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Diss Factsheets

Administrative data

short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
The test material contained 99.2 - 99.5% active ingredient as determined by gas chromatography with flame ionization detection (GC/FID) using an external standard, infrared spectroscopy (IR) and water determination by Marl Fischer titration.

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:
Male and female Fischer 344 rats were obtained from Charles River Research Laboratories, Kingston, NY. Rats used for the 28-day study were born on September 6, 1993 (calculated date); dosed between November 2, 1993 and November 30, 1993 and were sent to necropsy December 1, 1993. Extra animals were ordered to ensure that a sufficient number of animals of acceptable health and weight were available to conduct the study. The Fischer 344 rat strain was selected because of its general acceptance and suitability for toxicity testing, availability of historical background data, and the reliability of the commercial supplier.

Animal care facilities are fully accredited by the American Association for Accreditation of Laboratory Animal Care. Upon arrival at the laboratory, the rats were examined by the laboratory veterinarian. The animal rooms of the testing facility were designed to maintain adequate environmental conditions concerning temperature, humidity, photocycle and air exchanges. The relative humidity is maintained within a range of 40-70% for rats. The building alarm system is set to activate if relative humidity is below 39% or above 71%. The room temperature is maintained at 22+/-1°C for rats. The building alarm system is set to activate if the temperature is below 19°C or above 25°C. A 12 hour light/dark photocycle is maintained for all animal rooms with lights on at 7:00 a.m. and off at 7:00 p.m. Room air exchanges occur 15 times/hour, and water lines automatically bleed every six hours. Animals were provided Purina Certified Rodent Chow #5002 (Purina Mills, Inc., St. Louis, MO) in meal form. Tap water was provided ad libitum. Water and feed analyses are performed according to the Standard Operating Procedures of The Toxicology Research Laboratory. Animals used for the probe and 28-day portion of the study were acclimated to the laboratory environment for approximately 7 days prior to study initiation. Animals were identified by a uniquely numbered metal eartag and randomized by weight during the prestudy period. The feed was analyzed by the supplier to confirm the nutritional adequacy of the diet and to quantitate levels of selected contaminants. The drinking water was analyzed by the City of Midland and an independent laboratory in accordance with the Laboratory Standard Operating Procedures. Copies of these analyses are maintained in the Toxicology Research Laboratory. The results of the feed and water analyses were judged to be within acceptable limits.

Administration / exposure

Type of coverage:
Details on exposure:
In order to demonstrate the highest percent solution of TIPA that would not cause severe irritation, ten male Fischer 344 rats (2/dose level) were administered 4 mL/kg of a 15%, 30%, 45%, 60%, or 75% TIPA solution in distilled water dermally, for at least six hours per day, for four consecutive days. Patches containing the test solutions were applied to the back of the rat as noted. Each animal was observed and scored daily for signs of systemic toxicity and dermal irritation (see below for description of the dermal irritation grading scheme). No further data was collected from these animals.

Groups of five male and five female Fischer 344 rats per dose level were administered 4 ml/kg of a 0%, 7.5%, 25%, or 75% TIPA solutions corresponding to 0 (control; distilled water vehicle), 300, 1000, or 3000 mg TIPA/kg body weight/day. The high dose level was chosen based on results of previously conducted studies as well as the results of the probe study.

All animals were dosed 21 days during the 28-day interval (5 days/week). Patches were applied to the back of each rat and covered with nonabsorbent cotton. Each animal was observed daily and scored weekly following patch removal for signs of systemic toxicity and dermal irritation. Rats were dosed for two days immediately prior to necropsy.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The test material was delivered to each rat as an aqueous solution. Since TIPA is a solid at room temperature, the test material was warmed to approximately 60°C to facilitate the preparation of dosing solutions.

Dose solutions based upon percent TIPA were prepared approximately biweekly during the course of the study. The volume of percent dose solutions delivered dermally to each animal were adjusted weekly to maintain the dose levels on the targeted mg/kg body weight/day basis. Reference samples (1/dose/sex/solution) were retained and stored at ambient temperatures in a manner consistent with the sample retention policy of the laboratory.

