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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.99 to 99.995%
- Physical form: colorless liquid

Test animals

Species:
mouse
Strain:
other: Crl:CD-1 BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 30-32 days
- Weight at study initiation: 13-28 grams (males) and 12-24 grams (females)
- Housing: Mice were housed individually in stainless steel cages suspended above cage boards. Male and female mice were housed on separate cage racks.
- Diet: Mice were fed irradiated Purina Certified Rodent Chow #5002 ad libitum when they were not in the inhalation chamber.
- Water: Provided ad libitum by an automatic watering system. Water was not available during the inhalation exposures.
- Acclimation period: Animals were maintained for a quarantine period of approximately three weeks and selected animals were subjected to serological evaluation of blood for the presence of antibodies to selected rodent pathogens, prior to study start.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12-hour on/off cycle

IN-LIFE DATES: From: 4-28-91 To: 4-16-93 (mice and rats inclusive)

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Four 4 cubic meter chambers were dedicated to the study throughout the duration of the exposure period. The chambers were operated in a one-pass, flow-through mode with air flow rates adequate to provide sufficient oxygen for test animals to prevent contamination from volatiles derived from animal excretia, and to enable adequate distribution of the test material in the chambers. Air flow rates were continually monitored and were recorded at a minimum of one-hour intervals. During exposure, temperature and relative humidity of the chamber was monitored continuously and recorded at least once every 30 minutes. Chamber oxygen content was measured at least twice daily.

Male and female mice received a total of 381-383 exposures. All exposures were conducted during the same eight-hour period of the day. During exposure, mice were exposed in their individual housing cages. Cage positions within the exposure chambers were rotated daily. Before transfer to housing facilities at the end of each exposure, test groups were left in their respective exposure chambers for a period that allowed clearance of test material from the chamber atmosphere, as determined by atmospheric analysis.

The test material vapor for each test chamber was generated separately by metering the liquid test material into a glass J-tube filled with glass beads. Heated air (~100-130 deg C) was blown through the glass beads to evaporate the liquid test substance. The resulting vapor was diluted to the desired concentrations with filtered conditioned air for each of the three test chambers. Chamber concentrations were controlled by varying the test substance flow rates into the exposure chamber. Heated air alone was metered in an identical manner into the control chamber.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to study commencement, the distribution of the test material was determined by collecting samples from the top and middle of one test chamber and was found to be homogeneously distributed (i.e., coefficient of variation between sampling points was 4.0).

Chamber atmospheres were analyzed by gas chromatography at approximately 30-minute intervals during each six-hour exposure period. Samples were drawn by vacuum pump from representative areas of the chambers where rats were exposed. The samples from the chambers were collected in impingers containing approx. 1.0 mL of acetone. The liquid samples (approx. 1.0 uL) were analyzed with a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector. The temperature of the injection port and detector was 225 and 250 deg C, respectively. Nitrogen was utilized as the carrier gas at a flow rate of approx. 15 mL/min. Samples were chromatographed in a isothermally at 100 deg C on a 10 m X 0.53 mm (inside diameter) fused silica glass Carbowax 20M column with a film thickness of 1.33 um. The detector was supplied with air (approx. 350 mL/min), hydrogen (approx. 30 mL/min), and auxiliary nitrogen (approx. 15 mL/min). The atmospheric concentration of test material was determined by comparing the detector response of the chamber samples to that of liquid standards with the use of standard curves.
Duration of treatment / exposure:
18 months
Frequency of treatment:
5 days/week, 6hr/day
Doses / concentrations
Remarks:
Doses / Concentrations:
0.0, 25, 101.0, 350.5 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
78/sex/group
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: none

Examinations

Observations and examinations performed and frequency:
BODY WEIGHT: All animals were weighed once each week during the first three months and once every other week for the remainder of the study.

CLINICAL OBSERVATIONS AND MORTALITY: Cage-side examinations to detect moribund or dead animals and abnormal behavior and appearance among animals were conducted twice on exposure days and at least once on non-exposure days. Moribund animals were humanely sacrificed, and all animals sacrificed or found dead were submitted for pathological examination. At each weighing, each animal was individually handled and carefully examined for abnormal behavior and appearance.

