2,2-dichloro-1,1,1-trifluoroethane

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in compliance with OECD TG

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2-dichloro-1,1,1-trifluoroethane

EC Number:

206-190-3

EC Name:

2,2-dichloro-1,1,1-trifluoroethane

Cas Number:

306-83-2

Molecular formula:

C2HCl2F3

IUPAC Name:

2,2-dichloro-1,1,1-trifluoroethane

Constituent 2

Reference substance name:

Ethane, 2,2-dichloro-1,1,1-trifluoro

IUPAC Name:

Ethane, 2,2-dichloro-1,1,1-trifluoro

Details on test material:

- Name of test material: HCFC-123 (1,1-dichloro-2,2,2-trifluoroethane)
- Physical state: Colorless liquid
- Analytical purity: 99.8%
- Supplied by Allied-Signal Inc.
- Analytical purity of test material was determined by gas chromatography by Allied-Signal Inc.

Test animals

Species:

rat

Strain:

other: Crl:CD®BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: approximately 41 days
- Weight at study initiation: Males: 33.8-61.7 grams; Females: 23.6-49.8 grams.
- Housing: Rats were housed individually in stainless steel, wire-mesh cages suspended above Upjohn Deotized Animal Cage Boards or R-2 Reemay-backed cage boards.
- Diet: ad libitum - Purina Certified Rodent Chow #5002 (chunk)
- Water: ad libitum

BASIS FOR SPECIES SELECTION
- The Crl:CDBR rat was selected on the bases of extensive experience with this strain and its suitability with respect to hardiness, longevity, sensitivity, and low incidence of spontaneous diseases.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2
- Humidity (%): 55 +/- 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: no data

Details on inhalation exposure:

Vapor was generated by evaporating the liquid sample in a stream of metered air. The vapor was diluted with filtered air to the desired concentrations for each of the three test chamber. Filtered air alone was metered in an identical manner into the control chambers. During exposure, the relative humidity of the chamber air, the chamber temperature, and airflow rates were measured at approximately five-minute intervals. Chamber oxygen content was determined at least twice daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber samples of atmospheric HCFC-123 were taken from the animal breathing zone at approximately 30 minute intervals through Teflon lines leading to an automated gas chromatograph situated in a room adjacent to the inhalation chambers. Samples were analyzed with a Hewlett-Packard gas chromatograph, equipped with a flame ionizer detector. Chamber concentrations were determined by comparing the chamber sample GC response with that obtained from standard samples prepared daily in 50 liter gas bags.

Duration of treatment / exposure:

2 years

Frequency of treatment:

6 h/day, 5 days/week (weekends and holidays excluded)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 ppm
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 ppm
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
5000 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

80/sex/dose

Control animals:

yes, sham-exposed

Details on study design:

Two year mean concentrations for the 0, 300, 1000, and 5000 ppm dose groups were 0 ppm, 300 ppm, 1002 ppm, and 4997 ppm respectively.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Cage-side examinations to detect moribund or dead rats and abnormal behavior and appearance among rats were conducted at least once daily throughout the study. Moribund rats were sacrificed. Moribund and dead rats were given a pathological examination. At every weighing, each rat was individually handled and examined for abnormal behavior and appearance.

