio, Alkaline phosphatase, Aspartate aminotransferase, Calcium
Chloride, Creatinine, Fasting glucose, Globulin (calculated), Phosphorus, Potassium, Sodium, Total bilirubin, Total serum protein, Urea nitrogen

All animals were subjected to a complete gross necropsy at the time of death or sacrifice. The gross necropsy included examination of the
external surfaces of the body and all internal viscera. Surviving animals were sacrificed on study day 92 or 93 by exsanguination following carbon dioxide asphyxiation. Five anirnals/sex/group were sacrificed on each day, when possible. Fresh organ weights were obtained at scheduled
sacrifice for the liver, kidneys, testes, adrenals, brain and heart of surviving animals. Paired organs were weighed together. Photographic slides of the exposure sites were taken at scheduled necropsy. The following organs and tissues from surviving animals were preserved in 10% neutral
buffered formalin.
- Accessory genital organs (epididymides, seminal vesicles, prostate or uterus and vagina)
- Adrenals
- All gross lesions
- Aorta
- Brain (including medulla/pons, cerebellar cortex and cerebral cortex)
- Cecum
- Colon
- Duodenum
- Esophagus
- Exorbital lachrymal glands
- Eyes
- Femur (including articular surface)
- Heart with aorta
- Ileum
- Jejunum
- Kidneys
- Liver
- Lungs
- Mammary gland
- Mesenteric lymph node
- Ovaries
- Pancreas
- Peripheral nerve (sciatic)
- Pituitary
- Rectum
- Skeletal muscle (thigh)
- Skin (treated) - dorsal back
- Skin (untreated) - hip region
- Spinal cord (cervical, midthoracic and lumbar)
- Spleen
- Sternum with bone marrow
- Stomach
- Submaxillary salivary glands
- Testes
- Thymus
- Thyroid/parathyroid
- Trachea
- Urinary bladder

Tissues collected at necropsy from control animals, high-dose animals and all animals found dead or sacrificed during the study, as well as the
treated skin, untreated skin, lungs, liver, kidneys and gross lesions from all low and mid-dose animals, were processed for histopathological
examination. The tissues were trimmed, embedded in paraffin, sectioned and stained with hematoxylin and eosin. Histology was performed by
Histo Techniques, Powell, OH, and the tissues were examined microscopically by Dr. Robert G. Geil, a board certified veterinary pathologist.
Statistics:
Continuous data, including body weights, weight gain, food consumption, organ weights and clinical pathology, were analyzed by One Way
Analysis of Variance (ANOVA). When significance was observed with ANOVA, control to treatment group comparisons were performed using
Dunnett's Test or a modified version of Dunnett's Test. All tests were two-tailed with a minimum significance level of 5%.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Dose-related dermal irritation was observed during the study for males and females in the 10.0 and 25.0 mg/kg/day groups. The dermal findings in these groups included erythema and desquamation which progressed to eschar formation with subsequent eschar exfoliation. The incidence
and severity of the dermal irritation were increased in the 25.0 mg/kg/day group where the eschar formation progressed to cover >50% but
<75% of the application site in two males and five females. Ulcerations were also observed within the exposure site of three males and five females in the 25.0 mg/kg/day group. Eschar formation in the 10.0 mg/kg/day group progressed to cover >10% but <25% of the application site in one male and three females. No signs of dermal irritation were observed for males or females in the control or 2.5 mg/kg/day group.
Key result
Dose descriptor:
NOEL
Remarks:
(systemic)
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Dose descriptor:
NOEL
Remarks:
(local)
Effect level:
2.5 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
not specified

For the local skin irritation effect, a NOAEL is given as 2.5 mg/kg/day. For the purposes of DNEL derivation or risk characterisation to convert this into a mg/cm^2 can be done as follows:

Conc of PTAP in dosing solution = 0.42 mg/ml

Total amount of dosing solution applied/kg = 6 ml/kg

Estimated average weight of rat = 220g

Total amount of dosing solution applied per rat = (6/1000) x 220 = approx 1.3 ml/rat

Total in mg per rat = 1.3ml x 0.42mg = 0.5 mg/rat

Surface area applied = Dorsal region = 10% of total surface area of a 200g rat = approx 25cm^2 = approx 20 microg/cm^2

(This value is used in the endpoint summary for local effects)

