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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 July to 11 August 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study conducted similarly to OECD test guideline No. 407. Read-across substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Absence of storage stability and formulation analysis. Prostate, seminal vesicle, epididymides and thymus weights are missing. Vagina, eye, prostate, vesicle seminal, thymus, trachea, spinal cord were not examined. Neurobehaviour not assessed.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lorillard, Inc. / Lot. No. 1
- Physical state: Liquid
- Date of receipt: 17 June 1988

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored protected from light under refrigeration

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl: CD® (SD) BR
Details on species / strain selection:
Rats were selected as the test animal for this study based upon their historical use in safety evaluation studies, and because they are recommended by appropriate regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina.
- Age at study initiation: ca. 43 days
- Weight at study initiation: 206.2-249.3 g for the males; 142.8-191.2 g for the females
- Housing: Animals were housed individually in hanging stainless-steel wire-mesh cages
- Diet: Purina® Certified Rodent Chow 5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water is routinely analyzed on a retrospective basis for specified pesticides, heavy metals and microbes. None of the possible contaminants in animal feed or water were considered to be at levels sufficient to interfere with this study.

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 6 ° F
- Humidity: 50 ± 20 %
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 12 July to 11 August 1988

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of dosing was chosen because it is a generally accepted means of administration when used to assess the toxicity potential of test material.
Vehicle:
methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was assumed to be 100% pure for purposes of dosage calculations. The calculated amount of test substance was weighed into a beaker of appropriate size and then transferred into a pre-calibrated container using a rinsing process. Vehicle (1% methylcellulose) was added to achieve the appropriate volume and the container was placed on a magnetic stirrer and stirred for five minutes while being sonicated. The test mixtures were prepared fresh weekly and administered daily by gavage based on a dosing factor of 10 mL/kg bw.

VEHICLE
- Concentration in vehicle: 16, 40 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from each batch of test and vehicle control solution were reserved and stored at 5 °C. Samples of fresh formulations and vehicle control solution from both the first day and last day of mixing (Weeks 1 and 4) were taken and sent to the sponsor for verification of concentration analysis. Due to the necropsy schedule an extra day (Week 5) of mixing was necessary, this mixture was not sent to the sponsor.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: Following a 14-day acclimation period, a weight randomization computer program designed to ensure homogeneity of body weights was used to select and assign the rats to the different groups.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for mortality and moribundity, once in the morning and once in the afternoon approximately six hours apart. Daily cageside observations (approximately 1 hour post dose) for obvious indications of a toxic effect were recorded by exception. Physical examinations were performed and detailed clinical observations were recorded at each weekly weighing interval.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly.

FOOD CONSUMPTION:
Food consumption was recorded weekly.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to initiation, ten animals per sex were selected at random from the pool of healthy animals not selected for study. The animals were fasted overnight and blood samples for hematology and clinical chemistry were collected by orbital sinus venipuncture under ketamine anesthesia. At termination all surviving animals were fasted overnight prior to necropsy and blood samples for hematology and clinical chemistry were collected by orbital sinus venipuncture under ketamine anesthesia.
- Anaesthetic used for blood collection: Yes (Ketamine)
- Parameters checked:
Haematology: Leukocyte count (WBC), erythrocyte count (RBC), hemoglobin, hematocrit, platelet count, leukocyte differential count, cell morphology, myeloid/erythroid ratio (M/E)-(Groups 1 and 4 only at Week 5)
Clinical chemistry: Sodium, potassium, chloride, total protein, albumin, calcium, total carbon dioxide, total bilirubin, blood urea nitrogen, creatinine, glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT), alkaline phosphatase (ALK P)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE: After at least 28 days of treatment, all surviving animals were weighed and sacrificed by exsanguination under sodium pentobarbital anesthesia. Necropsies were conducted over a two-day period on study days 30 and 31.

GROSS PATHOLOGY: Yes ; external surfaces, all orifices, cranial cavity, carcass, nasal cavity and paranasal sinuses, cervical tissues and organs, external surface of the brain and spinal cord (at necropsy) ; the cut surfaces of the brain were examined at the time of tissue trimming, thoracic, abdominal and pelvic cavities and their viscera

ORGAN WEIGHTS:
For each terminally sacrificed animal, the following organs were weighed following careful dissection and trimming to remove fat and other contiguous tissue in a uniform manner:
Brain (including brainstem), spleen, liver, heart, kidneys, testes with epididymides, thyroid with parathyroids*, adrenals*, ovaries*, pituitary*

HISTOPATHOLOGY: Yes
Tissue Preservation: The following tissues from each animal were preserved in 10% neutral buffered formalin: femoral bone marrow$, lung (with mainstem bronchi)$, ovaries$, lesions$, kidneys$, adrenals$, testes with epididymides$, duodenum$, Jeiunum$, ileum$, brain with brainstem (medulla/pons, cerebellar cortex, cerebral cortex)$, thymus, esophagus, pancreas$, sciatic nerve, skeletal muscle, cervical, thoracic and lumbar spinal cord, urinary bladder$, pituitary$, uterus$, thyroid (parathyroids)$, heart$, liver$, spleen$, colon$, cecum$, rectum$, stomach$, salivary gland (mandibular), trachea, mesenteric lymph node$
Additionally, a femoral bone marrow smear was prepared from all sacrificed animals and preserved in methanol and stained with Wright stain.
Histopathology: The aforementioned tissues ($indicated) from all control and high-dose animals; the heart, liver, kidneys, stomachs and lesions from all low- and mid-dose animals, were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.