The stability of a 7.5% solution of TIPA in distilled water (low dose test solution) was determined to be at least 15 days. The mixing method that was used to produce a homogeneous dose solution was validated analytically. Analyses of the dose solutions to verrfy concentration of the dose solutions indicated an acceptable agreement between the observed versus targeted concentrations of TIPA in the dose solution preparations.
Duration of treatment / exposure:
5 days/week for 28 days
Frequency of treatment:
21 applications/dose
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
3 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
The dose solutions were delivered onto a gauze patch and then applied to a clipped area on the back of each rat. A dosing volume of 4 mL/kg/day was used with the volume/animal dose adjusted each week based upon the most recent body weight data. Distilled water was administered to control animals in a dosing volume similar to the treated animals. Distilled water was used as the dosing vehicle since TIPA was water soluble, water does not produce skin irritation, and its absorption would not confound the results of the study. The homogeneity and stability of TIPA in water was determined concurrent with the conduct of the study using appropriate analytical methods. The concentration of TIPA in dosing solutions was determined concurrent with the initiation and mid-point through the study. Probe and repeated dose animals were acclimated to elastic wrap, used to hold the test material dressing in dermal contact, for at least 6 hours for three days prior to the probe study initiation. The test material solution (or distilled water for controls) was dosed onto an absorbent gauze patch and then applied to a 5x5 cm clipped area (approximately 10% body surface area) on the back of each rat. The dosed patch was covered with nonabsorbent cotton held in place with non-irritating cloth tape and then covered with elastic wrap and tape. The dosed patches and wrap were removed approximately 6 hours after application. The treated area was wiped with a soft water-dampened disposable towel to remove any residual test material.
Positive control:


Observations and examinations performed and frequency:
In-Life Observations. A careful clinical examination was conducted on all animals prior to the start of the study, at weekly intervals during the study, and one day prior to necropsy, coincident dermal scoring. This examination included thorough, hands on, evaluations of the skin, fur, mucous membranes, respiration, central nervous system function (including tremors, convulsions and diarrhea), swelling, masses and animal behavior. A daily visual, cageside examination was also made each day (5-days/week during which, to the extent possible, the above parameters were evaluated. A cageside examination was not performed when clinical examinations were performed.

An additional observation for morbidity, mortality, and the availability of feed and water was made each day of the work-week as well as twice daily on weekends.

Evaluation of Dermal Application Site. In addition to daily cageside and clinical examinations, the condition of the dermal test site was subjectively evaluated weekly following five consecutive days of dosing and on the day prior to necropsy using this laboratory's modification of the acute dermal irritation scoring system recommended by the Organisation for Economic Cooperation and Development (OECD, 1981):
Grading of Dermal Exam
Erythema and Eschar Grade
Within normal limits ......................................................................................... 0
Very slight erythema (barely perceptible) .................................................... 1
Well-defined erythema .................................................................................... 2
Moderate to severe erythema .........................................................................3
Severe erythema to slight eschar formation ............................................... 4
Within normal limits ........................................................................................ 0
Very slight (barely perceptible). .................................................................... 1
Well-defined (edges raised). .......................................................................... 2
Moderate (raised approximately 1 millimeter) ............................................ 3
Severe (raised more than 1 millimeter). ....................................................... 4
Scaline and Fissuring:
Within normal limits ........................................................................................ 0
Slight scaling .................................................................................................... 1
Moderate - severe scaling .............................................................................. 2
Slight fissuring ................................................................................................. 3
Moderate - severe fissuring ........................................................................... 4
In addition, necrosis, scabs and/or scars were noted if present; however, they were not graded.

Blood samples were collected from all rats at the scheduled necropsy. Rats were fasted overnight, anesthetized by inhalation of methoxyflurane vapors and blood samples collected by venapuncture of the post-orbital venous plexus. The following hematologic parameters were evaluated for each animal: hematocrit (HCT), hemoglobin concentration (HGB), erythrocyte count (RBC), total leukocyte count (WBC), and platelet count (PLAT). Automated differential counts of leukocytes were also conducted, including neutrophil (NEUT), lymphocyte (LYMP), monocyte (MONO), eosinophil (EOS), basophil (BASO), and large unstained cells (LUC) and an assessment of erythrocyte, leukocyte and platelet morphology. Parameters were measured using a Technicon H-1E (Miles Instruments, Tarrytown, NY). Samples were mixed with EDTA and blood smears were prepared and stained with Wright's Stain and retained in the files.

