Registration Dossier

Administrative data

Description of key information

There are repeated dose studies with a structural analogue of N-Methyldiisopropanolamine, tris-(2-hydroxypropyl)-amine available: 
In a sub-chronic oral toxicity study in dogs, performed according to FDA guidelines, NOAELs of 272 mg/kg bw/day for males and 288 mg/kg bw/day for females were established, the highest doses tested.
In a sub-acute dermal toxicity study in rats, performed according to OECD TG 410, a NOAEL of 3000 mg/kg bw/day (the highest dose tested) for systemic toxicity was established.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP
Qualifier:
according to
Guideline:
other: U.S. FDA Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food-21 CFR 314.50(d)(2)
Principles of method if other than guideline:
U.S. FDA Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food-21 CFR 314.50(d)(2)
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
18 male and 18 female beagle dogs, born between 8/3/86 and 8/16/86 (average birth date 8/10/86), were acquired from Marshall Research Animals, North Rose, New York on 1/8/87. All dogs had been individually identified by means of ear tattoos by the supplier. The beagle dog was selected on the bases of extensive experience with that strain and its suitability with respect to hardiness, longevity, sensitivity, and low incidence of spontaneous diseases. Upon arrival at Haskell Laboratory, all dogs were housed individually in stainless steel cages. During a five-week acclimation period, each dog was fed approximately 350 g ground Purina® Certified Canine Diet #5007 each day, and provided tap water ad libitum. All dogs were weighed the day after arrival and approximately once every week thereafter, throughout the acclimation period. The dogs were observed at least twice per day for eating habits and any gross signs of disease or injury. Twice during the acclimation period, all dogs were subjected to clinical laboratory examinations as described in Materials and Methods, Section G. All dogs also underwent a pretest ophthalmoscopic examination. After the acclimation period, and on the basis of pretest clinical pathology measurements, body weights, ophthalmoscopic examination, and a general physical examination, the dogs were divided into four groups of four males and four groups of four females. Differences in these parameters between each group within a sex were minimized. In addition to the ear tattoo, each dog was identified with a collar bearing the individually assigned Haskell animal number. The approximate age of each dog at start of feeding of TIPA (2/13/87) was 27 weeks. The two remaining dogs (one male and one female) which were not selected for the palatability study or main study, were subsequently used on a palatability study for another material. During the pretest period and throughout the study, the animal room was maintained on a 12-hour light/12-hour dark cycle. The target room temperature and relative humidity were 23±2°C and 50±10%, respectively.
Route of administration:
oral: feed
Vehicle:
water
Details on oral exposure:
During the test period, dogs in each group were fed ground Purina@ Certified Canine Diet #5007 (GPCCD) that contained 0, 500, 2000 or 7500 ppm TIPA. Individual diet allotments of approximately 350 g/dog were given to the dogs each day and tap water was provided ad libitum. Before preparing diet, TIPA was dried with Drierite drying agent under vacuum for several days. All compound was stored in containers which were purged with nitrogen every time after being opened. For diet preparation, TIPA was completely dissolved in 200 ml of distilled water, added to the GPCCD, and thoroughly mixed with a Hobart high speed mixer (model VCM-60E) for three minutes to assure a homogeneous distribution in the diet. During the first two weeks of diet preparation, a Brinkmann polytron homogenizer (model# PT 10-35) was used to dissolve the TIPA in the water. Thereafter, a magnetic stirring platform was used instead of the polytron. Magnetic stirring was found to dissolve the sample better and more rapidly than the polytron without aerating the solution. Control diets also had 200 ml distilled water added and were mixed for the same period of time as the test diets. All diets were prepared weekly and refrigerated until used. On test days 0 and 91, samples (approximately 50 g each) of freshly prepared diet from each treatment level (excluding control level) were collected and analyzed for homogeneity and concentration/stability of TIPA in the diet. An additional set of homogeneity samples was collected at day 14 because the method of diet preparation was changed (from polytron to magnetic stirrer for dissolving TIPA).