Registration Dossier

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:
comparable to guideline study
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 %


Test animals

Species:
rat
Strain:
other: Crl: CD BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 22-25 days
- Weight at study initiation: 42 to 82 grams (males) and 33 to 65 grams (females)
- Housing: Rats were housed individually in stainless steel cages suspended above cage boards. Male and female rats were housed on separate cage racks.
- Diet: Rats 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: 4-27-91 To: 4-16-93

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Four 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 rats received a total of 506-507 exposures. Female rats received a total of 494-495 exposures. All exposures were conducted during the same eight-hour period of the day. During exposure, rats were housed two or three per cage, with sexes separate. 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 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:
2 years
Frequency of treatment:
5 days/week, 6hr/day
Doses / concentrationsopen allclose all
Dose / conc.:
25.2 ppm
Remarks:
analytical conc.
Dose / conc.:
101 ppm
Remarks:
analytical conc.
Dose / conc.:
350.5 ppm
Remarks:
analytical conc.
No. of animals per sex per dose:
87/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 per species were conducted by a veterinary ophthalmologist. Both eyes of all rats 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 8) and 1% tropicamide for the final exam (test day 683).

CLINICAL LABORATORY EVALUATIONS: Conducted approximately 3 months (test days 95 and 96), 6 months (test days 186 and 187), 12 months (test days 375 and 376), 18 months (test days 550 and 551), and 24 months (males only, test day 726) after study initiation. At the beginning of the study, ten male and ten female rats were randomly selected from each test group for the clinical evaluation. Where possible, the same ten rats/group were used for the 3-, 6-, and 12-month evaluations. These rats were sacrificed at the 12-month evaluation. Ten rats/group were randomly selected from the survivors at the 18- and 24-month clinical evaluations. Rats that had died prior to any of the clinical evaluations were replaced.

Two days prior to collection of blood samples for clinical evaluation, the rats were placed in metabolism cages and allowed to acclimate overnight. On the day prior to blood collection, the rats were returned to cages for exposures, allowed access to food and water for at least 2 hours after the exposure period, then returned to metabolism cages. Only water was available during urine collection. Urine was collected from each rat for approximately 14 hours overnight prior to blood collection. Blood samples were collected from the orbital sinus while the rat 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 rat at each sampling time, but evaluation was not necessary to substantiate or clarify the results of hematologic evaluation. Blood smears were also prepared from rats 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.

CLINICAL CHEMISTRY: Blood serum from rats was evaluated for 5'-nucleotidase activity, alanine aminotransferase activity, aspartate aminotransferase activity, sorbitol dehydrogenase activity, and concentrations of blood urea nitrogen, total serum protein, albumin, globulin (calculated), creatinine, cholesterol, glucose, calcium, sodium, potassium, phosphate, chloride, and total bilirubin.

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 12 months (test days 376 and 377) after study initiation, the ten rats/group designated for clinical evaluation were sacrificed and necropsied. All surviving males were sacrificed on test days 738 and 740. Due to low survival in the control group, surviving females were sacrificed and necropsied on test day 720 and 721. All rats that died or were sacrificed in extremis before the end of the study were necropsied. Rats were sacrificed either by pentobarbital overdose and exsanguination or by carbone 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), 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 rats 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 28, 89 and 384, five randomly selected rats of each sex per concentration group were evaluated for hepatic cell proliferation. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The dorsal lumbar area of each rat was shaved and a small incision in the skin was made to allow the subcutaneous insertion of an osmotic minipump 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 Section O (page 36) of the study report.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Male and female rats in the 350 ppm dose group had a significantly lower incidence of skin sores compared to controls. Although the reason is unknown, it is considered to be compound related. There were no compound-related effects on the total number of rats with grossly observable masses, or on the distribution of masses over all sites in males at any exposure concentration. Females exposed to 350 ppm had a lower incidence of grossly observable masses, although the differences from control were not statistically significant. The incidence of grossly observable masses located in the perineum and on the side or sides of the females was significantly lower for 350 ppm females compared to control. However, the incidence of grossly observable masses located in the inguinal area was significantly higher for 350 ppm females compared to control. Masses in the inguinal area would most likely correlate with mammary tumors, and the incidence of mammary tumors in females was similar to control for all exposure concentrations. As a result, the increased incidence of grossly observable masses in the inguinal area for 350 ppm females was considered to be a spurious finding, and is not biologically significant.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no adverse, compound-related effects on survival in either males or females for any exposure concentration.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Females: Female rats in the 350 ppm dose group had compound-related lower body weight and body weight gain compared to controls. During the first 11 months of the study, there appeared to be no compound-related body weight effects at any exposure concentration. At approx. test day 351, the weight curve for the female 350 ppm dose group began to diverge from those of the control and other concentration groups, due to lower body weights compared to control values. Mean body weights were significantly lower than control values on test days 365, 393-421, 449, and 477-505. Body weights for the 350 ppm dose group continued to be lower through the remainder of the study, although the differences were not statistically significant. The lower body weights for the 350 ppm females were considered to be an adverse compound-related effect because the difference from control was greater than 10% (body weight gain was 17% lower than the control value over the interval of test days 1-715), they exhibited a dose-response relationship, they were consistent with the effects observed in the 350 ppm male rats, and they were consistent with the effects observed in a previous two-year study in which DMAC was administered to rats in drinking water. Although both statistically significant higher and lower values for body weight and/or body weight gain for the 25 and 100 ppm females were observed, no dose-related differences were evident and were therefore not considered to be compound related.