BODY WEIGHT: Yes
- Time schedule for examinations: Once per week during the first three months and once every other week for the remainder of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE : The amount of food consumed by each group over each weighing interval was determined throughout the study. For the first three months of the study, the amount of food consumed over a one week period was divided by the weekly body weight data. For the remaining 21 months of the study, the amount of food consumed over a two week period was divided by the body weight determined every other week. These data were used to calculate mean individual daily food consumption and food efficiency.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Three ophthalmological examinations were conducted during the study by a veterinary ophthalmologist. At least one hour before each examination, one or two drops of 1% atropine sulfate solution were placed in each eye of every rat. Both eyes were examined by focal illumination and indirect ophthalmoscopy. The examinations were conducted under subdued lighting. The initial examination was performed during the pretest period, on test day -10, on all rats recieved for the study. One male rat and one female rat were excluded from consideration for use on the study when the examination revealed pre-existing ophthalmological abnormalities.The other two examinations were performed prior to the scheduled one-year sacrifice on test days 355 and prior to the 24-month sacrifice on test day 698.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 6, 12, 18, and 24 months after initiation of the study.
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes
- How many animals: 10 per group
- Parameters examined: Erythrocyte, leukocyte, differential leukocyte, and platelet counts; hemoglobin; hematocrit; mean corpuscular hemoglobin; mean corpuscular volume; and mean corpuscular hemoglobin concentration.
-Blood smears for reticulocyte counts were prepared from each rat at each sampling time.
-Bone marrow smears were prepared from all rats at the 12-month interim and 24-month terminal sacrifices.
-Blood smears were prepared from all rats that were sacrificed in extremis and sacrificed by design.
-Blood samples for hematological analyses were collected from the orbital sinus of each rat while the rat was under light carbon dioxide anesthesia.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinical laboratory evaluations were conducted approximately 6, 12, 18, and 24 months after initiation of the study.
- Animals fasted: Yes. However, at the six-month clinical evaluation ten rats from each group were not fasted and bled to determine the effects of fasting.
- How many animals: 10 per group were randomly selected for the first evaluation at 6-months, and the same ten rats per group were used for the 12-month evaluation and were designated for the interim sacrifice on test day 367 (males) and 368 (females). Ten rats from each group were randomly selected for the 18-month clinical evaluation and ten rats from each group were randomly selected for the 24-month clinical evaluation.
- Parameters examined: Gamma glutamyl transferase, alkaline phosphatase, alanine aminotransferase, aspirate aminotransferase, lactate dehydrogenase, and creatine kinase activities, and concentrations of blood urea nitrogen, total serum protein, albumin, globulin (calculated), creatinine, cholesterol, triglycerides, glucose, calcium, sodium, potassium, phosphate, chloride, and total bilirubin.
- Prior to the 18-month clinical evaluation, six rats per sex from the control group and the high-concentration group were fasted for approximately 16 hours and blood samples were collected. Serum triglyceride concentration was measured by the standard clinical chemistry method (automated colorimetric assay) and by an alternative method (thin layer chromatography and high pressure liquid chromatography).
-Blood samples for measurement of plasma fluoride concentration were collected at necropsy from all rats selected for the 12-month interim sacrifice and the surviving rats for the 24-month terminal sacrifice.
-Blood samples for clinical chemical measurements were collected from the orbital sinus of each rat while the rat was under light carbon dioxide anesthesia.

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes for overnight urine collection.
- Animals fasted: Yes
- Parameters checked: volume, pH, osmolality, fluoride ion, and semi-quantitative 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:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

All rats that were found dead, accidentally killed, and were sacraficed were necropsied. After approximately 12 months on test day 367 for males and 368 for females, the ten rats per group designated for the 12-month clinical evaluation were sacrificed and necropsied. All rats surviving the 24-month test period were sacrificed and necropsied on test days 736 and 737 for males and test days 738 and 739 for females. The order of sacrifice for both pathological evaluations was random among all groups within a sex.

The lungs, brain, heart, liver, spleen, kidneys, ovaries, adrenals, and testes were weighed wet at necropsy. Organ weight/final body weight ratios were calculated.

Tissues for pathology (collected from all rats):  bone marrow, skin, lymph nodes (mandibular and mesenteric), spleen, thymus, aorta (thoracic), heart, trachea, lungs, nose (4 cross sections), larynx, pharynx, salivary glands, esophagus, stomach, liver, pancreas, small intestine (duodenum, jejunum, and ileum), large intestine (cecum, colon, and rectum), tongue, kidneys, bladder, pituitary, thyroid-parathyroid, adrenals, prostate, testes, epididymides, seminal vesicles, mammary gland, ovaries, uterus, vagina, brain (includes sections of medulla/pons, cerebellar cortex, cerebral cortex), spinal cord (3 levels), peripheral nerve (sciatic), muscle (thigh), bone (femur, including joint, and sternum), eyes, exorbital lacrimal glands, harderian glands, and all gross lesions. Collected tissues were fixed in 10% neutral buffered formalin (except testes, epididymides, eyes, skin, mammary gland and bone which were fixed in Bouin's solution).

All tissues were processed to the block stage. All tissues collected from rats in the high-concentration groups, control groups, and from rats that were found dead (tissue integrity permitting) or were sacrificed, were further processed to slides, stained with hematoxylin and eosin, and examined microscopically. The nose, lungs, liver, kidneys, and all gross lesions, from rats in the low- and intermediate-concentration groups were also processed to slides and examined microscopically. Pancreas and eyes in males and females, and testes in males were also examined in all rats that died or were sacrificed by design after the 12-month sacrifice.

Two additional samples of liver were collected from each rat at the 12-month sacrifice. One of the two samples was frozen in liquid nitrogen and stored frozen for possible determination of peroxisome proliferation. The second sample was fixed by immersion in a mixture of glutaraldehyde (2.5%) and paraformaldehyde (2%) in a 0.1M phosphate buffer; samples were then refrigerated. The first five rats from each group designated for the 12-month sacrifice were also evaluated for cell proliferation.

Bone marrow smears were prepared from all rats at the 12-month interim and 24-month terminal sacrifices.

