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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not applicable
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and reported study according to GLP and the guideline of the day.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Principles of method if other than guideline:
No guideline information provided
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name : Flux Oil (MCS 2170 or Therminol ®59)
Description: Light yellow liquid of alkyl substituted aromatics
Specific Gravity: 0.97
Lot Number: NBP 2933500
EHL Substance Identification No.: T850028
Source: Monsanto Chemical Company

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Male and female Sprague-Dawley rats (Crl : CD® (SD) BR) were obtained from the Charles River Breeding Laboratory (Portage, Michigan). Test animals were held and observed in quarantine for fourteen days prior to release for study. At the start of the study, animals weighed on the average of 245 grams for males and 165 grams for females. On first day of study, males and females were 52 days of age. Each rat was identified uniquely by ear tag
and bar-coded cage card.

Prior to study, animals were randomly allocated on the basis of body weight into 4 groups of 15 animals/sex/group. The randomization precluded significant differences among group mean body weights and inordinate variability of individual body weights within the groups.

Animals were housed individually in suspended stainless steel wire mesh cages and, during non-exposure periods given, ad libitum, Purina Laboratory Certified Rodent chows (5002) and tap water (St. Louis City, MO), conditioned by an ion exchange water softener using sodium zeolite. Animal rooms were maintained routinely at 70-74 degrees F and 35-60% relative humidity with a 12-hour light/l2-hour dark cycle.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: The particle size of the test material ranged from 3.45 to 4.41 µm mass median aerodynamic diameter (MMAD) with geometric standard deviations between 1.98 and 2.57 µm in the mid exposure level and 3.22 to 4.13 um MMAD with geometric standard deviations between 2.02 and 2.22 µm in the high exposure level during the study period.
Details on inhalation exposure:
Four 10 m3 New York University-style stainless steel chambers with pyramidal tops and bottoms were used for the exposure. The test material was metered from a tank using a capillary restrictor to a Laskin-style nebulizer (I), which was used to generate the test atmosphere. The concentration of test material in the inhalation chamber was controlled by regulating the pressure in the tank headspace. The nebulizer was positioned in the side of a vertical particle-size separator which was connected to the air inlet of the inhalation chamber. One nebulizer was used in each of the generation systems. The particle-size separator prevents most of the large, non-respirable aerosol particles from entering the chamber with the large particles collecting in the bottom of the separator. Airflow in the chamber was nominally maintained at 1700 liters per minute.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal atmospheric concentration measurements were determined daily for each exposure level. This concentration was calculated as the net amount of test material (amount delivered to the nebulizer minus amount recovered in the bottom of the separator) entering the air inlet of the inhalation chamber per unit time. The samples for analytical measurements of the test atmosphere were obtained by drawing ten liters of chamber atmosphere through a single impinger containing iso-octane. Samples were drawn at a port located at the lower center of the door on the side of the chamber holding the animal cage rack. At least three samples per day were obtained from each chamber, except the control level. Additional atmospheric concentrations were measured twice during the study period from five specific locations in each chamber to demonstrate the uniformity of distribution of the flux oil atmosphere.

Particle-size analysis was performed for the mid and high exposure levels once daily during the first week of the study and weekly thereafter using an Andersen Cascade impactor. A sample was drawn for a known time at a flowrate of approximately 1 CFM. The mass of material collected on each stage was determined gravimetrically and was used to determine mass median aerodynamic diameter, geometric standard deviation, and percentage of particles less than 10 microns.

A procedure for assaying flux oil for stability assessment was developed with the test material being assessed prior to and after the termination of the study.
Duration of treatment / exposure:
6 hours per day, 5 days per week for a total of at least 64 exposures over the period of 14 weeks
Frequency of treatment:
daily (not including weekends)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 100, 400 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: No data
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: second and fifth hours of each exposure


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Following each exposure all animals were also individually examined for gross clinical signs following each exposure and checked for mortality preceding each exposure and on non-exposure days. They were also given a weekly thorough examination for gross signs of toxicity after weighing.


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly each animal was weighed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the last study week.
- Dose groups that were examined: Control and high level exposure groups


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to each animal's termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals:All groups


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to each animal's termination
- Animals fasted: Yes
- How many animals:All groups



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Blood Sample Collection for Clinical Pathology:Blood for complete hematologic and serum biochemical analyses was collected from the posterior vena cava of each animal of all groups just prior to each animal's termination. Animals were fasted overnight prior to collection.

