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Description of key information

As the NOAEL of 300 mg/kg/d is well above the CLP guideline concentration values for specific target organ toxicity for repeat dose exposure, the substance can be considered not classified for this endpoint.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 14, 1989 - March 15, 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards, well documented and acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
One-hundred twenty-six (63 males and 63 females) 4-week-old Sprague-Dawley rats, Crl:CD•BR, •(birth date 1/02/89), were received from Charles River Laboratories, Raleigh, North Carolina, on January 31, 1989. The animals were housed individually in hanging stainless-steel, wire-mesh cages with Purina Certified Rodent Chowe 5002 and tap water available ad libitum.
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. Results of all analyses of feed and water are on file with Hazleton Laboratories America, Inc. None of the possible contaminants in animal feed or water were considered to be at levels sufficient to interfere with this study. From initiation of dosing to study termination, environmental conditions were maintained within protocol-specified limits of temperature within 72 ± 6°F and the relative humidity within 50 ± 20%. Temperature and humidity were recorded using a sling psychrometer. A 12-hour light/12-hour dark cycle was maintained.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The test material was assumed to be 100% pure for purposes of dosage calculations. The calculated amount of test material was weighed out into a pre-calibrated container on an appropriate balance and the vehicle (corn oil) was added to reach the final volume. The purity of the test vehicle was assumed to be 100%. A stir bar was added to the contents and the container was placed on a magnetic stir-plate and the mixture was stirred for approximately five to seven minutes until a solution was achieved. Samples of fresh formulations and vehicle control solution from both the first day and last day of mixing were taken for stability and homogeneity analysis and sent to the sponsor.
The test mixtures were prepared fresh weekly and administered by gavage once daily, seven days a week, for 29 days. The dosing volume was based on a dosing factor of 10 ml/kg.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
29 days
Frequency of treatment:
administered by gavage once daily, seven days a week, for 29 days
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
0 mg/kg/d (vehicle control)
Basis:
actual ingested
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
300 mg/kg/d (low)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1500 mg/kg/d (mid)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
3000 mg/kg/d (high)
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
As per guideline
Positive control:
No
Observations and examinations performed and frequency:
Observations and Records
Mortality and moribundity checks were made twice daily (morning and afternoon). Daily cage side observations for obvious indications of a toxic effect were recorded (approximately one hour post-dose) by exception. Body weights and food consumption were recorded weekly. Physical examinations were performed and detailed clinical observations were recorded at each weekly weighing interval.
Sacrifice and pathology:
-Clinical Pathology-
Prior to initiation of dosing, ten animals per sex were selected at random from the pool of healthy animals available for study (identified as Group 5). These animals were fasted overnight and blood samples for baseline hematology and chemistry values were obtained by orbital sinus venipuncture under ketamine anesthesia. After blood collection the animals were euthanized and discarded without necropsy.
At termination all surviving animals were fasted overnight prior to necropsy. Animals were injected with ketamine and blood samples for haematology and chemistry were collected by orbital sinus venipuncture. The following haematology and clinical chemistry parameters were analyzed:
---Haematology---
leukocyte count (WBC)
erythrocyte count (RBC)
haemoglobin (HGB)
haematocrit (HCT)
platelet count (PLATELET)
leukocyte differential count
cell morphology
myeloid/erythroid ratio (M/E)

---Clinical Chemistry---
sodium (SODIUM)
potassium (POTAS)
chloride (CHLORIDE)
total protein (T PROT)
albumin (ALBUMIN)
calcium (CALCIUM)
total carbon dioxide (T C02)
total bilirubin (T BILl)
blood urea nitrogen (BUN)
creatinine (CREAT)
glucose (GLUCQSE)
alanine aminotransferase (ALT)
aspartate aminotransferase (AST)
gamma glutamyltransferase (GGT)
alkaline phosphatase (ALK P)

---Gross Necropsy---
Gross necropies were performed on all found dead or moribund sacrificed animals. After 29 days of treatment, all surviving animals were weighed and sacrificed by exsanguination under sodium pentobarbital anaesthesia. Necropsies were performed on all animals by trained personnel using procedures approved by board-certified pathologists. The necropsy included examination of the following, all findings were recorded:
external surfaces
all orifices
cranial cavity
carcass
thoracic, abdominal and pelvic cavities and their viscera
cervical tissues and organs
nasal cavity and paranasal sinuses
external surface of the brain (at necropsy); the external surface of the spinal cord cut surfaces of the brain and spinal cord were examined at the time of tissue trimming.