-Males: Male rats in the 350 ppm dose group also had lower mean body weight and body weight gain compared to controls. During the first 15 months of the study, there were no apparent compound-related effects on body weight for male rats at any exposure concentration. At approx. test day 491, the body weight curves for the 100 and 350 ppm males diverged from the control and 25 ppm males, reflecting a lower body weight compared to control values. At approximately test day 547, the curve for the 350 ppm males diverged from the curve for the 100 ppm males reflecting the statistically lower body weights for 350 ppm males on test days 659 and 687. The lower body weight/body weight gain for the 350 ppm males was considered to be an adverse, compound-related effect since the difference from control (16% for body weight gain over the interval of test days 1-729) was greater than 10%, exhibited a dose-response relationship, was consistent with the effects observed in 350 ppm females, and was consistent with the effects observed in a previous 2-year study. Although 100 ppm males also exhibited a tendency toward lower body weight, the differences from control (body weight gain was 8% lower over the interval of test days 1-729) was not considered to be adverse since it was less than 10%. Although there were instances of both significantly higher and lower mean body weight gain values for 25 ppm males, no dose-related differences were evident and were therefore not considered to be compound 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:
effects observed, non-treatment-related
Description (incidence and severity):
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 study, the incidences of male rats with eye lesions were 28%, 28%, 35%, and 12% for the 0, 25, 100, and 350 ppm groups, respectively. The incidences of female rats with eye lesions were 13%, 21%, 16%, and 15% for the 0, 25, 100, and 350 ppm groups, respectively. The most frequent findings were pale ocular fundi and superficial corneal vascularization which are common in rats of this strain and age.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no compound-related effects on hematology parameters in either male or female rats at any exposure concentration. Male rats exposed to 350 ppm had significantly lower hemoglobin and hematocrit at the 18- and 24-month evaluations, however, the differences from control were small, and there was no evidence for a dose-related decrease in red blood cell count at the 24-month evaluation and therefore, the lower hematocrit and hemoglobin were not considered to be compound-related. Male rats exposed to 25, 100, or 350 ppm also had significantly lower platelets at the six-month and 18-month evaluations (for 25 ppm males, platelets were statistically significant only at 18 months). However, these values were within the range of normal biological variation and were not considered to be compound related. Females exposed to 350 ppm had lower white blood cell counts and lymphocytes at the 3-month and 6-month evaluations (statistically significant only at the 3-month evaluation). At the 12-month evaluation, these parameters continued to be lower than control values, but a dose-related trend was not evident. However, the magnitude of differences between the 350 ppm females and controls was small, and was considered to be secondary to the stress associated with an inhalation exposure regimen. All other statistically significant differences noted in the study were within the expected range of normal biological variation and were not considered to be biologically significant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were several compound-related changes in clinical chemistry parameters. Male and female rats exposed to 350 ppm had increased serum sorbital dehydrogenase activity at the 3-month evaluation, and 6-month evaluation for 350 ppm males only. These results indicate mild hepatocyte membrane injury had occurred. Serum cholesterol concentrations were significantly increased in 100 and 350 ppm females at the 3-, 6-, and 12-month evaluations and were considered to be compound-related since they occurred over several time intervals and exhibited a dose-response relationship. A statistically significant decrease in serum cholesterol was also observed at the 6-month evaluations for 25 ppm females, however, since the values were similar to control at other time points, and it was within the range of biological variation, it was not considered to be compound-related. Serum glucose concentration was also significantly higher for 100 and 350 ppm females at the 3-, 6-, and 12-month evaluations (the data were not statistically significant for 100 ppm at the 3-month evaluation). This effect was considered to be compound-related since it occurred over several time intervals and exhibited a dose-response relationship. Although the magnitude of the changes in serum cholesterol and serum glucose for 100 and 350 ppm females was not considered to be biologically adverse, it was considered to be indicative of a compound-related, toxicologically significant change in energy metabolism. Males in the 350 ppm dose group also had higher cholesterol compared to control values at the 18-month and 24-month evaluations (statistically significant only at 18 months), however, the magnitude of the increase was small and it was not considered to be biologically adverse. All other statistically significant differences in clinical chemistry parameters were within the expected range of normal biological variation or did not exhibit dose-response relationships and were not considered to be compound related, and were not considered to be biologically significant.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no compound-related effects on urinalysis parameters in either males or females at any exposure concentration.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the 12-month interim sacrifice, 100 and 350 ppm females had significantly higher mean relative liver weight compared to control values. Absolute liver weights were also higher (14.5% and 18.5%, for 100 and 350 ppm, respectively) compared to control, although this was not statistically significant. Although there were no compound-related morphological changes observed at the 12-month interim sacrifice, the changes in absolute and relative liver weight exhibited a dose-response relationship. There were no compound-related changes in absolute or relative organ weight for males at any exposure concentration at the 12-month interim sacrifice.