OPHTHALMOLOGICAL EVALUATION: Two ophthalmological examinations pere species were conducted by a veterinary ophthalmologist. Both eyes of all mice were examined by focal illumination and indirect ophthalmoscopy. Examinations were conducted under subdued lighting after mydriasis was produced with a solution of 1% atropine for the pretest exams (~day-9) and 1% tropicamide for the final exam. All surviving animals were examined prior to the 18 month sacrifice (day 537).

CLINICAL LABORATORY EVALUATIONS: A hematological evaluation was conducted on mice approximately 3 months (test days 96 and 97), 6 months (test days 187 and 188), 12 months (test days 369 and 370) and 18 months (test days 551 and 552) after initiation of the study. Prior to each evaluation, ten male and ten female mice were randomly selected from each test group. Blood was drawn from unfasted mice. Samples were collected from the orbital sinus of each mouse while it was under light carbon dioxide anesthesia.

HEMATOLOGY: Parameters included erythrocyte, leukocyte, differential leukocyte, and platelet counts, hemoglobin concentration, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration, and relative numbers of neutrophils, band neutrophils, lymphocytes, atypical lymphocytes, monocytes, eosinophils, and basophils. Blood smears for reticulocyte counts were prepared from each mouse at each sampling time, but evaluation was not necessary to substantiate or clarify the results of hematologic evaluation. Blood smears were also prepared from mice sacrificed by design, but evaluation was not necessary to substantiate or clarify the results of histomorphologic findings. Bone marrow smears were prepared from all animals at the interim and terminal sacrifices, but evaluation was not necessary to substantiate or clarify the results of hematologic evaluation.


URINE ANALYSES: Urine collected from rats during the 14 hours preceding blood collection was measured for the following: volume, pH, osmolality, and semiquantitative measures of glucose, protein, bilirubin, urobilinogen, ketone, and occult blood. Urine color and transparency were recorded, and sediment from each urine sample was microscopically examined.
Sacrifice and pathology:
Approximately 18 months after study initiation (test days 555 to 563), all surviving mice were sacrificed and necropsied. The order for sacrifice was random. All mice that died or were sacrificed in extremis before the end of the study were necropsied. Mice were sacrificed either by pentobarbital overdose and exsanguination or by carbon dioxide anesthesia and exsanguination.

The following tissues were collected from all animals (tissue integrity permitting): skin, bone marrow (femur, sternum), lymph nodes (mandibular and mesenteric), spleen, thymus, aorta (thoracic), heart, trachea, lungs* (inflated), nose (4 cross sections, including paranasal sinuses), larynx/pharynx, salivary glands, esophagus, stomach, liver*, pancreas, small intesting (duodenum, jejunum, and ileum), large intestine (cecum, colon, and rectum), gall bladder, kidneys*, bladder, pituitary, thyroid - parathyroid, adrenals, brain* (includes sections of medulla/pons, cerebellar cortex, cerebral cortex), spinal cord (cervical, thoracic, lumbar), peripheral nerve (sciatic), muscle (thigh), bone (femur, sternum), eyes, exorbital lacrimal glands, harderian glands, all gross lesions and tumors. The following tissues were collected from males: prostate, testes*, epididymides, and seminal vesicles. The following tissues were collected from females: mammary gland, ovaries, uterus, vagina. All organs indicated with a "*" were weighed.

All tissues were fixed in 10% neutral buffered formalin except testes, epididymides, eyes, skin (males), and skin with mammary gland (females), which were fixed in Bouin's fixative. The lungs were inflated with formalin at the time of necropsy. All tissues collected from mice in the control and 350 ppm groups, and from animals that were found dead, accidentally killed, or sacrificed in extremis, were further processed to slides, stained with hematoxylin and eosin, and examined microscopically. Lungs, liver, kidneys, and all gross lesions from animals in the low- and intermediate-concentration groups that were sacrificed by design were also processed to slides and examined microscopically.
Other examinations:
On test day 26, 87 and 384, five randomly selected mice of each sex per concentration group were evaluated for hepatic cell proliferation. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital. The dorsal lumbar area of each mouse was shaved and a small incision in the skin was made to allow the subcutaneous insertion of an osmotic mini-pump loaded with BrdU. Livers collected from animals in the control and the 350 ppm groups were evaluated for cell proliferation (cells in the S-phase). Approximately 1,000 nuclei from each tissue were counted and evaluated. The labeling index was the number of S-phase cells in each tissue, expressed as a percentage of the total number of nuclei counted.
Statistics:
Sufficient information regarding the statistical analysis methods for body weights, body weight gain, clinical observations, clinical laboratory data, organ weights, etc. was provided in study report.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
MEAN BODY WEIGHTS AND BODY WEIGHT GAINS:
Male mice exposed to 100 ppm and 350 ppm had a tendency toward higher body weight over the course of the 18-month exposure period. In addition, total body weight gain over the interval of test days 1-547 was 15% higher for both 100 and 350 ppm males, compared to control values, and was statistically significant for 100 ppm males. Total body weight gain for the first nine months of the study (days 1-267) was similar for all exposure concentrations. However, males exposed to 350 ppm had significantly higher total body weight gain for the interval of the test days 267-547. Total body weight gain over the interval of test days 267-547 was variable for 25 ppm and 100 ppm males, although higher than the control values. However, a dose-response relationship with respect to body weight and body weight gain was not evident, with the total weight gain for both 100 and 350 ppm males being 15% higher than control values and therefore, these differences are not considered to be compound related.