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Methanol was added to each diet sample and TIPA was extracted by sonication, filtered, evaporated under nitrogen, and derivatized with Pyridine and N-Trimethylsilylimidazole (TMSI). The reaction was completed by warming the solutions to 80°C for 10 (0, 500 ppm) or 20 minutes (2000, 7500 ppm). Recovery efficiencies were determined at the 500 ppm level by adding 5 mL of a 1000 µg/mL TIPA stock solution to control diet and at the 2000 and 7500 ppm levels by adding 20.3 and 75.9 mg H-16,648, respectively, to 10.0 g control diet. Extraction and preparation of the recovery samples were performed as described above. Calibration curves were created using standarrd solutions of TIPA or TEA. Peak area ratios were calculated (TIPA to TEA) and used for quantitation by linear regression analysis. The dietary concentration of TIPA was determined by multiple injection with a Hewlett-Packard 5880A gas chromatograph.
Duration of treatment / exposure:
102-104 days
Frequency of treatment:
Continuous
Remarks:
Doses / Concentrations:
0; 500; 2,000 and 7,500 ppm TIPA
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
The mean daily intake of TIPA in the 0; 500; 2000 and 7500 ppm diets was 0, 16.8, 71.2, and 272 mg TIPA/kg body weight/day for males and 0, 19.7, 78.3, and 288 mg/kg for females, respectively
Basis:
other: mean daily intake
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels for this study were based upon acute and subchronic studies in rats conducted with TIPA and after consultation with the FDA. Studies with dogs were not available. On an acute basis, the single dose oral LD50 of TIPA in rats was 5994 mg/kg. When rats were given 140 to 1350 mg TIPA/kg/day in drinking water for 30 days, 1350 mg/kg/day resulted in growth reduction and decreased food consumption. A dose level of 260 mg/kg/day produced unspecified histopathologic changes. In a 90-day study in rats, dose levels of 770 mg TIPA/kg/day in drinking water produced kidney effects consisting of dilation of Bowman's capsule and convoluted tubules, as well as marked cloudy swelling of liver parenchyma. A level of 220 mg/kg/day produced unspecified pathological changes in some animals. Dosages of 140 mg/kg/day for 30 days or 110 mg/kg/day for 90 days revealed no microscopic pathologic changes. Dietary levels for this study were selected to be consistent with a 90-day rat feeding study which was also being conducted on this material. The two highest dose levels for the rat study (2,000 and 7,500 ppm in diet) were selected to produce mean daily intakes in rats similar to those at which pathological changes were seen in a previous rat drinking-water study (220 and 770 mg/kg/day). Further, while lower mean daily intakes were expected for dogs than for rats, a dietary level of 7500 ppm was expected to produce mean daily intake in dogs of greater than 200 mg/kg/day, a level associated with some pathology in rats.
Prior to starting the 90-day study, one male and one female beagle dog were fed 7500 ppm TIPA for ten days to determine palatability of the test material. Procedures for feeding and weighing were the same as for the main study. At the end of a ten-day feeding period they were sacrificed without pathological examination. Over the ten-day period, the male dog gained 0.7 kg (initial weight, 8.0 kg; final weight, 8.7 kg). The female dog gained 0.4 kg (initial weight, 7.1 kg; final weight, 7.5 kg). Food consumption was 320 and 238 g/day for the male and female dog, respectively, versus 314 and 266 g/day in the four-day period immediately prior to feeding TIPA. Neither dog displayed any abnormal clinical signs. Since food consumption was adequate, and in the absence of severe weight decreases, 7500 ppm was determined to be an acceptable level for the conduct of the 90-day study on the basis of palatability.
Positive control:
None
Observations and examinations performed and frequency:
Clinical Observations and Mortality
All dogs were observed at least twice daily for any abnormal behavior and appearance.