Urine samples were obtained by external manual compression of the bladder from all nonfasted surviving rats during the week prior to necropsy. If an insufficient quantity was collected from a particular rat, a second urine collection was attempted as soon as possible after the first attempt. The following parameters were assayed using a Clinitec 200 (Ames, Elkhardt, IN): specific gravity, pH, bilirubin, glucose, protein, ketones, blood and urobilinogen. In addition, microscopic examination of sediment in a pooled sample from each group were performed.

Blood samples were collected from all fasted rats at the scheduled necropsy by venapuncture of the post-orbital venous plexus. Samples were held on ice and the serum was harvested. The following parameters were evaluated for each animal: alkaline phosphatase activity (AP), alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), urea nitrogen (UN), creatinine (CREAT), total protein (TP), albumin (ALB), globulin (GLOB), glucose (GLUC), total bilirubin (TBILI), phosphorus (PHOS), calcium (CALC), sodium (Na), potassium (K) and chloride (Cl). All analyses, with the exception of GLOB, which was calculated, were conducted using a Monarch 2000 Chemistry System (Instrument Laboratory Inc., Lexington, MA).
Sacrifice and pathology:
All rats were presented for necropsy approximately 24 hours after the final dermal application. A fasting terminal body weight of each rat was recorded prior to being anesthetized by inhalation of methoxyflurane vapors. Following the collection of blood samples for hematologic and clinical chemistry examinations, the trachea was exposed and clamped, the rat euthanized by decapitation, and then exsanguinated. A complete gross examination of tissues as per guidelines was conducted on all animals by a veterinary pathologist. The necropsy included an in situ examination of the eyes by visual inspection of the cornea, lens and other internal components via placement of a moistened glass slide on the corneal surface under fluorescent light illumination. Following inspection of the externum and body orifices, the cranial, oral, thoracic, and abdominal cavities were opened and the visceral organs were examined both in situ and following dissection. The digestive tract and larger visceral organs were incised for examination. The adrenal glands, brain, heart, kidneys, liver, and testes or ovaries were dissected and weighed for each animal and the ratio of organ weight to terminal body weight was calculated. The lungs were distended to their approximately normal inspiratory volume with phosphate-buffered 10% formalin. The nasal cavity was flushed with this solution delivered via syringe through the nasopharynx. A complete set of tissues, as per guideline, was retained in fixative from all rats. Special attention was given to the skin at the site of application of the test material or vehicle control. The skin sample included both the entire shaved area from the dorsal trunk of the rat and the adjacent unshaved area posterior to it. A second skin specimen was collected from the inguinal region to include the mammary gland. The latter skin was not used for comparison between treated and untreated skin.

Tissues were prepared for light microscopic evaluation by standard procedures, sectioned at approximately 6 microns and stained with hematoxylin and eosin. Histopathologic examination was conducted on the liver (3 sections), kidneys (2 sections), the treated skin site, the untreated skin site, and all gross lesions except those considered normal physiologic changes: (i.e. uterus distended with clear fluid) or artifacts from necropsy (i.e. lungs with aspirated blood secondary to decapitation) from all control and rats given 3000 mg/kg/day. Sections of treated skin, untreated skin and gross lesions were examined from all rats receiving 300 or 1000 mg/kg/day. The sections were evaluated light microscopically by a veterinary pathologist. When deemed appropriate, histopathologic changes were graded as to:
symmetry - unilateral or bilateral;
distribution - focal, multifocal or diffuse;
and severity - very slight, slight, moderate or severe.
The grade of severity was established based upon the extent of parenchyma involved, which required correlation with gross observations, and the degree to which the parenchyma was altered by the change. Very slight and slight lesions were both minimal grades and were not expected to affect the health of the animal nor the function of the involved organ, although the difference was distinguished to reflect any possible difference that could be attributable to treatment. These grades were differentiated based upon the amount of parenchyma involved and the extent of involvement. Specifically, the effect at the treated skin site, termed epidermal hyperplasia, was graded as to severity by the following criteria. Normal skin from the dorsal trunk consists of only about two layers of nuclei in the stratum germinativum, a thin inconspicuous stratum granulosum with only a very few nuclei present, and a modest but irregular stratum corneum. Very slight hyperplasia was defined as a generalized, but irregular, increased cellularity of the stratum germinativum averaging less than four nuclei thick. Frequently the stratum granulosum was also slightly thickened and the stratum corneum may also have been equivocally thickened. Slight epidermal hyperplasia was defined as greater thickening of the epidermis such that the stratum germinativum in the treated site averaged about four cells thick with some areas focally increased to about seven cells thick.