At the 24-month sacrifice, 350 ppm males had significantly higher absolute and relative liver and kidney weights. Absolute and relative liver weights were also higher for the 100 ppm males, although this effect was not statistically significant. The higher absolute and relative liver weights for 100 and 350 ppm males at the 24-month sacrifice were considered to be compound related since they exhibited a dose-response relationship. The liver weight increases in both males and females were most likely the result of enzyme induction associated with metabolism of the compound since there were no histological changes that would correlate with an increase in liver weight. The increased kidney weight in 350 ppm males was also considered to be compound related since it was associated with an increase in severity of chronic progressive nephropathy. Other statistically significant differences in absolute or relative organ weights in either males or females were not associated with morphological changes and were considered to be spurious.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No compound-related gross morphological changes were noted at the 12-month interim sacrifice in either males or females at any exposure concentration. After approximately two years of exposure to the test substance, 350 ppm males had an increased incidence of large kidneys and chronic progressive nephropathy, small testes, and large parathyroid glands. The morphological changes in the kidney were considered to be compound related, and the changes in the testes and parathyroid were considered to be secondary to the effects on the kidney. The incidence of gross morphological changes in females was similar to control for all exposure concentrations.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No compound-related microscopic morphological changes were noted at the 12-month interim sacrifice in either males or females at any exposure concentration. After approximately two years, compound-related microscopic morphological changes were observed in the liver of male and female rats exposed to 100 or 350 ppm doses. Males exposed to 100 or 350 ppm had a statistically significant, dose-related increase in the incidence of hepatic focal cystic degeneration. The severity of this lesion was generally minimal in all groups. The 350 ppm males had an increased incidence of biliary hyperplasia. The severity was generally minimal in all groups. Males exposed to 350 ppm also had a significantly increased incidence of hepatic peliosis characterized by irregular, dilated sinusoids lined by a single layer of well-differentiated endothelium. The incidence of this lesion was also increased in 100 ppm males, although the difference was not statistically significant, and was considered to be compound related. The severity of this lesion was minimal for all groups. Males and females in the 350 ppm exposure group had an increased incidence of pigment in the Kupffer cells which appeared to be hemosiderin and lipofuscin. The severity of the pigment accumulation was primarily minimal in both sexes. The presence of increased lipofuscin suggests an autocatalytic process has occurred. Accumulation of hemosiderin generally occurs in the liver as a result of exposure to iron-containing compounds or following breakdown of red blood cells. In this study, compound-related hematological changes did not occur, however, iron is one of the contaminants in the test substance that was reported by the supplier and may have contributed to the increased accumulation of hemosiderin.

A compound-related increase in the severity of chronic progressive nephropathy was observed in 350 ppm males; however, the incidence of this lesion was similar for all groups. The change in severity is reflected by the number of animals with severe (grade 4) nephropathy (9/61, 9/62, 12/62, and 20/62 for 0, 25, 100, and 350 ppm males, respectively). There were increases in a number of additional lesions which were secondary to the effects of kidney dysfunction in the following tissues: renal vein, aorta, heart, spleen, stomach, parathyroid, seminal vesicles, femur/joint, sternum, nose, testes, and epididymides. Although survival was slightly higher in 350 ppm males, the severity of the nephropathy was greater than that which could be attributed to the slightly greater survival.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
25 ppm
Sex:
male/female
Basis for effect level:
other: Effects on body weight, clinical chemistry parameters, organ weight parameters, and/or morphological changes at 100 and 350 ppm doses.