Female mice exposed to 350 ppm had several instances of significantly higher body weight compared to control values over the interval of test days 36-183. In addition, total body weight gain for 350 ppm females was significantly higher for the first 9 months of the study (test days 1-267). However, during the remainder of the study, body weight and body weight gain for 350 ppm females were comparable to control values. Since the significantly higher body weight and body weight gain for 350 ppm females did not persist over the remainder of the study and were not correlated with any pathological changes, these differences were not considered to be compound related.

Over the course of the 18-month exposure period, there were sporadic instances of either significantly lower or higher body weight or body weight gain for all three exposure concentrations. However, these differences represent normal biological variation and were not considered to be compound related.

CLINICAL OBSERVATIONS AND MORTALITY:
Males exposed to 100 or 350 ppm had an increased incidence of diarrhea. There were no morphological changes which correlated with the increased incidence of diarrhea. In addition, although it appears to be compound-related, the diarrhea was transient, did not affect body weight or survival, and therefore, was not considered to be an adverse effect.

Female mice exposed to 350 ppm had a significantly higher incidence of ruffled fur compared to controls. Although it appears to be compound-related, there were no morphological changes, effects on survival, or adverse effects on body weight which correlated with the increased incidence of ruffled fur, and therefore, it was not considered to be adverse. Females exposed to 100 and 350 ppm had a significantly decreased incidence of stained fur compared to control. The lower incidence of stained fur in 100 and 350 ppm females was also considered to be a compound-related, although beneficial, effect.

There were no compound-related effects on the total number of male or female mice with grossly observable masses or in the distribution of masses over all sites at any exposure concentration.

There were no compound-related effects on mortality in either male or female mice at any exposure concentration. Males exposed to 25 ppm had slightly higher survival compared to controls, however, since a dose-response trend was not present, this was not considered to be compound related. Female mice exposed to 350 ppm had slightly lower survival compared to controls (80% and 66% survival for controls and 350 ppm females respectively.) Since there were no compound-related pathological findings that could be associated with the lower survival in 350 ppm females, this difference in survival was not considered compound related.

OPHTHALMOLOGICAL EVALUATIONS
There were no compound-related effects on ophthalmoscopically visible structures of the eye in either males or females at any exposure concentration. Near the end of the 18 month exposure period, the incidences of male mice with eye lesions were 3%, 3%, 8%, and 4% for the 0, 25, 100, and 350 ppm groups respectively. The incidences of female mice with eye lesions were 6%, 8%, 12%, and 7% for the 0, 25, 100, and 350 ppm groups, respectively. The most frequent findings were cataracts (including anterior and posterior pole cataracts). Since a dose-response relationship was not evident for either the incidence or types of lesions observed, they were considered to be spontaneous. The 350 ppm females had an increased incidence of bilateral diffuse retinal atrophy (determined by light microscopy). This lesion was not detected by ophthaloscopic evaluation.

HEMATOLOGY EVALUATIONS
There were no compound-related effects on hematology parameters in either male or female mice at any exposure concentration. All statistically significant differences noted were within the expected range of normal biological variation or did not exhibit dos-response relationships and were not considered to be compound-related.

PATHOLOGICAL EVALUATIONS
Organ Weights: In male mice, there were no compound-related effects on absolute or relative organ weight at any exposure concentration.