Other grades indicating greater severity were available to the pathologist but were not required for the diagnoses for any organ. The definitions for these grades were: Moderate lesions would involve a greater amount of organ parenchyma or the area involved would be substantially altered from normal microscopic appearance. Lesions of this severity might affect organ function although not to the point of organ failure and they would generally be considered not to be life threatening. Severe lesions would be distinguished by substantial if not entire involvement of the organ. They would be expected to have an effect upon organ function and, depending upon the organ involved, the lesion might affect the health of the animal if not be responsible for its death or moribund condition.
Other examinations:
In-Life Observations. A careful in-life observation for signs of toxicity was conducted each working day throughout the probe portion of the study. Two additional daily observations were limited to animal husbandry procedures to ensure the availability of feed and water.

Evaluation of Dermal Application Site. The condition of the dermal test site was subjectively evaluated daily following removal of wrap during the probe study using the same irritation scoring system as found in the design of the 28-day study.
All parameters examined statistically were first tested for equality of variance using Bartlett's test. When the results from Bartlett's test were significant, then the data for the parameters were subjected to a transformation to obtain equality of the variances. The transformations that were examined were the common log, the inverse, and the square root, in that order with a Bartlett's test following each transformation. When Bartlett's test was satisfied no further transformations were applied, or, if none of the transformations resulted in homogeneous variances, the transformed data or raw data with the lowest Bartlett's statistic was used.

In-life body weights were evaluated using a three-way repeated measures (RM) analysis of variance (ANOVA) for time (the repeated factor), sex, and
dose (Winer, 1971). In the three-way RM-ANOVAs, a linear contrast will be used to compare the control group to the dosed groups when a statistical
difference in the time by dose interaction effect exists.

Terminal body weight, organ weight (absolute and relative, excluding ovaries and testes), hematologic parameters (excluding differential WBC and RBC indices), clinical chemistry parameters, and urine specific gravity were evaluated using a two-way ANOVA with the factors of sex and dose; differences between the groups were primarily detected by the dose factor. For these parameters, the first examination was whether the sex-dose interaction
was significant; if it was, a one-way ANOVA was done separately for each sex. Comparisons of individual dose groups to the control group were made with Dunnett's test only when a statistically significant dose effect existed; this was subsequent to the evaluation of the sex-dose interaction. The form of the ANOVA, one-way or two-way, was determined by whether or not the analysis was separated by sex or not.