Body Weights
All dogs were weighed once a week during the study.

Food Consumption, Food Efficiency, and Intake of TIPA
The amount of food consumed by each dog was determined on a weekly basis throughout the study. From these determinations and body weight data, mean individual daily food consumption, food efficiency, and intake of test material were calculated.

Ophthalmoscopic Examinations
A veterinary ophthalmologist conducted an ophthalmoscopic examination on each dog before the initiation of the study and prior to the termination of the study. At each examination, one or two drops of Pharmafair 1% Tropicamide Ophthalmic Solution USP-Sterile were placed into each eye and both eyes were examined for abnormalities. Room lighting was subdued after eye drops were administered and for the remainder of the day.

Clinical Laboratory Evaluation
Two times during the pretest period and approximately one, two, and three months after initiation of the study, each dog was fasted for approximately 16 hours. Urine was collected from each dog during this period. At the conclusion of this period, blood samples for hematological and clinical chemistry measurements were collected from the jugular vein of each dog.

Hematology
The hematological parameters examined at each sampling time consisted of erythrocyte, leukocyte, differential leukocyte and platelet counts, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. Blood smears for reticulocyte counts were prepared from each dog at each sampling time and bone marrow smears were prepared from all dogs at the final sacrifice.

Clinical Chemistry
In conjunction with each hematological examination, blood serum was evaluated for concentrations of sodium, potassium, chloride, calcium, glucose, blood urea nitrogen, phosphorous, cholesterol, bilirubin, creatinine, total serum proteins, albumin, globulin (calculated), and activities of alkaline phosphatase, alanine aminotransferase, creatine kinase, and aspartate aminotransferase. Blood phosphorous was inadvertently not analyzed at the pretest sampling period. This is not considered to have adversely affected the outcome of the study.

Urine Analyses
Urine collected during each fasting period from each dog was subjected to quantitative measures of volume, pH, and osmolality and semi-quantitative measures of protein, glucose, urobilinogen, ketone, bilirubin, and occult blood. Urine color and transparency were recorded, and sediment from each urine sample was microscopically examined.

Methemoglobin Determinations
Methemoglobin was determined at two- and three-month sampling periods by a standard spectrophotometric method.
Sacrifice and pathology:
The final sacrifice for all dogs occurred between test days 102 and 104. The order of sacrifice was random within a sex. Each dog was fasted for at least sixteen hours prior to sacrifice. Sacrifice was performed by intravenous injection of thiobarbiturate (sodium thiamylal) and exsanguination. A section of rib from each dog was submitted to the Clinical Pathology Section for collection of bone marrow.

The following tissues were collected from all dogs:
Skin (ventral), rib (with costochondral junction), sternum, skeletal muscle (thigh), trachea, lungs (two sections), thymus, spleen, bone marrow, lymph nodes (mesenteric and mandibular), heart* (four sections), aorta (thoracic), salivary glands, tonsil, esophagus, stomach (cardiac, fundic, and pyloric), gallbladder, liver* (two sections), pancreas, small intestine (duodenum, jejunum, and ileum), large intestine (cecum, colon, and rectum), kidneys*, bladder, pituitary, thyroid-parathyroids*, adrenals*, prostate, mammary gland, testes*, ovaries, epididymides, uterus, vagina, brain* (three levels), spinal Cord (two levels), peripheral nerve (sciatic), eyes, and all gross lesions.
* Organs weighed and organ weight/body weight ratios calculated.

All tissues were fixed and stained with appropriate agents, processed by conventional methods and evaluated by light microscopy.
Statistics:
Body weights, body weight gains, food consumption data, organ weights, clinical laboratory measurements, and blood methemoglobin values were analyzed by a one-way analysis of variance. When the test for differences among test group means (F test) was significant, pairwise comparisons between test and control groups were made with the Dunnett's test. Significance was judged at alpha = 0.05. Homogeneity of variances of organ weights and clinical laboratory data were analyzed with the Bartlett's test (alpha = 0.005). When the results of Bartlett's test were significant (variance was not homogeneous), the Mann-Whitney U test was employed instead of Dunnett's test for comparison of means (alpha = 0.05).
Details on results:
No dogs died during the study. There were no biologically significant or compound-related effects found at any level in clinical observations, body weights and body weight gains, food consumption and efficiency, ophthalmoscopic examinations, hematology, serum chemistry, urinalysis, methemoglobin determinations, organ weights, or gross or microscopic pathology.
Dose descriptor:
NOAEL
Effect level:
> 7 500 other: ppm (272 mg/kg bw/day for males; 288 mg/kg bw/day for females)
Sex:
male/female
Basis for effect level:
other: No treatment-related effects were observed up to the highest dose tested.
Critical effects observed:
not specified
Conclusions:
The NOAEL for the study was 7,500 ppm for both male and female dogs.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
272 mg/kg bw/day
Study duration:
subchronic
Species:
dog
Quality of whole database:
The study was conducted according to FDA test guidelines and in accordance with GLP.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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.
Type of coverage:
semiocclusive
Vehicle:
water
Details on exposure:
Probe Study.
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.