Female mice exposed to 350 ppm for approximately 18 months had significantly increased absolute and relative liver weight compared to control values. The increased absolute and relative liver weights in 350 ppm females were considered to be compound related and were most likely the result of enzyme induction associated with metabolism of the compound since there were no histological changes that correlated with an increase in liver weight. Absolute and relative kidney weights were also increased in 350 ppm females, and relative kidney weights were increased in 25 ppm females. However, these data did not exhibit a dose-response relationship, and therefore, they were considered to be spurious.

INCIDENCES OF GROSS OBSERVATIONS
The incidences of gross morphological changes were similar to control in both males and females for all exposure concentrations. In addition, no compound-related gross lesions were observed in males or females sacrificed for cell proliferation at 1 week, 3 months or 12 months.

INCIDENCES OF MICROSCOPIC OBSERVATIONS
There were several compound-related effects observed in the liver in male and female mice exposed to the test substance for approximately 18 months. Male mice exposed to 100 or 350 ppm and 350 ppm females had an increased incidence of pigment accumulation in the Kupffer cells. The incidences were statistically significant only for 100 and 350 ppm males; however, the incidence in 350 ppm females was also considered to be biologically significant. The severity of pigment accumulation was generally minimal in both males and females. The staining reaction of the pigment suggested the presence of both hemosiderin and lipofuscin. In addition, 350 ppm males had an increased incidence of minimal to mild hepatocellular hypertrophy. The hypertrophy is probably the result of enzyme induction and is not considered to be an adverse effect. Female mice exposed to 350 ppm had a statically significant increased incidence of minimal to mild individual hepatocellular necrosis. Males exposed to 100 or 350 ppm also had an increased incidence of individual hepatocellular necrosis; and although the incidences were not statistically significant, they were considered to be biologically significant.

The incidence of diffuse, bilateral retinal atrophy was significantly increased in 350 ppm females. However, the lesions were generally minimal to mild in severity in all groups. It is possible that the increased incidence of retinal atrophy is secondary to the compound-related effects on liver function rather than a direct compound-related effect on the retina.

The test substance was not oncogenic at any exposure concentration in either males or females. However, 350 ppm females had a significantly higher incidence of lymphoma compared to controls (5%, 2%, 5%,15%, for 0, 25, 100, and 350 ppm respectively). The historical control range for lymphoma at this laboratory was 3.3% to 23.8% and the average incidence for historical controls was 15.5%. Since the incidence of lymphoma in 350 ppm females is nearly identical to the average incidence of the historical controls, and a dose-response relationship was not present, it was not considered to be a compound-related effect.

There were no compound-related effects on hepatic cell proliferation rate in either male or females. Males exposed to 350 ppm had a significantly higher cell proliferation rates on test day 26 evaluation. However, the cell proliferation rate was similar to control on test day 87 and 384 and the cell proliferation rate for 100 ppm males was also similar to control on test day 26. Considering the histopathological findings after 18-months of exposure and the lack of effects at other time points, the increased rate observed on test day 26 is considered to be a spurious finding.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
25 ppm
Sex:
male
Basis for effect level:
other: Clinical signs of toxicity and/or morphological changes at 100 and 350 ppm doses.
Dose descriptor:
NOAEL
Effect level:
100 ppm
Sex:
female
Basis for effect level:
other: Clinical signs of toxicity and morphological changes at 350 ppm.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Clinical pathology was evaluated at 3,6,12,18 months; hepatic cells proliferation was examined at two weeks and three and twelve months. No compound-related effects on survival were observed. There were no compound-related effects on body weight or weight gain at any concentration. There were no compound-related adverse effects on the incidence of clinical signs of toxicity. No hematological changes were observed. Compound-related morphological changes were observed in the liver. Exposure to 100 or 350 ppm produced increased absolute and/or relative LIVER WEIGHT (350 ppm female only), accumulation of LIPOFUSCIN/HEMOSIDERIN in KUPFFER CELLS and CENTRILOBULAR SINGLE CELL NECROSIS. Female mice exposed to 350 ppm had an increase incident of BILATERAL, DIFFUSE RETINAL ATROPHY. No increase in hepatic cells proliferation was seen at any exposure concentration.

Applicant's summary and conclusion

Conclusions:
The NOAEL for toxicity for male mice is 25 ppm based on effects on clinical signs of toxicity and morphological changes at 100 and 350 ppm. The NOAEL for toxicity for female mice is 100 ppm based on clinical signs of toxicity and morphological changes at 350 ppm. The study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).