Other examinations:

Determination of peroxisome proliferation: Liver samples were collected from designated rats for the 12 month sacrifice at the time of necropsy, frozen, and maintained at -85°C until analyzed. Samples were analyzed for peroxisomal Beta-oxidation activity of fatty acids by the method of Lazarow. Each sample was thawed, homogenized separately in a tris-and-sucrose buffer solution, followed by centrifugation to remove connective tissue, nuclei, and intact cells. The remaining supernatant from each sample was centrifuged, and the pellet containing the peroxisomes was retained. Each pellet was resuspended in a buffer and incubated with the reaction mixture containing [14C]palmitoyl-CoA. The reaction was suspended by transferring the reaction vessel to an ice bath and adding perchloric acid. Following centrifugation, the radioactivity of the supernatant (formation of acid-soluble [14C]acetyl-CoA) was measured by liquid scintillation spectometry.

Determination of Cell Proliferation: On the day of the 12-month sacrifice, five rats per sex per group were injected intraperitoneally with 100 mg/kg 5-bromo-2'-deoxuridine (BrdU). The order of injection was random among all groups within a sex. Approximately two hours after BrdU injection, the rats were sacrificed by pentobarbital anesthesia and exsanguination, and necropsied. Liver tissue from the control and high-concentration groups was processed for immunohistochemical analysis of BrdU incorporation and evaluated for cell proliferation. Tissues collected from rats in the control and high-concentration groups were evaluated for cell proliferation (cells in S-phase). Approximately 1,000 cells were counted from each tissue and evaluated. The number of cells in S-phase in each tissue was expressed as a percentage of the number of cells counted. This value was termed the "labeling index."

Statistics:

Body weights, body weight gains, organ weights, clinical laboratory measurements, hepatic Beta-oxidation activity data, and hepatic labeling index data were analyzed by a one-way analysis of variance. When the test for differences among test group means (F-test) was significant, pairwise comparisons between test and control groups were made with the Dunnett’s test. Bartlett’s test for homogeneity of variance was also performed on the clinical chemistry data. When the results of the Bartlett’s test were significant (p
Incidence of clinical observations were evaluated by the Fisher’s Exact test with a Bonferroni correction and the Cochran-Armitage test for trend. Survival among groups was evaluated with the Cochran-Armitage test for trend and the Fisher’s Exact test with a Bonferroni correction. Incidences of gross lesions, microscopic lesions, and tumors were evaluated by the Fisher’s Exact test and the Cochran-Armitage test for trend. Since there was an apparent compound-related effect on survival at the end of the two-year study, additional statistical analyses were conducted on selected tumors to adjust for the difference in survival. The incidences of hepatic hepatocellular adenomas, hepatocellular carcinomas, pancreatic acinar cell adenomas, pancreatic adenocarcinomas, pancreatic neurofibrosarcomas, testicular interstitial cell adenomas, and hepatic cholangiofibromas were analyzed by Cochran-Armitage test for trend, Bickis-Krewski Exact (BK-Exact) trend test, Peto’s Method, and Logistic Score test. The BK-Exact test was applied when the incidence of a lesion was 0% in 3 of the 4 groups analyzed. The logistic score test and Peto’s method are used for mortality adjusted statistical analyses. Except for Bartlett’s test, all other significance was judged at alpha = 0.05.

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):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

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:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY:
During exposure, rats visible through the window of the chambers were observed at approximately hourly intervals. During the observation interval, responsiveness to sound was assessed by tapping on the glass with a coin. During the exposure period, 5,000 ppm rats were generally less responsive to auditory stimuli compared to control rats. However, by the time residual test material was exhausted from the chamber and the rats returned to the animal room, the responsiveness of rats exposed to 5,000 ppm was similar to control. Males exposed to 5,000 ppm and females exposed to 1,000 or 5,000 ppm had a significantly increased incidence of stained fur. No morphological changes were correlated with this observation. In addition, 1,000 and 5,000 ppm females had a decreased incidence of skin sores. Females exposed to 5,000 ppm also had a higher incidence of wet inguen and wet perineum. A significantly lower incidence of colored discharge from the eye also occurred in 5,000 ppm females. Morphological correlates for these observations were not observed. Males exposed to 5,000 ppm had a significantly higher incidence of grossly observable masses in the inguinal area. However, the total number of male rats with masses was similar for all exposure concentrations. In contrast, females exposed to 1,000 or 5,000 ppm had a significantly lower incidence of masses in the inguinal area. In addition, the incidence of 5,000 ppm females which were noted to have masses was slightly lower compared to control. Although an increased number of inguinal masses was observed in 5,000 ppm males over the course of the study, a correlation with a particular morphological change was not apparent since many of the masses had resolved prior to necropsy. It is possible that some of the inguinal masses may have been transient swelling of the inguinal lymph nodes. The lower incidence of inguinal masses in 5,000 ppm females correlates with the lower incidence of mammary adenomas and carcinomas observed histologically. The lower incidence of mammary tumors may be related to the lower body weight of the 5,000 ppm females since lean rats have been demonstrated to have a lower incidence of neoplasms. The lower incidence of inguinal masses in 1,000 and 5,000 ppm females is considered to be compound related. A statistically significant trend for greater survival was observed in females exposed to 1,000 and 5,000 ppm. Similarly, males exposed to 1,000 or 5,000 ppm had slightly higher survival compared to controls. The increased survival was correlated with a decreased incidence of spontaneous lesions in aged rats (mammary and pituitary tumors in females and glomerulonephropathy and pituitary tumors in males). Increased survival is often observed in conjunction with lower body weight, which correlates with the lower body weight observed at 1,000 and 5,000 ppm for females and 5,000 ppm for males. The increased survival is considered to be compound related.

