Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/Guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2007
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
minimum purity 98.8%

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Fischer 344 rats were selected because of their general acceptance, and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.

Supplier and Location
Charles River Laboratories Inc. (Raleigh, North Carolina).

Age at Study Start
Approximately 7 weeks of age.

Physical and Acclimation
Each animal was evaluated by a laboratory veterinarian to determine their general health status and acceptability for study purposes upon arrival at the laboratory. The animals were housed 2-3 per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately one week prior to the start of the study.

Housing
Animals were housed one per cage in stainless steel cages after assignment to the study. The room relative humidity and temperature were maintained within a range of 46.8 - 56.5% and 21 - 24.4 °C, respectively. A 12-hour light/dark photocycle was maintained for the animal room with lights on at 6:00 a.m. and off at 6:00 p.m., and room air was exchanged approximately 12 - 15 times/hour. Cages had wire-mesh floors that were suspended above cageboards and contained a feed crock and a water bottle.

Randomization and Identification
Animals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) which were correlated to unique alphanumeric identification numbers.

Feed and Water
Animals were provided LabDietâ Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility.

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Dose Levels and Justification
The high-dose, 1000 mg/kg bw/day, was chosen based on results of a 14-day study drinking water toxicity study of DIPA (McCollister et al., 1981) and is defined in several regulatory guidelines as a limit dose for subchronic toxicity studies. The high-dose was expected to produce minimally decreased water and feed consumption, some body weight depression, and possible renal effects. The mid-and low-dose levels were expected to provide dose response data for any treatment-related effects observed in the high-dose group. The low-dose was expected to be a no-observed-effect level (NOEL).

Dose Preparation and Analysis
The initial preparation of DIPA/drinking water solutions used historical body weight and water consumption data. Subsequently, the concentrations of DIPA in water were adjusted weekly based upon the most recent mean body weight and water consumption data from the combined 90-day dosing and 28-day recovery groups. The drinking water solutions were prepared weekly throughout the study by serially diluting the high-dose with municipal drinking water. The pH of each dose level was adjusted with concentrated HCl to match that of control water. The homogeneity of the low-dose female and the high-dose male drinking water solutions were determined pre-exposure and near the middle and end of the study.

Stability
A previous 14-day toxicity study has shown DIPA to be stable for at least 4 days in drinking water at targeted dose levels of 100 (0.097%) and 3000 (2.51%) mg/kg bw/day. Additional stability data was obtained concurrently with the conduct of the present study for the low dose-female (100 mg/kg bw/day) and high-dose male (1000 mg/kg bw/day) solutions under adjusted pH conditions in the presence and absence of rats. Two male and two female rats were provided the test material solutions for 7 days. The solutions were determined to be stable in the presence or absence of rats for 7 days. The solutions provided to rats for 7 days were maintained at room temperature and determined to be stable for at least 25 days.

Concentration Verification
Analyses of all dose levels and the control were conducted pre-exposure, near the mid-point in the dosing period, and at the end of the dosing period. The method used for analyzing the test material in water was high performance liquid chromatography (HPLC) with refractive index detection and external standards.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The 100 mg/kg bw/day female and 1000 mg/kg bw/day male solutions (which had the lowest and highest concentrations used in this study) were determined to be homogeneous, with relative standard deviations for all solutions sampled between 0.177% and 4.70%.

DIPA was determined to be stable for 7 days at the 100 mg/kg bw/day female and the 1000 mg/kg bw/day male dose levels, both for samples provided to rats as well as initial solutions. The range of day 7 samples varied from 99.6 to 105% of original concentrations. The samples provided to rats for 7 days were retained at room temperature and determined to be stable for up to 25 days with a range of 94.6 to 107% of original concentrations.

The concentrations of DIPA were determined for the control, 100, 500, and 1000 mg/kg bw/day drinking water solutions mixed on 5/7/02, 6/25/02, and 7/30/02 for male and female rats. HPLC analysis with refractive index detection and external standards indicated 96.4 to 108% of the target concentration was obtained for each individual sample. The mean concentrations for each dose level ranged from 99.2 to 104% of targeted concentration. No DIPA was found in the control drinking water.
Duration of treatment / exposure:
90 days
Frequency of treatment:
continuous in water
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 500, 1000 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Four groups of 10 male and 10 female Fischer 344 rats were given drinking water formulated to supply 0, 100, 500, or 1000 milligrams DIPA per kilogram body weight per day for at least 90 days to evaluate the potential for systemic toxicity (main group). Additional groups of 10 male and 10 female control and high dose groups of rats were given control water for at least an additional 28 days to evaluate the reversibility of potential effects induced during the 90-day dosing period (recovery group). Cage-side clinical observations, detailed clinical observations, eye exams, body weights, feed consumption, water consumption, hematology, clinical chemistry, urinalysis, and organ weights were conducted on all main group animals. In addition, a gross necropsy was conducted with extensive histopathologic examination of tissues. Test material administration for all animals began on May 9, 2002. Main group rats were necropsied on August 7 and 8, 2002, respectively (test days 91 and 92). Recovery group rats were necropsied on September 5, 2002 (test day 120).