Results for testes or ovaries weight (absolute and relative) were analyzed using a one-way ANOVA.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related observations suggestive of systemic toxicity in any treated animals.
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Localized signs of reaction to treatment consisted of dose-related dermal irritation at the test site. There was no evidence of dermal irritation in male and female rats administered 300 mg TIPA/kg bw/day and female rats administered 1000 mg TIPA/kg bw/day. The dermal application of TIPA resulted in very slight erythema (barely perceptible) and scabs at the dermal test site in two of five male rats administered 1000 mg TIPA/kg bw/day. One of the 1000 mg TIPA/kg bw/day rats had erythema observed on test day 4 (resolved by test day 11) and the second rat had erythema observed on the last day of application. Rats administered 3000 mg TIPA/kg bw/day had very slight erythema and scabs at the dermal test site in two male rats on test day 4 (resolved by test day 11), and 3 male rats on test day 18 (resolved by test day 25 in two of three rats and by termination of the study in the third rat) and one female rat on test day 25 (still evident at study termination)
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effect on mean body weights or cumulative body weight gains was observed in male or female rats at dose levels up to 3000 mg TIPA/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related effect on mean food consumption was observed in male or female rats at dose levels up to 3000 mg TIPA/kg bw/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Examination of the eyes pre-study and at study termination revealed no treatment-related effects.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related effect on haematology parameters were noted in male or female rats at dose levels up to 3000 mg TIPA/kg bw/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related effect on clinical chemistry parameters were noted in male or female rats up to 3000 mg TIPA/kg bw/day.
Urinalysis findings:
no effects observed
Description (incidence and severity):
A statistically identified decrease in specific gravity was observed in 1000 mg TIPA/kg bw/day female rats. Due to lack of dose response, the decreased specific gravity was considered not related to the administration of TIPA. No other changes in urinalysis parameters were observed.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related effects were noted in the organ and organ to body weight ratios in male and female rats dosed up to 3000 mg TIPA/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no lesions suggestive of a systemic response to the dermal application of TIPA. Very few, minute scabs were present at the dermal test site of one male administered 1000 mg TIPA/kg bw/day and one female administered 3000 mg TIPA/kg bw/day. Pale foci were noted in various liver lobes in one control male, one male administered 1000 mg TIPA/kg bw/day and 3 males administered 3000 mg TIPA/kg bw/day. While the incidence of these foci was somewhat increased at the highest dose level, their occurrence in a control suggests that they may be due to experimental conditions associated with preparation and bandaging of the treatment site. All other changes occurred with a low incidence, i.e. usually only a single rat in a dose group, and without any relationship to TIPA treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The only effect ascribed to treatment was thickening of the skin, termed epidermal hyperplasia, restricted to the treatment site. This effect overall was of a minimal degree, graded as very slight except for one rat/sex graded as slight, and involved 3 males and 4 females administered 3000 mg TIPA/kg bw/day. In one of the females administered 3000 mg TIPA/kg bw/day, epidermal hyperplasia was focal rather than generalized at the treated site. Two males and 1 female administered 1000 mg TIPA/kg bw/day also had epidermal hyperplasia of very slight degree as did one control female. Epidermal hyperplasia was characterized by slightly increased numbers of cells (nuclei) within the stratum germinativum and increased thickness or prominence of the stratum granulosum. Other responses to dermal irritation, i.e. increased inflammatory cells in the subcutis or hyperplasia of sebaceous glands, were not noted. Two small scabs, one of which overlaid a shallow ulcer, were found histologically in the treated skin of the one female administered 3000 mg TIPA/kg bw/day correlating with the minute scabs noted at necropsy. Very slight epidermal hyperplasia but no additional lesions were found for the one male administered 1000 mg TIPA/kg bw/day that had two minute scabs grossly. There were no lesions in any other organ examined attributed to TIPA treatment including the untreated skin site caudal to the treated site. The pale foci noted grossly in the livers of several males were found to be small foci of coagulation necrosis of hepatocytes and were considered to be unrelated to TIPA. They are considered to likely be of traumatic origin from handling or bandaging procedures. There was acute necrosis of the adrenal gland in one male administered 1000 mg TIPA/kg bw/day. The etiology of this lesion is unknown but it was not attributed to TIPA. All other lesions were of minimal severity and are considered spontaneously-occurring changes typically noted in rats of this strain, age and husbandry conditions.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs of severe irritation were not evident in the two male rats/dose group that were dermally administered 4 ml/kg of 15%, 30%, 45%, 60% or 75% TIPA solution for four consecutive days. Very slight erythema was observed at the application site of one of two rats following four doses of 75% TIPA solution. Based on the results of the probe study and previous study data, a 4 mL/kg dosing volume of a 75% TIPA solution corresponding to 3000 mg TIPA/kg/day was selected as the high dose for the 28-day study. TIPA solutions of 7.5% and 25% corresponding to 300 mg TIPA/kg bw/day and 1000 mg TIPA/kg bw/day, respectively, were selected to evaluate a potential dose-response relationship.

Effect levels

open allclose all
Dose descriptor:
systemic toxicity
Effect level:
>= 3 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
local toxicity
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
dermal irritation

Target system / organ toxicity

Critical effects observed:

Applicant's summary and conclusion