Conclusions:
PTAP did not produce systemic toxicity by the dermal route up to a top dose tested of 25 mg/kg/day (dosed in 6 mg/ml over an area of 10 cm^2).
PTAP did cause dermal irritation at a dose of 10 mg/kg/day and 25 mg/kg/day.
Executive summary:

This study was performed to evaluate the potential toxicity of Nipacide PTAP when administered dermally to Sprague Dawley rats over the course of 91 days. The study consisted of a control group and three treatment groups with ten animals per sex per group. The test article was diluted with the vehicle (50% v/v ethanol in distilled water) and administered five days/week, for thirteen weeks, at levels of 2.5, 10.0 and 25.0 mg/kg/day. All doses were given at a constant volume of 6.0 ml/kg. Control animals were administered with the vehicle under the same experimental conditions and at an equivalent dose volume. The rats were observed daily for clinical signs of toxicity and application sites were examined prior to dosing for adverse skin reactions.

No treatment-related mortality or clinical signs of toxicity were observed during the study. Substantial, dose-dependent dermal irritation was produced by the test article in the 10.0 and 25.0 mg/kg/day groups. The dermal irritation was characterized by

erythema, desquamation, eschar formation and subsequent eschar exfoliation. There were no apparent test article-related changes noted among the groups with respect to body weight, body weight gain, food consumption, ophthalmology, clinical pathology, necropsy or organ weight data. Similarly, no treatmentrelated histopathological changes were noted in any of the study animals beyond the dermal changes noted above.

Based on the above results, a dosage level of 25.0 mg/kg/day was considered a no observed-effect level (NOEL) for systemic toxicity.

A level of 2.5 mg/kg/day was considered a NOEL for dermal effects of PTAP in rats.

For the local skin irritation effect, a NOAEL is given as 2.5 mg/kg/day. For the purposes of DNEL derivation or risk characterisation to convert this into a microgram/cm^2 can be done as follows:

Conc of PTAP in dosing solution = 0.42 mg/ml

Total amount of dosing solution applied/kg = 6 ml/kg

Estimated average weight of rat = 220g

Total amount of dosing solution applied per rat = (6/1000) x 220 = approx 1.3 ml/rat

Total in mg per rat = 1.3ml x 0.42mg = 0.5 mg/rat

Surface area applied = Dorsal region = 10% of total surface area of a 200g rat = approx 25cm^2 = approx 20 microg/cm^2

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
20 µg/cm²
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1. Good quality OECD guideline study. Conversion from dermal NOAEL specified as 2.5 mg/kg/day to 20 microg/cm^2 is approximate and outlined in the additional information on the study record.

Additional information

ORAL: The recommended oral NOAEL for p-tert amyl phenol based on all the weight of evidence for repeat dose effects is 70 mg/kg bw/day. This value is from a 2-generation study on the one-to-one read across analogue p-tert butyl phenol and is recommended as a basis for the oral DNEL. In this longest term study available, p-tert butyl phenol was administered orally to Sprague Dawley rats (28 males and females F0 generation; 24 males and females F1 generation) at 0, 800, 2500 and 7500 ppm in diet yielding approximate intake levels of 0, 70, 200 and 600 mg/kg bw/day.There were signs of general toxicity in dams at >200 mg/kg/day, in the form of reduced body weight gains. No adverse systemic toxicity effects were seen in pups.

In a 90 day repeated dose study (according to US EPA OPPTS 870.3100), p-tert amylphenol was administered to 20 female and 20 male CD(SD) rats by oral gavage at doses of 50, 200 and 600 mg/kg/day. Minor test related clinical pathology changes (minimal prolongations in prothombin time, mild reductions in glucose, decreases in albumin and moderate increases in urine volume were observed. The changes were not considered adverse as not biologically relevant based on sporadic nature, small magnitude and lack of correlative findings. Decreases were observed with thymus weights in males, but these were not considered adverse as there were no microscopic changes. Test related changes in the stomach (both macroscopic and microscopic) were observed in males and females. The erosion/ulceration of the nonglandular stomach and mucosal hypertrophy of the glandular stomach at 600 mg/kg/day were considered adverse effects. No test article related effects were observed in thyroid assays, bone marrow parameters, estrous cycling or sperm parameters. The NOAEL is 200 mg/kg/d based on the stomach effects.