Hematology: Whole blood treated with anticoagulant (EDTA-pretreated commercial tubes) was processed on a coulterB S-Plus I1 blood cell counter using manufacturer's methods. For each submitted sample the total erythrocyte count (RBC, cells per mm3), the total leukocyte count (WBC, cells per mm3), the platelets (count per mm3) , the hematocrit (HCT, %) , the level of hemoglobin (HGB, =/dl), and the red blood cell indices (mean corpuscular volume -MCV, u3; mean corpuscular hemoglobin - MCH, pg; and mean corpuscular hemoglobin concentration - MCHC, gm/dl) were determined.

Thin blood smears on labelled glass slides were prepared and stained with Wright stain and a microscopic determination of the leukocyte differential was performed. A portion of the EDTA sample was mixed with vital stain. A labelled slide was then prepared and a reticulocyte count was made. Resulting test data were transcribed as required to a computer to perform statistical analyses.

Serum Biochemistry-
Serum was harvested by centrifugation from samples (collected same day) submitted in commercial clot tubes and assayed by standard manufacturer's methodology on a KDA.

Gross and Microscopic Pathology: Terminal body weights were obtained just prior to euthanasia. Detailed necropsies were performed on all rats at the termination of the study. Organ weights were taken. Tissues were saved and perserved. Tissues from the males and females of the conrol and high exposure levels were examined microscopically.




Statistics:
Noncategorical data (including terminal body weights and absolute organ weights) were examined statistically by Dunnett's test for the comparison of means of multiple treatments with control, and by inspection. Categorical data were examined to determine any remarkable group differences. Organ-to-body weight ratios were analyzed by the Mann-Whitney test with Bonferroni's inequality. Microscopic lesions were evaluated by the Fisher Exact Test with Bonferroni's inequality.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived the scheduled exposures. The only notable observation that was seen during exposure was labored breathing in the high exposure animals. Notable after exposure observations seen were nasal/respiratory irritation, hypoactivity, red discharge of eye, pale eyes, and skin and fur abnormalities. A higher incidence of after exposure observations were noted in the high exposure animals. Additional observations noted during weekly weigh periods were red/brown perinasal encrustation (mid and high exposure animals), salivation (high exposure males), and urine-stained hair (high exposure animals).

BODY WEIGHT AND WEIGHT GAIN
A significant (p ≤ 0.01) reduction in body weight occurred in the high exposure level animals beginning at Study Week 1 and continued until the end of the study. Mid exposure female animals displayed significant (p < 0.01) body weight reduction from Study Week 2 to the end of the study. The low exposure female animals had decreased body weight throughout the study period.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmic examinations of the control and high level animals showed no ocular changes which could be attributed to test material exposure.


HAEMATOLOGY/CLINICAL CHEMISTRY
Great variation was encountered in a number of hematologic and serum chemistry parameters. The changes that were statistically significant and considered possibly treatment-related were the following: depressed red cell numbers, total hemoglobin, and the hematocrit in the high exposure animals; elevated mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentrations (MCHC) in the high exposure males; depressed blood glucose in the high exposure animals; and depressed plasma cholinesterase in the mid and high exposure animals, but not statistically significant an the mid and high males. Total protein, globulin and creatinine were elevated in the high exposure level animals, and calcium and albumin were elevated in the high exposure males. Total bilirubin levels were elevated in high exposure females and elevated phosphorus levels occurred in all exposure level females.


GROSS PATHOLOGY/ORGAN WEIGHTS
A significant depression in terminal body weights occurred in the high exposure males and in all exposure level females. Absolute adrenal weights were significantly increased in high exposure level females and adrenal-to-body weight ratios were significantly increased in females from all
exposure levels. Absolute kidney weights were increased in high exposure level animals (signifieant only for males) and kidney-to-body weight ratios were significantly increased in high exposure level males and all exposure level females. In the high exposure level females and all exposure level males absolute liver weights, and liver-to-body weight ratios were significantly increased. In addition, for the mid exposure females, absolute
liver weights tended to be higher than those of control and liver-to-body weight ratios were significantly increased. Decreased absolute heart weight and increased organ-to-body weight ratios for brain, heart, and spleen were present in those animals of exposure groups which had substantially reduced body weights and were thus not interpreted as direct effects of compound exposure.

Very few gross lesions were observed and none could be considered related to test material expcsure. Various spontaneous lesions were encountered but neither the nature of the lesion or a relation to dosage suggested a treatment relationship. The focal pancreatic inflammation and associated acinar atrophy in two high exposure level males is a common spontaneous rodent disease and is considered unrelated to exposure to the test material.