BODY WEIGHT AND WEIGHT GAIN:
Male and female rats exposed to 5,000 ppm and females exposed to 1,000 ppm had significantly lower body weight compared to control values. Significantly lower body weight of males exposed to 5,000 ppm occurred generally from test day 104 and continued through test day 706. Although not statistically significant, body weight for 5,000 ppm males continued to be lower through the last weigh period on test day 734. For females exposed to 5,000 ppm, significantly lower body weight was generally evident from test day 20 through test day 734. Females exposed to 1,000 ppm also had significantly lower body weight from test day 62 through test day 636 (except for two weeks when the lower body weight was not statistically significant). Although the values were not statistically significant after test day 636, body weight for 1,000 ppm females continued to be lower than control values for the remainder of the study. In addition, female rats exposed to 300 ppm also had significantly lower body weight compared to control values beginning on test day 454 through test day 538. Body weight of 300 ppm females continued to be slightly lower than control values from test day 538 through test day 720; however, the values were not statistically significant.

Mean body weight gain was significantly lower for males exposed to 300, 1,000, or 5,000 ppm HCFC-123 in the first week of the study. Thereafter, sporadic instances of lower and higher body weight gain were observed in males exposed to 300 and 1,000 ppm. Numerous instances of lower body weight gain for 5,000 ppm males occurred throughout the first year of the study. Mean body weight gain was significantly lower in 5,000 ppm males over the intervals 1 - 356 days and 1 - 734 days. Females exposed to 5,000 ppm had numerous instances of lower body weight gain throughout the two-year period. Mean body weight gain was significantly lower in 5,000 ppm females over the intervals 1 - 356 days and 1 - 734 days. Females exposed to 1,000 ppm had lower body weight gain during the interval 1 - 356 days. The lower body weight and lower body weight gain observed in 5,000 ppm males, and 5,000, 1,000, and 300 ppm females is considered to be compound related.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Slightly higher food consumption occurred among male rats exposed to 5,000 ppm through test day 510. Female rats exposed to 5,000 ppm had instances of slightly higher and lower food consumption over the course of the study. However, food efficiency was lower for males and females exposed to 5,000 ppm over the interval of test days 1 - 356. For the interval of test days 356 - 734, food efficiency was similar to control values for all test groups. The lower food efficiency correlates with the lower body weight observed at 5,000 ppm.

CLINICAL CHEMISTRY:
At the 6-, 12-, 18-, and 24-month clinical evaluations, serum triglyceride concentrations in both male and female rats exposed to 300, 1,000, and 5,000 ppm continued to be significantly lower than control values. The greatest decrease in triglyceride concentrations was observed at the 12-month clinical evaluation. Triglycerides were 76, 82, and 98% lower than the 12-month control values for males exposed to 300, 1,000, and 5,000 ppm respectively. For females, triglyceride values were 65, 98, and 100% lower than the 12-month control values for the 300, 1,000, and 5,000 ppm groups, respectively. Although serum triglycerides continued to be significantly lower compared to control values in both males and females at all exposure concentrations at the 18- and 24-month evaluations (except the 300 ppm females at the 24-month evaluation which was biologically significant, but not statistically significant), the difference from control values was less than that observed at the 12-month evaluation which may indicate adaptation, or an age-related decrease in metabolism to a metabolite responsible for the effects on serum triglycerides. In order to eliminate the possibility that the test material or a metabolite interfered with the analysis of triglycerides, additional blood samples were collected from control and 5,000 ppm males and females at approximately 18 months, and analyzed by high pressure liquid chromatography (HPLC). The HPLC method measures the concentration of triglyceride specific fatty acids (lauric, palmitic, stearic, oleic, linoleic, and linolenic fatty acids). Palmitic, oleic, and stearic acids were the most prevalent fatty acids, comprising approximately 96% of the fatty acids in the male serum triglycerides and 90% of the fatty acids in female serum triglycerides. Palmitic acid was the most prevalent of the fatty acids, and calculations were made comparing the concentration of triglyceride-derived palmitic acid. By HPLC analysis, male rats exposed to 5,000 ppm had a 75% reduction in triglyceride-derived palmitic acid compared to control values, and females exposed to 5,000 ppm had a 72% reduction in triglyceride-derived palmitic acid. The blood samples were also analyzed by the standard clinical chemistry analysis (colorimetric), which yielded an 81% and 75% reduction in serum triglycerides in 5,000 ppm males and females respectively. These data indicate that the substance or its metabolite(s) do not interfere with the colorimetric analysis for triglyceride analysis in rat serum.

