Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity: 98.76%

Test animals

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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

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.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for the study was 7,500 ppm for both male and female dogs.