OTHER FINDINGS
No test material was detected in the sample taken from the control chamber. The nominal/analytical ratios indicate a loss of test material due to impaction within the particle-size separator and/or chamber. The results of the stability assessments indicate that the material was stable over the time frame of the study. The distribution of the exposure atmosphere was determined to be reasonably uniform for each of the exposure level chambers.

Effect levels

open allclose all
Dose descriptor:
LOAEC
Effect level:
20 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: Based on changes in bodyweight seen in all dose levels
Dose descriptor:
NOAEC
Remarks:
Local effects only
Effect level:
100 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: Based on signs of respiratory irritation at the highest dose group

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Gross signs of toxicity, observed almost exclusively in the high exposure level animals, indicate upper respiratory irritation due to the exposure to flux oil. High exposure animals, and the mid and low level females were exposure effected with reduced body weights.

Gross necropsy and microscopic findings indicate no evident changes as a result of test material exposure. The hematology and serum biochemistry data do suggest an exposure response. Depressed red cell numbers, hemoglobin, hematocrits (resulting to an increase in the calculated values for MCH and MCHC in high level males), and blood glucose in the high exposure level animals appear to be a direct effect of flux oil exposure. However, the mechanism for the mild anemia could not be determined.

The elevated parameters of total protein, globulin, creatinine in high level animals, calcium and albumin (high level males), and bilirubin (high level females) probably represent direct effects of test material exposure. Elevated serum phosphorus values for females could not be attributed to compound administration since the mean values were essentially within the historical control range for rats of this age. Depression of the plasma cholinesterase levels in the high and mid females (and probably high males) is apparently a treatment-related effect.

Increases in the absolute and relative weights of kidney in the high level animals (though absolute values were not statistically significant in the high females) and livers of the high level females and all exposure level males were attributed to compound exposure. Since there were no obvious microscopic lesions or any abnormalities in organ associated serum chemistry values, the biological significance of the apparent organ enlargements is unknown. The adrenal weight increases, both absolute and relative in females, are unexplained as no morphological changes were significant and the lymphocyte counts were elevated rather than depressed as would be expected with adrenal cortical hypertrophy and hypersecretion.

Applicant's summary and conclusion

Conclusions:
Even though there were a number of hematologic, serum chemistry, body and organ weight changes, there was a real absence of detectable morphologic changes in tissues. Interpretation of a "no-effect" level was therefore difficult to determine. There were definite toxic effects to rats exposed to flux oil at concentrations of 100 milligrams per cubic meter of air or greater. The reduced body weights of all the female exposure groups and the increased absolute and relative liver weights of all the male exposure groups preclude the low exposure level of 20 mg/m3, in this study, of being a "no-effect" level.
Executive summary:

Four groups of 15 male and 15 female Sprague- Dawley rats per group were exposed to mean analytical concentrations of 0, 20, 100 or 400 mg flux oil per cubic meter of air in 10 m3 inhalation chambers. A minimum of sixty-four 6-hour exposures were conducted over an approximate 14-week period. All animals survived the scheduled exposures. Labored breathing was observed in the high exposure animals during exposures. Nasal/respiratory irritation, hypoactivity, red discharge of eye, pale eyes, and skin and fur abnormalities were observed after exposures almost exclusively in the high exposure level animals. Additional observations noted during the weekly weigh periods were red/brown perinasal encrustation (mid and high exposure animals), salivation (high exposure males, and urine-stained hair

(high exposure animals). A significant reduction in body weight occurred in the high exposure level animals, and in the mid and low level females. Great variation was encountered in a number of hematologic and serum chemistry parameters. The changes that were statistically significant and considered possibly treatment related were depressed red cell numbers, hemoglobin, hematocrits, and blood glucose (high exposure level animals). Also noted were elevated parameters of total protein (high level animals), globulin (high level animals), creatinine (high level animals), albumin (high level males), bilirubin (high level female), and calcium (high level males); and depressed plasma cholinesterase levels (mid and high level females). Neither gross necropsy nor microscopic findings indicated any changes resulting from test material exposure. Absolute kidney weights were significantly increased in the high exposure males and kidney to body weight ratios were significantly increased in high exposure males and all exposed females. The absolute and relative liver weights were significantly increased in the high exposure animals and low and mid level males. The reduced body weights of all the female exposure groups and the increased absolute and relative liver weights of all male exposure groups preclude the low exposure level of 20 mg/m3, in this study, of being a "no-effect" level.