1,4-dihydroxy-2,2,6,6-tetramethyl piperidinium-2-hydroxy-1,2,3-propanetricarboxylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-02-16 to 1999-1999-05-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant OECD Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted July 27, 1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-370-5

EC Name:

-

Cas Number:

220410-74-2

Molecular formula:

C34H67N3O13

IUPAC Name:

tris(1,4-dihydroxy-2,2,6,6-tetramethylpiperidin-1-ium) 2-hydroxypropane-1,2,3-tricarboxylate

Test material form:

other: Ivory-peach flakes

Details on test material:

- Identification: TKA 45021
- Appearance: Ivory-peach flakes
- Lot/batch No.: pax 2068 rd 100
- Test Item arrived at Test Facility: 1998-11-25

Documentation concerning chemical identification, purity, strength, stability and other required data are the responsibility of the Sponsor. Details of the chemical characterization of the test material are confidential information of the Sponsor.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation: approximately seven weeks
- Weight at study initiation: ranging from 211 to 250 g for males and 151 to 193 g for females
- Fasting period before study: no
- Housing: 2 or 3 males or females per cage for six days following receipt to adjust to the automatic watering system. During remainder of acclimation and while on study: individually in suspended stainless steel cages
- Diet: PMI Certified Rodent Chow #5002 (Purina Mills, Inc.) ad libitum
- Water: municipal tap water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 65 to 79°F (18.3 °C to 26.1 °C)
- Humidity: 30 to 70%
- Air changes: 10 to 15 air changes per hour
- Photoperiod: 12-hour light / 12-hour dark cycle

IN-LIFE DATES: From: 1999-03-04 To: 1999-04-01 or 1999-04-14 (recovery groups)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Prior to weighing, the test article was ground using a mortar and pestle, then weighed into a beaker. Reverse Osmosis Deionized (RODI) water was added to the beaker to achieve the desired concentration, and stirred for 15 minutes. Prepared fresh weekly, dispensed into daily aliquots and stored refrigerated. The physical state of the vehicle and each test article dosing mixture was recorded during each preparation. Daily aliquots of the dosing mixtures were removed from the refrigerator, stirred continuously and allowed to equilibrate to room temperature prior to dispensing.

VEHICLE
- Amount of vehicle: 10 mL / kg bw
The vehicle control material used in the preparation of dosing mixtures and for administration to control animals was Reverse Osmosis Deionized (RODI) water.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration verification analysis was performed on the vehicle and each test article dosing mixture during weeks 1, 2, 3 and 4.

Prior to initiation of the 28-day study, homogeneity and stability analyses were performed on concentrations of the test article in the vehicle which bracketed the low- and high-doses administered in this study. Homogeneity analyses were performed on duplicate samples taken from the top, middle and bottom of the two mixtures. Stability of the test article in the vehicle was evaluated on duplicate samples at 0, 3 and 8 days following preparation and refrigerated storage.
The analytical results indicated that the doses were accurately formulated during the toxicity study. The results also confirmed that the formulations were homogeneous and stable from the time of preparation to completion of dosing.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily (7 days per week)

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 500, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

five males and five females, in addition 5 males and 5 females per recovery group (vehicle and high dose)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dosage levels were selected based on data from the range-finding study to produce graded responses to the test article.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during weeks -1, 1, 2 and 3 (days - 1 , 7, 14 and 21, respectively) for each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: -Day 2, 8, 15, 22 and 28 (during the treatment phase) and day 35 (during the recovery phase). In addition, a terminal body weight was recorded on the day of scheduled euthanasia (day 29 or 42) for calculation of relative organ weight data

FOOD CONSUMPTION: Yes
- Time schedule for examinations: -Day 2, 8, 15, 22 and 28 (during the treatment phase) and days 35 and 41 (during the recovery phase).

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: near the end of the treatment phase (day 26) and the recovery phase (day 37)
- Dose groups that were examined: all dose groups (surviving animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment phase (day 29) or the recovery phase (day 42)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving rats (incl. recovery groups)
- The following parameters were examined:
Erythrocyte count (RBC), hematocrit (Hct), hemoglobin concentration (Hgb), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), platelet count, total and differential leukocyte counts, reticulocyte count (reticulocyte slides were prepared, however, reticulocyte counts were not considered necessary by the Study Director)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment phase (day 29) or the recovery phase (day 42)
- Animals fasted: Yes
- How many animals: all surviving rats (incl. recovery groups)
- The following parameters were examined:
Alanine aminotransferase (ALT), albumin, albumin/globulin ratio (calculated), alkaline phosphatase, aspartate aminotransferase (AST), calcium, cholesterol, blood creatinine, gamma glutamyl transpeptidase (GGT), globulin (calculated), glucose, electrolytes (sodium, potassium and chloride), phosphorus, total bilirubin, total serum protein, triglycerides, urea nitrogen