In a 13 week combined repeat dose and reproductive toxicity study (according to OECD guideline 422) performed by the Japanese Ministry of Health (1996), p-tert butyl phenol was administered to13 female and 13 male Sprague Dawley rats by oral gavage at dose levels of 0, 20, 60, 200 mg/kg bw/day). Respiratory distress was observed in females at the top dose, but histopathology did not show respiratory tract irritation. Changes to clinical chemistry and haematology parameters were observed at the top dose (200 mg/kg/day) in males. Observations included general condition, food consumption, body weights, haematology, serum biochemistry, gross necropsy, and histopathology of several organs. The NOAEL from this subchronic 422 study on p-tert butyl phenol, for repeat dose oral systemic toxicity is 60mg/kg bw/day in males and females. It supports the choice of the NOAEL above for the DNEL calculation.  

There is further supporting evidence for the conclusion on systemic effects from a teratology study on p-tert amyl phenol by Siglin (1991), where there are no systemic toxicity effects seen at a NOAEL of 50 mg/kg bw/day. Treatment related clinical signs of maternal toxicity occurred at a dose of 200mg/kg/day but gross necropsy did not reveal any observable effects to organs. Histopathology of major target organs was not performed in detail in the reproductive and teratology studies, therefore the weight of evidence regarding histopathology on major organs is needed from the 422 study to comprehensively assess the potential for systemic toxicity by the oral route.  All 3 of these studies provide weight of evidence that a NOAEL for p-tert amyl phenol is in the region of 50-70 mg/kg/day. In defining the NOAEL as 70 mg/kg/day, the greatest weight has been placed on the longest term study.

The justification for the use of data on a one-to-one read-across analogues substance p-tert butylphenol is provided in an accompanying read-across report titled 'Read-across betweenp-tert-Amylphenol(CAS 80-46-6)and p-tert-Butylphenol (CAS 98-54-4) or sodium-p-tert-amylphenol (CAS 31366-95-7)'.

Dermal:

In a 90-day dermal repeat dose study, p-tert amylphenol was diluted with the vehicle (50% v/v ethanol in distilled water) and administered to male and female rats five days/week, for thirteen weeks, at levels of 2.5, 10.0 and 25.0 mg/kg/day. A control group and three treatment groups had ten animals per sex per group. Doses were given at a constant volume of 6.0 ml/kg. Control animals were administered with the vehicle under the same experimental conditions and at an equivalent dose volume. No treatment-related mortality or clinical signs of toxicity were observed during the study at any dose. Substantial, dose-dependent dermal irritation was produced by the test article in the 10.0 and 25.0 mg/kg/day groups.

The dermal NOEL for systemic effects is 25 mg/kg bw/day (top dose tested due to effects of irritation).  The dermal NOEL for repeat dose local irritation effects is 2.5 mg/kg/day.

For the purposes of DNEL derivation or risk characterisation to convert this into a microgram/cm2 can be done as follows:

Conc of PTAP in dosing solution = 0.42 mg/ml

Total amount of dosing solution applied/kg = 6 ml/kg

Estimated average weight of rat = 220g

Total amount of dosing solution applied per rat = (6/1000) x 220 = approx 1.3 ml/rat

Total in mg per rat = 1.3ml x 0.42mg = 0.5 mg/rat

Surface area applied = Dorsal region = 10% of total surface area of a 200g rat = approx 25cm2

Dose = 0.5mg/25cm2 = approx 20 microg/cm2

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:  Longest term oral study indicating a NOAEL for systemic effects, performed to OECD guidelines and GLP.  Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:  One dermal 90-day study on p-tert amylphenol. Justification for selection of repeated dose toxicity dermal - local effects endpoint:  One repeat dose dermal study for p-tert amylphenol available.

Justification for classification or non-classification

The effects seen in the oral data are for reduced maternal body weight and small but significant changes in some blood parameters. The observed signs of respiratory distress at the highest dose in females are seen related to the administration procedure (gavage). All effects occur at doses >/= 200 mg/kg/day and are not significant enough to warrant classification as a systemic toxicant by the oral and inhalation routes.

The dermal 90-day study did not show any effects at the top dose tested. The top dose used was limited by the local skin irritation of the substance.

There is no evidence to suggest that p-tert amylphenol should be classified as a systemic toxicant.

In general, the signs of respiratory distress observed in two different animal studies with the read across compound p.-tert.-butylphenol are not considered to justify classification, namely due to the high dose used in an acute inhalation study (5600 mg/m3) and due to the administration procedure (gavage) used in a repeated dose study, as mentioned above.