28-Day Repeated Dose Study.
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:
yes
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:
a total of 21 applications/dose
Remarks:
Doses / Concentrations:
0, 300, 1000 or 3000 mg TIPA/kg/day
Basis:
nominal per unit body weight
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:
no
Observations and examinations performed and frequency:
Probe Study.
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.

28-Day Repeated Dose Study.
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
Edema:
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.
Sacrifice and pathology:
Clinical pathology.
Hematology. 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.

Urinalysis. 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.

Clinical Chemistry. 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).

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.

Histopathology. 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:
no other examinations conducted
Statistics:
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.
Clinical signs:
effects observed, treatment-related
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
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:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Probe Study.
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/day and 1000 mg TIPA/ kg/day, respectively, were selected to evaluate a potential dose-response relationship.

28-Day Repeated Dose Study.
In-Life Observations. 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/day and female rats administered 1000 mg TIPA/kg/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/day. One of the 1000 mg TIPA/kg/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/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). There were no treatment-related observations suggestive of systemic toxicity in any treated animals.

Body Weights and Feed Consumption.
No treatment-related effect on mean body weights, cumulative body weight gains or food consumption was observed in male or female rats at dose levels up to 3000 mg TIPA/kg/day.

Ophthalmologic Exams.
Examination of the eyes prestudy and at study termination revealed no treatment-related effects.

Hematology.
No treatment-related effect on hematology parameters were noted in male or female rats at dose levels up to 3000 mg TIPA/kg/day.

Clinical Chemistry.
No treatment-related effect on clinical chemistry parameters were noted in male or female rats up to 3000 mg TIPA/kg/day.

Urinalysis.
A statistically identified decrease in specific gravity was observed in 1000 mg TIPA/kg/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.

Terminal Body and Organ Weights.
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/day.

Gross and Histopathology.
All rats survived the test period and 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/day and one female administered 3000 mg TIPA/kg/day. Pale foci were noted in various liver lobes in one control male, one male administered 1000 mg TIPA/kg/day and 3 males administered 3000 mg TIPA/kg/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.

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/day. In one of the females administered 3000 mg TIPA/kg/day, epidermal hyperplasia was focal rather than generalized at the treated site. Two males and 1 female administered 1000 mg TIPA/kg/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/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/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/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.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
3 000 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no systemic effects were reported
Dose descriptor:
NOAEL
Remarks:
local toxicity
Effect level:
300 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: slightly irritative effects: epidermal hyperplasia (300 mg/kg bw/day corresponds to 2.4 mg/cm2 assuming a body weight of 0.2 kg and as 25 cm2 skin was exposed)
Critical effects observed:
not specified

All rats survived the test period and there were no indications of systemic toxicity observed in treated animals. At the end of the in-life period, very slight erythema and scabs were observed at the dosing site of one male rat administered 1000 mg TIPA/kg/day and one female rat administered 3000 mg/kg/day. Histopathologically, the only effect attributed to treatment of TIPA was minimal thickening of the skin, termed epidermal hyperplasia, at the dosing site. This effect was noted in most, but not all, rats dosed with 3000 mg TIPA/kg/day. 

Under the conditions of this study, the repeated dermal administration of TIPA resulted in very slight irritative effects at the dosing site in rats administered 1000 or 3000 mg/kg/day, 5 days/week for 28 days (21 applications).
 There was no evidence of systemic toxicity in rats administered up to 3000 mg/kg/day. Therefore, the NOAEL for systemic toxicity was 3000 mg/kg/day, for male and female Fischer 344 rats following dermal administration.