*weighed post-fixation
Other examinations:
None
Statistics:
Mean body weight changes (Weeks 0-4), total food consumption (Weeks 1-4), clinical pathology data (except cell morphology), absolute organ weight and organ-to-body weight ratios of the control group were compared statistically to the data from the same sex of the treated groups. If variances of untransformed data were heterogeneous, analyses were performed on transformed data to achieve variance homogeneity. When the series of transformations were not successful in achieving variance homogeneity, analyses were performed on rank-transformed data. Group comparisons were performed at 5% two-tailed probability level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Isolated incidences of alopecia and sores were noted in both males and females of several groups. In vehicle control group, one male had a small movable head mass at Week 1, but this observation was not present thereafter.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One high-dose female was found dead on Day 2 of the study and was replaced on study with another animal which was dosed for the required period of time. One high-dose male was found dead on Day 9 of the study. However the death was attributed to be handling accident.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant changes in body weight was observed.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No statistically significant changes in food consumption was observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment related findings were noted in the clinical haematology.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related increases in total protein and serum albumin were observed in the mid- and high-dose males and the high-dose females. Serum calcium was also increased in these same treated groups, apparently as a secondary response to the increase in albumin, its major serum binding protein. The pathogenesis of these increases, however, is unknown.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A treatment-related increase in absolute and relative kidney weight was observed in the high-dose males and females, and in the mid-dose males and high dose males and females, respectively.
In the liver, a treatment-related increase in both absolute and relative liver weight was observed in the mid and high-dose males and females, While statistical significance was also noted in the absolute liver weight of the low-dose females, the corresponding relative liver weight, while elevated and consistent with a dose-related response, was not statistically significant. Therefore, a relationship to treatment in the low-dose females must be considered as equivocal. A modest increase in both absolute and relative liver weight was observed in the low-dose males. While these increases could be consistent with a dose-related response, the magnitude of the increases and the absence of statistical significance do not appear to attribute these increases to treatment.
While several instances of statistically significant decreases in thyroid/parathyroid weights were observed, these findings were considered incidental to treatment in the absence of a meaningful dose relationship.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related gross pathology findings and treatment-related increases in organ weights were generally observed in the kidney and liver of these same treated groups, and were considered as confirmation of the histopathological findings.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related periportal hepatocellular cytoplasmic vacuolization was noted in the liver of all treated female groups, with the effect observed in nine of the ten high-dose females. A treatment-related increase in degenerative lesions in the renal cortex was noted in the high-dose males. Treatment related lesions in the non-glandular region of the stomach were observed in the mid- and high-dose females. These lesions consisted of a low incidence of erosion, subacute inflammation and acanthosis, and were somewhat more prevalent in the high-dose females.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
There are no other impact such as an increase in hepatic enzymes (ALP, AST, GGT, GLDH...) or significant changes in other biomarkers indicating a liver dysfunction (Chol, bile acid, TG…) at the low dose both in females and males. Therefore, the effects reported in the females at low dose can be considered as an adaptive response to the test substance and the NOAEL can be established at 160 mg/kg bw/day.

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the NOAEL for test substance was determined to be 160 mg/kg bw/day in males and females.
Executive summary:

In a repeated dose toxicity study performed similarly to OECD test guideline No. 407 and in compliance with GLP, test substance was administered to Crl:CD(SD) rats (10/sex/dose) via oral (gavage) at 160, 400 and 1000 mg/kg bw/day for 28 days. Control animals received 1% methyl cellulose. During the study, clinical signs, mortality, body weight, food consumption, hematology, clinical chemistry, organ weight, gross and histopathology investigations were undertaken.

 

No treatment-related effects on survival, clinical observations, body weights, food consumption and hematology were observed.

Treatment-related increases in total protein and serum albumin were observed at 400 and 1000 mg/kg bw/day in males and at 1000 mg/kg bw/day in females. Serum calcium was also increased in these same treated groups, apparently as a secondary response to the increase in albumin, its major serum binding protein. The pathogenesis of these increases, however, is unknown.

Treatment-related lesions were noted histopathologically in the kidney at 1000 mg/kg bw/day in males, in the non-glandular region of the stomach at 400 and 1000 mg/kg bw/day in females, and in the liver at 1000 mg/kg bw/day in females. Similar lesions of the liver were also noted at 160 and 400 mg/kg bw/day in the females, but at a lower incidence. Treatment-related gross pathology findings and treatment-related increases in organ weights were generally observed in the kidney and liver of these same treated groups, and were considered as confirmation of the histopathological findings. There were no other impact such as an increase in hepatic enzymes (ALP, AST, GGT, GLDH...) or significant changes in other biomarkers indicating a liver dysfunction (Chol, bile acid, TG…) at the low dose both in females and males. Therefore, the effects reported in the females at low dose can be considered as an adaptive response to the test substance and the NOAEL can be established at 160 mg/kg bw/day.

 

Under the test conditions, the NOAEL for test substance was determined to be 160 mg/kg bw/day in males and females.