As previously observed at 90 days, males and females exposed to 300, 1,000, and 5,000 ppm had decreased serum glucose at the 6- and 12-month evaluations. At the 18-month evaluation, 1,000 and 5,000 ppm males and 5,000 ppm females had significantly lower glucose compared to control values. However, at the 24-month evaluation, glucose was similar to control values for all exposure concentrations.

Serum albumin was significantly higher in 1,000 and 5,000 ppm males at the 6-, 12-, 18- (5,000 ppm males only), and 24-month evaluations. In addition, serum globulin was significantly decreased in 1,000 and 5,000 ppm males at the 6-, 18-, and 24-month evaluations. Females exposed to 300, 1,000, or 5,000 ppm also had significantly lower serum globulin at the 6- and 24-month evaluations. In addition, 5,000 ppm females had lower serum globulin at the 12-month interval, and at the 18-month evaluation, 1,000 and 5,000 ppm females had lower serum globulin.

In an attempt to further elucidate the effect of the substance on lipid and carbohydrate metabolism, a group of nonfasted rats was evaluated for compound-related changes in clinical chemistry parameters at the 6-month sampling interval. Serum triglycerides were significantly lower in 5,000 ppm nonfasted males and in 300, 1,000, and 5,000 ppm nonfasted females. However, serum glucose was significantly higher compared to control values in 1,000 and 5,000 ppm nonfasted males and in 300, 1,000, and 5,000 ppm nonfasted females. Serum cholesterol was significantly lower in 300, 1,000, and 5,000 ppm nonfasted females. Although significantly lower than control, triglyceride concentrations of nonfasted rats were higher compared to fasted rats. Nonfasted male rats exposed to 1,000 or 5,000 ppm HCFC-123 also had higher serum albumin and lower serum globulin. Nonfasted females exposed to 5,000 ppm also had higher serum albumin, and 1,000 and 5,000 ppm nonfasted females had lower serum globulin. These results suggest a compound-related effect on lipid, protein, and/or carbohydrate metabolism/production.

Plasma fluoride concentrations at the 12-month sacrifice were significantly lower in males exposed to 300, 1,000, or 5,000 ppm; however, this difference is considered to be an incidental finding due to analytical variation since the metabolism of HCFC-123 yields free fluoride ions as demonstrated by its presence in the urine. Plasma fluoride values for males at the 24-month evaluation and for females at the 12- and 24-month evaluations were similar to control values.

HAEMATOLOGY:
Male rats exposed to 300, 1,000, or 5,000 ppm had a significantly lower red blood cell count at the 6-month sampling interval. In addition, 1,000 and 5,000 ppm males had lower hemoglobin and hematocrit. However, these differences were within the range of biological variation, were not observed at the 12-month interval, and therefore, were not considered to be compound related. A slight decrease in the number of neutrophils was observed in 5,000 ppm males at the 24-month evaluation, and in 5,000 ppm females at the 18- and 24-month evaluations. In addition, 1,000 and 5,000 ppm females had a slightly lower number of monocytes at the 24-month evaluation. Other hematology and clinical chemistry parameters with significant differences from control values for both fasted and nonfasted rats were within the normal reference ranges, or were the result of technical difficulties with the assay method (sodium, potassium, chloride) and were considered to be biologically insignificant.