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
100 ppm
System:
hepatobiliary
Organ:
liver
Treatment related:
yes

Any other information on results incl. tables

- Clinical  pathology was evaluated at 3,6,12,18,24 months; interim sacrifice was carried out at 12 months; hepatic cells proliferation was examined at two weeks and three and twelve months.- No compound-related effects on survival were observed. - Rats exposed to 350 ppm had lower body weight and/or body weight gain. - There were no compound-related adverse effects on the incidence of clinical signs of toxicity. No hematologic changes were observed.
- SERUM SORBITOL DEHYDROGENASE ACTIVITY was increased in rats exposed to 350 ppm. - SERUM CHOLESTEROL and GLUCOSE CONCENTRATIONS WERE SIGNIFICANTLY HIGHER IN 100 AND 350 PPM FEMALE RATS.
- Compound-related morphological changes were observed in the liver. Exposure to 100 or 350 ppm produced increased absolute and/or relative LIVER WEIGHT, HEPATIC FOCAL CYSTIC
DEGENERATION, HEPATIC PELIOSIS, BILIARY HYPERPLASIA (350 ppm only), and LIPOFUCSIN/HEMOSIDERIN accumulation in KUPFFER
CELLS. Male rats exposed to 350 ppm had higher KIDNEY WEIGHT correlated with the gross and microscopy changes resulting from a compound-related increase in severity of CHRONICPROGRESSIVE NEPHROPATHY.
- No increase in hepatic cells proliferation was seen at any exposure concentration.

Applicant's summary and conclusion

Conclusions:
The NOAEL for toxicity for male and female rats is 25 ppm based on effects on body weight, clinical chemistry parameters, organ weight parameters, and/or morphological changes at 100 and 350 ppm.
Executive summary:

The purpose of the study was to evaluate the oncogenic potential of the test substance in rats when administered by inhalation. Groups of 87 male and 87 female Crl:CD®BR rats were exposed to either 0, 25, 100, or 350 ppm test substance for six hours per day, five days per week, for up to two years. Chamber atmospheres were analysed during the exposure period for test substance concentration, temperature, humidity, and airflow rate. Body weights were obtained weekly during the first three months of the study and every other week for the remainder of the study. Clinical signs of toxicity were monitored throughout the study. An ophthalmological examination was performed on all animals prior to study start. All surviving rats were examined prior to their respective final sacrifices. At 3, 6, 12, 18, and 24 months, haematology, clinical chemistry, and urinalysis parameters were measured for ten rats per sex per concentration (females were not evaluated at 24 months). Interim sacrifices to determine the rate of hepatic cell proliferation were conducted on approximately five rats per sex per concentration and occurred on test days 28, 89, and 384. Ten rats per sex per concentration also underwent a 12-month interim sacrifice. All surviving rats underwent gross necropsy after approximately 24 months of exposure. Selected tissues were weighed and tissues were collected for microscopic evaluation.

The overall mean concentrations of test substance in the exposure chambers for the two-year test period were 0.0, 25.2, 101.0, and 350.5 ppm. Mean chamber temperatures generally ranged between 16-29°C. Mean chamber relative humidity generally ranged between 20-65%. Mean chamber air flow ranged between 738-1046 L/min.

There were several compound-related effects observed in rats exposed to the test substance. Male and female rats exposed to 350 ppm had lower body weight and/or body weight gain. There were no adverse, compound-related effects on the incidence of clinical signs of toxicity, on the incidence of ophthalmoscopically observable ocular lesions, or on survival in rats of either sex at any exposure concentration.

Compound-related effects on haematology parameters on urinalysis parameters in rats were not observed. However, male and female rats exposed to 350 ppm test substance had compound-related increases in sorbitol dehydrogenase activity that were indicative of minimal to mild hepatocellular injury. In addition, serum cholesterol and serum glucose concentrations were significantly higher in 10 and 350 ppm females, and were considered to be compound related.

There were several compound-related effects observed in the livers of rats exposed to the test substance. Male and female rats exposed to 100 or 350 ppm test substance had higher absolute and/or relative liver weight at either the 12-month or the 24-month sacrifice. The changes in liver weight most likely represent enzyme induction associated with metabolism of the test substance. Male rats exposed to 100 or 350 ppm had an increased incidence of hepatic focal cystic degeneration and hepatic peliosis, and the incidence of biliary hyperplasia was also elevated in 350 ppm males. In addition, male and female rats exposed to 350 ppm of the test substance, and 100 ppm females had an increased incidence of hemosiderin and lipofuscin pigment accumulation in the hepatic Kupffer cells.

In addition to the compound-related changes on the liver, 350 ppm male rats exhibited significantly higher absolute and relative kidney weights which correlated with the gross and microscopic changes resulting from a compound-related increase in severity of chronic progressive nephropathy.

In rats, exposure to the test substance for 2 years did not produce an oncogenic response in the liver or any other tissue of either sex at any exposure concentration. In addition, there were no compound-related effects on hepatic cell turnover in rats of either sex at any exposure concentration. Therefore, the no-observable-adverse-effect level (NOAEL) for oncogenicity was 350 ppm in rats.

The NOAEL for toxicity for male and female rats is 25 ppm based on effects on body weight, clinical chemistry parameters, organ weight parameters, and/or morphological changes at 100 and 350 ppm.