Male and female F344 rats were received from Charles River Labs, INC. at 7 weeks of age. They were acclimated to the lab for one week prior to dosing. They were housed singly in wire mesh cages, in rooms designed to maintain adequate environmental conditions for rats. Feed and water were provided ad libitum. They were stratified by body weight using a computer program, randomized into dose groups, and identified by a subcutaneously-implanted transponder correlated to unique alphanumeric identification numbers.
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
Twice each day a cage-side examination was conducted and to the extent possible the following parameters were evaluated: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behavior, moribundity, mortality, and the availability of feed and water.

Detailed clinical observations (DCO) were conducted at pre-exposure and weekly throughout the study. The DCO was conducted on all animals, at approximately the same time each examination day according to an established format. The examination included cage-side, hand-held and open-field observations.
Sacrifice and pathology:
Anatomic Pathology
Fasted rats submitted alive for necropsy were anesthetized by the inhalation of CO2, weighed, and blood samples were obtained from the orbital sinus. Their tracheas were exposed and clamped, and the animals were euthanized by decapitation. A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary, and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened glass slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened, and the viscera examined. All visceral tissues were dissected from the carcass, re-examined, and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate buffered 10% formalin using a hand-held syringe and blunt needle. The urinary bladder was distended with formalin via transmural injection from a syringe and 21 gauge needle and subsequently ligated at the urethral orifice. The brain, liver, kidneys, heart, adrenal glands, testes, epididymides, uterus, ovaries,
thymus, and spleen were trimmed and weighed immediately. The ratios of organ weight to terminal body weight were calculated. Representative samples of tissues as per guidelines were collected and preserved in neutral, phosphate-buffered 10% formalin. Transponders were removed and placed in jars with the tissues.

The number of sections from all preserved tissues as per guidelines were processed by standard histologic procedures from control- and high-dose group animals. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope. The following tissues from the remaining groups were processed and histopathologically examined: kidneys, liver, lungs, urinary bladder, and relevant gross lesions.
Other examinations:
All rats were weighed during the pre-exposure period and weekly during the remainder of the study. Body weight gains were also calculated.

Feed consumption data were collected at least weekly for all animals.

Clinical Pathology
Blood samples were collected from the orbital sinus of all fasted animals, anesthetized with CO2, at the scheduled necropsy.

Hematology
Sample Preparation
Blood samples for a complete blood count from the main group were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared stained with Wright’s stain and archived for potential future evaluation if warranted. Hematologic parameters were assayed using a Technicon H·1E Hematology Analyzer (Bayer Corporation, Tarrytown, New York).
Assays
Hematocrit (Hct)
Hemoglobin (Hgb) concentration
Red blood cell (RBC) count
Total white blood cell (WBC) count
Platelet (PLAT) count
Differential WBC count
RBC indices (MCH, MCV and MCHC)
Coagulation
Sample Preparation
Blood samples for coagulation from the main group were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an ACL9000 (Instrumentation Laboratory, Lexington, Massachusetts).
Assay
Prothrombin time (PT)
Clinical Chemistry
Sample Preparation
Blood samples from the main group were collected in glass tubes and sera were separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyzer Boehringer-Mannheim, Indianapolis, Indiana).
Enzyme Activities of:
Alkaline phosphatase (AP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Concentrations of:
Albumin (ALB)
Cholesterol (CHOL)
Creatinine (CREAT)
Electrolytes (Na, K, PO4, Cl and Ca)
Glucose (GLU)
Total bilirubin (TBILI)
Total protein (TP)
Urea nitrogen (UN)