URINALYSIS:
The concentration of urinary fluoride was significantly higher in 1,000 and 5,000 ppm males at the 6-month interval and in 300, 1,000, and 5,000 ppm males at the 12- and 18-month intervals. Similarly, urinary fluoride was higher at the 6-, 12- (except 1,000 ppm which was not statistically significant), and 18-month evaluations for females exposed to 300, 1,000, and 5,000 ppm BCFC-123. At the 24-month evaluation, urinary fluoride was slightly higher for the 1,000 and 5,000 ppm males, and for the 300, 1,000, and 5,000 ppm females; however, the differences from control values were not statistically significant. Urine volume was slightly increased and osmolality slightly decreased for 1,000, and 5,000 ppm males at the 6-month evaluation, and in 300, 1,000, and 5,000 ppm males at the 12-month evaluation. Similarly, 300, 1,000, and 5,000 ppm females had higher urine volume and slightly lower urine osmolality at the 6-month interval. The differences in urine volume and osmolality may be a secondary effect of the test material related to the higher concentration of fluoride ion in the urine. The incidence of female rats observed to have ketone bodies in the urine was higher in the 1,000 and 5,000 ppm groups at 6 months and in the 5,000 ppm group at 18 months. Similarly, there was a higher incidence of 1,000 and 5,000 ppm males with urinary ketone bodies at the 12- and 18-month evaluations. This observation may be correlated with the differences in serum glucose and triglycerides.

ORGAN WEIGHTS:
At the 12-month interim sacrifice, male and female rats exposed to 5,000 ppm had significantly higher mean relative liver weight. In addition, 5,000 ppm males had significantly higher relative adrenal weights, higher relative lung weights, and lower absolute kidney weights which were considered to be the result of lower body weight. At the 24-month sacrifice, 5,000 ppm males had increased mean relative liver weight and decreased absolute kidney weight. Females exposed to 5,000 ppm had decreased mean absolute kidney weight, lung weight, and heart weight. In addition, 1,000 ppm females had decreased absolute kidney weight. The decrease in kidney weight was attributed to the reduction in the incidence and severity of spontaneously occurring glomerulonephropathy. The decreases in lung and heart weights of the 5,000 ppm females were secondary to their lower body weight. The higher relative liver weights of the 5,000 ppm males were considered to be compound related and correlate with the increased incidence of gross and microscopic morphological changes in the liver.

GROSS PATHOLOGY:
No compound-related gross or microscopic morphological changes were observed at any exposure concentration in either males or females at the 12-month sacrifice. Female rats exposed to 5,000 ppm had a higher incidence of grossly observed ovarian cysts; however, this is a spurious finding since the microscopic incidence of ovarian cysts was similar to control. The lack of compound-related morphological changes at this time point is consistent with previous studies in which compound-related morphological changes were not observed.

At the 24-month sacrifice an increased incidence of compound-related gross morphological changes were observed. Male rats exposed to 5,000 ppm had an increased incidence of grossly observed large and discolored livers. These effects correlated with the increased incidence of microscopically observed foci of cellular alteration and hepatocellular adenomas. Female rats exposed to 5,000 ppm had an increased incidence of grossly observed hepatic masses and a slightly higher incidence of liver discoloration which correlated with microscopic observations of hepatocellular adenomas and hepatic foci of cellular alteration. In addition, one or more test groups had decreased incidences of spontaneous age-related lesions which included mesenteric nodules, skin nodules and masses, large or deformed kidneys, large parathyroids, ulcers and/or erosions on limbs, periocular chromodacryorrhea, large spleens, large mammary glands, brain compression, and pituitary masses. The decrease in age-related spontaneous lesions was considered to be related to the lower mean body weights and/or lower serum lipid levels.

Compound-related increases in the incidences of several microscopic lesions were observed in 300, 1,000, and 5,000 ppm male and female rats at the terminal sacrifice. Male rats exposed to 5,000 ppm and females exposed to 300, 1,000, and 5,000 ppm had an increased incidence of hepatocellular adenomas (the incidence was not statistically significant for the 1,000 ppm females). However, when the incidence of total hepatocellular adenomas in males was analyzed by mortality adjusted statistics, it was not statistically significant. Analyses of the incidence of hepatocellular adenomas in females by mortality adjusted statistics did not alter the statistical significance of the data. Basophilic foci of cellular alteration were also significantly increased in both males and females at 1,000, and 5,000 ppm. Cholangiofibromas and cholangiofibrosis were increased in 5,000 ppm females. An increased incidence of other foci of cellular alteration (which includes clear cell, eosinophilic cell, fatty, or mixed) was also observed in one or more treatment groups of both sexes. Males exposed to 5,000 ppm also had a compound-related increased incidence of hepatic focal necrosis. The incidence of hepatic centrilobular fatty change was also increased in 5,000 ppm males and females. Females exposed to 5,000 ppm also had an increased incidence of sinusoidal ectasia. In addition, 1,000 ppm and 5,000 ppm males and 5,000 ppm females had an increased incidence of hepatic cystic degeneration.