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment phase (day 29) or the recovery phase (day 42), overnight, prior to scheduled euthanasia
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food withheld when in metabolic cage, water provided)
- The following parameters were examined:
Overnight volume, color and appearance, pH, specific gravity, protein, glucose, ketones, urobilinogen, nitrites, bilirubin, occult blood, leukocytes, microscopy of spun deposit

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during weeks 4 and 6 (days 27/28 and 40/41, respectively).
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength and motor activity

Sacrifice and pathology:

- Euthanized by carbon dioxide Inhalation followed by exsanguination

GROSS PATHOLOGY: Yes, on all animals (incl. recovery groups)
- Parameters: external surfaces of the body and all viscera
- Fresh organ weights: liver, kidneys, adrenal glands, testes with epididymides, ovaries, spleen, thymus, thyroids, brain and heart of all animals. Paired organs were weighed together.
- Preserved from all animals: accessory genital organs (epididymides, seminal vesicles and prostate or uterus and vagina), adrenals, all gross lesions, aorta, brain (including sections of medulla/pons, cerebellar cortex and cerebral cortex), cecum, colon, duodenum, esophagus, exorbital lachrymal glands, eyes with optic nerve, femur (including articular surface) and bone marrow, heart, ileum, jejunum, kidneys, liver (3 sections collected), lungs (infused with formalin) with bronchi, mammary gland, mandibular lymph node, mediastinal lymph node, mesenteric lymph node, pancreas, peripheral nerve (sciatic) pituitary, rectum, skeletal muscle (thigh), skin, spinal cord (cervical, midthoracic and lumbar), spleen, sternum with bone marrow, stomach (glandular/nonglandular), submaxillary salivary gland, testes/ovaries, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder


HISTOPATHOLOGY: Yes
Investigated: All tissues and organs collected at necropsy (days 29 and 42) from all animals in the control and high-dose groups

Statistics:

- One-way analysis of variance (ANOVA): body weights, weight gain, food consumption, hematology, coagulation, biochemistry, organ weights and appropriate urinalysis parameters
- Tukey-Kramer Test (group comparisons when significance was observed)
- Kruskal-Wallis Test (rank and count data)
- Fisher's Exact Test (Descriptive (categorical) and quantal data)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mild, dose-dependent clinical abnormalities

Mortality:

mortality observed, treatment-related

Description (incidence):

Mild, dose-dependent clinical abnormalities

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slight, dose-dependent decrease in mean body weights

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A dose-dependent trend towards increased spleen weights was noted primarily in the 500 and 1000 mg/kg bw / day males at the end of the dosing phase of this study.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Mild, dose-dependent clinical abnormalities were noted following test article administration in the 500 and 1000 mg / kg bw / day males and females (salivation and apparent blood around the facial area, neck and forelimbs). The clinical abnormalities resolved during recovery phase; no mortality occurred.

BODY WEIGHT AND WEIGHT GAIN
A slight, dose-dependent decrease in mean body weights (i.e., 3%, 4% and 6% for the 100, 500 and 1000 mg / kg bw / day groups, respectively) was noted in treated males by the end of the dosing phase. The biological impact of this change was considered marginal as the decrease in mean body weights for the 1000 mg / kg bw / day males (i.e., 6%) was well below 10%.

FOOD CONSUMPTION - no notable abnormalities

OPHTHALMOSCOPIC EXAMINATION - no notable abnormalities

HAEMATOLOGY and CLINICAL CHEMISTRY
Slight treatment-related effects: decreased RBC counts, hemoglobin concentration and hematocrit, increased serum bilirubin in the 500 and 1000 mg / kg bw / day groups.

URINALYSIS -no notable abnormalities

NEUROBEHAVIOUR - no notable abnormalities

ORGAN WEIGHTS
A dose-dependent trend towards increased spleen weights was noted primarily in the 500 and 1000 mg/kg bw / day males at the end of the dosing phase of this study.

HISTOPATHOLOGY:
A reversible minimal to mild increase in the congestion of the red pulp of the spleen was observed microscopically in several of the 1000 mg / kg bw / day males and females at the end of the dosing phase.

GROSS PATHOLOGY - no notable abnormalities

HISTORICAL CONTROL DATA: no notable abnormalities when compared to results of study

Effect levels

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Primarily induced by mildly and sporadically abnormal parameters regarding clinical signs, haematology, organ weight and histopathology at 500 and / or 1000 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:

not specified