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Repeated dose toxicity: inhalation

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Administrative data

sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
study report
Report Date:

Materials and methods

Principles of method if other than guideline:
This study was performed to reveal any toxicological effects which might result from repeated long-term exposure to airborne concentrations of the test substance.
GLP compliance:
not specified
Limit test:

Test material

Test material form:

Test animals

Fischer 344
Details on test animals and environmental conditions:
Male rats weighed approx. 115 mg and females approx. 77 mg. All animals were selected quarantined and examined. Rats were exposed in compartmented stainless-steel wire mesh cages. Each rat cage compartment measured 7-1/2 in by 3-1/2 in. The compartments were labeled with a number so that each animal was in the same cage and compartment for the daily exposures. Following the 6-hour exposure, the caged animals were removed from the chamber, placed on carts and carried to adjoining holding rooms. One room was for control and another room was for exposed animals. The animals were removed from the exposure cages and placed in individually labeled stainless steel mesh holding cages. Rat holding cages measured 9-1/2 in. x 6-3/4 in. x 7 in. Food and water was available to the animals only while in the holding cages and not during exposures to the test substance. The animals were sex-segregated at all times. The exposed and control animals were observed daily for toxic signs and death. They were weighed weekly and at that time were scrutinized more criticially for toxic signs. The animals used were weighed individually at approx. 2-week intervals.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Details on inhalation exposure:
Exposures were conducted in two 3000-liter dynamic flow stainless-steel chambers operated under negative pressure. Air was drawn into the system and the test substance aerosol was dispersed into the intake air and passed through collective protective filters before exiting into the atmosphere. Chamber design assured that the airborne concentration of the test substance was uniform. The chamber airflow was held constant throughout the test. The flow rate was used to calculate the nominal concentration. Air pressure through the generators was adjusted to achieve the different test concentrations of the test substance.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 h/d, 5 d/wk
Doses / concentrationsopen allclose all
Dose / conc.:
0.1 mg/L air (nominal)
Dose / conc.:
1 mg/L air (nominal)
No. of animals per sex per dose:
A total of 216 rats were required, 72 to be exposed to the high dose level, 72 to the low dose level and 72 to serve as controls. Each of these groups were divided into three subgroups:
(a) 6-week exposure group of 24 animals
(b) 13-week exposure group of 24 animals
(c) a group of 24 animals to be held for 30 days after the 13-week exposure.
Control animals:
yes, concurrent vehicle