At the terminal sacrifice, 300, 1,000, and 5,000 ppm males had a compound-related increase in the incidence of pancreatic acinar cell adenomas (incidence at 300 ppm was not statistically significant). Although the incidence was not statistically significant, 300 and 5,000 ppm females also had a slightly higher incidence of pancreatic acinar cell adenomas. The incidence of focal acinar cell hyperplasia was also increased in 1,000, and 5,000 ppm males and females and slightly increased (not statistically significant) in 300 ppm females. Since acinar cell adenomas are a continuum of hyperplasia, the adenomas observed in females were also considered to be compound-related. Analyses of the incidence of pancreatic acinar cell adenomas by mortality adjusted statistics did not alter the statistical significance of the data. However, the incidences of acinar cell atrophy and polyarteritis were significantly lower for the 300, 1,000, and 5,000 ppm males.

Males had a compound-related increased incidence of testicular interstitial cell adenomas at 300, 1,000 (not statistically significant), and 5,000 ppm. Focal interstitial cell hyperplasia in the testes was also increased at 1,000 and 5,000 ppm. Application of two types of mortality adjusted statistical analyses to the incidence of total interstitial cell adenomas yielded p-values of 0.059 (Peto analysis) and 0.043 (Logistic Score) indicating equivocal statistical significance. Thus, when the time course of the tumors is taken into account, the results are not as significant as indicated by the analysis of the lifetime incidence. In addition, 5,000 ppm males had an increased incidence of unilateral tubular atrophy which was the result of tumor induced pressure atrophy. Bilateral tubular atrophy was significantly decreased in 5,000 ppm males, and testicular polyarteritis was decreased in all test groups.

At the terminal sacrifice, the incidence of diffuse retinal atrophy was increased in males and females at all exposure concentrations; however this was considered to be an indirect compound-related effect due to several factors. This lesion is spontaneous in aged rats, and may be modulated by light, sex, pituitary and ovarian hormones, and decreased levels of retinal taurine. In addition, the incidence is widely variable. Therefore, the lesion was considered to be an indirect, compound related effect since there was a lack of a clear dose response in females, variability in survival between treated and control groups, and nutritional differences (as indicated by reduced body weight) between treated and control groups.

Males exposed to 5,000 ppm had an increased incidence of focal degeneration of the adrenal cortex; however, it was minimal to mild in severity and was considered to be a compound-related effect of minimal or no biological significance. In the 5,000 ppm females, the incidence of ovarian cysts was significantly increased; however, since they were minimal to mild in severity, these cysts were considered to be of minimal or no biological significance.

At the terminal sacrifice, the incidences of several lesions were decreased in one or more groups of male and female rats exposed to the substance and were considered to be beneficial effects related to decreased body weight and/or lower serum lipid levels. Males exposed to 300, 1,000, or 5,000 ppm had decreased incidences of fibrous osteodystrophy of the nose and chronic glomerulonephropathy. In addition, the incidence of uremic gastritis, mineralization of coronary vessels, polyarteritis in the stomach, fatty change in the adrenal cortex, skin fibromas, inflammation of the limbs/tail/footpads, and fibrous osteodystrophy of the femur and sternum was decreased in 5,000 ppm males. Females exposed to 300, 1,000, and 5,000 ppm had a decreased incidence of chronic glomerulonephropathy, and 1,000 and 5,000 ppm females had a decreased incidence of limbltaillfootpad inflammation. In addition, 5,000 ppm females had a decreased incidence of mammary fibroadenomas, mammary adenocarcinomas, hyperplasialdilation of mammary gland ducts, and focal liver necrosis (not statistically significant).

OTHER FINDINGS:
Hepatic Peroxisome Proliferation: Males exposed to 300, 1,000, or 5,000 ppm for one year had significantly (2.3, 3.1, or 4.0 fold respectively) enzyme activity compared to controls. Females exposed to 1,000 or 5,000 ppm had significantly higher (1.7 and 3.1 fold respectively) enzyme activity compared to controls. These data indicate that exposure to 300, 1,000, or 5,000 ppm HCFC-123 caused induction of hepatic peroxisome proliferation in both male and female rats.

Hepatic Cell Proliferation: Compound-related differences in the rate of cell proliferation (labeling index) of the liver were not observed at any exposure concentration at the 12-month sacrifice. These results suggest that HCFC-123 did not induce an increase in regenerative repair of the liver that was evident at the 12-month sacrifice. These results are consistent with the microscopic evaluation of the liver at the 12-month sacrifice.