---Organ Weights---
All animals were weighed prior to necropsy. For each terminally sacrificed animal, the following organs were weighed after careful dissection and trimming to remove fat and other contiguous tissue in a uniform manner:
brain (including brainstem)
spleen
liver
heart
kidneys
thymus*
testes with epididymides
thyroid with parathyroids*
adrenals*
ovaries*
pituitary*
*= Weighed post-fixation.

---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, jejunum, ileum
thymus
brain with brainstem (medulla/pons, cerebellar cortex, cerebral cortex)
esophagus
pancreas
sciatic nerve w/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-Giemsa stain.

---Histopathology---
The aforementioned tissues, except the esophagus, from all control and high-dose animals; and •the heart, liver, kidneys, and gross lesions from all low- and mid-dose animals, were embedded in paraffin, sectioned, stained with haematoxylin and eosin, and examined microscopically.
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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See other information and additional tables.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
See other information and additional tables.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See other information and additional tables.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
See other information and additional tables.
Food efficiency:
not examined
Description (incidence and severity):
See other information and additional tables.
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
See other information and additional tables.
Ophthalmological findings:
not examined
Description (incidence and severity):
See other information and additional tables.
Haematological findings:
not examined
Description (incidence and severity):
See other information and additional tables.
Clinical biochemistry findings:
not examined
Description (incidence and severity):
See other information and additional tables.
Urinalysis findings:
not examined
Description (incidence and severity):
See other information and additional tables.
Behaviour (functional findings):
not examined
Description (incidence and severity):
See other information and additional tables.
Immunological findings:
not examined
Description (incidence and severity):
See other information and additional tables.
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
See other information and additional tables.
Gross pathological findings:
no effects observed
Description (incidence and severity):
See other information and additional tables.
Neuropathological findings:
not examined
Description (incidence and severity):
See other information and additional tables.
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
See other information and additional tables.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
See other information and additional tables.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A NOEL of 300 mg/kg/day of B212.
Details on results:
See other information and additional tables.
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
other: At 300 mg/kg/day there were insufficient findings to suggest a relationship to treatment.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
other: Whole body
Organ:
other:
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
not specified

Mortality. Weekly Clinical and Daily Cageside Observations

Four animals either died or were sacrificed moribund prior to study termination (three high-dose females and one high-dose male). On the basis of findings for the high-dose animals, the deaths were considered treatment-related. However, a diagnosis of the cause of death was not reached.

Isolated incidences of alopecia (paws, legs), salivation, soft faeces, urine stains and sores (mouth) were the only clinical signs noted. None of the signs were considered treatment related.

After two days of dosing one or more of the following daily cage side observations were exhibited in the high-dose animals and in only a few of the mid-dose animals: languid behaviour, prostration, hunched appearance, ataxia, and excessive salivation. These observations were observed for the duration of the study and were considered to be treatment related.

Body Weights and Food Consumption

Statistical analysis of the mean body weight change Weeks 0-4 revealed a significant decrease for the high-dose males when compared to the control. The effect was considered treatment related. Evaluation of the mean total food consumption values revealed no significance differences in either the treated males or females as compared to the control animals.

Clinical Pathology

Evaluation of the haematology data revealed significant increases for bone marrow (myeloid to erythroid ratio) in the mid-dose males, and platelet count in the high-dose males; and significant decreases for the red blood cells and haematocrit of the high-dose females. Evaluation of the clinical chemistry data revealed significant increases in blood urea nitrogen, creatinine, and alkaline phosphatase in the high-dose males; total protein (mid-dose females, and high-dose males and females); total bilirubin (low-dose females) and albumin (mid- and high-dose males and females). Significant decreases were noted for chloride (mid-dose females and high-dose males); glucose (mid-dose females); and total carbon dioxide (high-dose females). Only the alkaline phosphatase, albumin and total protein findings could be ascribed to treatment.