Observations and examinations performed and frequency:
The animals were sex-segregated at all times. The exposed and control animals were observed daily for toxic signs and death. The animals used were weighed individually at approximately 2-week intervals. At the end of the exposure, the rats were taken to the laboratory where they were weighed, returned to their home cages and allowed to acclimatize overnight. During the following 2 days they were examined and tested according to the guidelines published by Cummings et al. These included measurements of temperature, EKG, heart rate, blood pressure, ventilation, physical performance and reflex activity. EKGs were analysed for wave amplitudes, intervals, duration, rhythm and axis. Pulmonary ventilation was analysed for frequency, volume, any changes due to breathing 6% carbon dioxide. The measurements described were all performed on anaesthetised and comfortably restrained rats. Groups of males and females were euthanatised following 6 and 13 weeks to the test substance aerosol. Another group was euthanatised 30 days after exposure to the test substance.
Sacrifice and pathology:
At termination and necropsy, tissues were evaluated grossly and preserved in 10% buffered formalin. The tissues were embedded in paraffin and subsequently processed for staining with hematoxylin and eosin. Tissues form the following were evaluated macroscopically: brain, eye, adrenal, thyroid, trachea, lung, turbinates, kidney, liver, spleen, heart, esophagus, stomach, small intestine, large intestine, pancreas, urinary bladder, testes, ovaries, and uterus. Before fixing the hearts, lungs, livers, kidneys and gonads, they were weighed, and organ-to-body weight ratios were calculated. Rats evaluated for physiological and behavioural effects were not routinely necropsied. Some were examined if the results of the physiological evaluation warranted pathological verification of compound effects. Blood chemistry and haematological evaluations were only performed on the rats normally scheduled for necropsy. Pathological and haematological examinations were performed on the respective groups at the termination of the exposure-holding periods. The animals were anesthetised with pentobarbital sodium and blood samples taken by cardiac puncture. The following hematological blood parameters were evaluated: red blood cells, white blood cells, differential white count, hemoglobin and hematocrit. The following blood chemistry parameters were evaluated: glucose, urea nitrogen, creatinine, sodium, potassium, chloride, carbon dioxide, uric acid, total protein, albumin, globulin calcium, phosphate, cholesterol, triglycerides, alkaline phosphatase, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, lactic dehydrogenase, and total bilirubin.
The data were compared statistically using an analysis of variance (ANOVA) which distinguish differences according to sex, exposure level, and the effect of dose within sexes.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No obvious pattern of overt toxic signs was seen exposed to either 100 or 1000 mg/m3 of the test substance. After each exposure, the fur of the animals exposed to the high dose appeared oily.
no mortality observed
Description (incidence):
None of the rats died during the 13-week exposure or the 30-day post-exposure observation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Both males and females, sowed no definite pattern as to weight loss or gain. At the end of the post-exposure period, the rats weighed as much as, or slightly greater than, the controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
During the 13-week exposure or the 30-day post exposure period there were no consistently significant changes in red blood cell count, total and differential white blood cell count, haematocrit, or haemoglobin.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no consistently significant changes in rat blood chemistry at the end of the 5- or 13-week exposures or the 30-day post exposure period.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The organ body weight ratios in rats at all concentrations and for the 6- and 13-week exposure periods and the 30-day post-exposure period showed no pattern of significance that could be related to treatment.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- 6 weeks: Mild to moderate proliferative interstitial pneumonia was seen in 9 rats (3 control males, 5 exposed males (2 low- and 3 high-dose), and one female in the low dose. Acute to subacute inflammation of the turbinates was observed in 1 control female and 2 exposed low dose males.
- 13 weeks: Infiltration of foamy macrophages occurred in the lungs of 1 exposed high dose male. Suppurative inflammation of the turbinates was seen in a control female. A renal cortical cyst was found in one control female.
- 13 weeks exposure + 30-day post exposure: Infiltration of foamy macrophages occurred in 3 females (1 control, 1 low and 1 high dose). One female exposed to the low dose exhibited mild turbinate inflammation. Inflammation of the renal pelvis was seen in one exposed high dose female.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Female rats exhibited a higher rectal temperature and a slightly lower systolic blood pressure. At the 6-week exposure point, the low dose female rats showed a higher systolic blood pressure than the controls. Since this did not occur in the high-dose animals and was not repeated in subsequent exposures, it appeared to be a chance occurrence. In male rats exposed for 13 weeks to the high dose, there appeared to be a slight decrease in the P-R interval in the EKG compared to the control animals. The usual abnormality associated with the P-R interval is an increase in duration, which indicates a slowing of the arterial conduction system. This interval will vary inversely with heart rate, but heart rate did not appear to be a factor in the present study. In any event, the decrease was not associated with any abnormality and may be considered inconsequential.
The results of the pulmonary tests include the respiratory rate, estimated pulmonary resistance and respiratory flow rate. The results indicate that these dose levels of the test substance for a period of 13 weeks had no effect on the pulmonary function of the rat. Female rats exhibited some change after the 6-week exposure to 1000 mg/m3; the statistical differences were not significant. However, 4 out of the 5 animals showed a decrease in respiratory rate and an increase in estimated pulmonary resistance. These changes were apparently transient since they were not apparent in the males, nor at 13 weeks and 30 days postexposure in the females.

Effect levels

Dose descriptor:
Effect level:
1 mg/L air
Based on:
test mat.
Basis for effect level:
other: overall effects

Target system / organ toxicity

Critical effects observed:

Applicant's summary and conclusion