Ophthalmological Evaluations:
After approximately 12 months, a second ophthalmoscopic examination was conducted on all surviving rats. The incidences of grossly observable abnormalities in male rats were 1%, 7%, 7%, and 5% in the 0, 300, 1,000, and 5,000 ppm groups respectively. The incidences of abnormalities in female rats were 0%, 1%, 3%, and 5% in the 0, 300, 1,000, and 5,000 ppm groups respectively. At the 12-month examination, all abnormalities were considered to be spontaneous for this strain. The third ophthalmoscopic examination was conducted on all surviving rats prior to the final sacrifice. The incidences of grossly observable abnormalities in males exposed to 0, 300, 1,000, and 5,000 ppm were 9%, 12%, 9%, and 11% respectively. The incidences of grossly observable abnormalities in females were 9%, 34%, 18%, and 4% respectively. The ophthalmologist reported that a compound-related effect on ocular tissue was not evident from gross examination. However, histological evaluation of the retina indicated a significantly increased incidence of diffuse retinal atrophy in both males and females at all exposure concentrations.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Mortality and clinical signs: Females exposed to 1,000 ppm or 5,000 ppm also had significantly greater survival, and 1,000 and 5,000 ppm males had slightly greater survival compared to controls. The increased survival can be attributed to the beneficial effects of lower body weight and serum triglycerides/lipids. Ophtalmoscopic examination did not reveal any treatment-related abnormality.

Stained fur, wet inguen and wet perineum were observed in males exposed to 5000 ppm.

Bodyweight: Male and female rats exposed to 5,000 ppm and females exposed to 1,000 ppm had lower body weight and body weight gain.

Blood Chemistry: Serum triglyceride concentrations were significantly decreased compared to control values at all exposure concentrations, and in both sexes. Serum glucose concentrations were significantly decreased at all exposure levels at 6- and 12 -month evaluations. By 24 -month evaluation, glucose concentrations were similar to controls at all exposure concentrations. Serum cholesterol was also significantly lower in females exposed to 300, 1,000, or 5,000 ppm and in males exposed to 5,000 ppm. Serum albumin was significantly higher in males exposed to 1000 and 5000 ppm and serum globulin was significantly lower in the same groups.

Organ weight: at the 12 -month sacrifice, increased liver weight was observed at 5000 ppm in both sexes. at 24 -month examination increased liver and kidney weight were observed at 5000 ppm in both sexes.

Gross pathology: at 24 -month, large and discoloured livers were observed in males exposed to 5000 ppm and liver masses in 5000 ppm females.

Histopathology:

Non-hyperplastic changes: Basophilic alterations were observed in liver in both sexes at all exposure levels. Furthermore, males showed a treatment-related increased incidence in focal hepatic focal necrosis. Females exposed to 5000 ppm showed an increased incidence of sinusoidal ectasia. males exposed to 1000 and 5000 ppm and females at 5000 pm showed an increased incidence of hepatic cystic degeneration, and hepatic centrilobular fatty change was observed in both sexes at 5000 ppm.

Neoplastic changes: At the 24 -month sacrifice, there were compound-related increases in incidences of benign hepatocellular adenomas in 5,000 ppm dose males and in all test groups of females, in benign hepatic cholangiofibromas in 5,000 ppm females, in benign pancreatic acinar cell adenomas in all test groups of males, and in benign testicular tumors in all test groups of males. Acinar cell hyperplasia of the pancreas was increased in the 1,000 and 5,000 ppm males and females. Since pancreatic acinar cell adenomas are a continuum of acinar cell hyperplasia, the incidence of acinar cell adenomas and hyperplasia in all test groups of females is also considered to be compound related.

Enzymetic activity: Males exposed to 300, 1,000, or 5,000 ppm and females exposed to 1,000 or 5,000 ppm HCFC-123 had higher hepatic Beta-oxidation activity compared to controls. The higher enzyme activity indicates an induction of hepatic peroxisome proliferation, which may be related to the formation of liver tumors. Hepatic cell proliferation may accompany an increase in hepatic Beta-oxidation activity; however, a sustained increase in cell proliferation was not evident at the 12 -month interim sacrifice at any exposure concentration.

A no-observable-effect level was not achieved for this study based on the effects in clinical chemistry parameters at all concentrations, lower body weight and body weight gain at 300, 1,000, and 5,000 ppm, organ weight changes at 5,000 ppm, increased incidence of neoplastic and non-neoplastic morphological changes, and higher hepatic peroxisomal Beta-oxidation activity at all concentrations.

Applicant's summary and conclusion

Conclusions:

A no-observable-effect level was not achieved for this study based on the effects in clinical chemistry parameters at all concentrations, lower body weight and body weight gain at 300, 1000 and 5000 ppm, organ weight changes at 5000 ppm, increased incidence of neoplastic and non-neoplastic morphological changes, and higher hepatic peroxisomal beta-oxidation activity at all concentrations.