Gross Pathology

In the four high-dose animals that died prior to study termination, gross pathology lesions were noted for the lungs (dark/pale areas and mottled); spleen (pale and small); liver (mottled); kidney (dark/pale areas and pale in appearance); and in the glandular and non-glandular regions of the stomach (dark areas).

In the animals sacrificed at study termination the most frequently noted gross pathology lesion was in the glandular and non-glandular regions of the stomach (thickened, pitted and rough mucosa; prominent vessel and raised areas) of the high-dose animals.

Other incidences were noted throughout the groups but were sporadic and not considered to be treatment related.

Organ Weight Data

A summary of statistically significant findings for the absolute and relative organ weight data is presented below:

Males

Females

Group:

2

3

4

2

3

4

Dose Level (mg/kg):

300

1500

3000

300

1500

3000

Terminal Body Weight

Absolute Kidney

Relative Kidney

Absolute Adrenal

Relative Adrenal

Absolute Liver

Relative Liver

Absolute Thyroid/parathyroid

Relative Thyroid/parathyroid

Relative Brain w/stem

Relative Testls/epidid

  KEY:=Significantly greater than control.

=Significantly less than control.

 

The renal and hepatic organ weight results (Groups 3 and 4) were considered of toxicological significance as they were accompanied by clinical chemistry findings and/or morphological changes. The other organ weight results were not considered to be of toxicological significance.

Histopathology

Hepatic, renal and abdominal effects were produced by B-212 administration. Minimal centrolobular to diffuse hepatocellular hypertrophy which was accompanied by decreased periportal vacuolization and increased density of hepatocellular cytoplasm, increased alkaline phosphatase, increased total protein and albumin levels, and increased liver weights were produced by 3000 mg/kg/day. At 1500 mg/kg/day the hepatic effects were also exhibited in both sexes and were considered to exhibit effects patterns related to dose when compared to the responses at 3000 mg/kg/day. At 300 mg/kg/day, the magnitude of the hepatic effects was very slight and did not evidence a treatment relationship.

Renal findings included increased incidences and severity of protein resorption droplets in the tubular cells of all groups of treated males. The kidney weights (both relative and absolute) were significantly increased and the changes exhibited patterns related to dosage.

The non-glandular region of the stomach in Group 4 animals exhibited acanthosis compared to control animals and the change was considered treatment related.

It was concluded that the primary toxicological findings included the liver and kidney of both sexes of mid- and high-dose animals. In the low-dose animals only the males exhibited renal findings. The abdominal findings were considered, on a comparative basis, to be of secondary importance.

Other sporadic changes observed in the treatment groups were considered consistent with the test species in our laboratory and were not considered treatment related.

DISCUSSION AND CONCLUSION

Three groups of Sprague-Dawley rats received B-212 in corn oil by gavage for 29 days at dose levels of 300, 1500 or 3000 mg/kg/day.

One male and three females, receiving 3000 mg/kg/day, died as a result of treatment during the study. Other signs associated with treatment in this group included languid behavior, prostration, hunched, ataxia and excess salivation. A treatment-related decrease in mean body weight change (Weeks 0-4) was exhibited by the male rats. Clinical and anatomical pathology findings indicated treatment-related changes in the liver and kidney. Alkaline phosphate levels (males), total protein (both sexes) and albumin (both sexes) levels were increased for treated animals compared to controls. The absolute and relative, liver and kidney mean weights were increased for both sexes compared to controls. These findings were accompanied by minimal centrilobular to diffuse hepatocellular hypertrophy, decreased periportal vacuolization and increased density of hepatocellular cytoplasm in the liver. In the kidney, the male rats exhibited increased incidences and severity of protein resorption droplets in the tubular cells.

The non-glandular region of the stomach of the 3000 mg/kg/day animals exhibited acanthosis; the finding was considered treatment-related, but of secondary importance.