Conclusions:
Under the conditions of this study, the repeated dermal administration of TIPA resulted in very slight irritative effects at the dosing site in rats administered 1000 or 3000 mg/kg/day, 5 days/week for 28 days (21 applications). There was no evidence of systemic toxicity in rats administered up to 3000 mg/kg/day. Therefore, the NOAEL for systemic toxicity was 3000 mg/kg/day, for male and female Fischer 344 rats following dermal administration.
Executive summary:

Triisopropanolamine (TIPA), containing 99.2 or 99.5% active ingredient, respectively, was evaluated for potential systemic toxicity following repeated dermal administration. Groups of ten Fischer 344 rats (five/sex/dose) were administered 0 (control), 300, 1000 or 3000 mg TIPA/kg body weight/day, dermally, 21 times over a 28-day interval. The highest concentration tested was a 75% TIPA solution. Data was collected on the following: clinical appearance and behavior; in-life and terminal body weights; dermal irritation at the dosing site; clinical chemistry and hematologic parameters; organ weights; and gross and histopathologic appearance of tissues. All rats survived the test period and there were no indications of systemic toxicity observed in treated animals. At the end of the in-life dosing period, very slight erythema and scabs were observed at the dosing site of one male rat administered 1000 mg TIPA/kg/day and one female rat administered 3000 mg/kg/day. Histopathologically, the only effect attibuted to treatment of TIPA was minimal thickening of the skin, termed epidermal hyperplasia, at the dosing site. This effect was noted in most, but not all, rats dosed with 3000 mg TIPA/kgBW/day.

Under conditions of this study, the repeated dermal administration of TIPA resulted in very slight irritative effects at the dosing site in rats administered 1000 or 3000 mg TIPA/kg body weight/day, 5 days/week for 28 days (21 applications). There was no evidence of systemic toxicity in rats administered up to 3000 mg TIPA/kg body weight/day. Therefore, the NOAEL for systemic toxicity was considered 3000 mg TIPA/kg body weight/day, for male and female Fischer 344 rats following dermal administration.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
3 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was conducted according to test guidelines and in accordance with GLP.

Additional information

Currently, there is no repeated dose toxicity study with1,1’-(methylimino)-dipropane-2-ol available. Nevertheless, a subacute as well as a subchronic study via the dermal (OECD TG 410) and oral route of exposure (performed according to FDA guidelines) were carried out with tris-(2-hydroxypropyl)-amine (CAS 122-20-3) and can be used for cross reading.

1,1’-(methylimino)-dipropane-2-ol and tris-(2-hydroxypropyl)-amine are structurally very similar low molecular weight tertiary amines, which differ only in a methyl group and an isopropanol moiety respectively.They are low volatile (vapour pressure ≤ 0.015 hPa) organic compounds with similar specific gravity (1,1’-(methylimino)-dipropane-2-ol: 147.22 g/mol and tris-(2-hydroxypropyl)-amine: 191.27 g/mol) and log Pow values between -0.03 for 1,1’-(methylimino)-dipropane-2-ol and -0.02 for tris-(2-hydroxypropyl)-amine. Moreover, the comparison of the available toxicity data for both compounds indicates a similar toxicological profile: the substances are showing low acute toxicity by the oral route (i.e. oral LD50values for1,1’-(methylimino)-dipropane-2-ol of 2150 mg/kg bw and beyond 5000 mg/kg bw for tris-(2-hydroxypropyl)-amine).Tris-(2-hydroxypropyl)-amine is irritating to the eyes, whereas 1,1’-(methylimino)-dipropane-2-ol shows more potent and corrosive properties.The chemical difference between those two structures apparently do not affect the anticipated toxicity. For a detailed read across justification see also attached assessment reports (Reporting Format for the analogue approach of 1,1’-(methylimino)-dipropane-2-ol– with comparison to tris-(2-hydroxypropyl)-amine).