Urinalysis
Urine was collected from all non-fasted animals from the main group during the week prior to necropsy by placing each animal overnight in a metabolism cage (~ 16-hour period). Urine was collected in a glass container and the following assays conducted:
Assays
Color, appearance and specific gravity (refractometer), and urine volume.
Semiquantitative analysis (MultistixÒ Reagent Strips, Bayer Corporation, Elkhardt, Indiana on the Clinitek 200+) of:
pH
Bilirubin
Glucose
Proteins
Ketones
Semiquantitative analysis (MultistixÒ Reagent Strips, Bayer Corporation, Elkhardt,
Indiana on the Clinitek 200+) of:
pH
Bilirubin
Glucose
Proteins
Ketones
Blood
Urobilinogen
Urine was also collected by manual compression of the bladder prior to the necropsy for characterization of the microsediment using a pooled sample from each dose group/sex.
Statistics:
Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, water consumption, organ weights, urine volume, urine specific gravity, clinical chemistry data, coagulation and appropriate hematologic data were evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA). If significant at alpha = 0.05, the ANOVA was followed respectively by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with a Bonferroni correction (Miller, 1966) for multiple comparisons to the control. The experiment-wise alpha level was reported for these two tests. DCO incidence scores were statistically analyzed by a z-test of proportions comparing each treated group to the control group (alpha = 0.05; Bruning and Kintz, 1987). Data collected at different time points were analyzed separately. Descriptive statistics only (means and standard deviations) were reported for body weight
gains, RBC indices, and differential WBC counts. Statistical outliers were identified by a sequential test (alpha = 0.02; Grubbs, 1969), but routinely excluded only from feed and water consumption calculations. Outliers were excluded from other analyses only for documented, scientifically sound reasons.

Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) will be greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
There was no mortality noted, no observations made during clinical exams or detailed clinical observations, no notations during ophthalmologic exams, and no statistically-significant changes in body weight or food consumption that were attributed to DIPA toxicity. Water consumption decreased slightly in treated groups, likely due to water aversion than DIPA toxicity. Test material consumption was within 6% of targeted dose levels throughout the study. There was no change in prothrombin times or any hematologic parameters. WBC counts were unaffected. Serum cholesterol, albumin, and phosphorous of males at the high dose were different than controls, but are considered to be of little clinical significance due to the small magnitude of the change. Urine specific gravity was increased for both sexes at 1000 mg/kg bw/day, and urine volume decreased for females at this dose level. Both findings are consistent with decreased water intake, secondary to DIPA administration, and was considered an adaptive effect. The only organ affected by DIPA was the kidney. Absolute and relative kidney weights were both increased in 500 and 1000 mg/kg bw/day dose groups. Absolute kidney weights of males at 100 mg/kg bw/day were increased. There were no gross pathological findings that were attributed to DIPA, nor did histopathological exams reveal any findings. Although there were no effects attributed to DIPA during the 90 days, the kidneys of the recovery group were examined. Only slight renal tubular degeneration with regeneration was found, with the incidence similar between controls and groups given 1000 mg/kg bw/day.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
LOAEL = 1000 mg/kg bw/day

The only effect found in the 500 mg/kg bw/day group was increased relative and absolute kidney weights without any histopathologic correlate. Therefore,

NOAEL = 500 mg/kg bw/day for females
NOAEL = 100 mg/kg bw/day for males
Executive summary:

Ten male and ten female Fischer 344 rats per group were given drinking water formulated to supply 0, 100, 500, or 1000 milligrams diisopropanolamine (DIPA) per kilogram body weight per day (mg/kg/day) for at least 90 days to evaluate the potential for systemic toxicity of DIPA. Standard toxicologic parameters were evaluated during the in-life phase of the study with clinical and anatomic pathology investigations at termination. Additional groups of ten/sex were maintained on untreated drinking water for at least an additional 28 days after initially receiving the control or high-dose water (0 or 1000 mg/kg bw/day) for at least 90 days to assess recovery from effects induced by DIPA. Rats of either sex given 1000 mg DIPA/kg bw/day for at least 90 days had few effects, all of which were of minimal degree, attributed to DIPA. While there were no treatment-related clinical signs, rats given this dose level drank slightly less water (females had a greater decrement than males) with corresponding decrements of feed consumption and body weights considered secondary to the water aversion. The body weight decrements were only ~ 2% at termination and were never statistically identified. Urine specific gravity was increased in both sexes while urine volume was decreased for females; both of which were adaptive effects to the decreased water consumption. Serum cholesterol was slightly increased while serum phosphorus was slightly decreased for male rats given 1000 mg/kg bw/day. These effects were not present after the 28-day recovery period. Kidney weights, both absolute and relative to body weight, were increased with the males affected to a greater degree (male relative kidney weight increased ~ 21% and female increased ~ 14%). However, histopathologic effects were not found. After four weeks drinking untreated water, the increase in kidney weight was about one-half of that present at the end of the dosing period for both males and females. This limit dose level, 1000 mg/kg bw/day, was considered to be a LOAEL. The only effect found in rats given 500 mg/kg bw/day was increased absolute and relative kidney weights with males again having a greater increase than females (male relative kidney weight increased ~ 12% and female increased ~ 7%), also without any histopathologic correlate. The absolute kidney weight of males given 100 mg/kg bw/day was also increased and was statistically identified; however, these rats weighed more than controls and the relative kidney weight was similar to controls. Thus, the 500 mg/kg bw/day dose level was considered the NOAEL for females, while 100 mg/kg bw/day was the NOAEL for males.