p-(1,1-dimethylpropyl)phenol

Repeated dose toxicity: dermal

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

para-t-amyl-phenol

IUPAC Name:

para-t-amyl-phenol

Test material form:

other: granules

Details on test material:

para-t-amyl-phenol. Supplied by owner company and verified analytically.
White granules were dissolved in 200 Proof Ethyl Alcohol

Test animals

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.

Administration / exposure

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.

Doses / concentrations

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

Examinations

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%.

Results and discussion

Results of examinations

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.

Effect levels

1

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'

2

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'

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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