 

Oral

Beagle dogs (4/sex/dose) were administered tris-(2-hydroxypropyl)-amine in their diet at 0, 500, 2000 or 7500 ppm (approximately 0, 16.8, 71.2 and 272 mg/kg bw/day in males or 0, 19.7, 78.3 or 288 mg/kg bw/day in females) for 102 - 104 days (DuPont, 1987; performed according to FDA guidelines). Ophthalmology, hematology, serum chemistry, urinalysis, blood methaemoglobin, macroscopic and microscopic examinations, and organ weight evaluations showed no treatment-related effects, resulting in a NOAEL of 272 -288 mg/kg bw/day (the highest dose tested).

 

Dermal

Fischer 344 rats (5/sex/dose) were administered tris-(2-hydroxypropyl)-amine on the skin (semiocclusive) at 0, 300, 1000 or 3000 mg/kg bw, 5 days/week for 28 days (OECD guideline 410, GLP). Urinalysis, hematology, clinical chemistry, macroscopic and microscopic examinations, and body and organ weights showed no treatment-related systemic effects. Regarding local dermal effects, slightly irritative effects were observed. Erythema and scabs were noted in one animal at each of the highest two dose groups. Most animals of the 3000 mg/kg bw/day group had epidermal hyperplasia in the form of minimal thickening. Therefore, the NOAEL for systemic effects were determined to be 3000 mg/kg bw/day (Dow, 1994).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
1,1’-(methylimino)-dipropane-2-ol and tris-(2-hydroxypropyl)-amine (CAS 122-20-3) are structurally very similar low molecular weight tertiary amines, which differ only a methyl group and an isopropanol moiety respectively. They are low volatile (vapour pressure ≤ 0.015 hPa) organic compounds with similar specific gravity (1,1’-(methylimino)-dipropane-2-ol: 147.22 g/mol and tris-(2-hydroxypropyl)-amine: 191.27 g/mol) and log Pow values between -0.03 for 1,1’-(methylimino)-dipropane-2-ol and -0.02 for tris-(2-hydroxypropyl)-amine. Moreover, the comparison of the available toxicity data for both compounds indicates a similar toxicological profile: the substances are showing low acute toxicity by the oral route (i.e. oral LD50values for1,1’-(methylimino)-dipropane-2-ol of 2150 mg/kg bw and beyond 5000 mg/kg bw for tris-(2-hydroxypropyl)-amine). Tris-(2-hydroxypropyl)-amine is irritating to the eyes, whereas 1,1’-(methylimino)-dipropane-2-ol shows corrosive properties. The chemical difference between those two structures apparently do not affect the anticipated toxicity. Therefore, tris-(2-hydroxypropyl)-amine is regarded as appropriate for read across.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
1,1’-(methylimino)-dipropane-2-ol and tris-(2-hydroxypropyl)-amine (CAS 122-20-3) are structurally very similar low molecular weight tertiary amines, which differ only a methyl group and an isopropanol moiety respectively. They are low volatile (vapour pressure ≤ 0.015 hPa) organic compounds with similar specific gravity (1,1’-(methylimino)-dipropane-2-ol: 147.22 g/mol and tris-(2-hydroxypropyl)-amine: 191.27 g/mol) and log Pow values between -0.03 for 1,1’-(methylimino)-dipropane-2-ol and -0.02 for tris-(2-hydroxypropyl)-amine. Moreover, the comparison of the available toxicity data for both compounds indicates a similar toxicological profile: the substances are showing low acute toxicity by the oral route (i.e. oral LD50values for1,1’-(methylimino)-dipropane-2-ol of 2150 mg/kg bw and beyond 5000 mg/kg bw for tris-(2-hydroxypropyl)-amine). Tris-(2-hydroxypropyl)-amine is irritating to the eyes, whereas 1,1’-(methylimino)-dipropane-2-ol shows corrosive properties. The chemical difference between those two structures apparently do not affect the anticipated toxicity. Therefore, tris-(2-hydroxypropyl)-amine is regarded as appropriate for read across.

Justification for classification or non-classification

Based on the results of the oral and dermal repeated dose toxicity studies for tris-(2-hydroxypropyl)-amine, 1,1’-(methylimino)-dipropane-2-ol does not need to be classified according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.