Animals receiving 1500 mg/kg/day all survived to termination. Many of the physical signs exhibited by the 3000 mg/kg/day animals were also exhibited in the 1500 mg/kg/day animals. A dose response relationship was noted in results at 1500 mg/kg/day for total protein and albumin values and hepatic and renal histopathology findings. The results indicated this level was the lowest observable effect level for both sexes in this study.

At 300 mg/kg/day B212 there were insufficient findings to suggest a relationship to treatment. On this basis it was concluded that the level represented a no observable effect level for both sexes in this study.

Conclusions:
At 300 mg/kg/day B212 there were insufficient findings to suggest a relationship to treatment. On this basis it was concluded that the level represented a no observable effect level for both sexes in this study.
Executive summary:

The test material, B-212, was administered daily for 29 days to male and female Sprague-Dawley rats by oral gavage in order to evaluate the subchronic toxicity. Corn oil was used as the vehicle. Dose levels were 0, 300, 1500 and 3000 mg/kg/day (Groups 1 through 4, respectively). Criteria evaluated included mortality, cage side and detailed clinical signs, body weight, food consumption, clinical haematology, clinical chemistry, organ weights, organ-to-body weight ratios, gross pathology, and histopathology.

At 3000 mg/kg/day, signs of treatment included death (one male, three females), languid behavior, prostration, hunched, ataxia, and excess salivation. Mean body weight change (Weeks 0-4) was decreased in males compared to control males; the effect was associated with treatment. Other findings included increased alkaline phosphatase (males), total protein (both sexes) and albumin levels (both sexes), and increased liver and kidney weights (absolute and relative, both sexes). Microscopic evaluations revealed minimal centrilobular to diffuse hepatocellar hypertrophy which was accompanied by decreased periportal vacuolization and increased density of hepatocellular cytoplasm. In addition, the kidneys of male rats exhibited increased incidences and severity of protein resorption droplets in the tubular cells. The non-glandular region of the stomach for both sexes exhibited acanthosis, however, the finding was considered of secondary importance compared to other findings.

Animals receiving 1500 mg/kg/day exhibited similar findings (except death) and the incidence and severity of the findings generally followed

At 300 mg/kg/day, the findings were so minimal that it was concluded the level was probably a no effect dosage.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Three groups of Sprague-Dawley rats received B-212 in corn oil by gavage for 29 days at dose levels of 300, 1500 or 3000 mg/kg/day.

One male and three females, receiving 3000 mg/kg/day, died as a result of treatment during the study. Other signs associated with treatment in this group included languid behavior, prostration, hunched, ataxia and excess salivation. A treatment-related decrease in mean body weight change (Weeks 0-4) was exhibited by the male rats. Clinical and anatomical pathology findings indicated treatment-related changes in the liver and kidney. Alkaline phosphate levels (males), total protein (both sexes) and albumin (both sexes) levels were increased for treated animals compared to controls. The absolute and relative, liver and kidney mean weights were increased for both sexes compared to controls. These findings were accompanied by minimal centrilobular to diffuse hepatocellular hypertrophy, decreased periportal vacuolization and increased density of hepatocellular cytoplasm in the liver. In the kidney, the male rats exhibited increased incidences and severity of protein resorption droplets in the tubular cells.

The non-glandular region of the stomach of the 3000 mg/kg/day animals exhibited acanthosis; the finding was considered treatment-related, but of secondary importance.

Animals receiving 1500 mg/kg/day all survived to termination. Many of the physical signs exhibited by the 3000 mg/kg/day animals were also exhibited in the 1500 mg/kg/day animals. A dose response relationship was noted in results at 1500 mg/kg/day for total protein and albumin values and hepatic and renal histopathology findings. The results indicated this level was the lowest observable effect level for both sexes in this study.

At 300 mg/kg/day B212 there were insufficient findings to suggest a relationship to treatment. On this basis it was concluded that the level represented a no observable effect level for both sexes in this study.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
one-generation reproductive toxicity study, performed to recognised standards and well documented

Justification for classification or non-classification

As the NOAEL of 300 mg/kg/d is well above the CLP guideline concentration values for specific target organ toxicity for repeat dose exposure, the substance can be considered not classified for this endpoint.